Encyclopedic Reference of Immunotoxicology - Part Lextras.springer.com/.../Itox-2005-04-04_Part13.pdf · A non-heme β-globulin that acts as an iron-transport-ing protein. 3 Respiratory

L

Lactate Dehydrogenase (LDH) and/orAdenylate Kinase (AK) Leakage

Lactate dehydrogenase (LDH) and adenylate kinase(AK) are both enzymes which are essential for normalcell metabolism. Upon cell damage these proteins arereleased from the cytosol into the cell culture medium.There, the activity of these enzymes is detected withspecial measurement procedures and can finally beused as non-destructive parameters for the determina-tion of cytotoxicity.

3Three-Dimensional Human Skin/Epidermal Modelsand Organotypic Human and Murine Skin ExplantSystems

Lactoferrin

A non-heme β-globulin that acts as an iron-transport-ing protein.

3Respiratory Infections

Lamina propria

Layer of connective tissue between epithelium of theintestines and the underlying muscle layer of the epi-thelium—the muscularis mucosae. Site of lymphocyteextravasation and primary infections.

3Immunotoxic Agents into the Body, Entry of

3Mucosa-Associated Lymphoid Tissue

Langerhans Cells

Bone-marrow derived epidermal cells with a dendriticmorphology expressing a CD1 marker in humans, andcontaining a cytoplasmic organelle called the Birbeckgranule. They express class II MHC antigen and arethe principal antigen-presenting cells of the skin,which emigrate to local lymph nodes to become den-

dritic cells. They are very active in presenting antigento T cells.

3Skin, Contribution to Immunity

3Local Lymph Node Assay

3Local Lymph Node Assay (IMDS), Modifications

Large Granular Lymphocyte

A lymphocyte lineage characterized by large size anddistinct cytoplasmic granules. This cell usually isthought to represent a natural killer cell.

3Lymphocytes

Laser Capture Microdissection (LCM)

A technique for isolating single cells from tissuesusing a laser, yielding a pure population of cells forthe analysis of molecular function. LCM is especiallyuseful for removing tumor cells from surrounding tis-sue.

3Polymerase Chain Reaction (PCR)

Lateral Line

A sensory organ of fish perceiving low frequency vi-brations. The organ consists of a canal running downthe side of the body that.communicates to the outsidevia pored scales.

3Fish Immune System

LDA

3Limiting Dilution Analysis

Lectins

A family of carbohydrate-binding proteins found inanimals and plants. Different types of lectins recognizespecific carbohydrate ligands.

3Cell Adhesion Molecules

3Mitogen-Stimulated Lymphocyte Response

Leukemia

Leigh Ann Burns-Naas

Pfizer Global Research & Development10777 Science Center Dr.San Diego, CA 92121USA

Synonyms

Acute lymphocytic leukemia, acute myelogenous leu-kemia, chronic lymphocytic leukemia, chronic myel-ogenous leukemia, biphenotypic leukemia; prolym-phocytic leukemia, hairy cell leukemia

Definition

Leukemia is a heterogeneous group of malignancies ofthe bone marrow and blood that affect individuals ofall ages. As a result of hematopoetic dysregulation,these cancers can result in conditions such as 3ane-mia, 3thrombocytopenia, and 3neutropenia, and in-crease the risk for 3opportunistic infection in affectedindividuals. Though there are numerous types of leu-kemias, these disorders can be divided broadly intofour major types:* acute lymphocytic leukemia (ALL)* acute myelogenous leukemia (AML)* chronic lymphocytic leukemia (CLL)* chronic myelogenous leukemia (CML).

ALL is the most common acute form in children,AML being the predominant acute form in adults.Chronic leukemias are uncommon in childhood andteenage years, but increase in incidence with increas-ing age. Biphenotypic leukemias have morphologicalcharacteristics of both myelogenous and lymphoblas-tic leukemias. Prolymphocytic and hairy cell leuke-mias are rare.

Characteristics

Acute leukemia is a rapidly progressing disease re-flecting an imbalance between proliferation and differ-entiation that results in the accumulation of immaturecells in the bone marrow and in the blood. Over time,these immature cells accumulate to an extent that can

suppress the normal hematopoetic mechanisms, and asa result anemia, thrombocytopenia, and neutropeniacan occur. Chronic leukemias represent an expandedpopulation of cells that proliferate, but also can differ-entiate. Chronic leukemias generally progress moreslowly and allow greater numbers of more mature,functional cells to be made. Leukemias are diagnosedvia evaluation of the blood and often the bone marrow.In the acute forms of leukemia, red cell counts andplatelet counts are decreased and there is an increasein the number of abnormal white cells (leukemicblasts) in the blood. This is confirmed by examinationof the marrow, which almost always demonstrates thepresence leukemia cells. 3Immunophenotyping and

3cytogenetic analyses can be used to confirm the dis-ease. AML has several diagnostic subtypes defined bythe identity and proportions of abnormal cells. Thisagain reflects the heterogeneous nature of the disease.In CLL, there is also an increase in the number ofwhite cells (lymphocytes) in the blood and bone mar-row. These malignant cells are most oftenB lymphocytes (approximately 95% of CLL cases),but may also be T lymphocytes or natural killer(NK) cells. Low platelet counts and low red cellcounts in the blood may be observed, but these cellsare usually only slightly decreased in the early stage ofthe illness. Abnormal expression and regulation ofgenes (e.g. bcl2) that control normal cell death (apop-tosis) appears to be malignant mechanism in the ma-jority of CLL cases. Individuals with CLL may alsohave other immunological abnormalities, including au-toimmune hemolytic anemia or immune-mediatedthrombocytopenia.Blood from patients with CML exhibits a few blastsand promyelocytes and a larger number of maturingand fully matured myelocytes and neutrophils. 3Cy-togenetic analysis of bone marrow, and polymerasechain reaction (PCR) or fluorescence in situ hybridiza-tion (FISH) analysis of blood or bone marrow from anindividual with CML would reveal the presence of anabnormal reciprocal translocation between pieces ofchromosomes 9 and 22 (the Philadelphia chromo-some). This chromosomal abnormality is characteristicof CML and results in expression of a chimeric onco-gene, bcr-abl, that encodes a protein, tyrosine kinasecontrolling a variety of signaling pathways involved incell proliferation.There are other less common forms (or variants) ofchronic leukemias. All result from a malignant trans-formation of a lymphocyte and the accumulation ofthese cells primarily in the marrow, blood, and thespleen. Large granular lymphocytic (LGL) leukemiais another type of chronic leukemia. The immunophe-notype is either a T cell or an NK cell, but not a B cell.Unlike cells in other types of chronic lymphocytic leu-kemia, LGL leukemia is characterized by larger lym-

374 Lectins

phocytes containing granules. Hairy cell leukemia is achronic leukemia of lymphocytes, usually of a B cell.The malignant cells are larger than regular lympho-cytes and have irregular, hair-like projections onthem. These cells infiltrate the bone marrow and redpulp of the spleen. Prolymphocytic leukemia is a vari-ant of CLL that displays larger than normal, mature-looking lymphocytes. Prolymphocytic leukemia dif-fers from CLL by the presence of intense surface im-munoglobulin staining, among other surface markers.The disease progresses more rapidly than the chronicform but more slowly than the acute form.

Preclinical RelevanceAlterations in 3cell-mediated immunity (CMI) areoften considered to facilitate the development of neo-plastic disease. This arm of the immune system isinvolved in controlling spontaneous tumors and infec-tions among other things. The destruction of tumorcells can result from the cytolytic action of specific(cytotoxic) T lymphocytes (CTL), macrophages, andNK cells. These cells recognize specific antigens ontumor or virally infected cells and cause their death byone of several mechanisms considered here.In cell-mediated cytotoxicity, the effector cell (CTL orNK) binds in a specific manner to the target cell. CTLrecognize either foreign major histocompatabilitycomplex (MHC) class I on the surface of allogeneiccells, or antigen in association with self MHC class I(e.g. viral particles), while NK cell recognition of tar-get cells involves the binding of the Fc portion ofantigen-specific antibody coating a target cell to theNK cell via its Fc receptors. This NK cell mechanismof killing is also referred to as antibody-dependentcellular cytotoxicity (ADCC). Once the CTL or NKcells interact with the target cell, they undergo cyto-plasmic reorientation so that cytolytic granules are or-iented along the side of the effector that is bound to thetarget. The effector cell then releases the contents ofthese granules onto the target cell. The target cell maybe damaged by the perforins or enzymatic contents ofthe cytolytic granules. In addition, the target is inducedto undergo programmed cell death (apoptosis). Once ithas degranulated, the CTL or NK cell can release thedying target and move on to kill other target cells.The role of the macrophage in cell-mediated cytotox-icity involves its activation by T cell-derived cytokine(e.g. IFN-γ) and subsequent recognition of comple-ment-coated target cells via complement receptorspresent on the surface of the macrophage. The resultis enhanced phagocytic ability and the synthesis andrelease of hydrogen peroxide, nitric oxide, proteases,and tumor necrosis factor, all of which serve cytolyticfunctions. Macrophages may also kill tumor or in-fected cells via ADCC in a manner similar to thatdescribed for NK cells.

Drugs and non-drug chemicals and agents that altercell-mediated immunity have the potential to causean increased risk of opportunistic infections (e.g.viral, bacterial, parasitic) and development of neoplas-tic disease. By evaluating such things as the ability ofthe immune system to recognize and destroy tumorcells such as the P815 mastocytoma (used in theCTL assay) or YAC-1 cell (used in the NK assay) aswell as evaluating proliferative ability to mitogens,cytokines, or allogeneic (foreign; non-self) cells, andevaluation of allograft rejection, immunotoxicity test-ing in rodents has identified many agents capable ofcausing suppression of cell-mediated immunity. A fewof these agents are considered below.Therapeutic agents have been developed that specifi-cally inhibit several immune endpoints. These drugsare often used to treat symptoms associated with au-toimmunity, in transplantation to prevent immune-mediated graft rejection, or to treat individuals withsignificant hypersensitivity responses. Cyclophospha-mide is the prototypical member of a class of drugknown as alkylating agents. Experimentally, cyclo-phosphamide is often used as a positive immunosup-pressive control in immunotoxicology studies becauseit can suppress both humoral immunity and cell-mediated immune responses. Cell-mediated immuneactivities that are suppressed include the delayed hy-persensitivity response (DTH), the cytotoxicT lymphocyte response (CTL), graft vs host (GVH)disease, and the mixed lymphocyte response (MLR).The immunosuppressive action of corticosteroids isalso well known. Following binding to an intracellularreceptor, these agents produce profound lymphoid celldepletion in rodent models and lymphopenia, asso-ciated with decreased monocytes and eosinophils andincreased PMN, in primates and humans. Corticoster-oids induce apoptosis and T cells are particularly sen-sitive. In general, corticosteroids suppress the genera-tion of CTL responses, the MLR, NK activity, andgeneral lymphoproliferation. A large range of cell-mediated immune reactivity is also reduced by aza-thioprine immunosuppressive treatment including theDHR, the MLR, and GVH disease. Although T cellfunctions are the primary targets for this drug, inhibi-tion of NK function and macrophage activities has alsobeen reported. Cyclosporin A is an important immu-nosuppressant that acts preferentially on T cells byinhibition of IL-2 gene transcription and subsequentinhibition of T cell proliferation.In addition to therapeutic chemicals, an immunosup-pressive effect on cell-mediated immunity by occupa-tional and environmental chemicals has also beendemonstrated. By far the most well characterized im-munotoxic effects are those produced by benzene. Inanimal models, benzene induces anemia, lymphocyto-penia, and hypoplastic bone marrow. In addition, it has

Leukemia 375

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been suggested that this myelotoxicity may be a resultof altered differentiative capacity in bone marrow-de-rived lymphoid cells. Benzene exposure alters bothhumoral and cell-mediated immune parameters. Thepolycyclic aromatic hydrocarbons (PAHs) are a ubi-quitous class of environmental contaminants that havebeen studied extensively regarding their immunotoxicpotential. PAHs have been found to be potent immu-nosuppressants, having effects on humoral and cell-mediated immunity, as well as on host resistance toinfection. A great deal of interest in recent years hasbeen drawn to the untoward effects of cigarette smok-ing. Cigarette smoke contains literally hundreds ofchemicals that the smoker inhales, many of whichare known carcinogens, genotoxins, and/or immuno-suppressants. Although the primary site of exposure ofthe immune system to cigarette smoke is the lung,other systemic immune parameters have been shownto be altered in smokers, including decreased serumimmunoglobulin levels and lower NK cell activity.There are numerous examples of drug and non-drugchemicals with the potential to alter immunocompe-tence in a manner that might influence susceptibility tospontaneous (primary or secondary) neoplasms andopportunistic infection, or reactivation of latent patho-gens. This section has only considered a very few ofthese agents, but those which have limited or sufficientevidence for an association between suppression ofimmune function and predilection for leukemia.Clearly, though, other contributing factors (e.g. geno-toxicity) cannot be excluded.

Relevance to HumansUncontrolled proliferation of specific lymphoid cells isthe hallmark of the leukemias. Dysregulation of themechanisms controlling proliferation and differentia-tion of these cells is likely to result from a series oftransformations that affect chromosomes and the inap-propriate expression of 3oncogenes and their abnor-mal proteins. There is evidence that supports a geneticpredisposition to the development of some acute formsof leukemia. This evidence includes an increased in-cidence of leukemias in individuals with congenitaldisorders such as Down’s syndrome, Fanconi’s ane-mia, and ataxia telangiectasia, though a multistep pro-cess to malignancy is still likely to be in place. Butpredisposition resulting from congenital disorders can-not fully explain the overall incidence of leukemiasobserved. Other factors, such as therapeutic interven-tions and environmental influences that cause geneticdamage and/or that suppress the immune system, arelikely to play a role.There is epidemiological evidence supporting an asso-ciation between a variety of other agents and an in-creased risk for developing leukemia. Perhaps themost striking example involves the survivors of the

atomic bomb explosions in Japan. In Hiroshima radi-ation victims were 30 times more likely to developALL, AML, or CLL than non-exposed individuals,and in Nagasaki the incidence of AML was even high-er. Though these represent tremendous doses of radi-ation, there is some evidence that lower doses of io-nizing radiation can also predispose individuals to thedevelopment of leukemia. Higher than expected inci-dences of AML have been also observed in patientsreceiving low doses of therapeutic radiation as well asindividuals working at radium plants. While this ob-served increase in risk is probably due primarily togenetic damage from the radiation, there are indica-tions that some forms of radiation are capable of caus-ing varying degrees of systemic 3immunosuppres-sion, particularly of cell-mediated immunity.Altered immunocompetence has been associated withan overall increased risk for the development of leu-kemia. Individuals with acquired immunodeficiencysyndrome (AIDS) have increased incidences of a va-riety of cancers, including leukemia, most likely as aresult of the loss of the ability of the host to identifyand eradicate neoplastic cells, particularly those in-fected with herpes simplex or Epstein-Barr viruses.A similar effect has been observed in transplant reci-pients receiving chemotherapy with immunosuppres-sive drugs. AML is one of the most common 3sec-ondary neoplasms after chemotherapy and radiationtreatment for other cancers or after autologous trans-plantation. Typical drugs used in these treatments in-clude alkylating agents and immunosuppressants. Thealkylating agents have been clearly shown to suppressthe immune system and sufficient evidence exists re-garding their association with an increased risk of leu-kemia. The risk for developing AML appears to berelated to cumulative doses of the alkylating agent (s)and may be enhanced by the addition of radiationtherapy to the treatment regimen.In addition to therapeutic interventions, exposure tosome environmental chemicals has been associatedwith an increased risk for the development of leuke-mias. Among these, benzene (a widely used solvent) isprobably the most studied and well recognized chem-ical leukemogenic agent. Benzene has been shown tobe immunosuppressive and is associated with an in-creased incidence of AML in exposed individuals.Other industrial or agrochemicals also appear to conferan increased risk for developing acute leukemias in-cluding some pesticides, PAHs, ethylene oxides, andembalming fluids. Additionally, while there is a clearlink between smoking and the development of neo-plastic disease in the lung, it has been suggested thatchronic cigarette smoking may increase the risk fordeveloping AML. Of interest, cigarette smoke con-tains various levels of known immunosuppressive che-

376 Leukemia

micals such as benzene and/or its metabolites, and thePAHs.There is a clear association between suppression ofimmune function and an increased incidence of infec-tious and neoplastic disease in humans. Agents thatproduce immunotoxicity in animals have the potentialto produce immune effects in the human population,and these effects may occur in the absence of obser-vable disease. Of the agents described here that areassociated with an increased risk for the developmentof leukemias, no direct and specific causal relationshipbetween the development of cancer and the immuno-suppressive action by these drugs and non-drug che-micals/agents has been demonstrated. However, a pre-ponderance of epidemiological evidence exists show-ing that exposure to various immunotoxic chemicals isassociated with increased risk for malignancies likeleukemia and lymphoma that are also known tooccur in immunocompromised patients. Thus, it is rea-sonable to conclude that alteration of immune functionmay contribute to the observed increase in risk.

Regulatory Environment

Because of the concerns regarding the potential fordrugs and non-drug chemicals to cause any numberof cancers, including leukemias, global regulatorybodies have established guidance and test guidelinesfor assessing this potential. These include specific as-sessment of carcinogenic potential (e.g. lifetime stu-dies in rodents) as well as assessment of the mutagenicand/or clastogenic potential (short-term in vivo and/orin vitro tests) of chemicals. Though not directly relatedto genotoxicity and carcinogenicity assessment, guide-lines regarding the assessment of immune status fol-lowing repeated exposure to drug and non-drug che-micals have recently been and are continuing to be putinto place. Of interest, not all immunotoxicology guid-ance/guidelines require the evaluation of cell-mediatedimmunity. Some examples of these guidances and testguidelines are provided in Table 1.

References1. International Programme on Chemical Safety (1996)

Health impact of selected immunotoxic agents. In:Environmental Health Criteria 180: Principles and Meth-ods for Assessing Direct Immunotoxicity Associated withExposure to Chemicals. World Health Organization,Geneva, pp 85–147

2. Leukemia & Lymphoma Society (2002) Public literature.The Leukemia and Lymphoma Society, White Plains

3. Luster MI, Simeonova P, Germolec DR, Portier C,Munson AE (1996) Relationships between chemical-induced immunotoxicity and carcinogenesis. Drug Info J30:281–285

4. Miller KB, Grodman HM (2001) Leukemia. In: LenhardRE Jr, Osteen RT, Gansler T (eds) Clinical Oncology.American Cancer Society, Blackwell Science, Malden,pp 527–551

5. Pitot HC, Dragan YP (2001) Chemical carcinogenesis.In: Klaassen CD (ed) Casarett and Doull’s Toxicology:The Basic Science of Poisons, 6th ed. McGraw-Hill, NewYork, pp 280–286

Leukemia-Initiating Cells (L-IC)

The hematopoietic stem cells of the leukemia. Notethat only a small subfraction of all leukemic cellscan self-renew in an organism. In human leukemias,the incidence of the L-IC subfraction was found evenlower than the incidence of normal hematopoietic stemcells occurring in bone marrow samples from healthyindividuals.

3Hematopoietic Stem Cells

Leukocyte

White blood cell.

3CD Markers

Leukocyte Culture: Considerationsfor In Vitro Culture of T cells inImmunotoxicological Studies

Maciej Tarkowski

Nofer Institute of Occupational MedicineLodzPoland

Synonyms

Characteristics

The the immune system comprises of innate and ac-quired mechanisms of defense against invading patho-gens. Through the help of innate, non-antigen-specificmechanisms utilizing, for example, professionalscavenging cells such as macrophages, it helps toclear mucosal tissues, to fight off the skin pathogens.The second line of defense consists of cells thatthrough fetal–neonatal “education” can eliminate for-eign for the host antigens and not react to those thatare recognized as self. Both mechanisms of defensecan be compromised by toxic effects of chemicals inoccupational and non-occupational settings. The theimmune system can be affected by chemicals towhich we are exposed through inhalation, by foodconsumption or by skin contact. A growing numberof allergies, development of autoimmune diseases, and

Leukocyte Culture: Considerations for In Vitro Culture of T cells in Immunotoxic 377

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compromised defense mechanisms to fight off infec-tions could result from this exposure to toxicants. De-pending on the exposure route differenT cells of theimmune system can be involved, but antigen-specificreactions will always occupy dendritic cells and lym-phocytes.Recognizing the toxic effects of chemicals on the im-mune system is one of the main goals of toxicologicalassessment of the risk that they may pose for humanhealth. Efforts to establish adequate tests to assessthese immunotoxic effects are still ongoing. Thosethat are implemented, in general, test for the immuno-toxic effects on innate, cell-mediated, or humoral-

mediated defense mechanisms. They use different an-imal models and in vitro tests, as well as exposure-controlled human studies. One of the main interests forstudying immunotoxic effects of chemicals is to rec-ognize whether they can induce antigen-specific reac-tions or—as is the case for most of chemicals—hap-ten-specific immune responses, and what the conse-quences of these effects are.As indicated earlier these responses are mediated bylymphocytes and antigen-presenting cells (APCs). Theantigen-specific response of lymphocytes results in in-duction of their proliferation and differentiation. Inparticular, the analyses of proliferative responses

Leukemia. Table 1 Examples of regulatory guidance and test guidelines for assessment of immunotoxicity,genotoxicity, or carcinogenicity (where available, only harmonized guidance/guidelines are presented)

Regulatory body Endpoint Guidance/test guideline

US Food and Drug Administration(FDA)

Immunotoxicity Center for Food Safety and Nutrition (CFSAN)Redbook 2000: Immunotoxicity studiesCenter for Devices and Radiological Health (CDRH)Immunotoxicity testing guidanceCenter for Drug Evaluation and Research (CDER)Immunotoxicology evaluation of investigational new drugs

Genotoxicity Center for Food Safety and Nutrition (CFSAN)Redbook 2000: Bacterial reverse mutation testIn vitro mammalian chromosome aberration testIn vitro mouse lymphoma TK+/- gene mutation assayIn vivo mammalian erythrocyte micronucleus testCenter for Drug Evaluation and Research (CDER)See ICH

Carcinogenicity Center for Food Safety and Nutrition (CFSAN)Carcinogenicity studies with rodentsCombined chronic toxicity/carcinogenicity studies withrodentsCenter for Drug Evaluation and Research (CDER)See ICH

Committee for Proprietary Medici-nal Products (CPMP)

Immunotoxicity Note For Guidance on Repeated Dose Toxicity

Genotoxicity See ICH

Carcinogenicity See ICH

Japanese Ministry of Health, Labor,and Welfare (MHLW )

Immunotoxicity Immunosuppression, Draft Guidance

Genotoxicity For Pharmaceuticals, see ICH

Carcinogenicity For Pharmaceuticals, see ICH

International Conference on Har-monization (ICH)

Genotoxicity ICH S2: GenotoxicityIncludes the standard battery of tests and guidance onspecific aspects of testing

Carcinogenicity ICH S1: CarcinogenicityIncludes the need for carcinogenicity studies, approachesto evaluating carcinogenic potential, and dose selection

378 Leukocyte Culture: Considerations for In Vitro Culture of T cells in Immunotoxic

have found applications in in vivo and in vitro immu-notoxicologic tests. The in vivo 3local lymph nodeassay (LLNA) (1) uses this phenomenon to test forskin-sensitizing properties of chemicals, whereas invitro proliferation tests were proven to help identifyberyllium-specific responses of T cells isolated frombronchoalveolar lavage of beryllium-sensitized peo-ple (2).Culture of cells in in vitro conditions is a techniqueused in many disciplines of research including immu-notoxicology. Presently many different cell types ofhuman or animal origin can be maintained in theseconditions due to the recognition of their nutritionaland other cell-specific requirements. Once the cellsbecome available there are several aspects that haveto be recognized before starting in vitro cell culture.The type of cells and the test for which they are going

to be used will determine the specific culture condi-tions, thus the following must be considered:* cell type nutritional requirements* duration of cell culture* presence of other cells (feeding cells) or/and soluble

components.* incubation requirements* adequate culture vessels.

Two major divisions of cell types are used that deter-mine specific culture conditions. There are cells grow-ing in suspension, or in adherent conditions, which arereflected by the basic morphological and physiologicalcharacteristics of the cells. Lymphocytes grow in sus-pension, whereas cells such as keratinocytes, epithelialor endothelial cells grow as adherent cells. Cell culturecan be short term or it may require a longer period ofincubation and several passages. Some experimentslike those assessing cell proliferation of lymphocytes


US Environmental ProtectionAgency (EPA)

Immunotoxicity Health Effects Test Guidelines870.7800 ImmunotoxicityBiochemicals Test Guidelines880.3550 Immunotoxicity880.3800 Immune response

Genotoxicity Health Effects Test Guidelines870.5100 Bacterial reverse mutation test870.5140 Gene mutation in Aspergillus nidulans870.5195 Mouse biochemical specific locus test870.5200 Mouse visible specific locus test870.5250 Gene mutation in Neurospora crassa870.5275 Sex-linked recessive lethal test in Drosophilamelanogaster870.5300 In vitro mammalian cell gene mutation test870.5375 In vitro mammalian chromosome aberration test870.5380 Mammalian spermatogonial chromosomal aber-ration test870.5385 Mammalian bone marrow chromosomal aberra-tion test870.5395 Mammalian erythrocyte micronucleus test870.5450 Rodent dominant lethal assay870.5460 Rodent heritable translocation assays870.5500 Bacterial DNA damage or repair tests870.5550 Unscheduled DNA synthesis in mammalian cellsin culture870.5575 Mitotic gene conversion in Saccharomycescerevisiae870.5900 In vitro sister chromatid exchange assay870.5915 In vivo sister chromatid exchange assay

Carcinogenicity Health Effects Test Guidelines:870.4200 Carcinogenicity870.4300 Combined Chronic Toxicity/Carcinogenicity

Leukemia. Table 1 Examples of regulatory guidance and test guidelines for assessment of immunotoxicity,genotoxicity, or carcinogenicity (where available, only harmonized guidance/guidelines are presented) (Continued)


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can require isolation of peripheral blood mononuclearcells (PBMCs) and their in vitro stimulation with theantigen for 3–10 days, depending on the antigen. Theycan also be used to assess the production of cytokinesin cultured supernatants. This direct use of cells in testdiffers from other prolonged cultures in which, first,cells of interest have to be grown to reach the appro-priate cell number, confluence or differentiation pro-file. This is the case in most of the adherent cellswhich have to be grown to reach the appropriate cellnumber and confluence. The need for prolonged cul-tures is also needed when growing cells which areneeded to differentiate, as in the case of establishing,for example, specific T cell lines, clones or differenti-ation of monocytes into dendritic cells.Regardless of the cell type, for in vitro cultures spe-cific conditions have to be met. One is to provide cellswith culture media formulated the way it meet the cellsoptimal growth support. Following are the basic ele-ments of the culture media.Nutritional requirements of different cell types are ful-

filled by culture media usually supplemented withserum and other specific components.Culture media consist of basic elements such as ba-lanced salts that provide adequate buffering condi-tions. There are culture media which utilize Hank’sor Earle’s salts depending whether the equilibrationis required with the atmospheric or gas phase contain-ing 5% or 10% of CO2, respectively. These salt solu-tions provide conditions to grow the cells at a pHvalue that is appropriate for the cell type and to pre-vent pH fluctuations that may occur during the cellgrowth and/or metabolism. Usually, in addition tothe culture media HEPES is added, which improvesthe control of pH over the range of 6.7–8.4. However,care must be taken because of its toxicity fordifferent cell types. Adequate concentrations shouldbe used, usually up to 25 mM. Additionally culturemedia are supplemented with essential amino acids,vitamins, and carbohydrates.Depending on the cell type that is cultured they maybe supplemented with different media.


Organization for Economic Coordi-nation and Development (OECD)

Genotoxicity OECD 471 Bacterial reverse mutation testOECD 473 In vitro mammalian chromosomal aberrationtestOECD 474 Mammalian erythrocyte micronucleus testOECD 475 Mammalian bone marrow chromosomal aber-ration testOECD 476 In vitro mammalian cell gene mutation testOECD 477 Sex-linked recessive lethal test in DrosophilamelanogasterOECD 478 Rodent dominant lethal testOECD 479 In vitro sister chromatid exchange assay inmammalian cellsOECD 480Saccharomyces cerevisiae gene mutationassayOECD 481Saccharomyces cerevisiaemiotic recombinationassayOECD 482 DNA damage and repair, UDS in mammaliancells in vitroOECD 483 Mammalian spermatogonial chromosome ab-erration testOECD 484 Genetic toxicology: mouse spot testOECD 485 Genetic toxicology: mouse heritable transloca-tion assayOECD 486 Unscheduled DNA synthesis test with mam-malian liver cells in vivoOECD Draft In vitro Syrian hamster embryo (SHE) celltransformation assay

Carcinogenicity OECD 451 Carcinogenicity studiesOECD 453 Combined chronic toxicity/carcinogenicitystudies

Leukemia. Table 1 Examples of regulatory guidance and test guidelines for assessment of immunotoxicity,genotoxicity, or carcinogenicity (where available, only harmonized guidance/guidelines are presented) (Continued)


Serum

Supplements include fetal bovine serum, newborn calfserum, horse serum, and human serum. However, dif-ferent batches of serum are available from differentvendors and they can contain proteins, growth factors,hormones, amino acids, lipids, vitamins, carbohy-drates that may affect cell culture. Thus it is importantto test several different sera from different sourcesbefore starting the cell culture, especially when noprevious results on cell growth are available. Insome cases of culturing human cells, to minimizeany unwanted background measurements generatedfrom calf serum, AB or autologous serum can beused. For some experiments, especially those usingblood mononuclear cells, serum has to be heat inacti-vated to prevent complement-dependent cell lysis. Inseveral cases serum-free media is used, for examplefor keratinocytes or hepatocytes that are often used intoxicological studies.

l-Glutamine

This amino acid belongs to the essential componentsof culture media. It may be utilized by some cells,especially those that quickly divide, in a short periodof time. Thus adequate concentrations of it have to beused, otherwise the growth arrest may occur. Also, it isimportant to supplement culture media with fresh l-glutamine since it easily hydrolyzes and its by-pro-ducts can even be toxic for cells.

Non-Essential Amino Acids

These are especially useful as supplements in culturemedia for long-term cell cultures.

Sodium Pyruvate

Sodium pyruvate is usually used when a higher energysource is required. It acts as an intermediate in sugarmetabolism where it is converted to acetaldehyde andcarbon dioxide.

Growth Factors

These include cell-specific factors for which growth ofthe cells can be highly dependent. They include endo-thelial cell growth factor, epidermal cell growth factor,nerve growth factor, and certain cytokines.

Antibiotics

These supplements include the mixture of streptomy-cin, penicillin and gentamicin. They are used to pre-vent infections with gram-positive or gram-negativebacteria, and Mycoplasma.

Attachment Factors

They are needed for surface adhesion and growth ofsome of the adherent cell types. Examples are fibro-nectin, laminin, and collagen.

Another important factor in cell growth is the useappropriate tissue vessels. The use of the particulartype of the culture vessel will depend on the cellline, its growth extent and the type of the test thatthe cells will be used for. In the case of adherenttypes of cells, first they have to be grown to reachconfluency before being tested. Their spread over thesurface provides the means for their growth, and thustheir increased numbers. This step usually is achievedby using flasks of different sizes. Once the cells reachconfluence they can be transferred to the next flask forfurther growth (this is called the passage) or to othervessels, if used for the test.For suspension of cells, the factor that determines thesize of the vessel, or flask, is the concentration [ofcells] in the culture medium and thus the volume.No less important than the size is the material fromwhich the vessels are made. Most cells are grown inpolystyrnee built surfaces but some cells may needspecial conditions, for example addition of matrix pro-teins to support adhesion and growth of adherent cells,or specifically formulated surfaces for growth of neu-rons.Cell growth may depend also on specific requirementsof the biology of the cell. T cells are dependent on thepresence of antigen and APCs for differentiation andproliferation. Thus for in vitro propagation of T cells,not only do adequate culture conditions have to be metbut also the requirements that stem from the biology ofthese cells.Animal or human studies on T cell reactions to tox-icants frequently involve analysis of their proliferativeresponses. The T cell proliferation test is used as anindicator of the immune response of the host to thetoxicant it was exposed to. In this case, PBMCs frompeople or animal cells (obtained by draining the lymphnodes at the site of exposure) are usually used. In somecases however, human cells of bronchoalveolar lavageare used, as in the case of sensitization to beryllium.This test uses the process of antigen-specific responseof T cells and their subsequent proliferation to confirmthe engagement of the host immune system in re-sponse to toxicant exposure. In this test isotope-la-beled (H3-labeled) thymidine is usually used which,during cell proliferation, incorporates into newlysynthesized DNA. The in vitro analysis of prolifera-tion of the cells indicated above does not require ad-ditional sources of APCs due to the fact that usuallythey are present in the cell preparations and they take3–10 days depending on the antigen.Another endpoint of T cell activity includes the mea-surement of the release of soluble mediators—cyto-kines—and the expression of surface receptors. How-ever, to increase the sensitivity of the antigen-mediatedreactions that could be detected in in vitro conditions itmight be necessary to propagate antigen specific


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T cells, to mimic an in vivo processes that lead toproliferation and differentiation of T cells.T cell lines in humans can be derived from PBMCs.The chance of success increases with a high proportionof in vivo antigen-specific T cells. For example, highernumbers of pollen allergen-specific T cells will existduring the pollen season; also high numbers of tuber-culin-purified protein derivative can be expected inpatients immunized against tuberculosis. The cultureand establishment of T cell lines specific for the anti-gen of interest can be achieved by their culture inappropriate conditions that provide cells with suffi-cient growth factors but also renewed sources of anti-gen and APCs. A general schematic presentation ofthis method is shown in Figure 1, and Figure 2shows the preparation of APCs.

Although the source of APCs shown in Fig. 2 isB cells, some scientists prefer to use dendritic cells,which are differentiated from monocytes. These cellsconsist of a professional pool of APCs which can moreeffectively present antigen to T cells. Thus in somecases use of these cells rather than B cells may beneeded, and may have more advantages.The technique of preparing B cells shown in Figure 2is the most widely used one. It uses the property ofT cells to express the cluster determinant (CD) 2 re-ceptor, which binds sheep red blood cells (SRBC).B cells do not possess this receptor and thus willstay in the interphase between the gradient and thebuffer. To obtain pure populations of B cells it is ne-cessary to incubate with SRBC twice. Because T cellsare the ones whose activity is tested, B cells added to

Leukocyte Culture: Considerations for In Vitro Culture of T cells in Immunotoxicological Studies.Figure 1 Preparation of antigen-specific T cell lines.


the culture need to be inactive in terms of their ownprotein synthesis, which is usually inhibited by γ-ir-radiation.Once the population of T cells is obtained they can bethe source for the analysis of antigen-specific reac-tions. These can include cytokine production, expres-sion of cell surface receptors, and intracellular signal-ing mechanisms. Cytokine analysis, especially ofT helper (Th) type 2 cells or Th type 1, may provideimportant information about the properties of the an-tigen. Preferential increase of Th2 cytokines, such as

interluekin(IL)-4 or IL-13, can indicate that antigenmay induce a humoral type of immune response, andlead to allergic sensitization.These cytokines provide signal for B cells to undergoimmunoglobulin isotype switch to heavy-chain classE. Preferential production of IL-12 in cultures ofmononuclear cells and interferon (IFN)-γ of T cells,on the other hand, can suggest that antigen may inducecell-mediated immune responses.Also of Importance is the analysis of cell receptor ex-pression. Analysis of the expression of chemokine re-

Leukocyte Culture: Considerations for In Vitro Culture of T cells in Immunotoxicological Studies.Figure 2 Preparation of antigen-presenting cells.


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ceptors, 3adhesion molecules, the tumor necrosis fac-tor receptor family, and cytokine receptors may pro-vide important information about the activity of anti-gen-mediated stimulation of T cells. The expression ofsome of the receptors indicate important changes incell migration, proliferation, inactivation, cytokineproduction, and others. Most of these can be measuredby cytofluorometry, which can show qualitative andquantitative differences in receptor expression.

Pros and Cons

The study of T cell activity is one of the major fields inbasic immunology. Rapid advances have been and stillare being made in this field, making it easier to under-stand the immunological effects of toxic agents. Rec-ognition of T cell reactions to the antigen of exposuremay provide important information for risk assess-ment. The proliferation of T cells to toxic agents hasalready been established as a fundamental element inthe detection of chemicals with skin sensitizing prop-erties (1).Further research may bring important information onthe activity of these cells and respiratory sensitizationor autoimmune reactions. Thus the technique of estab-lishing antigen-specific T cells in an in-vitro systemmay provide researchers with an important tool forinvestigating immunotoxic effects, and may in the fu-ture create markers for easy identification of these ac-tions.Chemicals are usually haptens which by themselvescan not induce an immune response—they requirebinding to host proteins. Antigen-specific T cells aredifficult to establish in in-vitro conditions for suchantigens; there is even evidence that the hapten in-duces a specific immune responses. Therefore, fornow, high-molecular-weight antigens are being studiedin the main. These studies, due to their complexity,will not be used as methods for measuring immuno-toxic potentials of chemicals, but they may lead tofinding easy determination markers.

References

1. Kimber I, Dearman RJ, Basketter DA, Ryan CA,Gerberick GF (2002) The local lymph node assay: past,present and future. Cont Dermat 47:315–328

2. Frome EL, Newman LS, Cragle DL, Colyer SP,Wambach PF (2003) Identification of an abnormalberyllium lymphocyte proliferation test. Toxicology183:39–56

Leukocyte Differentiation Antigens

3CD Markers

Leukocyte Emigration

A highly selective and specific process involving therecruitment of leukocytes to a site of inflammation.The sequential events include rolling, activation, ad-hesion and transendolthelial cell migration of leuko-cytes.

3Inflammatory Reactions, Acute Versus Chronic

Leukocyte Function-AssociatedAntigen-3 (LFA-3)

Leukocyte function-associated antigen-3 (LFA-3) is aco-stimulatory molecule whose ligand is the CD2 mol-ecule expressed on T lymphocytes. LFA-3 interactionsco-stimulate cytokine secretion from T lymphocytes. Itis also known as CD58.

3Interferon-γ

Leukocyte Margination and Adhesion

Specific events associated with leukocyte emigrationthat can be observed histologically at the site of aninflammatory response.

3Inflammatory Reactions, Acute Versus Chronic

Ligand Blotting

Ligand blotting is a modification of western blot anal-ysis, in which protein receptors, following electropho-retic separation and transfer onto a blotting membrane,are detected by the specific binding of labeled ligands.

3Western Blot Analysis

Ligand Passer

Ligand passer is one of several hypotheses put forwardto explain the pro-apoptotic influence of TNF-R2 inthe absence of a death inducing signaling complex. Ithas been suggested that the high affinity and slow on/off kinetics of TNF-R2 for TNF-α may result in se-questration of biologically active TNF-α possible li-gand passing to TNF-R1 thus facilitating TNF-R1-in-duced apoptosis.

3Tumor Necrosis Factor-α

384 Leukocyte Differentiation Antigens

Ligand Passing

A cytokine bound to a soluble receptor or to one typeof a membrane-inserted cytokine receptor may leavethe complex and bind to the signaling transmembranecytokine receptor (i.e. from the p75 receptor for tumornecrosis factor to the p55 receptor).

3Cytokine Receptors

Ligand Traps

Ligand traps are recombinant molecules which containthe ligand binding domains of the α-chain and the β-chain of a heterodimeric cytokine receptor fused to theconstant parts of a human immunoglobulin G mole-cule. Ligand traps are artificial binding moleculeswhich bind their corresponding cytokine with highspecificity and with a higher affinity than a homodi-meric fusion protein containing only two α-chains ofthe cytokine receptor.

3Cytokine Receptors

Limiting Dilution Analysis

Georg Brunner

Fachklinik Hornheide an der Universität MünsterDorbaumstrasse 300D-48157 MünsterGermany

Synonyms

LDA, limiting dilution analysis

Short Description

Limiting dilution analysis is a method in cell biologyfor estimating the frequency of a specific cell type in acomplex cell mixture. This cell type is identified by itsresponse to an activation signal, which can induce cellproliferation, differentiation, or the expression of spe-cific cellular functions in the 3responder cells, such as

3cytotoxic activity or the release of cytokines or anti-bodies. To set up limiting dilution analysis, a dilutionseries of the cell mixture is prepared, and a large num-ber of 3replicate cultures at each dilution is assayedfor their response to the activation signal. The culturesare scored as responding or nonresponding. Mathema-tical analysis of the percentage of nonresponding cul-tures at each dilution allows the estimation of the

3responder cell frequency in the original cell popula-tion.

CharacteristicsLimiting dilution analysis is a powerful technique tocharacterize quantitatively and qualitatively definedcellular subpopulations present in an unfractionatedcell mixture, based on their functional response to spe-cific stimulation. A number of different cell types canbe analyzed responding to a variety of signals. Theanalysis is most commonly used to investigate T-and B-lymphocyte repertoires. It is particularly usefulin evaluating the role of specific cell types in immuneresponses as well as in detecting responder cell typespresent at low frequency. Apart from applications ofthis technique in the analysis of lymphocyte reper-toires, the quantitation of hematopoietic stem cells iscommonly achieved by limiting dilution analysis invivo or in vitro (1).For a standard limiting dilution analysis, replicate cul-tures are set up at various dilutions of the cell popu-lation to be analyzed, in the presence and absence of aspecific stimulus (e.g. antigen), and, if appropriate, inthe presence of exogenously added cytokines (e.g. in-terleukin-2 or T cell growth factor) (2). The larger thenumber of replicates set up at each dilution, the moreprecise is the estimate of the responder frequency.Therefore, a minimum of 24 (up to 96) replica culturesis commonly used. The culture conditions should beoptimal for the cell population analyzed such that thefrequency of responders is the only limiting factor inthe assay. This includes addition of an optimal con-centration of cytokines as well as optimal time of in-cubation. Depending on the type of assay and the out-put signal measured, cultures are incubated for periodsranging between 3 and 18 days.The most commonly used output signals are measure-ments of cytotoxic activity, cell proliferation, or cyto-kine production.Analysis of cytotoxic activity is used in assays deter-mining the frequency of cytotoxic T lymphocytes(CTLs), natural killer (NK) cells, lymphocyte-acti-vated killer (LAK) cells, or antibody-dependent cellu-lar cytotoxic (ADCC) cells. This effector function ismeasured via the release of 51Cr from labeled 3targetcells. The target cell types used depend on the specifi-cities of the cell populations assayed. To confirm thespecificity of cytotoxic analyses, multiple target cellscan be used ( 3split-well analysis).A less specific output signal is the measurement of cellproliferation. Proliferative cells are usually monitoredby measuring the incorporation of tritiated thymidineinto DNA. In this case, measurements need to be cor-rected for baseline proliferation in the cell populationin the absence of specific stimulation as well as forproliferative activity due to cytokines added to thecultures (e.g. interleukin-2).An alternative output signal is the production of spe-cific proteins, e.g. lymphokines or antibodies, which

Limiting Dilution Analysis 385

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can be measured in immunochemical assays such asthe enzyme-linked immunosorbent assay (ELISA).Following incubation, the replicate cultures are scoredas positive (responsive to stimulation) or negative (un-responsive). While positive cultures may arise fromthe presence of one or more responder cells, negativecultures unequivocally demonstrate the absence of re-sponders. Therefore, the fraction of unresponsive cul-tures is determined for each dilution of the cell popu-lation assayed. These values are then used to estimatethe frequency of responder cells (n) by plotting the logof the fraction of negative cultures (F) against the cellnumber in the respective replicate cultures (Fig. 1).The zero term of the Poisson equation (F = e−n

where e is the base of the natural logarithm) predictsthat an average of one responder cell per culture(n = 1) will result in a fraction of 0.37 (1/e) of thereplicate cultures being negative (37%). Thus, the fre-quency of responder cells in the cell population as-sayed can be read directly from the graph (Figure 1).The most informative range of this analysis is betweennegative fractions 0.1–0.37 (3). Outside this range, it isadvisable to extend the analysis to larger fractions ofnegative cultures.Limiting dilution analysis of unfractionated cell popu-lations may result in nonlinear kinetics. This is usuallycaused by the interaction of two subpopulations ofresponder cells, present at distinct cell frequencies,responding to the same stimulatory signal (4). Thecell subpopulations do not behave independently butinteract with each other and compete for cytokines.This exemplifies the versatility of limiting dilutionanalysis in monitoring, apart from cellular frequencies,complex cellular interactions in a biologically relevantsetting.The principle of limiting dilution of a cell populationis also used preparatively to isolate single cells( 3limiting dilution cell cloning). In this technique,cells are plated at very low cell densities (between0.25 and 1 cells/culture), usually on top of a feedercell layer. The output signal measured is cell prolifer-ation, and positive cultures at the lowest cell densityare likely to be derived from single cells. Since a smallchance remains, according to Poisson statistics, thatpositive cultures arise from two cells rather than one,limiting dilution is usually repeated to verify clonality.Apart from the characterization of cell populations,limiting dilution analysis is also being used in molec-ular biology to quantitate DNA in complex biologicalsamples. This is achieved by amplifying the targetDNA sequence in the sample by 3limiting dilutionpolymerase chain reaction (PCR) (5). For this tech-nique, PCR is optimized such that the amplificationof endogenous DNA will take place in an all-or-noth-ing fashion, even at very low target DNA frequencies.By performing multiple replicate PCR reactions at se-

rial dilutions of the sample, the frequency of the targetDNA can be estimated from the fraction of negativereactions (no DNA product amplified) at each sampledilution using Poisson statistics as described above(see Figure 1).

Pros and ConsLimiting dilution analysis allows the monitoring of alarge number of variables in complex cell mixturescontaining several types of responder cells. The cellpopulation analyzed does not need to be fractionated,which enables cellular and molecular interactions tooccur mimicking the in vivo situation.The analysis is noninvasive and can easily be repeatedmultiple times. The results obtained with immune cellsof healthy individuals are reproducible over a rela-tively long time period.Reliability of the results is limited to a relatively nar-row range of dilutions of the cell population, which isusually not known at the beginning of a new experi-mental series.To obtain statistically significant results, relativelylarge numbers of replicate cultures (at least 24) arerequired for each dilution of the cell population.The data obtained from limiting dilution analyses ofunfractionated, mixed cell populations may deviatefrom linearity, which is usually due to the presenceof more than one responder cell type in the sample.These responders interact with each other resulting instimulatory and/or inhibitory effects, depending on thedilution of the sample (3).

PredictivityReliable information on cellular frequencies in mixedcell populations is only obtained in a relatively narrowrange of dilutions. The most informative value is be-tween multiplicities (responder frequency per well) of2.5 and 1 (i.e. at dilutions of the cell population result-ing in 10%–37% of the replicate cultures being unre-sponsive). At dilutions outside this range, reliability oflimiting dilution analysis drops sharply. As a compro-mise, it is recommended to design the assays such thatthey cover lower (< 1) rather than higher multiplicities.The precision of limiting dilution assays correlateswith the number of replicate cultures set up for eachdilution of the cell population. A replicate number of24 is minimally required. Larger replicate numberswill yield greater precision.Limiting dilution analysis has been shown to be repro-ducible, yielding similar frequencies of antigen-re-sponsive cells in humans over a time period of severalmonths. Limiting dilution analysis can be used to de-termine the frequency of functionally correspondingcell types in both animal models and humans. How-ever, the translation of data from animal models intothe human system is limited by the complexity and

386 Limiting Dilution Analysis

species-specific properties of immune responses. Thisis exemplified by the T lymphocyte response to HIV-1in an animal model for acute infection, which is muchmore pronounced than in humans (6).

Relevance to Humans

Limiting dilution analysis is of outstanding clinicalimportance, since it is the only technique allowingthe quantitation of immune responses at the single-cell level. It is being used to assess T cell responsesin humans, e.g. to different pathogens. The higher thefrequency of antigen-specific T cells, the stronger theimmune response will be following antigenic chal-lenge. Such information is important for attempts toenhance the efficiency of vaccine design.Limiting dilution analysis is also particularly useful ina variety of additional clinical settings involving thequantitation of immune or hematopoietic cells re-sponding to a specific stimulus. Examples are the im-munological monitoring of transplant recipients andcancer patients to assess their response to allo-MHC(major histocompatability complex) antigens of a par-ticular organ or bone marrow donor and to specificantigens of the patient’s tumor, respectively. In thecase of organ or bone marrow transplantation, this ishelpful in judging the patient’s risk of rejecting thegraft. Analysis of the immune response of cancer pa-tients can be helpful in predicting and/or monitoringthe success of therapeutic approaches involving thestimulation of the patient’s immune system. The suc-

cess of bone marrow transplantation significantly de-pends on the number of hematopoietic stem cells pres-ent in the graft. Limiting dilution analysis is widelyused to determine the stem cell frequency in bonemarrow grafts.Limiting dilution PCR (polymerase chain reaction) hasbeen used to assess the therapeutic success and thelevel of minimal residual disease in leukemia patientsby quantifying leukemic lymphocytes in the blood. Inaddition, this technique has been evaluated for poten-tial application in the detection of genetically modifiedorganisms in food.


Not applicable.

References

1. Sieburg HB, Cho RH, Müller-Sieburg CE (2002) Limit-ing dilution analysis for estimating the frequency ofhematopoietic stem cells: Uncertainty and significance.Exp Hematol 30:1436–1443

2. Sharrock CEM, Kaminski E, Man S (1990) Limitingdilution analysis of human T cells: A useful clinical tool.Immunol Today 11:281–286

3. Fazekas de St Groth S (1982) The evaluation of limitingdilution assays. J Immunol Meth 49:R11–R23

4. Dozmorov I, Eisenbraun MD, Lefkovits I (2000) Limit-ing dilution analysis: From frequencies to cellularinteractions. Immunol Today 21:15–18

5. Sykes PJ, Neoh SH, Brisco MJ, Hughes E, Condon J,Morley AA (1992) Quantitation of targets for PCR by useof limiting dilution. BioTechniques 13:444–449

6. Kent SJ, Corey L, Agy MB, Morton WR, McElrath MJ,Greenberg PD (1995) Cytotoxic and proliferative T cellresponses in HIV-1-infected Macaca nemestrina. J ClinInvest 95:248–256

Limiting Dilution Cell Cloning

This method in cell biology is used to isolate singlecells from complex cell mixtures.


Limiting Dilution Polymerase ChainReaction

This method in molecular biology is an application ofDNA amplification by standard polymerase chain re-action (PCR) for estimating the frequency of specificDNA sequences in complex biological samples.


Limiting Dilution Analysis. Figure 1 Example of agraphical analysis of LDA data. The log of the fractionof negative cultures (F) is plotted against the cellnumber in the cultures. The zero term of the Poissonequation predicts that, at F=0.37, there is on averageone responder cell per culture with the specificitytested. This allows the estimation of the responderfrequency in the original cell mixture (in this exampleone responder cell in 2.5 × 104 cells).

Limiting Dilution Polymerase Chain Reaction 387

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Lipid Rafts

Lipid rafts are sphingolipid/cholesterol-enriched mem-brane domains found in all mammalian cell types. Inunactivated lymphocytes they are small, but upon ac-tivation they cluster together to form large rafts. Theyare though to have a role in the initiation and organi-zation of the signaling cascades, which lead to theactivation of B cells and T cells by pre-assemblingthe signaling proteins in restricted areas of the mem-brane close to the receptor complexes. This clusteringis believed to allow quick and efficient connection tosignaling cascade upon receptor engagement.

3Signal Transduction During Lymphocyte Activation

Lipids

3Fatty Acids and the Immune System

Lipopolysaccharide (LPS)

Refers to lipid-containing polysaccharides found in thecell wall of Gram-negative bacteria. The lipopolysac-charide derived from Escherichia coli stimulates non-specifically the proliferation of B cells of several spe-cies. It is widely used in immunology for studies in-volving lymphocyte proliferation. The lipid A compo-nent mediates toxicity and cytokine productionwhereas the polysaccharides are responsible for immu-nogenicity. Also called endotoxin. LPS can induce ac-tivation of proinflammatory cytokines including IL-1β, TNF-α and IL-6.

3Lymphocyte Proliferation

3Polyclonal Activators

3Interleukin-1β (IL-1β)


3Cytokines

3Neonatal Immune Response

Listeria Monocytogenes

A facultative intracellular bacterium.

3Host Resistance Assays

Live-Death Discrimination

3Viability, Cell

Live Rate

3Viability, Cell

LLNA Challenge Experiment

An LLNA challenge experiment can be conductedwhen questionable results are obtained with an induc-tion phase LLNA. It consists of two phases, the induc-tion or sensitization phase, and the challenge or elici-tation phase. Challenged groups are compared with theinduction control in order to discover an allergy-rele-vant change in reactivity to the test chemical.

3Local Lymph Node Assay (IMDS), Modifications

Local Immune System

3Skin, Contribution to Immunity

Local Lymph Node Assay

Ian Kimber . Rebecca J Dearman

Syngenta Central Toxicology LaboratoryAlderley Park, MacclesfieldCheshireSK10 4TJUK

Short Description

The murine local lymph node assay (LLNA) was de-veloped initially as a method for the identification ofchemicals that have the potential to cause skin sensi-tization and allergic contact dermatitis. Since its firstdescription the LLNA has been the subject of detailedassessments in the context of national and internation-al collaborative trials, and of extensive comparisonswith both other predictive test methods and humandata. The origins, development, evaluation and even-tual validation of the LLNA have recently been de-scribed comprehensively elsewhere (1).In contrast to guinea pig tests, the LLNA identifiescontact allergens as a function of events induced dur-ing the induction phase of skin sensitization—specifi-cally the stimulation of proliferative responses indraining 3lymph nodes.

Characteristics

The LLNA is based on an appreciation of the events

388 Lipid Rafts

that characterize the induction phase of skin sensitiza-tion. Following topical exposure to a skin sensitizingchemical molecular and cellular processes are pro-voked that act in concert to elicit a cutaneous immuneresponse. Among the key events are the mobilizationof epidermal 3Langerhans cells (LC), and their mi-gration from the skin to draining lymph nodes, via the

3afferent lymphatics. While in transit from the skinthese cells undergo a functional maturation and loca-lize in the paracortical region of the lymph nodeswhere they present antigen to responsiveT lymphocytes. Antigen-activated T lymphocytes arestimulated to divide and differentiate, the former re-sulting in a selective clonal expansion of allergen-spe-cific cells. This is the cellular basis of sensitization. Ifthe now sensitized subject encounters the same aller-gen again—at the same site or a different skin site—then the expanded population of responsiveT lymphocytes will mount an accelerated and moreaggressive secondary immune response at the pointof exposure. This will, in turn, provoke the dermalinflammatory reaction that is recognized clinically asallergic contact dermatitis (2). A unifying and manda-tory characteristic of contact allergens is, therefore, thestimulation of T lymphocyte responses in lymph nodesdraining the site of skin exposure, and this providesthe mechanistic rationale for the LLNA.Methodological descriptions of the LLNA are avail-able elsewhere (3). Briefly, the standard assay is con-ducted as follows. Groups of mice (CBA strain) areexposed topically, on the dorsum of both ears, to var-ious concentrations of the test chemical, or to an equalvolume of the relevant vehicle alone. Treatment isperformed daily for 3 consecutive days. Five daysfollowing the initiation of exposure mice receive anintravenous injection of [3H] thymidine (3H-TdR). An-imals are killed 5 hours later and the draining (auric-ular) lymph nodes are excised. These are pooled foreach experimental group, or alternatively are pooledon a per-animal basis. Single cell suspensions oflymph node cells (LNC) are prepared and incorpora-tion of 3H-TdR measured by β-scintillation counting.Interpretation of results is based upon derivation of astimulation index (SI). For each concentration of testchemical an SI is calculated relative to the concurrentvehicle control. Skin sensitizers are defined as chemi-cals which—at one or more test concentrations—in-duce at least a 3-fold increase in LNC proliferationcompared with the vehicle control (an SI of 3 or more)(see Figure 1).There have been descriptions of proposed modifica-tions to the standard LLNA—some relatively minor inscope, others more substantial (1). A consideration ofthese alternatives is beyond the scope of this essay (see

3Local Lymph Node Assay (IMDS), modifications).The standard assay as described above was developed

specifically for the purposes of hazard identification,and the performance of the assay in this context isaddressed below. However, the LLNA is now usedalso for the measurement of relative potency in thecontext of risk assessment. This application is predi-cated on an understanding that the vigour ofT lymphocyte proliferation induced in draininglymph nodes correlates closely with the extent towhich sensitization will be acquired. The thesis is,therefore, that activity in the LLNAwill be informativealso of potency. For this purpose an EC3 value isderived—the Estimated Concentration (EC) of chem-ical required to induce an SI of exactly 3 (4).

Advantages and Disadvantages

In the context of hazard identification the LLNA offersa number of important advantages compared withguinea pig tests. Exposure is via the relevant route,there is no requirement for adjuvant, and the read-out is objective and quantitative. Moreover, it is ac-knowledged that the LLNA also provides for signifi-cant animal welfare benefits with regard to both a re-duction in the number of animals required and thetrauma to which they are potentially subject.The LLNA also permits accurate measurement of re-lative 3skin sensitization potency as a first step in therisk assessment process, in a way that was not usuallypossible with the standard guinea pig assays.

PredictivityAs indicated above the LLNA has been evaluated ex-haustively and has been found to provide a robust andreliable method for the identification of skin sensitiz-ing chemicals that can serve as a stand-alone alterna-tive to guinea pig test methods. In common with alltoxicity test methods the accuracy of the LLNA is notperfect. Thus it has been shown, for instance, thatsome—but by no means all—skin irritants may pro-voke low level responses. However, it has been dem-onstrated that the overall performance of the LLNA isat least comparable with (and in most instances supe-rior to) methods such as the guinea pig maximizationtest and the occluded patch test of Buehler (5). Themethod has been endorsed in the USA by the Inter-agency Coordinating Committee on the Validation ofAlternative Methods (ICCVAM) and in Europe by TheEuropean Centre for the Validation of AlternativeMethods (ECVAM).

Relevance to HumansClearly an important—indeed critical—aspect of theevaluation and validation of the LLNA was establish-ing that the method is able to identify accurately che-micals that are known to cause skin sensitization inhumans. As confirmed during validation, this is thecase.

Local Lymph Node Assay 389

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It has been demonstrated also that measurement ofrelative potency using the LLNA reflects clinical ex-perience. Thus, derived EC3 values have been shownto correlate well with what is known of the potencywith which chemical allergens cause skin sensitizationin humans (6,7).


In the period following endorsement of the method byICCVAM (in 1999) the LLNA has been adopted byseveral regulatory agencies in the USA. The methodhas also now been incorporated into a new test guide-line (number 429) entitled Skin Sensitization: LocalLymph Node Assay by the Organization for EconomicCooperation and Development (OECD). This wasadopted formally in April 2002. In parallel, the Eur-opean Union (EU) has prepared a new test guideline

for the LLNA (B.42) entitled Skin Sensitization: LocalLymph Node Assay which has now been published inthe Official Journal of the EU.

Concluding Comments

The LLNA provides an accurate method for the iden-tification of chemicals that have the potential to causeskin sensitization. Moreover, the method is findingincreasing application for the measurement of relativerisk. There will undoubtedly be opportunities forfurther refinement of the LLNA that will enhancefurther characterization of hazards and risks associatedwith skin sensitization.

References

1. Kimber I, Dearman RJ, Basketter DA, Ryan CA,Gerberick GF (2002) The local lymph node assay: past,present and future. Cont Derm 47:315–328

2. Kimber I, Basketter DA, Gerberick GF, Dearman RJ(2002) Allergic contact dermatitis. Internat Immunophar-macol 2:201–211

3. Dearman RJ, Basketter DA, Kimber I (1999) Locallymph node assay: Use in hazard and risk assessment. JAppl Toxicol 19:299–306

4. Kimber I, Basketter DA (1997) Contact sensitization: Anew approach to risk assessment. Human Ecolog RiskAssess 3:385–395

5. Gerberick GF, Ryan CA, Kimber I, Dearman RJ, Lea LJ,Basketter DA (2000) Local lymph node assay: Validationassessment for regulatory purposes. Amer J Cont Derm11:3–18

6. Basketter DA, Blaikie L, Dearman RJ et al. (2000) Use ofthe local lymph node assay for the estimation of relativecontact allergic potency. Cont Derm 42:344–348

7. Gerberick GF, Robinson MK, Ryan CA et al. (2001)Contact allergenic potency: correlation of human andlocal lymph node assay data. Amer J Cont Derm 12:156–161

Local Lymph Node Assay. Figure 1 A schema of thestandard local lymph node assay. Mice (CBA strain)receive three consecutive daily applications of 25 μl ofvarious concentrations of the test material, or of therelevant vehicle alone, to the dorsum of both ears. Fivedays after the initiation of exposure, mice receive anintravenous injection, via the tail vein, of 20 μCi of 3H-thymidine (3HTdR) in 250 μl of phosphate-bufferedsaline. 5 hours later draining auricular lymph nodes areexcised and pooled for each experimental group, or ona per-experimental-animal basis, and are processed forβ-scintillation counting.

390 Local Lymph Node Assay

Local Lymph Node Assay (IMDS),Modifications

Hans-Werner Vohr

PH-PD, ToxicologyBayer HealthCare AGAprather Weg 18a42096 WuppertalGermany

Peter Ulrich

Preclinical Safety, MUT2881.329Novartis Pharma AGAuhafenstrasseCH-4132 MuttenzSwitzerland

Description

In 1998, after a decade of scientific evaluation andextensive interlaboratory validation, the local lymphnode assay (see LLNA) was peer reviewed and was,in principle, endorsed as a stand-alone test by an in-dependent panel of experts on behalf of the US Inter-agency Co-ordinating Committee on the Validation ofAlternative Methods (ICCVAM) in 1999. However,improvements were suggested by the peer reviewpanel concerning the discrimination of contact aller-genic from irritation potential. Some irritating andphototoxic compounds were shown to cause a false-positive response, since they induced a vigorous cellproliferation in the ear-draining lymph nodes (1) or amarked LN 3hyperplasia (2,3). The occurrence offalse-positive results is an obvious consequence ofthe tight physiological connection between inflamma-tory tissue processes and specific immune reactions,with the latter being more vigorous, when the first hasreached a level sufficient to provide all adjuvant-likesignals to the immune system. Thus, the proliferationof lymphocytes in the ear-draining lymph node is amandatory, but not sufficient requirement for the in-duction of a contact allergic response (such as 3aller-gic contact dermatitis). A further reason for alternativeLLNA protocols are the opinions of many researchersconsidering the use of radioactivity in the "classical"LLNA as not appropriate for various reasons.

Characteristics

Chemicals applied onto the skin and penetratingthrough the stratum corneum may induce two formsof skin reactions. First a compound specific immunereaction may be induced by binding to self-proteins,and second an acute inflammatory reaction by cyto-toxic (irritating) properties of the chemical. The anti-

gen-specific immune reaction is characterised by thedevelopment of memory T cells and B cells. Suchmemory cells are responsible for the elicitation of apronounced skin reaction after re-exposure of low con-centrations of the test chemical during the challengephase. No such memory cells are induced in course ofan acute skin reaction due to cell destruction (irri-tancy), although most of the skin sensitizing chemicalsdo express slight irritating properties in addition.Additional endpoints were included in the test protocolwith the aim to provide more information about theirritation potential of chemicals, which can be directlyrelated to the reactivity in the LN. Homey et al (1998)(2) and Vohr et al. (2000) (4) reported that measure-ment of ear swelling in the LLNA can be used toassess the irritation potential of chemicals. Followinga comparison of chemical-induced effects in the earskin and the ear-draining LN, they concluded that LNhyperplasia in the absence of a significant primary earirritation points to an allergic process, whereas thefinding of LN hyperplasia together with ear irritationrequires a more careful evaluation of the nature of thereactivity—(photo)allergy or true 3(photo)irritation.In a recent paper by Ulrich et al, the weight determi-nation of circular biopsies of the ears has proven to bea useful marker for skin irritation (5).The experimental set-up of the alternative LLNA issimilar to the radioactive LLNA described by Kimberet al. (cf. related essay in this book), with the excep-tion that the alternative approach does not require a 2-day resting phase between the 3-day treatment andkilling the animals without being less sensitive. Thisstudy protocol was subject to an interlaboratory vali-dation with the aim of establishing the measurement ofLN hyperplasia and skin irritation as alternative end-points (6). The interlaboratory validation thus usedtwo modifications compared to the "classical"LLNA. These modifications measure lymph nodeweights and cell counts instead of radioactive labeling,and take the acute skin reaction (irritancy) into con-sideration for the judgment of the effects seen.

Irritancy

While measurement of cell counts (instead of incorpo-ration of radioactive material) is already widespread(see below), measurement of the acute skin reactiontoo was introduced, as originally developed by Homeyet al. (2). The reason for this modification was to flagup "false-positive" results due to skin irritation, as forexample measured after sodium dodecyl sulfate (SDS)application. This modified LLNA was called the "in-tegrated model for the discrimination of skin reac-tions" (IMDS).Inclusion of the ability to detect skin irritancy in themodel is one main advantage of the modified test. Theirritating effect of chemicals can be taken into consid-

Local Lymph Node Assay (IMDS), Modifications 391

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eration by determining the acute skin reaction (in-crease in ear thickness and/or ear weight). Althougha moderate irritation will also result in cell prolifera-tion in the skin draining lymph node (false positive) astrong irritating, and therefore cytotoxic, reaction willinduce more influx of lymph fluid, and by this anincrease in lymph weight instead of cell proliferation(see also Table 1).There is a dramatic difference in the cytokine patternexpressed during the induction phase of an immuno-logical skin reaction, or after epidermal cell destruc-tion by cytotoxicity. This cytokine balance, however,has an important impact on the skin reaction observed.During the onset of a specific (immunological) skinreaction the cytokines induced will cause migration ofthe 3Langerhans cells into the draining lymph nodes.In contrast to this mechanism the cytokines releasedduring irritating/cytotoxic activation will attract leuco-cytes into the skin, reduce the tissue integrity at theinflammatory side, and increase the permeability forbody fluids. In addition, the cytotoxic amounts of thetest item reaching the draining lymph node can alsoresult in cell destruction, and this process in somecases exceeds the cell proliferation/activation. Takentogether, with increasing irritancy, decreased cell pro-liferation and relative increased lymph node weightswill be observed.There are several examples in the literature about sucha "negative" influences on cell activation by increasingirritancy of a chemical or by pretreatment with sodiumlauryl sulfate (SLS).

Cell Counts

In contrast to the consideration of the irritating poten-tial in the LLNA, alternate endpoints (especially cellcounting) have been described from the very begin-ning. All results published so far verify that there is no

difference between determining proliferation in thedraining lymph node on the basis of the cell countsor 3H-thymidine incorporation. The sensitivity of bothmethods is comparable (Figure 1). It has to be clari-fied, however, that the “ 3positive level” or thresholdvalue as defined for the radioactive method, that is, theEC3 value (see LLNA), is exclusively defined for themethod and mouse strain used for LLNA. Such posi-tive limits have to be calculated for each endpoint andstrain of mice individually (see also below).A modification of the assay which involves measuringcell counts instead of radioactive labeling provides notonly comparable sensitivity but also has the advantagethat the cell suspension can be further analyzed bydifferent methods (flow cytometry, chemilumines-cence, immunofluorescence) to gain an insight intomechanistic events.By comparing the specific immune reaction inducedby the test item in the draining lymph nodes (LN cellcounts, LN weights) with the immediate unspecificacute skin reaction (ear swelling, ear weight) it is pos-sible to discriminate the irritating potential from thesensitizing potential of the compound tested. Interna-tional standards have successfully used this modifica-tion, and such modifications are also authorized in theNote of Guidance SWP/2145/00 of the CPMP (2001)and OECD Guideline No. 429.

International Catch-up Validation

The results of the interlaboratory validation of themodified LLNA in a working group of nine Europeanlaboratories have been published (6). This validationstudy has been conducted under the conditions recom-mended in the relevant OECD guidance, whereby thestudy was supervised by an independent coordinator,samples were blinded, and statistics were performedby an external independent statistician. This interna-

Local Lymph Node Assay (IMDS), Modifications. Table 1 Contact and irritant chemicals induce increases inacute ear reaction and in lymph node (LN) cell counts. On three consecutive days, five NMRI mice per group weretopically treated with either 1% oxazolone, 1% croton oil, or vehicle on the dorsal surface of both ears. On day 3,ear thickness of both ears was measured, the local draining lymph nodes of the ears were removed, and individuallymph node cell counts were determined. Data indicate mean ear swelling or lymph node cell count indices whichwere defined as ratio of mean values from test groups to mean of the vehicle-treated control group. The maximumincreases of stimulation indices were set to 2 for ear swelling and to 5 for cell counts, respectively.

Acute ear Reaction [Index] Lymph Node Cell Counts [Index]

Vehicle 1 1

1% Oxazolone 1.09* (≙9% increase) 4.13** (≙78% increase)

Vehicle 1 1

1% Croton oil 1.59** (≙59% increase) 2.58** (≙39% increase)

* P < 0.05.** P < 0.01.

392 Local Lymph Node Assay (IMDS), Modifications

tional validation study using NMRI and BALB/c miceshowed that the cell count index is a robust and reli-able parameter for detecting skin sensitizing propertiesof test material. In addition, this parameter againproved to be as reliable as radioactive labelling. Thedata obtained for the acute skin reaction (ear weight,ear swelling) prevented all participating labs fromclassifying SDS as a sensitizing compound or over-estimating the stimulation indices of test items withpronounced irritating properties.The evaluation is based on the statistical comparisonof the results of the test groups to the vehicle controlusing the Wilcoxon test. To this end, the parametersare measured for each individual animal. If statisticallysignificant changes in lymph node parameters are ob-tained, these are related to the ear weights to accountfor the contribution of the primary skin irritation. Thisis quite another approach as used for the radioactivemethod, which calculates a 3stimulation index out ofthe radioactivity determined in group-wise pooledlymph nodes, without taking account of possible der-mal irritation.Pronounced irritation could cover a slight skin sensi-tizing property of the test item. For verification of suchquestionable results it is possible to perform a so-called 3LLNA challenge experiment using test chem-

ical concentrations, which cause mild to moderate LNhyperplasia for induction and a lower concentrationfor challenge. In cases of contact allergy, the responsein the challenged group will be more vigorous than theinduction control, which received the compound thefirst time. In addition, the clinical manifestation ofcontact allergy in terms of increased ear weights (orswelling) can be set into relation to the response in theLN (2, 4).

Potency

Of special interest is the fact that during recent yearsevery effort has been made to rank sensitizing com-pounds according to their potency for inducing allergicskin reactions. Three or four categories of skin sensi-tizing compounds have been proposed, namely(weak), moderate, strong, and (extreme). In case ofthe LLNA the cut-offs for these categories is the so-called EC3 value (7). This "positive level" is the stim-ulation index of 3 based on radioactively labelling ofthe lymph node cells.It is evident that compared to the cell counts muchhigher stimulation indices can be reached by the ra-dioactive method. However, the individual standarddeviation is also higher than for the non-radioactivemethod. Therefore, the "positive level" for the cellcounts as determined on the basis of historical dataas well as in the international validation study is 1.4

Local Lymph Node Assay (IMDS), Modifications.Figure 1 Comparison of threshold values obtainedwith (i) the radioactive version of the LLNA (whitecolumns) and (ii) the alternative LLNA as described inthe text. EC3: effective concentration, which causes a3-fold increase in radioactivity in the LN preparations ofchemical-treated groups in comparison to the vehiclecontrol. This is the level considered to indicate apositive response in the radioactive LLNA. TC1.4:threshold concentration, which induces a 1.4-foldincrease in the lymph node cellularity in comparison tothe vehicle control.

Local Lymph Node Assay (IMDS), Modifications.Table 2

LN Ear

0,001 0,001

100 100

MBT 8,7500 100,0000

Eugenol 6,2000 100,0000

Isoeugenol 9,9000 100,0000

CinAld 5,4000 47,6200

OXA 0,0280 0,6250

DNCB 0,0105 0,3600

DNFB 0,0078 0,0560

GA 1,5000 0,5300

FA 24,0000 9,2000

CroOil 0,0240 0,0550

SDS 15,4000 15,0000

TCSA 0,0400 0,8150

TCSA/UVA 0,0260 0,2990

Acr 1,2220 100,0000

Acr/UVA 0,1175 0,0405

8-MOP/UVA 0,0078 0,0025

Local Lymph Node Assay (IMDS), Modifications 393

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for NMRI and 1.55 for BALB/c, respectively. On thebasis of these values a ranking of the sensitizing stan-dards has been made which shows exactly the sameorder as described for the LLNA based on the radio-active labelling (Figure 1).The interdependency of irritancy and sensitization forsome international standard compounds is given inFigure 2. There are characteristic clusters of com-pounds which reveal a striking association of sensitiz-ing potential and skin irritation. Chemicals with low orno skin irritation potential are at the same time weaksensitizers in the LLNA, and chemicals with a higherpotential to irritate the skin bear a stronger sensitiza-tion potential. However, some irritants and photoirri-tants appear also in the cluster of chemicals with highLN activation potential.

Pros and Cons

The principal advantages of the LLNA in comparisonto the classical GP assays have been discussed else-where (see LLNA). The addition of skin irritation as afurther endpoint contributes to the larger specificity ofthis alternative LLNA. Some chemicals may be pro-blematic when tested in an induction phase assay. Insuch cases, if there is no other information about thechemical, like the mode of action ( 3hapten theory) orthe structure-activity relationship, a biphasic (second-ary response) LLNA may be indicated, unless a certaindegree of uncertainty in the positive classification ofthe chemical as a contact allergen is tolerable.There are some technical limitations of the LLNA,which have to be considered. One problem is thatthe test item to be applied on the ear must be a solutionor a fine suspension in a small volume. In addition, thetest item must be prevented from dripping off the skin,and the vehicle should enhance the skin penetration ofthe test item because of the non-occlusive protocol.Therefore, the choice of vehicle is quite different tothat for guinea pig assays, and aqueous formulationsare not to be used without further modification of theprotocol.

Predictivity

The predictivity of the modified LLNA (IMDS) issimilar to that of the radioactive LLNA. However,its specificity could be improved by the introductionof skin irritation as an endpoint. Hence, the evaluationof both skin irritation and LN hyperplasia potentials ofa chemical provides a better foundation for the distinc-tion of contact allergenic activity from true skin irrita-tion. Structure-activity relationships and a hypothesison the mode of chemical action ( 3hapten theory)complete the evaluation and support the interpretationof LLNA results. In some cases the conduction of abiphasic LLNA consisting of an sensitization and an

elicitation phase (secondary response), may be appro-priate to bring final clarification.Benzalkonium chloride (BKC) is a good example forthe importance of measuring both the irritating proper-ties as well as the sensitizing properties of a test com-pound. BKC is a strong irritant but with clear skinsensitizing properties. BKC produced a bell-shapedconcentration-response curve for both the radioactiveindex (8) and the LN cell count index (Figure 3). Incontrast to LN hyperplasia, the skin irritation ex-pressed as ear weight index in the modified LLNAis increasing over a concentration range up to 10%(Figure 3). The conclusion from this pattern is thatsensitization occurs at lower and less irritable concen-trations of BKC, whereas at high concentrations thestrong irritation counteracts the onset of a contact al-lergic response in the LN. This is exactly what isfound in humans. Therefore, a recommendation existto use BKC at concentrations below 1% in humanpatch tests to avoid non-specific irritant reactions.

Relevance to HumansSee also Predictivity. Potency assessment with LLNAis very enticing because of the objective nature of theendpoints. However, one has to consider that thestrength of allergic response depends not only on thechemical features of the test compound, but also on thereadiness to react of the individual's immune system.Furthermore, the choice of vehicle has a considerableinfluence on the strength of the response in an animaltest. Thus, the conditions of human exposure shouldbe carefully evaluated before extrapolating absoluteanimal data.

Regulatory Environment* The LLNA had been accepted as a stand-alone test

for skin sensitization first by the OECD (no. 429:Skin Sensitization: Local Lymph Node Assay bythe Organisation for Economic Cooperation andDevelopment). This was adopted formally inApril 2002.

* In parallel, the European Union (EU) has prepared anew test guideline for the LLNA (B.42 Skin Sensi-tization: Local Lymph Node Assay) that has nowbeen published in the Official Journal of the EU.

* In 2003 the US-EPA (Environmental ProtectionAgency) adopted its Skin Sensitization guidelineto accept the lymph node assay as a stand alonetest (OPPTS 870.2600).

* The LLNA is also described in the following guide-lines of the EU: CPMP Note for guidance on non-clinical local tolerance testing of medicinal pro-ducts. Adopted February 2001. CPMP/SWP/2145/00, and CPMP Note for guidance onphotosafety testing. Adopted June 2002. CPMP/SWP/398/01.

394 Local Lymph Node Assay (IMDS), Modifications

* The FDA provides also a guideline with a discus-sion of the LLNA: Guidance for Industry: Immu-notoxicology evaluation of investigational newdrugs. CDER 2002.

References

1. Scholes EW, Basketter DA, Lovell WW, Sarll AE,Pendlington RU (1992) The identification of photoaller-gic potential in the local lymph node assay. Photoderma-tol Photoimmunol Photomed 8:249–254

2. Homey B, von Schilling C, Blumel J et al. (1998) Anintegrated model for the differentiation of chemical-induced allergic and irritant skin reactions. Toxicol ApplPharmacol 153:83–94

3. Ulrich P, Homey B, Vohr HW (1998) A modified murinelocal lymph node assay for the differentiation of contactphotoallergy from phototoxicity by analysis of cytokineexpression in skin-draining lymph node cells. Toxicology125:149–168

4. Vohr HW, Blumel J, Blotz A, Homey B, Ahr HJ (2000)An intra-laboratory validation of the Integrated Model forthe Differentiation of Skin Reactions (IMDS): Discrim-ination between (photo)allergic and (photo)irritant reac-tions in mice. Arch Toxicol 73 (10–11):501–509

5. Ulrich P, Streich J, Suter W (2001) Intra-laboratoryvalidation of alternative endpoints in the murine locallymph node assay for the identification of contact allergicpotential: Primary ear skin irritation and ear-draininglymph node hyperplasia induced by topical chemicals.Arch Toxicol 74:733–744

6. Ehling G, Hecht M, Heusener A, Huesler J, Gamer AO, vLoveren H, Maurer T, Riecke K, Ullmann L, Ulrich P,Vandebriel R, Vohr H-W (2004) An European Inter-Laboratory Validation of Alternative Endpoints of theMurine Loacl Lymph Node Assay. 1stROUND (and 2ndROUND) Toxicology, submitted

7. ECETOC (2003) Technical Report Number 87. ContactSensitization: Classification According to Potency. Brus-sels: European Centre for Ecotoxicity and Toxicology ofChemicals

8. Gerberick GF, Cruse LW, Ryan CA (1999) Local lymphnode assay: Differentiating allergic and irritant responsesusing flow cytometry. Methods 19:48–55

Local Lymph Node Assay (LLNA)

This uses a murine model to test for skin sensitizationproperties of chemicals.

3Leukocyte Culture: Considerations for In Vitro Cul-ture of T cells in Immunotoxicological Studies

3Hapten and Carrier

Local Skin Immune Hyperreaction

3Contact Hypersensitivity

Local Lymph Node Assay (IMDS), Modifications. Figure 2 Relation of primary ear skin irritation and LNactivation induced by chemicals. Threshold concentrations for primary LN activation and ear irritation werecalculated for each chemical by applying curve-fitting algorithms to the concentration-response curves. Thethreshold indices for LN activation (1.3) derived from cell count data and ear irritation (1.1) assessed by weightmeasurement were approximated from the lowest applied concentrations of the chemicals leading to statisticallysignificant responses. To support the definition of threshold concentrations a large set of historical data wasincluded in the survey. Data taken from Ulrich et al. (5).

Local Skin Immune Hyperreaction 395

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LPS


LTT

3Lymphocyte Transformation Test

Lung Sensitization Test

3Animal Models for Respiratory Hypersensitivity

Lupoid Hepatitis

3Hepatitis, Autoimmune

Lymph

A clear, yellowish fluid found in intercellular spacesand in the lymphatic vessels.

3Lymph Nodes

Lymph Flow

3Lymph Transport and Lymphatic System

Lymph Gland

3Lymph Nodes

Lymph Node

An encapsulated and organisedcollection of immuno-logically competent lymphoid cells that receive lymphand antigen from local tissues via afferent lymphatics.Primary immune responses to antigen encountered inthe surrounding tissues are induced in regional lymphnodes.

3Local Lymph Node Assay

Lymph Nodes

C Frieke Kuper

Toxicology and Applied PharmacologyTNO Food and Nutrition ResearchZeistThe Netherlands

Synonyms

lymph node, lymph gland, nodus lymphaticus

Definition

Lymph nodes are secondary lymphoid organs (periph-eral lymphoid organs) located in the course of 3lym-phatic vessels. They filter the 3lymph during its pas-sage from the tissues to the thoracic duct and initiateimmune reactions.

Characteristics

General

Lymph nodes are found in mammals as rounded tobean-shaped lymphoid structures. They are sur-rounded by a collagenous capsule from which trabec-ulae enter into the node, and connected by lymphvessels. The basic anatomical features are the cortex/paracortex and medulla (1). The cortex/paracortex isthe site of antigen encounter and initiation of immunereactions. The products of an immune response (acti-vated cells, effector lymphocytes, inflammatory med-iators) are generated in the medulla. The cortex con-sists of follicles, mainly just underneath the lymphnode capsule, and interfollicular areas which extendto the medulla and are known as paracortex (Figure 1).The follicles contain mostly B cells, whereas T cellsare the major lymphocyte population in the paracortex.The paracortex is easily distinguished by the presenceof specific blood vessels, the so-called high endothe-lial venules. The arterial blood supply, entering thenode at the medulla, ends in the cortex/paracortex asarteriolar capillaries. The capillary bed feeds venuleswhich are lined with endothelial cells that become cu-boidal (“high”) when activated. Lymphocytes migratethrough these high endothelial venules following ad-herence to the endothelium by specific receptor-ligandinteractions. The specificity of these receptor-ligandinteractions is different between lymph nodes drainingthe ‘internal’ organs and tissues and those drainingsecretory surfaces and mucosa-associated lymphoidtissues (MALT). This way, by using the same transportsystem (blood circulation), lymphocytes “belonging”to one of these systems can specifically access theinternal or mucosal lymphoid nodes. After migrationinto the parenchyma, the cells move to their microen-vironment in follicles or paracortical areas.

396 LPS

Reactions after Antigen Contact

The major route of entry for antigens and pathogens isby the afferent lymph flow, which ends in the subcap-sular area in most animals (in pig the flow ends in themedulla). From there, antigens, either free or pro-cessed by macrophages (“veiled macrophages” in theafferent lymph), move to the paracortex. The subcap-sular area is rich in macrophages (sinus macrophages)that can phagocytose free antigen. In the paracortex,antigens are presented to T lymphocytes for the initia-tion of the immune response. The main antigen-pre-senting cell population consists of interdigitating cells,which are a special type of cells closely related toLangerhans cells in the skin and veiled macrophagesin the afferent lymph. These cells are often surroundedby T cells. The antigen-presenting cells express MHC(major histocompatability complex) class II antigensin high density, enabling the T cells to recognize theantigenic determinant with their α-β TCR (T cell re-ceptor) and the polymorphic (self) MHC class II mol-ecule. In this cellular interaction, the immune responsestarts with synthesis of cytokines. For B-cell activa-tion, the process proceeds in the lymphoid follicle,which develops from a primary into a secondary fol-licle. The secondary follicle consists of a 3germinalcenter surrounded by a mantle. The activation andproliferation of B cells in the germinal center is ac-companied by an isotype switch of the immunoglob-ulin class synthesized by the B cell. Following activa-tion and clonal expansion in the germinal centers,B lymphocytes migrate to the medullary cords of thelymph nodes and either become plasma cells or exit

the lymph nodes as memory B cells. Since antigen canremain in the follicular microenvironment for a longtime, it causes a persistent activation of B cells and socontributes to immunological memory within theB lymphocyte compartment. After antigen disappear-ance, immunological memory in the B cells is short-lived and is taken over by the T cell population.The major site of effector immune reactions is themedulla. Medullary cords contain macrophages andmay contain activated effector cells, depending onthe response initiated in the cortex. Among the effectorcells are plasma cells and T cells, the latter bearing anα-β TCR that recognizes antigen in the context of thepolymorphic determinant of MHC class I molecules.In addition, the medulla houses T cells, involved in adelayed type hypersensitivity response and synthesiz-ing a variety of lymphokines. The reaction productsleave the lymph nodes from the medulla via the effer-ent lymph and blood circulation for other sites in thebody. For instance, plasma cell precursors home to thebone marrow, which supplies the major portion of in-travascular immunoglobulins.

Growth and Involution

The lymph node architecture is a dynamic rather than astatic feature. The unstimulated or resting lymphnodes, for example the popliteal or axillary lymphnodes, are very small, being a storage site for virginT and B cells; most follicles are primary. After stimu-lation the organ increases in size in a relatively shorttime, showing high proliferative activity of lympho-cytes and germinal center formation. After termination

Lymph Nodes. Figure 1 Schematic presentation of lymph node. •, ×, and ⊙ represent antigenic substances thatare drained from the respective tissues into the lymph node.

Lymph Nodes 397

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of the reaction or transfer of the reaction to the nextdraining node, it returns to its original size. Lymphnodes retain the ability to respond throughout life.An age-associated decrease in the number of germinalcentres and overall inactive appearance of the node hasbeen reported (2). Lymph nodes of aged animals canshow the same degree of reaction as those observed inyoung rats upon local or systemic antigenic stimula-tion.

Preclinical RelevanceThe lymph node is a lymphoid organ that drains spe-cific organs and tissues via the interstitial tissue fluidand lymph. Direct immunotoxic effects of a compoundmay be manifested in specific lymph nodes whereasindirect effects, via the thymus and/or bone marrow orvia systemic actions, may affect all lymph nodes moreor less in an equal manner. For immunotoxicity assess-ment, lymph nodes can be divided into those that drainthe site of exposure to the toxicants and those at somedistance. This does not imply that the nondraininglymph nodes are not stimulated, because mucosa-draining lymph nodes almost always demonstratesome level of activation due to continuous exposureto antigenic substances in the air or in the food. Forinstance, the superior cervical (mandibular) lymphnodes in rodents contain many germinal centres inthe cortex and plasma cells in the medullary cords,because they are continuously exposed to antigensvia the oronasal mucosae, and the superior mesentericlymph nodes house many macrophages, especially inthe sinuses and paracortex due to continuous exposureto antigens via the intestines. The microscopic appear-ance of lymph nodes varies widely depending on theplane of sectioning. This should be kept in mind whenevaluating these organs.

Relevance to HumansThe structure and function of lymph nodes varies onlyslightly between mammals. As with other lymphoidorgans such as the thymus and spleen, the universalityof the immune system observed in mammals and theresults of toxicity studies observed so far indicate thatdata from laboratory animals can be extrapolated quitewell to humans. Differences in immunotoxicity be-tween laboratory animals and man appear to dependpredominantly on differences in toxicokinetics andmetabolism of toxicants, and these aspects shouldtherefore be considered when immunotoxicity datafrom animals are extrapolated to humans.

Regulatory EnvironmentCurrent regulatory toxicity tests, e.g. for pharmaceuti-cals and industrial substances, include immune para-meters but are still under development. Several guide-lines require examination of both the lymph nodes that

drain the exposure site as well as examination of non-draining lymph nodes (see Table 1 for lymph nodesand the area they drain). The relevance of weighinglymph nodes is well recognized in certain guidelines,especially those dealing with pharmaceuticals andthose specifically designed for detection of immuno-modulating substances (local lymph node assay andpopliteal lymph node assay). The relevance and ap-plicability of weighing the mesenteric lymph nodesin oral studies is still under debate.

References

1. Sainte-Marie G, Belisle C, Peng FS (1990) The deepcortex of the lymph node: Morphological variations andfunctional aspects. In: Grundmann E, Vollmer E (eds)Reaction Patterns of the Lymph Node, Part I. Cell Typesand Functions. Springer Verlag, Berlin, pp 33–63

2. Losco P, Harleman H (1992) Normal development,growth and aging of the lymph node. In: Mohr U,Dungworth DL, Capen CC (eds) Pathobiology of theAging Rat, Volume I. ILSI Press, Washington DC, pp 49–75

3. Tilney NL (1971) Patterns of lymphatic drainage in theadult laboratory rat. J Anat 109:369–383

4. Kuper CF, Schuurman H-J, Vos JG (1995) Pathology inimmunotoxicology. In: Burleson GR, Dean JH, MunsonAE (eds) Methods in Immunotoxicology, Volume I.Wiley-Liss, New York, pp 397–437

Lymph Transport and LymphaticSystem

Anatoliy A Gashev . David C Zawieja

Department of Medical Physiology, College ofMedicine, Cardiovascular Research Institute Divisionof Lymphatic Biology, Texas A&M University SystemHealth Science Center336 Reynolds Medical BuildingCollege Station, TX 77843USA

Synonyms

lymph flow, lymphodynamics

Definition

The lymphatic system is an organized network of ves-sels and nodes, which are present in most tissues andorgans of the body. This system plays a vital role influid and macromolecular transport, lipid absorption,and immunity, and is involved in various pathologicprocesses of different origins. Effective functioning ofthe lymphatic system depends upon the central move-ment of lymph through its vascular and nodal network.

398 Lymph Transport and Lymphatic System

Characteristics

In spite of growing efforts to discover the physiolog-ical mechanisms of lymph flow, there is no commonlyaccepted concept of the regulation of lymph flow.However, investigations over the last decades havepresented many basic facts on lymph flow and its reg-ulation. The lymphatic transport system moves fluid,macromolecules, and formed elements from within theinterstitial spaces into the lymphatic capillaries in theform of lymph. To accomplish these tasks, the lym-phatic vessels must act as both fluid pumps and path-ways. Lymphatic vessels display genotypic and phe-notypic characteristics of vascular, cardiac, and viscer-

al myocytes, which are needed to fulfill the uniqueroles of the lymphatic system. Lymph formation inorgans and tissues occurs due to the differences ininterstitial and lymphatic capillary pressures (1).From the lymphatic capillaries, the lymph is propelledthroughout a complicated network of lymphatic ves-sels and numerous lymph nodes toward to the lym-phatic trunks and thoracic duct. Lymph enters the cen-tral veins from the lymphatic trunks and the thoracicduct which are functioning as the final outflow pathsfor the lymphatic circulatory pathways.Lymphatic vessels are made up of chains of sequen-tially located chambers called 3lymphangions (2). A

Lymph Nodes. Table 1 Drainage patterns of lymph nodes in the rat (adapted from (3,4))

Lymphnode(group)

Area drained Efferent drainage

Superficial cer-vical

Tongue, nasolabial lymphatic plexus Posterior cervical nodes

Facial Head; ventral aspect and sides of neck Posterior cervical nodes

Internal jugular Pharynx, larynx, proximal part of esophagus Posterior cervical nodes

Posterior cervi-cal

Superficial cervical, facial and internal jugular nodes, pharynx, larynx,proximal part of esophagus, NALT

Cervical duct

Brachial Upper extremities, shoulders, chest Axillary nodes

Axillary Upper extremities, trunk, brachial nodes Subclavian duct

Inguinal Thigh, haunches, scrotum, lateral tail Axillary nodes

Popliteal Foot, hind leg Lumbar, inguinal nodes

Gluteal Tail Caudal, lumbar, in-guinal, popliteal nodes

Parathymic Peritoneal cavity, liver, pericardium, thymus, lung Mediastinal duct

Posterior medi-astinal

Thoracic viscera, pleural space, pericardium, thymus Mediastinal duct

Paravertebral Diaphragm, thoracic viscera Posterior mediastinalnodes

Caudal Ventral tail, anus, rectum, gluteal nodes Iliac nodes

Iliac Pelvic viscera, popliteal, gluteal, caudal nodes Renal nodes

Para-aortic Pelvic viscera, popliteal, gluteal, caudal nodes Renal nodes

Renal Kidneys, suprarenal and lumbar lymphatics Renal duct to cisternachyli

External lumbar Fat pad, psoas muscles, pelvic viscera Lumbar lymphatics

Splenic Splenic capsule and trabeculae Posterior gastric nodes

Posterior gas-tric

Distal esophagus, stomach, pancreas, splenic node Portal nodes

Portal Liver, splenic, posterior and gastric nodes Portal duct to cisternachyli

Superior me-senteric

Small intestines, cecum, ascending and transverse colon, Peyer’spatches

Superior mesentericduct to cisterna

Inferior mesen-teric

Descending and sigmoid colon Inferior mesenteric ductto cisterna

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lymphangion is a morphological/functional unit oflymphatic vessels defined as the section of a lymphaticvessel between two adjacent lymphatic valves. Lym-phangions have highly organized endothelial andsmooth muscle layers in their walls. The presence ofspontaneous oscillatory contractile activity has beenobserved in lymphangions from many regions of thebody in many different species.The spontaneous contractile activity of lymphangionsis initiated by action potentials, apparently arisingwithin cells in the smooth muscle layer. The phasiccontractions of the lymphangions are termed the 3ac-tive lymph pump. It has been proven that this activepumping of the lymphatic vessels is a critically impor-tant mechanism for generating lymph flow. The phasesof the lymphangion contractile activity that lead to themovement of lymph toward the venous circulation aresimilar to the phases of contractile activity of the heartchambers (3). There are also several passive forcesthat affect lymph flow. The term "passive" reflectsthe origin of these forces, which are not generatedby active contractions of lymphatic muscle cells. Therate of lymph formation is the principal passive forcethat influences lymph flow in every lymphatic bed.Lymph formation depends on many factors and varieslocally in connection with the level of physiologicalactivity of the organ or tissue. The so-called 3passivelymph pump consists also of other passive lymph-driving forces: the contractions of skeletal muscles,

fluctuations in central venous pressure, the influenceof respiratory movements, pulsations of adjacent arte-ries, and influences of gravitational forces. Due tovarying anatomical positions in the body, differentlymphatic beds are affected differently by these forces.It is also known that lymphangions have inherent auto-regulatory mechanisms, such as stretch-dependent andshear-dependent responses.Two of the main physical factors that regulate lymphdynamics are 3intraluminal pressure and flow modu-late active lymph pumping (4).There are significant regional differences in the basiccharacteristics of lymphatic contractility. The regionalvariability in the active lymph pumps is likely prede-termined by different hydrodynamic factors and bydifferences in regional outflow resistances in the lym-phatics at their respective locations. The lymph pumphas very complicated systems of extrinsic control, bothneural and humoral (5), to match lymphatic pumpingto the different physiological activities in the differentparts of the body. Thus changes in the lymphatic con-traction/relaxation state via the intrinsic/extrinsicpumping mechanisms or neural/humoral controllerscan lead to changes in the lymph flow.

Preclinical RelevanceDamage to the transport capabilities of the lymphaticsystem causes lymphedema, which is associated withdifferent pathologies, such as inflammation, invasionsof parasites or bacteria, partial or full occlusions oflymphatic vessels and nodes after surgical manipula-tions or X-ray treatment of tumors. Despite of ongoingattempts to discover the mechanisms of and treatmentsfor lymphatic-involved diseases, there are a many dis-putable or unknown issues regarding the physiology oflymph transport.

Relevance to HumansThere is strong evidence for the presence of spontane-ous contractile activity in human lymphatic vessels.This evidence was obtained using many different tech-niques including visual observations, measurements oflymph pressure fluctuations in catheterized lympha-tics, and some limited data from isolated lymphaticsections in vitro (6). This spontaneous contractile ac-tivity presumably results in changes in lymph pressurethat are needed to produce lymph flow. The mecha-nism by which this pumping activity is regulated isstill not well understood. We know that some factorscan change lymph flow by modulating human lym-phatic contractility (e.g. adrenergic and nonadrenergicregulation); however, our knowledge of the mechan-isms which generate and regulate lymphatic pumpingactivity in humans is far from complete. In general,most of our understanding of the mechanisms respon-sible for controlling human lymphatic pump function

Lymph Transport and Lymphatic System.Figure 1 Microphotographs illustrating the lymphaticcontractile cycle. Two microphotographs were taken atthe end of diastole (relaxation phase, A) and at thepeak of contraction during systole (contraction phase,B) of isolated rat cervical lymphatic vessel. It is clearthat contraction caused a decrease in lymphatic diam-eter. The increase in intralymphatic pressure due to thecontraction led to the opening of the lymphatic valve (lv)and propulsion of fluid in the downstream direction(shown by arrows in area B).

400 Lymph Transport and Lymphatic System

is limited primarily to observational data and needsmore investigation.

References

1. Schmid-Schonbein GW (1990) Microlymphatics andlymph flow. Physiol Rev 70:987–1028

2. Mislin H, Rathenow D (1962) Experimentelle Untersu-chungen über die Bewegungskoordination der Lymphan-gione. Rev Suisse Zool 69:334–344 (in German)

3. McHale NG, Roddie IC (1976) The effect of transmuralpressure on pumping activity in isolated bovine lymphat-ic vessels. J Physiol 261:255–269

4. Gashev AA (2002) Physiologic aspects of lymphaticcontractile function: Current perspectives. Ann NY AcadSc 979:178–187

5. von der Weid P (2001) Review article: Lymphatic vesselpumping and inflammation—the role of spontaneousconstrictions and underlying electrical pacemaker poten-tials. Aliment Pharmacol Ther 15:1115–1129

6. Gashev AA, Zawieja DC (2001) Physiology of humanlymphatic contractility: A historical perspective. Lym-phology 34:124–134

Lymphadenopathy

This is a swelling of the lymph nodes, which can beclassified as localized or generalized. Causes mayrange from benign infections to serious underlying ill-nesses, such as lymphoma, metastatic cancer and ac-quired immunodeficiency syndrome.

3Interferon-γ

Lymphangion

Morphological/functional unit of lymphatic vesselsdefined as the section of a lymphatic vessel betweentwo adjacent lymphatic valves.


Lymphatic

Vessel that collects fluid from interstitial spaces andlead it via lymph nodes to the thoracic duct and blood.

3Lymph Nodes

Lymphoblastic Lymphoma

3Lymphoma

Lymphocyte-Activated Killer (LAK)Cells

LAK cells are cytotoxic cells with a relatively broadtarget cell specificity and develop from peripheralblood lymphocytes upon stimulation with interleu-kin-2.


Lymphocyte-Activating Factor

3Interleukin-1β (IL-1β)

Lymphocyte Activation Test


Lymphocyte Mitogenesis


Lymphocyte Proliferation

René Crevel

Safety & Environmental Assurance CentreUnilever ColworthSharnbrook, BedfordMK44 1LQUK

Synonyms

lymphocyte transformation, lymphocyte mitogenesis,lymphocyte proliferative response

Definition

Lymphocyte proliferation is defined as the processwhereby lymphocytes begin to synthesize DNA aftercross-linking of their antigen receptor either followingrecognition of antigen or stimulation by a polyclonalactivator (mitogen).

Characteristics.

Events in lymphocyte proliferation

Lymphocyte proliferation is a fundamental character-istic of the response of lymphocytes to antigenic stim-ulation. In physiological situations, contact between a

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lymphocyte and an antigen-presenting cell (APC) re-sults in the formation of an immunological synapse. Inthis synapse, binding to the 3 T cell receptor (TCR) ofthe peptide–MHC complex carried by antigen-present-ing cells, together with a costimulatory signal deliv-ered by interaction between 3 3CD28 and its ligand,initiates proliferation, while the synapse itself is stabi-lized by interactions between adhesion molecules andtheir ligands on the respective cells involved. The pro-cess can be short-circuited in vitro through the use ofpolyclonal activator (e.g. 3phytohemagglutinin,

3Concanavalin A (ConA) or anti-CD3 for T cells),which removes the requirements for antigen present-ing cells and costimulation. These events result in aincrease in the cytoplasmic concentration of the groupof molecules known as NFκB, which in turn, upontranslocating to the nucleus, initiates transcription ofgenes for IL-2 and its receptor, CD25. The increasedproduction of interleukin-2, through an autocrine pro-cess, further stimulates proliferation. While cross-link-ing of the antigen receptor is a critical event in lym-phocyte proliferation, other factors determine the out-come of the stimulatory process, in particular the sig-nals delivered by different types of antigen-presentingcells. Only antigen-presentation by professional anti-gen-presenting cells, such as dendritic cells, is capableof providing the accessory signals needed to driveproliferation of T cells leading to an effective immuneresponse to an antigen. Absence of some of thosesignals will lead instead to anergy or clonal deletion.The result of cell stimulation by antigen or mitogen isto shift the cell from the G0 (quiescent) phase of thecell cycle to the G1. At this particular phase of thecycle, regulation of progression through the cycle isgoverned by proteins which are involved in cell-cycleregulation in all cells (e.g. cyclins), so at this pointcontrol is no longer specific to cells of the immunesystem. Following activation and entry into G1, thelymphocyte will progress through the subsequentphases of the cycle—S phase (synthesis) whereDNA synthesis takes place, G2 phase (gap 2), andM phase (mitosis), where cell division actually takesplace, before returning to G1 (Fig. 1).

Time course of lymphocyte proliferation

The initial events of lymphocyte proliferation, namelyformation of the immunological synapse and the inter-action between APCs and lymphocytes, take placeover several hours, although there is debate over theactual time for which any one peptide–MHC complexneeds to bind to the TCR. Once the initial event hastaken place, proliferative activity peaks at differenttimes depending on the species, the antigen, or mito-gen concentration and—in vitro—the cell concentra-tion. Typically rodent lymphocytes will show peakmitogenic responses after 48 hours, while antigenicresponses require 72–96 hours. In man, the corre-sponding figures are 72 hours and 4–5 days, andsometimes longer.

Measurement of lymphocyte proliferation

Consideration of the events in cell proliferation showsthat the process can be measured at several points.However, DNA synthesis is the parameter that is stillmost commonly used to measure lymphocyte prolifer-ation. One of the reasons for preferring it to earlierphases is that it reflects a fundamental feature of theprocess, unlike some of the earlier events, and there-fore can be argued to have greater predictive value.One of the most common ways of measuring DNAsynthesis, and hence lymphocyte proliferation, isusing radiolabeled nucleotides, typically tritiated thy-midine. Lymphocytes are usually cultured in 96-wellmicroplates, although other formats (60-well and 384-well) are also used, for a duration appropriate to thespecies and the stimulus used (mitogen or antigen).Typically, for in vitro lymphocyte cultures, tritiatedthymidine is added up to 24 hours before terminationof the culture and is incorporated into the dividingcells in proportion to their number in the culture.After the end of the incubation period with labelednucleotide, the cells are harvested in such a way thatthe uptake of radioactivity by the cells can be mea-sured. As part of the harvesting process, the cell cul-tures are aspirated onto glass fiber filter papers, orother suitable supports, which are then air dried andplaced in vials containing a scintillation countingcocktail (when the radiolabel used is tritium). Thedisintegrations per minute (d.p.m.) reflect the intensityof proliferation in each culture. Results are expressedin a variety of ways, sometimes as d.p.m. corrected forbackground activity, and sometimes as a stimulationindex (ratio of d.p.m. of stimulated cultures to d.p.m.of control cultures). A similar method is used to mea-sure proliferation in vivo in tests such as the locallymph node assay, but the labeled nucleotide is inject-ed intravenously, and the lymphocytes harvested fromthe relevant lymph nodes at an appropriate time afterthe injection. The cell cultures are directly added to thescintillation cocktail and radioactivity measured.Lymphocyte Proliferation. Figure 1 The cell cycle.

402 Lymphocyte Proliferation

MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetra-zolium bromide) dye measurement relies on the incor-poration of the dye by viable cells. In proliferatingcultures, unstimulated cells tend to die off, thus theMTT method tends to measure survival as much asactual proliferation.Flow cytometric methods for measurement of lympho-cyte proliferation have been described. One uses theintracellular fluorescent dye carboxyfluorescein succi-nimidyl ester (CFSE). Indices of proliferation such aspercentage of blastic transformation and mitotic activ-ity correlated well with tritiated thymidine incorpora-tion, after 3CD3+ T cells were gated. Other variantsof the technique include analysis based on a dye in-corporated into the membrane of dividing cells, directcounting of cells, and analysis of the entry of cells intothe S phase of the cells cycle.Proliferative responses can be measured in differentsubpopulations of lymphocytes and in response topolyclonal lymphocyte activators (mitogens) as wellas to specific antigen, if the cells are from an immu-nized donor. T cell mitogens include Concanavalin A(Con A) and phytohaemagglutinin (PHA), which canbe used successfully with human cells, as well as thoseof several common laboratory species such as themouse and rat. In contrast, B cells from different spe-cies appear to respond to different mitogens. While

3pokeweed mitogen (PWM) stimulates human cells,murine cells respond to lipopolysaccharide (LPS),while rat cells can be stimulated with 3Salmonellatyphimurium mitogen (STM). The dose–response toCon A and PHA has been well investigated. Itshows an optimum dose beyond which the responsedeclines owing to toxicity of the mitogen to the cells(Fig. 2). This characteristic has implications for mea-surement of proliferation in immunotoxicity studies,inasmuch as measurement at a single dose may leadto misleading conclusions. Most antigens tend to pro-duce responses which plateau, thus assessment can be

limited to a dose which just gives a maximal response.The mixed leukocyte reaction (MLR) is also a form ofproliferative assay, in which allogeneic cells are thestimulus, rather than a soluble antigen or mitogen.

Preclinical RelevanceLymphocyte proliferation has been, and continues tobe used extensively as one of the immunotoxicity end-points. Recent studies incorporating it as an endpointinclude an investigation of the Gulf War Syndrome, ofthe effects of sodium bromate, a byproduct of waterdisinfection by ozone, and of the effect of N,N-diethy-laniline in mice. Its enduring popularity may be relatedin part to the fact that it does reflect a basic biologicalphenomenon in the immune system. Another advan-tage may be that direct comparisons can be made be-tween the activity of animal cells used in toxicity stu-dies and that of human lymphocytes exposed to thesame test material. Lymphocyte proliferation measure-ments can be applied in several ways to investigate thepotential immunotoxicity of test materials on immu-nocompetent cell populations. Probably the most com-mon consists of administering the test compound invivo, for instance in a standard repeat-dose study, andto examine the response to mitogens or specific anti-gen of suspensions of immunocompetent cells pre-pared from lymphoid organs removed at the end ofthe study. This type of study is relatively simple toperform in a laboratory familiar with cell culture tech-niques, but can be difficult to interpret in the absenceof supplementary data on the composition of cell po-pulations and other parameters of responsiveness, suchas cytokine profiles. This difficulty is increased furtherby the observation that the response of cell popula-tions from different lymphoid organs can be very dif-ferent. For instance, in early studies in which carra-geenan (a sulfated polysaccharide extracted from atype of seaweed) was administered orally to rats, theproliferative response of mesenteric lymph node cellswas depressed, while that of cells from cervical lymphnodes was increased (1). Luster et al (2) examined thesensitivity and predictive ability of different measures,and combinations of measures of immune responsive-ness, based on the extensive dataset generated in theB6C3F1 mice used in the US National ToxicologyProgram. They found that there was good concordancebetween lymphocyte proliferation to T cell mitogensand several other measures of immune responsiveness.However, they also found other combinations of teststo be more predictive and did not recommend the in-clusion of proliferative responses in standard immuno-toxicity test batteries, but this conclusion may dependon the exact experimental conditions. Lymphocyteproliferation in response to Con A and PHA wasalso included in the test battery used in the Interna-tional IPCS CEU Collaborative Immunotoxicity Study

Lymphocyte Proliferation. Figure 2 Dose responseof rat splenocytes to Concanavalin A.

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( 3ICICIS). This study found that lymphocyte prolif-eration was reasonably reproducible over several la-boratories, if a standard protocol was used, and was ofsimilar sensitivity to histopathologic methods forscreening immunosuppressive immunotoxicants(Fig. 3) (3). Lymphocyte proliferation ex vivo as ameasure of immune responsiveness also possessessome inherent weaknesses. The principal one is thatit relies on the cells not recovering from the immuno-toxic insult once they have been removed from theanimal and placed in culture. Clearly this will dependon the nature of the immunotoxicant and therefore theeffect that it has on cells in vivo. If, for instance, it isspecifically toxic to certain subpopulations, or is ableto prevent their activation, then this effect will be de-tectable ex vivo without the immunotoxicant in thecell culture, because, for instance, it will specificallyalter the balance of cells in the lymphoid organ used. Itis possible also to conceive of a situation where a testmaterial produces an effect on lymphocytes which re-quires continued exposure to manifest itself. An ex-ample might be that of a change in membrane fluiditythrough incorporation into the membrane. Such an ef-fect could rapidly wane once the cells had been placedin the culture environment.An alternative way in which lymphocyte proliferationhas been used is through exposure of immunocompe-tent cells to the putative toxicant in vitro. One advan-tage of this approach is that it permits a direct com-parison between the responses of human cells andthose of equivalent cells from experimental animals,exposed identically. It also permits the identification oftransient effects such as those postulated in the pre-ceding paragraph, although it is attended by otherdrawbacks. These include the potential for interactionswith the mitogens or antigens themselves, which couldresult in false positives. An additional requirement isto ensure that the doses used are not cytotoxic per se.If immunotoxicity is due to a metabolite, rather thanthe parent compound, the test is likely to give a false

negative result. A more serious difficulty is under-standing how to extrapolate the results obtained toan estimate of effect in vivo. For this reason, thevalue of exposure in vitro is probably limited toscreening assays and mechanistic studies.

Relevance to Humans

Lymphocyte proliferation is used as an index of im-mune function in many clinical applications. Specificinstances include measurement of responsiveness tomitogens and specific antigens to identify possible im-munodeficiency or hyporesponsiveness. One majorapplication continues to be the monitoring of immunestatus in HIV/AIDS patients and indeed efforts in thisarea have been focused on standardization of the pro-cedure to facilitate interlaboratory comparisons (4). Inmost instances, responses to mitogens rather than spe-cific antigens are measured, thereby avoiding the pro-blems associated with having to consider the patient'simmunization history, as well as the need for longerculture periods. Responsiveness to specific antigens isarguably more sensitive and relevant, and its feasibil-ity has been demonstrated, even in whole blood cul-tures.Other clinical applications of lymphocyte proliferationinclude the diagnosis of chronic beryllium disease andof pulmonary fibrosis induced by exposure to feathers,monitoring of the course of cutaneous T cell lympho-ma, and monitoring of the course of bee-venom aller-gen immunotherapy.Lymphocyte proliferation is also used widely as a clin-ical research tool. A few illustrative examples includeinvestigation of cellular immune responses to Try-panosoma cruzi proteins, of T cell function in tuber-culosis patients, and of autoimmune responses ofT cells in multiple sclerosis.Lymphocyte proliferation has been used to monitor theeffects of xenobiotics on the immune response ofhuman subjects since the beginning of immunotoxicol-ogy. Well-known early studies include those underta-

Lymphocyte Proliferation. Figure 3 ICICIS: proliferative response of rat spleen cells to concanavalin A indifferent laboratories (expressed as % change from control).

404 Lymphocyte Proliferation

ken by Bekesi et al (5) on the populations exposed topolybrominated biphenyls (PBBs) in Wisconsin, aswell as the investigations into the Yu-cheng (6) andYu-sho episodes resulting from contamination of ricebran oil with PCBs (Fig. 4). This method has also beenused as one of the immunological endpoints in aninvestigation of the immunomodulatory effects of con-jugated linoleic acid isomers (7).


Although widely used in clinical and preclinical trialsfor measuring disturbance of the activation of immu-nocompetent cells, lymphocyte proliferation assays areonly mentioned in some immunotoxicity guidelines.However, lymphocyte proliferation features as an end-point in the local lymph node assay (LLNA), a methoddeveloped to predict the potential of low-molecular-weight chemicals to induce allergic contact dermatitis(OECD 429). According to this guideline, the mea-surement of proliferation, using tritiated thymidine,is undertaken in vitro, even with radiolabeling takingplace in vivo. On the other hand this guideline alsomentions the possibility of measuring lymphocyte pro-liferation by other methods.

References

1. Bash JA, Vago JR (1980) Carrageenan-induced suppres-sion of T lymphocyte proliferation in the rat in vivosuppression induced by oral administration. J Retic Soc28:213–221

2. Luster MI, Portier C, Pait DG et al. (1992) Riskassessment in immunotoxicology. I. Sensitivity andpredictability of immune tests. Fund Appl Toxicol18:200–210

3. ICICIS Group Investigators (1998) Report of thevalidation study of assessment of direct immunotoxicityin the rat. Toxicology 125:183–201

4. Froebel KS, Pakker NG, Aiuti F et al. (1999) Standard-ization and quality assurance of lymphocyte proliferationassays for use in the assessment of immune function.European Concerted Action on Immunological andVirological Markers of HIV Disease Progression. JImmunol Methods 227:85–97

5. Bekesi JG, Roboz J, Fischblein A et al. (1985) Immu-nological, biochemical and clinical consequences ofexposure to polybrominated biphenyls. In: JH Dean etal. (eds). Immunotoxicology and immunopharmacology.Raven Press, New York, pp 346–406

6. Lee T-P (1992) The toxic effects of polychlorinatedbiphenyls. In: Miller K, Turk JL, Nicklin S (eds).Principles and practice of immunotoxicology. BlackwellScientific, London

7. Albers R, van der Wielen RP, Brink EJ, Hendriks HF,Dorovska-Taran VN, MohedeI C (2003) Effects of cis-9,trans-11 and trans-10, cis-12 conjugated linoleic acid(CLA) isomers on immune function in healthy men. Eur JClin Nutr 57:595–560

Lymphocyte Proliferation Test


Lymphocyte Proliferative Response3Lymphocyte Proliferation

Lymphocyte Transformation


Lymphocyte Transformation Test

Werner Pichler

Klinik für Rheumatologie & klinische Immunologie/AllergologieInselspital-Universtät Bern3010 BernSwitzerland

Synonymslymphocyte proliferation test, lymphocyte activationtest, LTT

Short DescriptionThe aim of the lymphocyte transformation test is todetermine, whether a patient has developed a T cell

Lymphocyte Proliferation. Figure 4 Proliferative re-sponses of individuals exposed to PBB.From Bekesi et al. (1985).

Lymphocyte Transformation Test 405

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response against a certain drug. Such T cell responsesare present in practically all types of allergic reactions( 3type I–IV reactions), which means also in antibody-mediated reactions (1). For that purpose, peripheralblood mononuclear cells obtained after density gradi-ent centrifugation (Ficoll-Hypaque) are cultured withdifferent concentrations of the incriminated drug for5–6 days (1–7). The drug concentration used in this invitro assay has previously been determined by estab-lishing toxic concentrations (e.g. by evaluating an in-hibition on PHA-stimulations). The proliferation of thecells can be measured by different means: most fre-quently 3H-thymidine incorporation is used, but eva-luation of T cell activation by flow cytometry (e.g.CD69 upregulation on T cells, intracellular stainingof newly synthesized cytokines) is also possible.The test is interpreted as positive if the proliferation isat least two times higher than the control culture,which does not contain the drug but was performedin the same media. This is given as stimulation index(SI). However, some authors use even lower or highercut-off values (SI 1.8–3.0). It is advisable to set thecut-off value for a novel drug by analyzing the reac-tivity to the drug in exposed, but not allergic, indivi-duals (n = 20).

Characteristics

For many years it has been well known that certaindrugs can induce the proliferation of some T cellclones bearing a specific T cell receptor. This prolifer-ation is drug specific and dependent on the available

T cells with a fitting T cell receptor repertoire, namelythe ability of the drug to interact with a certain T cellreceptor. These drug-specific T cells have been cloned(2–7). Both CD4 and CD8 cells react, but the majorityof cells obtained in such proliferation assays are CD4positive and express the αβ-T cell receptor. TheT cells recognize the drug as a hapten, which meansthat it is bound to a certain carrier protein, or the drugcan directly stimulate the T cell receptor, whereby in-teraction with the major histocompatability complex(MHC) is still required to completely activate thecell (8).The drug-reactive T cells secrete high amounts of var-ious cytokines, in particular the 3interleukin IL-5,which leads to 3eosinophilia—a typical hallmark ofdrug hypersensitivity. Measurement of IL-5 has beenproposed as alternative read-out system. The type ofT cell reaction corresponds to the clinical picture (8).Since the precursor frequency of drug-reactive T cellsis often low, special care has to be given to optimalculture conditions. Too many macrophages may blockproliferation by high prostaglandin PGE2 secretion,and it is also advisable to perform the test both inautologous plasma as well as AB serum (20% of themedia), since differences are not uncommon.

Pros and ConsThe pros of this test include the fact that it is easy toperform and can be rapidly adapted to new com-pounds. In addition:* it is a test based on human cells and it is an in vitro

test* it can be performed with frozen cells* high SI are clear indications of a sensitization.

Lymphocyte Transformation Test. Table 1 Diagnostic procedures in drug allergy – sensitivity and specificity (1)

(Epicutaneous) skin tests Lymphocyte transformation test

Retrospective: Sensitivity: 64% Sensitivity 78/100 = 78%

Specificity: 85% Specificity 87/102 = 85%(falsely+: mainly NSAID)

Prospective: 6/14 = 43% 13/19 = 68%

Lymphocyte Transformation Test.Table 2 Lymphocyte transformation test-suitable drugs

- pure substance antibiotics- antiepileptics- antihypertensives- NSAID- contact allergens- others, e.g. hydroxymethylcellulose- almost all substances, if soluble, pure and not toxic

Lymphocyte Transformation Test.Figure 1 Proliferation of T-cells in cell culture to thedrug.Interpretation:SI > 3 (stimulation index=cpm plus drug/cpm minusdrug) is indicatind a sensitization to β-lactam-antibio-tics.SI > 2 is indicating a sensitization to other drugs.

406 Lymphocyte Transformation Test

The specificity of the test seems to be very good: quitea few studies show that positive results with drugssuch as amoxicillin, lamotrigine, and carbamazepine,can only be obtained in sensitized individuals. How-ever, non-steroidal anti-inflammatory drugs (NSAIDs)could reduce PGE2 synthesis and thus enhance theproliferation. However, this pharmacological mecha-nism is not seen in all individuals.Disadvantages of the use of this test include the fol-lowing facts:* it is dependent on patients' blood cells, which have

been sensitized previously in vivo* it cannot predict the immunogenicity of a drug, as

non-sensitized individuals do not react* some drugs are difficult to dissolve and need spe-

cial solvents (e.g. DMSO) which must be tested aswell

* this biological test is based on the in vitro prolifer-ation of cells obtained from peripheral blood, so itis highly variable and it is problematic to set acorrect cut-off point

* the test is highly dependent on optimal cell compo-sition and serum supplementation of the media, andwith time the presence of sensitized cells in theperipheral blood may go down

* the sensitivity and specificity are debatable, becausesome groups report a relatively high sensitivity for

an allergy test (60%–70%) (1) and others do not,which might be because of the selection of differentpatients and of different drugs; a high sensitivitywas reported for anti-epileptic-induced drug hyper-sensitivity reactions (> 95%) (6,7).

Predictivity

The test is based on the evaluation of already sensi-tized individuals. Therefore, it cannot be used in non-sensitised individuals where it is—by definition—neg-ative. It could be used to follow people treated inphase 1–4 studies, by analyzing the proliferative re-sponse of exposed persons to the drug (± side effects).

Relevance to humans

There are not many tests available to indicate a drughypersensitivity reaction and to pinpoint the relevantdrug in drug hypersensitivity reactions (1). Thereforeeven a suboptimal test such as the LTT is considered tobe useful. The growing recognition for a role of T cellsin such hypersensitivity reactions makes the LTT evenmore attractive, as it is positive in a wide variety ofhuman hypersensitivity reactions. However, as withother immunological tests, sensitization is not neces-sary associated with clinical symptoms.

Lymphocyte Transformation Test. Table 3 Lymphocyte transformation test – positivity in drug allergic diseases

+ –

maculopapular exanthemabullous exanthemadrug feverAGEPeosinophilia, DHS/DRESShepatitispancreatitisnephritisinterstitial lung diseaseurticaria, anaphylaxis

macular exanthema?blood dyscrasia like ITP, hemolytic anaemiaGuillain-Barré?TEN (– > +)vasculitis (– > +)

Lymphocyte Transformation Test. Table 4 Drug allergy tests for T cell reactions

+ –

in vitro regulatory and inflammatory T-cell reactionsdetectablenot dependent on strong inflammatory responsepositive with IgE mediated reactionssensitivity and specificity satisfactoryopen for new developmentopen for research (cytokine determination,...)possible with frozen cells

complicated, long lastingnot in acute reaction, results available long afterreactiondependent on fresh cells (24–36 hours) standard-izationpure drugs necessaryfalse reactions with tablets„distant“ to clinicTEN, vasculitis often negative metabolism(?)

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References1. Pichler WJ, Tilch J (2004) The lymphocyte transforma-

tion test in the diagnosis of drug hypersensitivity. Allergy59:809–820

2. Nyfeler B, Pichler WJ (1997) Sensitivity and specificityof the lymphocyte transformation test to drugs. Clin ExpAllergy 27:175–181

3. Neukomm C, Yawalkar N, Helbling A, Pichler WJ (2001)T-cell reactions to drugs in distinct clinical manifestationsof drug allergy. J Invest Allerg Clin Immunol 11:275–284

4. Maria VA, Victorino RM (1997) Diagnostic value ofspecific T-cell reactivity to drugs in 95 cases of drug-induced liver injury. Gut 41:534–540

5. Tsutsui H, Terano Y, Sakagami C, Hasegawa I,Mizoguchi Y, Morisawa S (1992) Drug-specific T-cellsderived from patients with drug-induced allergic hepati-tis. J Immunol 149:706–716

6. Naisbitt DJ, Britschgi M, Wong G et al. (2003) Hyper-sensitivity reactions to carbamazepine: characterization ofthe specificity, phenotype, and cytokine profile of drug-specific T cell clones. Mol Pharmacol 63:732–741

7. Naisbitt DJ, Farrell J, Wong G et al. (2003) Characteriza-tion of skin homing lamotrigine-specific t-cells fromhypersensitive patients. J Allergy Clin Immunol111:1393–1403

8. Pichler WJ (2003) Delayed drug hypersensitivity reac-tions. Ann Int Med 139:683–693

Lymphocytes

Brad Bolon

GEMpath Inc.2540 N 400 WCedar City, UT 84720-8400USA

Definition

Lymphocytes are a subclass of leukocytes (whiteblood cells) that are the principal elements controllingthe 3adaptive immune response, which develops inresponse to a particular antigenic stimulus. One speciallymphocyte variety—the 3large granular lymphocyte—also plays a role in the antigen-independent 3innateimmune response.

Characteristics

Lymphocyte Categories

In general, lymphocytes can be classified by eitherlineage (Figure 1) or function.The three principal lineages of lymphocytes areB lymphocytes (B cells), T lymphocytes (T cells),and natural killer cells (NK cells). Activated B cellsdifferentiate into 3plasma cells, which secrete the im-munoglobulins (antibodies) that drive humoral immu-nity and serve to destroy extracellular pathogens andtheir products. Activated T lymphocytes power cell-

mediated immunity, either by killing damaged cellsdirectly—chiefly those expressing tumor antigens orproducts derived by intracellular pathogens (especiallyviruses)—or by regulating the activities of other im-mune effector cells (including B cells). Both B cell-mediated and T cell-mediated functions are controlledby antigen-specific receptors, which originate duringthe evolution of adaptive immunity to a specific anti-gen. In contrast, natural killer cells do not have anti-gen-specific receptors, but instead serve as compo-nents of the innate immune response that kill tumorcells or cells infected with intracellular pathogens (par-ticularly viruses).Functional distinctions also can be used to classifylymphocytes into various groups. Naive B andT cells are relatively inactive (or resting) until theyencounter an appropriate target antigen, after whichthey proliferate and differentiate into antigen-specificeffector lymphocytes and, in some cases, into immu-nological memory cells. The primary function of allB cell-derived effector and memory cells is antibodyproduction. In contrast, different classes of activatedT cells perform diverse tasks; cytotoxic T lymphocytesdestroy diseased cells, while helper T lymphocytesand suppressor T lymphocytes activate or dampen,respectively, the operation of other leukocyte classes(particularly B lymphocytes and macrophages) as wellas certain types of activated non-immune cells (e.g.fibroblasts).These different lymphocyte classes can be identifiedby their CD surface markers. For example, in humanscytotoxic and suppressor T cells bear CD8, while help-er T cells express CD4. Given populations of T cellsmay be divided further based on their specific tasks.Thus, CD4-positive T cells that activate macrophagesand promote digestion of intracellular bacteria are saidto facilitate the T helper type 1 (Th1) response, whilethose CD4-positive T cells that kill infected cells anddirect the destruction of extracellular pathogens byactivating B cells are said to promote the T helpertype 2 (Th2) response. The Th1 and Th2 reactionsare driven by divergent but overlapping groups of cy-tokines, which typically are secreted by T cells (i.e.

3lymphokines) and macrophages (monokines).

Lymphocyte Distribution

Lymphocytes are generated in the central or primarylymphoid organs, the bone marrow, and thymus. Lym-phocytes first arise in bone marrow from 3pluripotenthematopoietic stem cells (HSCs), which give rise toother partially committed HSCs, including the com-mon lymphoid progenitor cell. The lymphoid progen-itor cells differentiate in the marrow into immatureB cells and immature T cells. The B cells undergofurther maturation in the marrow into naive B cells,hence the designation "B cell," for "bone marrow-de-

408 Lymphocytes

rived." In contrast, immature T cells migrate to thethymus before maturing into naive T cells (thus thelabel "T cell" for "thymus-derived."). Naive B andT cells subsequently relocate to the peripheral or sec-ondary lymphoid organs, where adaptive immune re-sponses are initiated and resting lymphocytes aremaintained in close proximity to nascent antigen-pre-senting cells (chiefly 3dendritic cells). These second-ary lymphoid organs include the lymph nodes, various

3mucosa-associated lymphoid tissues (e.g. bronchiand gut), spleen, and tonsils. All lymphoid tissuescontain myriad lymphocytes framed by numerousnon-lymphoid cells and connective tissue fibers. Thestructures of secondary lymphoid organs are designedto filter circulating antigens from the blood or lymph,offer them to antigen-presenting cells (APCs), andthen to facilitate the interaction between the APCsand naive lymphocytes in 3germinal centers. Ex-changes between lymphocytes and the adjacent non-lymphoid cells (particularly antigen-presenting cells inthe secondary lymphoid organs) direct later stages oflymphocyte development, lymphocyte activation, andlong-term lymphocyte maintenance and retention.Lymphocytes also are common in many non-lymphoidorgans. 3Ectopic lymphoid tissues may form in dis-eased tissue during some chronic inflammatory condi-

tions. These structures consist of poorly circumscribedlymphocyte aggregates (chiefly T cells), with or with-out germinal centers, which are formed to provide aconcentrated focus of antigen-specific effector cells.Lymphocytes also are found frequently in capillaries,particularly the winding sinusoids in organs that filterblood (e.g. kidney and liver) and in the submucosalconnective tissues in organs with a direct connectionto the outside environment (particularly the digestive,genitourinary, and respiratory tracts). In the blood,these lymphocytes typically are either naive (unstimu-lated) cells or memory cells (antigen-specific elementsengaged in surveillance) and are merely passingthrough—rather than engaged in—a tissue-specificimmune response. In contrast, lymphocytes in the sub-mucosal connective tissue regulate the immune reac-tion to exogenous antigens that approach or penetratethe mucosa. A component of this response includes themovement of individual cells into the mucosa to serveas intraepithelial lymphocytes.

Lymphocyte Morphology

As shown in Figure 1, lymphocytes have a stereotypi-cal appearance that has earned them (along with mac-rophages) the moniker 3mononuclear leukocyte (asapposed to 3granulocytes, which comprise the

Lymphocytes. Figure 1 All leukocyte (white blood cell) lineages are derived from partially committed stem cellsthat arose from pluripotent hematopoietic stem cells. The common progenitor cells undergo further differentiationinto various classes of lymphocytes (blue cells on the left) or myeloid cells (pink cells on the right). For each lineage,fully differentiated effector cells (labels in red) participate in either the adaptive immune response (labelsunderlined) or the innate immune responses, or both (labels in boxes). For detailed descriptions of the biology forthe various leukocyte types, see the relevant entry.

Lymphocytes 409

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3polymorphonuclear leukocytes). Prior to activation,unstimulated lymphocytes are small cells with few cy-toplasmic organelles and relatively inactive nuclearchromatin. These features impart a unique aspect toboth the B cell and T cell lineages: cells with a dark,round, dense nucleus surrounded by a thin rim of cy-toplasm. Upon activation, T lymphocytes generallymaintain their initial appearance. In contrast, activatedB lymphocytes develop into plasma cells and assume adistinctive profile characterized by eccentric nucleuswith a few radiating chromatin stripes (a so-called"cartwheel" nucleus) and a prominent perinuclearpale zone (the enlarged Golgi apparatus). Natural kill-er cells are large granular lymphocytes that are distin-guished by their bigger overall size, enhanced quantityof cytoplasm, and prominent cytoplasmic granules.

Lymphocyte Physiology

Leukocytes—including large granular lymphocytes—engaged in the innate immune response bear severaldifferent cell surface receptors, each of which recog-nizes a different feature shared by many pathogens. Incontrast, most lymphocytes efficiently participate inthe adaptive immune response against a specific for-eign antigen because each lymphocyte forms a uniqueantigen receptor during maturation by rearranging itscomplement of receptor genes. Collectively, the entireuniverse of T and B lymphocytes bears an enormousrepertoire of receptors with highly diverse antigen-binding sites. When their specific antigen is presentedto them, naive lymphocytes become activated, expressmany new cell surface antigens, and proliferate, there-by forming myriad clones that will seek to neutralize asingle antigen. This process of clonal expansion toform an effector lymphocyte population takes severaldays upon the first exposure to an antigen (either byinfection or vaccination). Most effector lymphocytesundergo apoptosis once their target antigen has beeneliminated, but the persistence of memory cells afterantigen clearance has occurred leads to a more rapidreaction upon subsequent exposures. Lymphocyteswith receptors that recognize endogenous (self) anti-gens typically are deleted during development, therebyensuring 3tolerance of the body to its own tissues andpreventing the initiation of autoimmune disease.

Preclinical RelevanceLymphocytes are found in all mammals. Thus, theworking assumption is that preclinical evaluation oflymphocyte function in mammalian models will reca-pitulate the effects (including those endpoints relevantto immunotoxicology) that manifest in human beings.Typical endpoints used to evaluate lymphocyte phys-iology in animals and humans are a routine hematolo-gic assessment (complete blood count, with whiteblood cell differential count and cytologic evaluation

of blood smears) and various in vitro function tests,such as the lymphocyte transformation assay.Immune function testing in animal models typically isperformed to define the role of various lymphocyteclasses in the pathogenesis of disease (i.e. the "toxic-ity" of lymphocytes to tissues) rather than to examinethe toxicity of xenobiotics or other types of insults tolymphocytes. Hypotheses usually assess the molecularand cellular mechanisms responsible for the disease(1,2) and/or test potential therapies that might preventor reduce the tissue destruction (2,3).Lymphocyte numbers and/or activities in animals maybe substantially reduced or markedly enhanced byspontaneous or engineered mutations. For example,many inherited rodent models of immunodeficiencyhave been reported to result, at least in part, from ab-errant lymphocyte functions (4). Selected mouse mod-els characterized by deficient lymphocytes include bg/bg (beige), which harbor defective cytotoxic T cellsand NK cells; nu/nu (nude), which lack T cells; ob/ob(obese), which have less active T cells but hyperactiveNK cells (on the C57BL/6J genetic background); scid/scid (severe combined immunodeficiency), which lackboth B cells and T cells; and xid/xid (X-linked immu-nodeficiency), which lack B cells. The nude rat (rnu/rnu) has a T cell deficit equivalent to that in the nu/numouse (4). In contrast, lymphocyte numbers and func-tions are greatly elevated in MRL/Mp-lpr/lpr (lym-phoproliferation) mice, which develop generalized hy-perplasia of lymphocytes and plasma cells (4), and inanimals with lymphoma, a malignancy in which thetumor cells arise from a lymphocytic lineage (typicallyB cells or T cells). A novel means of examining theimpact of human lymphocytes in vivo using an animaltest system is to transplant human cells (isolated fromthe 3buffy coat of a routine blood sample), lymphoidtissue fragments, or wedges of lymphocyte-rich dis-eased tissue (e.g. synovial biopsy from an arthritis pa-tient) into an immunodeficient rodent (5).Other models with reduced or exaggerated lymphocyt-ic activity have been generated in immunocompetentanimals. Rodent models of autoimmune diseases ex-hibit many clinical and morphologic features that re-semble those of the corresponding human conditions.Potential lymphotoxic effects may be evaluated byadministering chemicals, such as alkylating agents(e.g. cancer chemotherapies), diethylstilbestrol, gluco-corticoids, and certain heavy metals, as well as bydelivery of ionizing radiation (6). Preclinical examina-tion of immunotoxicity testing typically is performedusing such immunocompetent subjects, both becausetheir intact immune systems more closely resemblethat of humans and because the studies will be morecost-effective as they do not require the special hus-bandry practices needed to maintain animals with im-munologic defects.

410 Lymphocytes

Relevance to Humans

Human disease counterparts have been discovered formany rodent models, especially those for autoimmunedisease (1) and primary immunodeficiency (7). Auto-immune diseases in humans are known to be incitedby certain xenobiotics, apparently as the result—atleast in part—of enhanced function by B cells or help-er T cells, or reduced control by suppressor T cells; theimmune system in animals behaves in a similar fash-ion. Both animal and human immune systems are af-fected in a comparable manner by exposure to manyimmunosuppressive agents. The concordance betweenthe response between the immune systems of immu-nocompetent animals and humans suggests that immu-notoxicity data—including information with regard topotential lymphotoxic effects—will be correlated to ahigh degree across species.


Due to the many structural and functional similaritiesbetween vertebrate immune systems, preclinical effi-cacy and safety studies in immunocompetent animalsare routinely employed as surrogates to define poten-tial lymphocytotoxic events that might be initiated inhuman beings exposed to xenobiotics. However, pre-clinical efficacy and safety studies are notoriously dif-ficult to perform when the candidate therapeutic agentis a human protein, as many will not cross-react withelements in animal immune systems. Alternativemeans of assessing immunotoxic risk for such proteinsinclude testing in non-human primates or investigatinghomologous molecules in the appropriate animal spe-cies. Evaluation of in vivo immunotoxicity towardhuman lymphocytes also might be examined usinghuman tissue xenografts in one or more immunodefi-cient rodent models, but at present such assays are tootechnically difficult and expensive to perform on aroutine basis. All these tests are surrogates to testingthe clinical candidate in humans, and as such each ofthese options will have its own set of caveats. How-ever, "humanized" immunodeficient mice should pro-vide a reasonable alternative in the future, especiallyfor immunotoxicity protocols in which the mouse re-sponse is well characterized.

References

1. Farine J-C (1997) Animal models in autoimmune diseasein immunotoxicity assessment. Toxicology 119:29–35

2. van den Berg W (2000) What we learn from arthritismodels to benefit arthritis patients. Baill Clin Rheumatol14:599–616

3. Bendele A, McComb J, Gould T et al. (1999) Animalmodels of arthritis: Relevance to human disease. ToxicolPathol 27:134–142

4. ILAR (Institute for Laboratory Animal Research of theNational Research Council) Committee on Immunolog-ically Compromised Rodents (2002) Hereditary immu-

nodeficiencies. In: Immunodeficient rodents: A guide totheir immunobiology, husbandry, and use. The NationalAcademies Press, Washington DC, pp 36–139

5. Cleland LG, Fusco M, Proudman SM, Wing SJ, SpargoLDJ, Mayrhofer G (2001) Recruitment of mononuclearleucocytes to osteoarthritic human synovial xenografts inthe ears of SCID mice. Immunol Cell Biol 79: 309–319

6. ILAR (Institute for Laboratory Animal Research of theNational Research Council) Committee on Immunolog-ically Compromised Rodents (2002) Induced immuno-deficiencies. In: Immunodeficient rodents: A guide totheir immunobiology, husbandry, and use. The NationalAcademies Press, Washington DC, pp 140–147

7. NICHD (National Institute of Child Health and HumanDevelopment), National Institutes of Health, USA (2002)Primary immunodeficiency. http://www.nichd.nih.gov/publications/pubs/primaryimmunobooklet.htm (last ac-cessed May 12, 2004)

Lymphocytes

Antigen-specific leukocytes such as B cells, T helpercells and cytotoxic T cells.

3Natural Killer Cells

Lymphodynamics


Lymphoid Organ Compartments

Compartments within lymphoid organs/tissues featureone or more specific functions, and each houses lym-phoid and non-lymphoid cells of different lineages andin different ratios.

3Histopathology of the Immune System, Enhanced

Lymphokine

A cytokine secreted by a lymphocyte to control theresponses of various immune (lymphocytes, macro-phages) and non-immune (chiefly cells of the fibro-blast lineage) effector cells. Now replaced by the moregeneral term cytokines.

3Cytokines

3Lymphocytes

Lymphokine 411

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Lymphoma

Leigh Ann Burns-Naas

Pfizer Global Research & Development10777 Science Center Dr.San Diego, CA 92121USA

Synonyms

Hodgkin’s lymphoma, Hodgkin’s disease, non-Hodg-kin’s lymphoma (NHL), Burkitt’s lymphoma, lympho-blastic lymphoma, marginal zone lymphoma, anaplas-tic large cell lymphoma

Definition

Lymphomas are diverse neoplasms that begin by themalignant transformation, usually of a T cell, B cell, ornatural killer (NK) cell, in the lymphatic system.These diseases result from damage (typically a muta-tion) to the cell’s DNA that results in uncontrolled andexcessive growth and confers a survival advantage onthe malignant lymphocyte and the cells that are formedfrom its proliferation. Accumulation of these dividingcells results in the lymphadenopathy, often an initialindicator of the disease. While similar to leukemia inits general nature (malignant transformation of lym-phocytes), lymphomas differ from leukemias in thatthey typically originate in the lymphatic system (e.g.lymph nodes) instead of the bone marrow.

Characteristics

Thirty or more subtypes of specific lymphomas orclosely related lymphocytic leukemias have been ca-tegorized. Biopsies are usually required to make a de-finitive diagnosis of lymphoma. Tissue from the biop-sy is examined microscopically to determine the pat-tern of the abnormalities and types of cells involved.Biopsied cells may be evaluated using immunopheno-typing and cytogenetic analysis. 3Immunophenotyp-ing may be used to provide additional diagnostic con-firmation and to determine if the malignant cells areT cells, B cells, or NK cell types. 3Cytogenetic anal-ysis is used to identify chromosomal abnormalitiescharacteristic of certain of the lymphomas. Together,these diagnostics may help in the choice of drugs usedfor treatment.Like the leukemias, non-Hodgkin lymphoma (NHL) isa diverse group of hematopoetic neoplasms, with thedistinctions between types based on the characteristicsof the neoplastic cells involved. Each histologicgrouping is diagnosed and treated differently. Of inter-est, in many cases of diagnosed NHL, a translocationbetween certain sets of chromosomes is observed uponcytogenetic analysis. Several genes have been closed

and sequenced at the breakpoints of these transloca-tions (bcl genes). These genes encode specific disease-related proteins that alter the normal growth and pro-liferation of the cells. For example, one of the better-known genes is bcl-2, an 3oncogene whose overex-pression can prevent normal programmed cell death.This gene is expressed in over 80% of cases of follic-ular NHL.Over half of hematopoetic neoplasms are lymphomaswith Hodgkin’s disease representing about one-tenthof those diagnosed lymphomas. Hodgkin’s disease is aunique form of lymphoma identified by the presenceof a special cell type known as the Reed-Sternbergcell, a large multinucleated cell containing extensiveeosinophilic cytoplasm and a large blue nucleoli. Oneof the important features of Hodgkin’s disease is adecrease in the functional capacity of the immunesystem. Cell-mediated immune function, T cell-mediated immunity in particular, appears to be im-paired and as a result, affected individuals with Hodg-kin’s disease are more susceptible to certain types ofinfection. The disease is more prevalent among ado-lescents and with diagnosis and proper treatment has avery high cure rate. Conversely, the incidence of NHLincreases with increasing age and is slightly more pre-valent in men than in women.Marginal zone lymphomas include mucosal-associatedlymphoid tissue (MALT) NHL (MALT-omas), mono-cytoid B-cell NHL, and primary splenic lymphomas.MALT-omas are extranodal neoplasms potentially in-volving one or several organ systems and may beassociated with the presence of autoimmune disordersor Helicobacter pylori infection. Cells in spleen lym-phomas are similar to other marginal zone lymphomas,however they have a distinguishing villous appearancethat can be confused with hairy cell leukemia. Ana-plastic large cell lymphomas have characteristic chro-mosomal abnormalities associated with overexpres-sion of nucleoplasmin-anaplastic lymphoma kinase,as well as unique surface protein expression indicatingthe origin of the cell to be most likely from the T cellor null cell lineages.

Preclinical RelevanceWhile the etiology of the majority of lymphomas hasyet to be determined, there are some associations thatcan be made (Table 1). Cancer patients undergoingtreatment with cytotoxic drugs are more likely to de-velop secondary neoplasms in the years followingtherapy. Additionally, transplant patients on highdoses of immunosuppressive drugs used to preventgraft rejection also appear to be at higher risk fordeveloping blood cancers with the predominant formbeing NHL. A similar trend is noted in immunocom-promised patients (e.g. HIV). One common thread inthis increased susceptibility to lymphomas is a de-

412 Lymphoma

crease in 3cell-mediated immunity in the susceptibleindividual.

The cell-mediated arm of the immune system is in-volved in controlling spontaneous tumors and infec-tions, among other things. The destruction of tumorcells can result from the cytolytic action of specific(cytotoxic) T lymphocytes (CTL), macrophages, andNK cells. These cells recognize specific antigens ontumor or virally infected cells and cause their death byone of several mechanisms considered here.

In cell-mediated cytotoxicity, the effector cell (CTL orNK) binds in a specific manner to the target cell. CTLrecognize either foreign major histocompatabilitycomplex (MHC) class I on the surface of allogeneiccells, or antigen in association with self MHC class I(e.g. viral particles), while NK cell recognition of tar-get cells involves the binding of the Fc portion ofantigen-specific antibody coating a target cell to theNK cell via its Fc receptors. This NK cell mechanismof killing is also referred to as antibody-dependentcellular cytotoxicity (ADCC). Once the CTL or NKcells interact with the target cell, they undergo cyto-plasmic reorientation so that cytolytic granules are or-iented along the side of the effector which is bound tothe target. The effector cell then releases the contentsof these granules onto the target cell. The target cellmay be damaged by the perforins or enzymatic con-tents of the cytolytic granules. In addition, the target is

induced to undergo programmed cell death (apopto-sis). Once it has degranulated, the CTL or NK cell canrelease the dying target and move on to kill other tar-get cells.The role of the macrophage in cell-mediated cytotox-icity involves its activation by T cell-derived cytokine(e.g. IFN-γ) and subsequent recognition of comple-ment-coated target cells via complement receptorspresent on the surface of the macrophage. The resultis enhanced phagocytic ability, and the synthesis andrelease of hydrogen peroxide, nitric oxide, proteases,and tumor necrosis factor, all of which serve cytolyticfunctions. Macrophages may also kill tumor or in-fected cells via ADCC in a manner similar to thatdescribed for NK cells.Drugs and agents that alter cell-mediated immunityhave the potential to cause an increased risk of 3op-portunistic infections (e.g. viral, bacterial, parasitic)and development of neoplastic disease. By evaluatingsuch things as the ability of the immune system torecognize and destroy tumor cells such as the P815mastocytoma (used in the CTL assay) or YAC-1 cell(used in the NK assay) as well as evaluating prolifer-ative ability to mitogens, cytokines, or allogeneic (for-eign; non self) cells, and evaluation of allograft rejec-tion, immunotoxicity testing in rodents has identifiedmany agents capable of causing suppression of cell-mediated immunity. A few of these agents are consid-ered below.Therapeutic agents have been developed that specifi-cally inhibit several immune endpoints. These drugsare often used to treat symptoms associated with au-toimmunity, in transplantation to prevent immune-mediated graft rejection, or to treat individuals withsignificant hypersensitivity responses. Cyclophospha-mide is the prototypical member of a class of drugknown as alkylating agents. It is often used as a pos-itive immunosuppressive control in experimental im-munotoxicology studies because it can suppress bothhumoral and cell-mediated immune responses. Cell-mediated immune activities that are suppressed in-clude the delayed hypersensitivity response (DTH),the cytotoxic T lymphocyte response (CTL), graft-ver-sus-host (GVH) disease, and the mixed lymphocyteresponse (MLR).In addition to alkylating agents, other drugs used tointentionally suppress the immune system also altercell-mediated immunity. The immunosuppressive ac-tion of corticosteroids is one example. Following bind-ing to an intracellular receptor, these agents produceprofound lymphoid cell depletion in rodent modelsand lymphopenia, associated with decreased mono-cytes and eosinophils and increased PMNs, in pri-mates and humans. Corticosteroids induce apoptosis,and T cells are particularly sensitive. In general, corti-costeroids suppress the generation of CTL responses,

Lymphoma. Table 1 Examples of agents associatedwith an increased risk for the development of primary orsecondary lymphomas

Chemotherapeutic agents

AzathioprineCyclosporin AFK-506RapamycinOKT3 monoclonal antibodyCyclophosphamideMelphalanBusulfanPhenytoinMethotrexate

Other agents

Infectious agentsReactivation of latent viruses (such as EBV)Helicobacter pyloriHerpes virus 8Hepatitis CHuman immunodeficiency virus / AIDS

Autoimmune diseases (such as rheumatoid arthritis)Environmental chemicals (?)

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the MLR, NK activity, and general lymphoprolifera-tion. A large range of cell-mediated immune reactivityis also reduced by azathioprine immunosuppressivetreatment including the DHR, the MLR, and GVHdisease. Although T cell functions are the primarytargets for this drug, inhibition of NK function andmacrophage activities has also been reported.Cyclosporin A and FK-506 are important immunosup-pressants that act preferentially on T cells by inhibitionof IL-2 gene transcription and subsequent inhibition ofT cell proliferation. Rapamycin is structurally relatedto FK-506, but inhibits T cell proliferation by blockingcell-cycle progression.There are numerous examples of drug and non-drugchemicals that have the potential to alter immunocom-petence in a manner that might influence one’s sus-ceptibility to spontaneous primary or spontaneous

3secondary neoplasms and opportunistic infectionor reactivation of latent pathogens. This section hasonly considered a very few of these agents, butthose which have limited or sufficient evidence foran association between suppression of immune func-tion and predilection for lymphomas. Clearly, though,other contributing factors (e.g. genotoxicity) cannot beexcluded.

Relevance to HumansA definitive cause for most lymphomas has not beenestablished, though exposure to some pathogens hasbeen implicated. Infection with some viruses such asthe Epstein-Barr virus (EBV), human immunodefi-ciency virus (HIV), and human T cell lymphocytotro-pic virus (HTLV) has been shown to be associatedwith an increased risk for the development of a varietyof lymphomas including Burkitt’s lymphoma, Hodg-kin’s disease, NHL, or T cell lymphoma. In fact, theincidence of lymphoma in HIV-infected persons hasbeen 50–100 times the incidence rate expected in theuninfected population since the apid rise in HIV infec-tion in the 1980s. Even bacterial infections such asH. pylori are associated with an enhanced susceptibil-ity to MALT-oma. Although alterations in DNA stillseem to be integral to the malignant transformation,the high frequency of infection may be a contributingfactor to the development of disease.Altered immunocompetence has been associated withan overall increased risk for the development of sec-ondary neoplasms such as lymphomas. Individualswith acquired immunodeficiency syndrome (AIDS)have increased incidences of a variety of cancers, in-cluding lymphoma, most likely as a result of the lossof the ability of the host to identify and eradicate neo-plastic cells, particularly those infected with pathogen-ic agents. A similar effect has been observed in trans-plant recipients and individuals with severe autoim-mune disorders receiving chemotherapy with cytotox-

ic and/or immunosuppressive drugs. These drugs areknown to alter cell-mediated immunity, an effect thatcan in effect increase the susceptibility to infectiousagents such as viruses. In many cases of significant

3immunosuppression in people, there is a reactivationof latent viruses such as EBV that is associated withthe development of secondary lymphomas. Of interest,there has been a suggestion that exposure to environ-mental chemicals such as pesticides or herbicides hascontributed to the increased incidence of lymphoma.While some of these agents have been demonstrated toalter both 3humoral immunity and cell-mediated im-munity, and some limited epidemiological studies sug-gests an increase in lymphoma in rural communitieswere farming is an important part of life, a clear asso-ciation has yet to be definitively established.

There is a clear association between suppression ofimmune function and an increased incidence of infec-tious and neoplastic disease in humans. Agents thatproduce immunotoxicity in animals have the potentialto produce immune effects in the human population,and these effects may occur in the absence of obser-vable disease. Of the agents described here that areassociated with an increased risk for the developmentof lymphomas, no specific causal relationship betweenthe development of cancer and the immunosuppressiveaction by these drug or non-drug chemicals/agents hasbeen clearly demonstrated. However, a preponderanceof epidemiological evidence exists showing that expo-sure to various immunotoxic chemicals is associatedwith increased risk for malignancies (e.g. leukemiaand lymphoma) that are also known to occur in im-munocompromised patients. Thus, it is reasonable toconclude that alteration of immune function may con-tribute to the observed increase in risk.

Regulatory EnvironmentBecause of the concerns regarding the potential fordrugs and non-drug chemicals to cause any numberof cancers, including lymphomas, global regulatorybodies have established guidance and test guidelinesfor assessing this potential. These include specific as-sessment of carcinogenic potential (e.g. lifetime stu-dies in rodents) as well as assessment of the mutagenicand/or clastogenic potential (short-term in vivo and/orin vitro tests) of chemicals. Though not directly relatedto genotoxicity and carcinogenicity assessment, guide-lines regarding the assessment of immune status fol-lowing repeated exposure to drug and non-drug che-micals have recently been and are continuing to be putinto place. Of interest, not all immunotoxicology guid-ance and guidelines require the evaluation of cell-mediated immunity. Some examples of these gui-dances and test guidelines are provided in Table 2.

3CD Markers

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References

1. International Programme on Chemical Safety (1996)Health impact of selected immunotoxic agents. In:Environmental Health Criteria 180: Principles and Meth-ods for Assessing Direct Immunotoxicity Associated withExposure to Chemicals. World Health Organization,Geneva, pp 85–147

2. Leukemia & Lymphoma Society (2002) Public literature.The Leukemia and Lymphoma Society, White Plains

3. Cheson BD (2001) Hodgkin’s disease and the non-Hodgkin’s lymphomas. In: Lenhard Jr RE, Osteen RT,Gansler T (eds) Clinical Oncology. American CancerSociety, Blackwell Science, Malden, pp 497–516

4. Luster MI, Simeonova P, Germolec DR, Portier C,Munson AE (1996) Relationships between chemical-

induced immunotoxicity and carcinogenesis. Drug Info J30:281

5. Pitot HC, Dragan YP (2001) Chemical carcinogenesis.In: Klaassen CD (ed) Casarett and Doull’s Toxicology:The Basic Science of Poisons, 6th edn. McGraw-Hill,New York, pp 280–286

6. Rosenthal DS, Schnipper LE, McCaffrey RP, AndresonKC (2001) Multiple myeloma and other plasma celldyscrasias. In: Lenhard Jr RE, Osteen RT, Gansler T (eds)Clinical Oncology. American Cancer Society, BlackwellScience, Malden, pp 517–525

Lymphoma. Table 2 Examples of regulatory guidance and test guidelines for assessment of immunotoxicity,genotoxicity, or carcinogenicity*

Regulatorybody

Endpoint Guidance/test guideline

US FDA Immunotoxicity Center for Food Safety and Nutrition (CFSAN)Redbook 2000: Immunotoxicity StudiesCenter for Devices and Radiological Health (CDRH)Immunotoxicity Testing GuidanceCenter for Drug Evaluation and Research (CDER)Immunotoxicology Evaluation of Investigational New Drugs

Genotoxicity Center for Food Safety and Nutrition (CFSAN)Redbook 2000: Bacterial Reverse Mutation TestIn Vitro Mammalian Chromosome Aberration TestIn Vitro Mouse Lymphoma TK+/− Gene Mutation AssayIn Vivo Mammalian Erythrocyte Micronucleus TestCenter for Drug Evaluation and Research (CDER)See ICH

Carcinogenicity Center for Food Safety and Nutrition (CFSAN)Carcinogenicity Studies with RodentsCombined Chronic Toxicity/Carcinogenicity Studies with RodentsCenter for Drug Evaluation and Research (CDER)See ICH

CPMP Immunotoxicity Note For Guidance on Repeated Dose Toxicity

Genotoxicity See ICH

Carcinogenicity See ICH

MHLW Immunotoxicity Immunosuppression, Draft Guidance

Genotoxicity For pharmaceuticals, see ICH

Carcinogenicity For pharmaceuticals, see ICH

ICH Genotoxicity ICH S2: Genotoxicity (includes the standard battery of tests and guidance onspecific aspects of testing)

Carcinogenicity ICH S1: Carcinogenicity (includes the need for carcinogenicity studies,approaches to evaluating carcinogenic potential, and dose selection)

US EPA Immunotoxicity Health Effects Test Guidelines870.7800 ImmunotoxicityBiochemicals Test Guidelines880.3550 Immunotoxicity880.3800 Immune Response

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Lymphotoxin

3Tumor Necrosis Factor-α

Lytic Unit (LU)

A value assigned to a quantity of effector cells neededto kill a certain percentage of a predetermined numberof target cells.

3Cytotoxicity Assays

Regulatorybody

Endpoint Guidance/test guideline

Genotoxicity Health Effects Test Guidelines870.5100 Bacterial Reverse Mutation Test870.5140 Gene Mutation in Aspergillus nidulans870.5195 Mouse Biochemical Specific Locus Test870.5200 Mouse Visible Specific Locus Test870.5250 Gene Mutation in Neurospora crassa870.5275 Sex-linked Recessive Lethal Test in Drosophila melanogaster870.5300 In vitro Mammalian Cell Gene Mutation Test870.5375 In vitro mammalian chromosome aberration test870.5380 Mammalian spermatogonial chromosomal aberration test870.5385 Mammalian bone marrow chromosomal aberraton test870.5395 Mammalian erythrocyte micronucleus test870.5450 Rodent dominant lethal assay870.5460 Rodent heritable translocation assays870.5500 Bacterial DNA damage or repair tests870.5550 Unscheduled DNA synthesis in mammalian cells in culture870.5575 Mitotic gene conversion in Saccharomyces cerevisiae870.5900 In vitro sister chromatid exchange assay870.5915 In vivo sister chromatid exchange assay

Carcinogenicity Health Effects Test Guidelines870.4200 Carcinogenicity870.4300 Combined chronic toxicity/carcinogenicity

OECD Genotoxicity OECD 471 Bacterial Reverse Mutation TestOECD 473 In vitro Mammalian Chromosomal Aberration TestOECD 474 Mammalian Erythrocyte Micronucleus TestOECD 475 Mammalian Bone Marrow Chromosomal Aberration TestOECD 476 In vitro Mammalian Cell Gene Mutation TestOECD 477 Sex-Linked Recessive Lethal Test in Drosophila melanogasterOECD 478 Rodent Dominant Lethal TestOECD 479 In vitro Sister Chromatid Exchange Assay in Mammalian CellsOECD 480 Saccharomyces cerevisiae, Gene Mutation AssayOECD 481 Saacharomyces cerevisiae, Miotic Recombination AssayOECD 482 DNA Damage and Repair, UDS in Mammalian Cells in vitroOECD 483 Mammalian Spermatogonial Chromosome Aberration TestOECD 484 Genetic Toxicology: Mouse Spot TestOECD 485 Genetic Toxicology: Mouse Heritable Translocation AssayOECD 486 Unscheduled DNA Synthesis Test with Mammalian Liver Cells invivoOECD Draft In Vitro Syrian Hamster Embryo (SHE) Cell Transformation Assay

Lymphoma. Table 2 Examples of regulatory guidance and test guidelines for assessment of immunotoxicity,genotoxicity, or carcinogenicity* (Continued)

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Encyclopedic Reference of Immunotoxicology - Part Lextras.springer.com/.../Itox-2005-04-04_Part13.pdf · A non-heme β-globulin that acts as an iron-transport-ing protein. 3 Respiratory