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β and MIP-1αInflammatory Protein (MIP)-1by the Chemokines MacrophageLymph Nodes During an Immune Response Regulation of T Lymphocyte Trafficking into

Andrew LloydDi Girolamo, Taline Hampartzoumian, Denis Wakefield and Nicodemus Tedla, Hong-Wei Wang, H. Patrick McNeil, Nick

http://www.jimmunol.org/content/161/10/56631998; 161:5663-5672; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 1998 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

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Regulation of T Lymphocyte Trafficking into Lymph NodesDuring an Immune Response by the Chemokines MacrophageInflammatory Protein (MIP)-1 a and MIP-1b1

Nicodemus Tedla, Hong-Wei Wang, H. Patrick McNeil, Nick Di Girolamo,Taline Hampartzoumian, Denis Wakefield, and Andrew Lloyd2

By virtue of their target cell specificity, chemokines have the potential to selectively recruit leukocyte subpopulations into sites ofinflammation. Their role in regulation of T lymphocyte traffic into lymph nodes during the development of an immune responsehas not previously been explored. The sensitization phase of contact hypersensitivity induced by the hapten, dinitrofluorobenzene(DNFB) in the mouse was used as a model of T lymphocyte trafficking in response to antigenic stimulation. Rapid accumulationof CD81 and CD41 T cells in the draining lymph nodes was closely associated with strongly enhanced expression of macrophageinflammatory protein (MIP)-1 a and MIP-1b mRNAs and proteins. Mast cells accumulating in the nodes during DNFB sensiti-zation were the predominant source of MIP-1b, whereas MIP-1a was expressed by multiple cell types. Neutralization of thesechemokines profoundly inhibited T lymphocyte trafficking into lymph nodes and altered the outcome of a subsequent challengeto DNFB. Thus, b-chemokines regulate T lymphocyte emigration from the circulation into lymph nodes during an immuneresponse and contribute significantly to the immunologic outcome.The Journal of Immunology,1998, 161: 5663–5672.

T he migratory properties of leukocytes have evolved to al-low efficient surveillance of tissues for infectious patho-gens and rapid accumulation at sites of injury or infection.

In contrast to neutrophils and monocytes, T lymphocytes may exitfrom the vascular compartment via specialized high endothelialvenules (HEV)3 in lymphoid organs and recirculate many thou-sands of times during their life span (1, 2). It has been estimatedthat one in every four lymphocytes leaves the circulation by cross-ing the HEV (3). After encountering Ag in lymph nodes, memoryT lymphocytes continually patrol the body for that Ag by recircu-lating from the blood, through tissues, into the lymphatic system,and back to the blood (1–4). T lymphocytes thus acquire a predi-lection, based on the environment in which they first encounter Ag,to home to, or recirculate through, that same environment (5, 6).Hence, relatively distinct subsets of T lymphocytes extravasatethrough the microvasculature in tissues such as skin and gut andacross HEV in lymph nodes (4–6).

The arrival of Ag, and hence the induction of an immune re-sponse in the node, greatly increases blood flow and traffic of lym-phocytes across HEV, coupled with a transient, sharp decrease inrecirculating lymphocyte output from the efferent lymphatics (7–9). In lymph nodes undergoing an immune response, lymphocyte

traffic across the HEV may increase substantially within 3 h afterantigenic stimulation and by as much as 10-fold over the first 48 hof the response (8, 9). This effect is not caused by lymphocyteproliferation within the node nor by increased numbers of cellsentering the lymph nodes from the lymphatics; instead,.95% ofthe effect is due to trafficking of cells from blood (8, 9).

At the molecular level, the leukocyte adhesion molecules, L-selectin, LFA-1 (CD11a/CD18), and VLA-4 (CD49d/CD29), me-diate T lymphocyte binding to peripheral lymph node HEVs byinteraction with glycosylation-dependent cell adhesion molecule-1(GlyCAM-1) (10) or CD34 (11) for L-selectin, with ICAM-1 andICAM-2 for LFA-1 (12, 13), and potentially with fibronectin forVLA-4 (14). Neutralizing antisera to L-selectin (15, 16), LFA-1(17), or VLA-4 (14) markedly reduce lymphocyte migration intoperipheral lymph nodes. Thus, lymphocyte recruitment into lymphnodes is likely to be a multistep process (similar to the processesof neutrophil and monocyte localization in inflammation) that re-quires L-selectin molecules to allow lymphocytes to “tether androll’’ via low affinity interactions and LFA-1 or VLA-4 to inducefirm adhesion to their counterreceptors (4, 18, 19). Activation ofL-selectin alone has been shown to trigger the high affinity state ofintegrins on naive T lymphocytes in vitro (20, 21), thus providinga potential mechanism for the preferential recirculation of thesecells through peripheral lymph nodes. However, pertussis toxintreatment abrogates LFA-1-dependent arrest of lymph node-de-rived lymphocytes (22) and may inhibit lymphocyte entry into sec-ondary lymphoid organs (23), suggesting a requirement for G pro-tein-linked signaling events in T lymphocyte trafficking intolymph nodes. Lymphocyte chemoattractants secreted from withinperipheral lymph nodes and their G protein-coupled receptors ex-pressed on lymphocyte subpopulations may provide this signal tostimulate integrin-dependent recruitment of lymphocyte subsets.

Members of theb-chemokine family are known to direct T lym-phocyte migration along a protein gradient (chemotaxis) (24, 25)and to induce adhesion to extracellular matrix proteins (26). Theb-chemokines MIP-1a, MIP-1b, monocyte chemotactic protein

Inflammation Research Unit, School of Pathology, University of New South Wales,Sydney, Australia

Received for publication April 14, 1998. Accepted for publication July 13, 1998.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work is supported by the National Health and Medical Research Council ofAustralia.2 Address correspondence and reprint requests to Dr. Andrew Lloyd, InflammationResearch Unit, School of Pathology, University of New South Wales, Sydney, NSW,2052, Australia.3 Abbreviations used in this paper: HEV, high endothelial venules; VLA, very lateAg; DNFB, 2,4-dinitrofluorobenzene; MIP, macrophage inflammatory protein; MCP,monocyte chemotactic protein; MMCP-5, mouse mast cell protease-5; PCNA, pro-liferating cell nuclear Ag; TBS, Tris-buffered saline; R-PE, R-phycoerythrin.

Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00

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(MCP)-1, MCP-2, MCP-3, and RANTES reportedly show chemo-tactic activity for T cells in vitro (27). Of these chemokines, somein vitro studies suggest that MIP-1a and MIP-1b preferentiallyattract CD81 and CD41 T cells, respectively (24, 25, 28).

We have recently reported the first evidence for a potential roleof chemokines in the regulation of lymphocyte traffic into lymphnodes in studies of nodes taken from HIV-1-infected patients andcontrol subjects (29). Strong expression of MIP-1a in the HIVlymph nodes was associated with the accumulation of CD81 Tcells in these tissues. The present experiments were designed tofurther define the potential in vivo role of MIP-1a and MIP-1b inrecruitment of T cell subsets to lymph nodes during an immuneresponse. The well-characterized murine model of contact hyper-sensitivity induced by dinitrofluorobenzene (DNFB) was chosenfor this study. It is believed that both CD41 and CD81 T cellsubsets are involved in the development of contact hypersensitivitymediated by DNFB, because studies to define the specific role ofeither subset have provided conflicting results (30, 31, 32). Thepattern of expression of MIP-1a and MIP-1b mRNAs and proteinsin draining lymph nodes of DNFB-painted mice was examined,and the kinetics of accumulation of T lymphocyte subsets in thenodes was determined.

Materials and MethodsMice

Six- to eight-week-old male and female C3H/HeN mice were bred in spe-cific pathogen free (SPF) conditions in the Animal Breeding and HoldingUnit at the University of New South Wales. SPF conditions were main-tained throughout the experimental phase of the study. For the experimentsdescribed, each sampling point consisted of four mice.

Sensitization of contact hypersensitivity

Twenty-five microliters of 0.5% 2,4-DNFB (Sigma, Sydney, Australia) in4/1 diluted acetone/olive oil (Sigma) was applied to the freshly shavedabdominal surface of mice on day 0 and day 1 for sensitization (33). Con-trol mice were painted on the shaved abdomen with 25ml of the vehiclealone.

Elicitation of contact hypersensitivity and evaluation of earswelling

Five days after sensitization, mice were challenged by applying 10ml of0.2% DNFB in acetone/olive oil to one ear. Control mice were similarlypainted with the vehicle alone. The degree of ear swelling was measuredbefore challenge and 24 h postchallenge using an engineer’s micrometer(Mitutoyo, Tokyo, Japan). Each earlobe was measured twice and contacthypersensitivity determined as the amount of swelling of the hapten-chal-lenged ear compared with the thickness of the vehicle-treated ear, ex-pressed in micrometers (mean6 SD). Mice that were challenged with thehapten without previous sensitization served as an additional negative con-trol.

Antichemokine treatment in vivo

Four hours before DNFB sensitization, neutralizing goat anti-mouse poly-clonal Abs directed against murine MIP-1a and/or MIP-1b were injectedi.p., diluted in sterile endotoxin-free saline, and control animals were in-jected with control goat IgG (R&D Systems, Minneapolis, MN) in com-parable doses to the experimental animals. Mice were coded so that cellcounts obtained during the sensitization phase and ear thickness measure-ments after challenge were made by an independent observer withoutknowledge of the status of the mouse.

Peripheral blood collection

Anticoagulated blood (200–500ml) was collected from the heart of eachmouse 1–2 min after the animals were sacrificed by CO2 inhalation, and avertical neck-to-tail skin incision was performed. Two sets of thin bloodfilms were made for differential counting after Giemsa-Grunwald staining,and the total cell count was measured by automated cell counter (SysmexNE 800, Australian Diagnostic Services, Sydney, Australia). Blood (50ml)was used to stain cell surface markers with mAbs in three-color flow cy-tometry (see below).

Lymph node excision

Both right and left inguinal lymph nodes were removed by carefully dis-secting the tissue to a radius of approximately 1 cm from the center of thenode. The right inguinal lymph node of each animal was used to prepare asingle-cell suspension, and the left inguinal lymph node was fixed in 10%buffered formalin (pH 7.0) and embedded in paraffin. Additional mice wereused to harvest the right inguinal node for extraction of protein from tissuelysates, and the left inguinal node was snap frozen by embedding the tissuein Tissue-Tek (OCT compound, Miles, Elkhart, IN) before storage at270°C for subsequent immunohistochemical studies.

Preparation of the single-cell suspension

A cell suspension from each right inguinal lymph node was prepared usinga modification of a previously published method (34). In brief, the lymphnode was weighed and washed twice in RPMI 1640/10% FCS supple-mented with penicillin/streptomycin andL-glutamine. The tissue wasminced to nearly complete dissociation using scissors and forceps in a6-well cell culture dish (Costar, Cambridge, MA) and resuspended in 4 mlof the above-mentioned medium. One mg/ml of collagenase type H(Boehringer Mannheim, Berlin, Germany) containing 1 mM calcium chlo-ride was added to the mince, which was then incubated at 37°C for 60 min.Digestion was then stopped by adding 1 ml of RPMI 1640/20% FCS andthe suspension filtered through a 40-mm nylon cell strainer (Becton Dick-inson, Mountain View, CA) into a 50-ml Falcon tube. A tuberculin syringeplunger was used to tease cells from tissue on top of the nylon mesh withrepeated rinsing in PBS. The cells were washed twice with PBS and re-suspended in this medium at 23 106/ml. The total cell count was enu-merated after assessment of viability with trypan blue.

Measurement of differential leukocyte counts in the lymph nodes

From each single-cell suspension, four air-dried smears were made. Oneslide from each mouse was then stained with Giemsa-Grunwald to deter-mine a differential leukocyte count. The remaining three slides were fixedin acetone and stored at220°C for immunohistochemical staining.

Three-color flow cytometry

The Abs use in flow cytometry included: anti-CD3-FITC, anti-CD4-R-phycoerythrin (R-PE), and anti-CD8a-RED163, which were rat anti-mousemAbs purchased from Life Technologies (Victoria, Australia). An irrele-vant negative control IgG with subclassesg1 andg2a (IgG1-FITC/IgG2a-PE) and the FACS lysing solution were purchased from Becton Dickinson.The wash solution consisted of PBS containing 2% BSA and 0.2% sodiumazide. A 1% paraformaldehyde solution in PBS was used to fix cells afterstaining.

mAb (4 ml) and;2 3 105 cells in 100ml were added to each labelingtube. After mixing, tubes were incubated in the dark at 4°C for at least 30min, then FACS lysing solution was added and tubes were further incu-bated at room temperature for 10 min followed by centrifugation. Thesupernatant was decanted and the cell pellet washed and then fixed inparaformaldehyde solution. A total of 10,000 events were acquired using aFACScan flow cytometer and data analysis performed with PC LYSIS IIsoftware (Becton Dickinson).

Analysis of proliferating lymphocytes in vivo

To determine the proportion of proliferating lymphocytes in response toDNFB sensitization, as opposed to the cells recruited into the lymph nodes,a mAb against proliferating cell nuclear Ag (PCNA) conjugated to FITCwas purchased from Boehringer Mannheim (Mannheim, Germany).Lymph node-derived single-cell suspensions (106/ml) were fixed for 2 minin 1% paraformaldehyde, followed by washing in cold PBS. Cells werethen incubated in 100% methanol at220°C for 10 min, centrifuged again,and washed in PBS containing 0.1% Triton X100 (Serva, Heidelberg, Ger-many). Subsequently, cells were incubated with anti-PCNA Ab (1.25mg in50 ml of 2% BSA in PBS) for 15 min at room temperature. After washingin PBS supplemented with 2% BSA, the cells were spun down and incu-bated with anti-CD4-R-PE and anti-CD8a-RED163 Abs for at least 30 minat 4°C in the dark. Cells were then washed twice in PBS/2% BSA andresuspended in PBS. As a positive control for this analysis, lymph node-derived mononuclear cells were stimulated in vitro with 10mg/ml of phy-tohemagglutinin (Wellcome Diagnostics, Charlotte, NC) before staining asdescribed above. Samples were analyzed on a FACScan (Becton Dickin-son) equipped with LYSIS II software.

In situ hybridisation

Single-stranded cRNA probes of 350 bp in both the antisense and senseorientation were prepared by in vitro transcription from murine MIP-1a

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and MIP-1b plasmid cDNAs using a nonisotopic probe-labeling technique(digoxigenin; Boehringer Mannheim). After a 2-h prehybridization at42°C, hybridization was performed overnight at the same temperature us-ing 100 ng of probe in 25ml of prehybridization solution on 4-mm thick,formalin-fixed, paraffin-embedded sections. This was followed by repeatedstringency washing at 42°C in 0.53 SSC. These hybridization and washingconditions were empirically determined to give optimal signal with theantisense probes, but minimal nonspecific signal with the control (sensestrand) probes. Probe detection was conducted according to the manufac-turer’s directions with an anti-digoxigenin mAb conjugated to alkalinephosphatase followed by an appropriate substrate (nitro blue tetrazolium159-bromo-4-chloro-3-indolyl phosphate (NBT1 BCIP); BoehringerMannheim). Control samples included sections of lymph nodes of theDNFB-treated mice hybridized without probe or without Ab detection, aswell as sections obtained from the lymph nodes of acetone-treated mice.

Immunohistochemical staining

A standard two-step streptavidin-horseradish peroxidase staining techniquewas performed to identify cell types on frozen sections and smears usingprimary rat mAbs against mouse T cell Ags CD3, CD4, CD8; a B cellmarker, CD40; and a marker for tissue macrophages (Mac-1); as well as anisotype-matched negative control Ab. A biotinylated rabbit anti-rat sec-ondary Ab was used. All of the above Abs were purchased from Serotec(Australian Laboratory Services, Sydney, Australia). A polyclonal rabbitanti-mouse Ab directed against mast cell protease-5 (MMCP-5; Ref. 35)was used to stain mast cells in formalin-fixed lymph node tissues after anenzymatic digestion and Ag retrieval by microwave treatment of the sec-tions in 0.01 M sodium citrate buffer, pH 6.0. A biotinylated goat anti-rabbit secondary Ab was used for this staining (Dako, Glostrup, Denmark).Immunohistochemical staining using primary goat anti-mouse polyclonalAbs directed against MIP-1a and MIP-1b (R&D Systems) and secondarydonkey anti-goat IgG directly conjugated to horseradish peroxidase (Sero-tec) were used to detect MIP-1a and MIP-1b proteins in formalin-fixedsections after a similar enzymatic digestion and Ag retrieval by microwaveof the sections in citrate buffer. An isotype-matched goat IgG (R&D Sys-tems) was used as a negative control.

A standard two-step streptavidin-horseradish peroxidase staining tech-nique was performed on formalin-fixed, paraffin-embedded lymph nodesections to localize proliferating cells using primary anti-PCNA mAb(Dako) and a biotinylated goat anti-mouse secondary Ab (Serotec).

Quantitative evaluation of MIP-1a and MIP-1b mRNAexpression

After a systemic sampling procedure as described elsewhere (29), comput-er-assisted morphometric analysis of lymph node sections was used toquantitate the number of cells expressing chemokine mRNAs as well as thetotal number of mast cells in the nodes (36). In brief, contiguous fieldsacross the whole section (on average, 10 fields) at a final magnification of2503 were assessed. After ensuring that the sections hybridized with thesense probes exhibited no significant signal, the number of positive cells(cytoplasmic blue staining) per field was enumerated. Although significantregional variations in staining were observed, the mean count for the wholesection is reported as a conservative measure of the signal for each probe.Morphologic analyses and immunohistochemical staining of adjacent sec-tions were used to determine the cellular sources of MIP-1a and MIP-1b.

Analysis of MIP-1a and MIP-1b production by Western blotting

Cell lysates from groups of three mice sacrificed 4, 24, and 96 h after DNFBrepainting, and 24 h after acetone/olive oil repainting (control mice), wereprepared using a standard method. To ensure equal loading of protein, theamount of total protein in the cell lysate was measured using the bicinchonicacid protein assay kit (Pierce, Rockford, IL). Lymph node-derived cell lysateswere electrophoretically separated on a 4% stacking and 10% resolving acryl-amide gel under nonreducing conditions and then transferred to Immobilon-Pmembranes (Millipore, Sydney, Australia) using a Trans-blot SemiDry Elec-trophoretic Transfer Cell (Bio-Rad, Sydney, Australia). After protein transfer,the membranes were washed twice in Tris-buffered saline (TBS) for 15 min atroom temperature. Membranes were then incubated overnight at 4°C with 50ml of blocking solution containing 3% BSA and 5% skim milk powder inTBS. The membranes were then rinsed twice with TBS for 5 min at roomtemperature and incubated with the primary goat anti-mouse MIP-1a orMIP-1b Ab (R&D Systems) for 1 h at room temperature with continuousgentle shaking. Primary Abs were reconstituted in sterile PBS at a concentra-tion of 1 mg/ml and used at a 1:500 dilution. After incubation with the primaryAb, the membrane was washed four times for 15 min with TBS and incubatedfor 1 h atroom temperature with a 1:500 dilution of donkey anti-goat second-

ary Ab (Serotec), which was directly conjugated to horseradish peroxidase.Membranes were then washed four times for 15 min in TBS, followed by theaddition of a chemiluminescence reagent (Renaissance, DuPont, Sydney, Aus-tralia). The membranes were finally exposed to X-OMAT-AR scientific im-aging film (Kodak, Sydney, Australia).

Metachromatic staining

After dewaxing and a 10-min incubation with 0.5 N hydrochloric acid,overnight staining with 1% toluidine blue in 0.5 N HCL was used as ametachromatic stain for mouse mast cells in the formalin-fixed lymph nodetissue sections. A standard hematoxylin and eosin staining method wasused for evaluation of the histopathologic changes in the lymph node.

Statistical analysis

A computer software program, Microsoft EXCEL, version 5.0 (Microsoft,Seattle, WA), was used to calculate means and SD. To assess the level ofstatistical significance, a two-tailed Mann-WhitneyU test was performedusing a statistical software program, SPSS for Windows, version 6.0(SPSS, Chicago, IL). Parameters of interest in each group of DNFB-treated

FIGURE 1. DFNB sensitization produces a rapid increase in the weightand cellularity of inguinal lymph node (A) and a decrease in the peripheralblood leukocyte count (B). The right inguinal lymph node collected fromanimals sensitized by painting with 0.5% DNFB and control animalstreated with vehicle alone (acetone) were weighed, minced, and digestedwith collegenase. Single-cell suspensions prepared from each node werecounted both manually and with an automated cell counter (Sysmex NE800). Cell smears prepared from the single-cell suspension were used fordifferential cell counting after Giemsa-Grunwald staining. A total leuko-cyte count was also performed, using the automated cell counter, in pe-ripheral blood collected from each animal. Each experimental group con-sisted of four animals, and each experiment was performed twice.“Acetone” on thex-axis represents pooled data from two sets of four con-trol animals that were sacrificed 4 and 24 h after repainting with vehiclealone. Results are presented as mean6 SD.

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animals were analyzed for statistical significance in comparison with con-trol animals. As several comparisons were performed, Bonferoni adjust-ments for statistical significance were made.

ResultsDNFB sensitization causes a rapid increase in cellularity andweight of inguinal lymph nodes

The lymph node weight increased significantly within 30 min ofrepainting with DNFB (p , 0.01), peaking at 12 h and graduallydecreasing thereafter (Fig. 1A). The lymph node weight remainedmildly increased above that of the control animals 1 mo after re-painting. The enlargement of the lymph nodes was accompaniedby a .10-fold increase in the number of nucleated cells in thenodes (Fig. 1A). Differential cell counting after Giemsa-Grunwald

staining of the cell smears showed that.90% of the cells in lymphnodes of DNFB-painted mice were lymphocytes, confirming thatthese cells represented the predominant leukocyte subpopulationaccumulating in the nodes. A substantial proportion of the increasein lymphocyte numbers was evident within 30 min, with;85% ofthe peak increase detected within 12 h (Fig. 1A). This rapid accu-mulation was statistically significant (p , 0.01) when comparedwith the control animals. The brisk kinetics indicate increased lym-phocyte traffic into the nodes (and perhaps reduced output) ratherthan in situ proliferation of lymphocytes.

DNFB repainting induces a rapid drop in PBL counts

Further evidence in support of the notion of increased trafficking oflymphocytes into lymph nodes was obtained from the rapid de-crease in lymphocyte numbers in peripheral blood coincident withtheir accumulation in the lymph nodes. Sensitization with DNFBproduced an;50% reduction in the total number of PBL withinthe first 30 min after repainting (Fig. 1B). The count graduallyreturned to normal values within 48 h after the treatment.

CD41 and CD81 T lymphocytes accumulate in lymph nodesafter repainting with DNFB

Three-color flow cytometric analysis of the draining lymph nodesof DNFB-treated mice showed a significant increase in the total(CD31) T lymphocytes in lymph nodes, which peaked within 12 to24 h after repainting (Fig. 2A). Both CD41 and CD81 T lympho-cyte subsets showed a similar rapid increase (p , 0.01 for each)following repainting with DNFB. After reaching a peak at 24 h, theCD41 and CD81 lymphocyte numbers in the nodes of DNFB-treated mice remained significantly higher than those of controllymph nodes 28 days after the repainting. The proportions ofPCNA-positive CD41 and CD81 T cell subsets, however, re-mained,2.5% (Table I) over the first 24 h, suggesting that themarked increase of both T cell subpopulations was predominantlyattributable to recruitment rather than proliferation in situ.

In the peripheral blood, there was a rapid and marked drop in thenumber of total T lymphocytes (CD31), .50% in the first 30 min,which gradually recovered within 24 to 48 h posttreatment (Fig.

FIGURE 2. DFNB sensitization produces a rapid increase in CD41 andCD81T lymphocytes in lymph nodes (A) and an associated decrease in theperipheral blood (B). Cells (23 105) of the single-cell suspension from theinguinal lymph node and heparinized whole blood obtained from eachanimal were stained with rat anti-mouse CD3-FITC, CD4-R-PE, and CD8-RED613 mAbs. To obtain the proportions of the T cell subsets, three-colorflow cytometric analysis was performed on the lymphocyte gate based onforward and side scatter as well as the T cell (CD3) gate. The absolutevalues for the T cell subsets were then obtained from the total leukocytecount, the proportions of total lymphocytes (obtained from the differentialcount), and the proportions of the T cell subsets (obtained from the flowcytometry). Isotype-matched irrelevant rat IgG Abs conjugated to the threedifferent fluorochromes were used as negative controls. Each experimentalgroup consisted of four animals, and each experiment was performedtwice. “Acetone” on the x-axis represents pooled data from two sets of fourcontrol animals that were sacrificed 4 and 24 h after repainting with vehiclealone. Results are presented as mean6 SD.

Table I. Induction of cell proliferation in draining lymph nodes ofmice treated with DNFBa

Treatment

MeanLymphocyteCount per

Lymph Node(3106)

% PCNA1b

CD41 CD81

Acetone 1.56 0.2 1.56 0.4 1.86 0.64 h after DNFB 3.46 0.5 2.46 0.5 2.76 0.824 h after DNFB 7.96 1.0 2.46 0.2 2.26 0.796 h after DNFB 3.26 0.5 14.36 2.7 10.16 0.4PHA-stimulated lymphocytes — 99.26 5.8 98.46 5.5

a Sensitization was performed on day 0 and 1 by painting hapten or acetone on theshaved abdomen as described elsewhere (seeMaterials and Methods). Lymphocyte-derived single-cell suspensions (13 106/ml) were fixed in 1% paraformaldehyde for2 min. After washing in cold PBS, cells were incubated in 100% methanol at220°Cfor 10 min and washed again with cold PBS containing 0.1% Triton X-100. Subse-quently, cells were incubated with FITC-conjugated anti-PCNA Ab (1.25mg in 50mlof 2% BSA in PBS) for 15 min at room temperature. After washing in PBS supple-mented with 2% BSA, cells were further incubated with anti-CD4-R-PE and anti-CD8-RED613 Abs at 4°C for 30 min in the dark. Cells were then washed twice inPBS/2% BSA and resuspended in 0.5 ml of PBS. As a positive control for thisanalysis, lymph node-derived mononuclear cells (.70% T lymphocytes) were stim-ulated for 3 days in vitro with 10mg/ml of PHA. Samples were analysed on aFACScan equipped with LYSIS II software. Each group consisted of four animlas.Results are expressed as mean6 SD.

b Represents the proportion of PCNA-positive cells (mean6 SD) of the totalCD41 or CD81 T cell populations.

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2B). This reduction included both the CD41 and CD81 popula-tions (Fig. 2B).

DNFB sensitization induces parafollicular hyperplasia and sinushistiocytosis of draining lymph nodes

During the period of lymph node enlargement after DNFB repaint-ing (0–4 days), histologic examination revealed prominent sub-capsular and medullary sinus expansion (data not shown). Theseregions were filled with a large number of leukocytes, predomi-nantly lymphocytes, macrophages, and mast cells, but also poly-morphonuclear cells. At later time points, there was also a signif-icant enlargement of the hilar region and a slight expansion themedullary regions of the nodes. A less pronounced expansion of

the paracortex was also noted. Increased activity of germinal cen-ters was visible in some nodes (data not shown).

MIP-1a and MIP-1b are rapidly induced during DNFBsensitization

In situ hybridization and immunohistochemical staining of thedraining lymph nodes demonstrated the induction of MIP-1a andMIP-1b mRNAs (Fig. 3) and abundant production of these pro-teins in animals sensitized with DNFB (Fig. 3). By contrast, lymphnodes obtained from acetone-painted (control) mice showed noexpression of these chemokine mRNAs (Figs. 3 and 5). Computer-assisted morphometric analysis of lymph node sections to quanti-tate the number of cells expressing chemokine mRNA, confirmed

FIGURE 3. DFNB sensitization rapidly induces expression of MIP-1a and MIP-1b mRNAs in draining lymph nodes. A and B, Sections (1603magnification) from animals sacrificed 4 and 24 h after DNFB repainting, showing intense blue staining of MIP-1b mRNA-expressing cells located in thesubcapsular and hilar regions. The background is lightly counterstained with neutral red. C, Lymph node section from a mouse sacrificed 4 h after repaintingwith DNFB, hybridized with the MIP-1a probe, showing abundant expression of the chemokine mRNA (blue staining) in the hilar and medullary regions.D, Section obtained 24 h after repainting with DNFB, showing substantial parafollicular staining of MIP-1a mRNA. Hybridization of adjacent sections tothose shown in A–D with the sense MIP-1b amd MIP-1a probes showedno signal (data not shown). E, A lymph node section from an acetone-painted(control) mouse hybridized with the atisense MIP-1a probe showing no mRNA signal. No positive signal was found in control lymph node sectionshybridized with the antisense MIP-1b probe (data not shown). F, Immunohistochemical stain (positive cells stained red) with anti-MIP-1a Ab on a sectionfrom an animal sacrificed 24 h after repainting with DNFB.

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the maximal induction of both chemokines at 4 h (Fig. 4A). Asexpected, the kinetics of induction of the chemokine proteins inWestern blot analyses was slightly delayed in comparison with themRNA. MIP-1b expression was faintly evident in control tissuesand was relatively constant from 4–96 h after DNFB treatment(Fig. 4B,upper panel), whereas MIP-1a expression appeared max-imal at 24 h (Fig. 4B, lower panel). Thus, the expression of thesechemokine proteins was coincident with the accumulation ofCD41 and CD81 T lymphocyte subsets, suggesting a functionalrole for these chemokines in the regulation of T lymphocyterecruitment.

MIP-1b is expressed predominantly by mast cells, whereasMIP-1a is expressed by a wide range of lymph node cells

Histomorphologic and immunohistochemical studies of lymphnode sections revealed the cellular sources of MIP-1b mRNA weresignificantly different from those of MIP-1a. At all time points, arange of cell types in the nodes was found to express MIP-1a,including macrophages, lymphocytes, and endothelial cells (figurenot shown). By contrast, a striking and novel finding was the dem-onstration that mast cells were the predominant source of MIP-1bmRNA in the lymph nodes of DNFB-treated mice (Fig. 5). Fur-thermore, computer-assisted quantitation in the nodes confirmed asignificant increase in the number of mast cells in the subcapsularand hilar regions of the draining nodes (54). This increase wasconcurrent with a decrease in the number of mast cells at the siteof sensitization in the skin, thus suggesting that mast cells travelfrom the skin via afferent lymphatics. Immunohistochemical de-tection of MIP-1b protein confirmed the mast cell localization ofthis chemokine. Furthermore, prominent staining of MIP-1b wasfound on endothelial cells (Fig. 5D), despite the absence of de-tectable mRNA in these cells. As the accumulation of MIP-1b-expressing mast cells coincided with the accumulation of T lym-phocytes in the nodes, this pathway may be critical to the observedrapid recruitment of T lymphocytes into the nodes after DNFBrepainting.

Treatment with anti-chemokine Abs abrogates T cell recruitmentto the draining lymph nodes

To test whether T lymphocyte recruitment in this model was de-pendent on MIP-1a and MIP-1b, mice were given an i.p. injectionof neutralizing anti-chemokine Abs 4 h before the first paintingwith DNFB (37, 38). The anti-chemokine Abs caused a dose-de-pendent inhibition of the recruitment of T lymphocytes to thedraining lymph nodes. Administration of 50, 200, or 500mg ofanti-MIP-1a Ab reduced CD31 T cell numbers, evaluated at 24 hafter repainting, by 16% (p , 0.05), 31% (p , 0.01), or 48%( p , 0.01), respectively, of the cell numbers in animals injectedwith 1000mg of control Ab. Similarly, anti-MIP-1b Ab treatmentin the same doses produced 10, 20, and 49% inhibition. The inhi-bition produced by pretreatment of mice with 500mg of eitheranti-MIP-1a or anti-MIP-1b Abs was significant for both CD41

and CD81 T cells (p , 0.01 for all four comparisons; Fig. 6).

Treatment with anti-chemokine Abs modifies a subsequentdelayed-type hypersensitivity response

To assess the effect of chemokine inhibition on the outcome of theDNFB-induced immune response, mice treated with a combinationof anti-MIP-1a (500 mg) and anti-MIP-1b (500 mg) Abs beforeDNFB sensitization were challenged 5 days later with 0.2% DNFBapplied to the left ear. Mice that were injected with control Abexhibited a good ear swelling response, comparable with that ofpositive control animals, whereas mice pretreated with a combi-nation of anti-MIP-1a and anti-MIP-1b Abs before hapten appli-

cation showed a modest, but significant, 19% inhibition of thecontact hypersensitivity reaction elicited by ear lobe challenge(Table II).

DiscussionA considerable body of in vitro and in vivo data highlights the roleof chemokines in the regulation of leukocyte emigration from thevascular compartment to sites of inflammation (27). This chemo-kine regulation hinges upon the ability of these cytokines to induceaffinity modulation of integrin adhesion molecules expressed onleukocytes (4, 13, 27, 39) and upon the target cell specificity of theindividual chemokines (27, 39–41). Like other leukocyte sub-populations, T lymphocytes appear to depend on chemokine-reg-ulated mechanisms for recruitment across vascular endothelium tosites of inflammation (23, 27).

The present findings provide clear in vivo evidence for an as-sociation between the production of theb-chemokines MIP-1aand MIP-1b and the recruitment of CD41 and CD81 T lympho-cytes into peripheral lymph nodes during an immune response. Inthis model of contact hypersensitivity, T lymphocytes accumulated

FIGURE 4. Chemokine mRNA and protein expression in draininglymph nodes increases dramatically during DNFB sensitization.A, In situhybridization was performed using MIP-1a or MIP-1b riboprobes. Con-tiguous high power fields across each whole section were examined beforethe mean number of cells per high power field (6SEM) was enumeratedwith a computer-assisted analysis system. Two lymph node sections fromeach mouse (four mice per time point) were analysed.B, Immunoblottingfrom chemokines in cell lysates prepared from mouse lymph nodes usinganti-MIP-1b Ab (upper panel) demonstrated the production of an ~8.5-kDaMIP-1b protein at 4, 24, and 96 h after repainting with DNFB. The 8.3-kDaband of the standard m.w. marker (Bio-Rad) is indicated on the left side ofthe membrane. Immunoblotting of cell lysates derived from mouse lymphnodes using an anti-MIP-1a Ab (lower panel) demonstrated the productionof an ~8.5-kDa protein at 4 h (lane 2) and 24 h (lane 3) but not at 96 h (lane4) after repainting with DNFB. Lysates from acetone-painted (control)mice (lane 1) showed no detectable MIP-1a. Each lane was loaded with anequal amount (20mg) of cell lysate derived from a pool of four to fiveanimals.

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very rapidly within the nodes and were predominantly PCNA neg-ative, thereby precluding any significant role for lymphocyte pro-liferation as an explanation for the 10-fold increase in T lympho-cyte numbers observed during sensitization.

The notion of altered trafficking of leukocytes into lymph nodesduring the induction of contact hypersensitivity was supported bythe rapid decrease in T lymphocyte numbers in the peripheralblood coincident with their accumulation in the lymph nodes. In asimilar fashion, a significant decrease in the PBL count coincidentwith accumulation of these cells in the lymph nodes was observedin mice that had received repeated i.p. injections ofCorynebacte-rium granulosum(42).

Three-color flow cytometric analysis of peripheral blood anddraining lymph node cells suggested that both CD41 and CD81 T

cell subsets had migrated into the nodes. This finding is consistentwith previous adoptive cell transfer studies indicating that bothCD41 and CD81 T lymphocyte subsets are mediators of DNFB-induced contact hypersensitivity (30, 31). By contrast, after stimula-tion with purified protein derivative of tuberculin, the large increase inT cell traffic through lymph nodes during the recruitment phase wasmostly due to CD41 memory phenotype T lymphocytes (8).

During the induction of contact hypersensitivity, abundant MIP-1aand MIP-1b mRNA and protein expression in the draining lymphnodes was detected. The kinetics of the protein expression of MIP-1aand MIP-1b was coincident with the recruitment of CD41 and CD81

T lymphocyte subsets, suggesting a functional role for these chemo-kines in the regulation of T cell recruitment. Although both chemo-kines were produced as early as 4 h after repainting, there was a

FIGURE 5. Mast cells are the predominant source of MIP-1b in draining lymph nodes during DNFB sensitization.A andB, Adjacent sections of thesubcapsular region (2503magnification) of a lymph node from a mouse sacrificed 4 h after DNFB treatment, showing expression of MIP-1b mRNA (bluestaining with background neutral red counterstaining;A), which colocalized with cells immunostained for MMCP-5, indicative of mast cells (red stainingwith hematoxylin counterstaining;B). The arrows indicate at least two of the mast cells, which can confidently be identified in both sections.C andF,Adjacent sections of the subcapsular region of a lymph node from an acetone-treated (control) mouse illustrating the absence of MIP-1b mRNA expression(F), despite the presence of MMCP-5-positive mast cells (red staining;C) in the adjacent section.D, Lymph node section from a mouse sacrificed 24 hafter DNFB treatment, immunostained with an anti-MIP-1b Ab showing widespread staining (in red) of the lumenal surface od endothelial cells (arrows).E, An adjacent section toD, stained with control goat IgG (negative control).

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difference in the kinetics of expression, with MIP-1b protein expres-sion being maximal at 4 h following DNFB repainting and persistingat high levels beyond 96 h, which was earlier and more prolongedthan the expression of MIP-1a. Chemokine expression in the lymphnodes was not examined at time points in between the first and secondpaintings with DNFB; however, given the rapid kinetics of mRNAexpression, it is likely that some induction of chemokine gene expres-sion occurs before the second painting.

The prominent expression of the protein, but not mRNA, ofMIP-1b by endothelial cells in the DNFB-sensitized lymph nodessuggests translocation of this chemokine protein. This may occurvia the recently described transendothelial chemokine transportmechanism (43), following initial synthesis by mast cells and mac-rophages in the nodes. In addition, the protein may be immobilizedby proteoglycans on the surface of the endothelial cells to facilitateinteraction with circulating lymphocytes (25, 44).

The kinetics and magnitude of lymphocyte accumulation in theregional lymph nodes demonstrated following Ag challenge in thisstudy were very similar to previously published reports (8, 9, 42).Although prior studies have indicated the involvement of draininglymph nodes in both the sensitization and elicitation phases ofDNFB-induced contact hypersensitivity (30–33), there are no re-ports on the histologic changes in the lymph nodes in this model.At the early time points, the nodes showed marked expansion ofthe subcapsular and medullary sinuses, which were filled with alarge number of cells, predominantly lymphocytes, macrophages,and mast cells. This extensive expansion of the sinuses may reflectan increased flow of lymph and inflammatory cells from the skinvia the afferent lymphatic vessels into the draining nodes. At latertime points, increased activity of germinal centers was visible insome nodes, indicating a proliferative response to the Ag.

The significance of MIP-1a and MIP-1b in this model was fur-ther confirmed by substantial inhibition of T lymphocyte recruit-ment into draining lymph nodes following administration of eitheranti-MIP-1a or anti-MIP-1b Abs. The reported preferential activ-ity of these chemokines on CD41 or CD81 T lymphocyte subsetwas not observed, as the accumulation of both T cell subsets wassimilarly inhibited by either anti-MIP-1a or anti-MIP-1b Abs.However, the combination of both Abs produced a significantlygreater reduction in CD81 ( p , 0.05) than CD41 T lymphocyterecruitment. This suggests that in CD41 T lymphocytes the twochemokines may act via a receptor (such as CCR5) that binds bothligands, whereas in CD81 T lymphocytes, additional receptors(such as CCR1, which is not responsive to MIP-1b) may also beutilized (27).

Other recently identifiedb-chemokines such as secondary lym-phoid tissue chemokine (SLC) and EBI1-ligand chemokine (ELC)have been proposed to be relevant to T lymphocyte recruitmentinto lymphoid organs (40, 45). These chemokines are constitu-tively expressed in lymphoid tissues and are chemotactic for lym-phocytes (45). However, there are no in vivo data regarding thefunction of these molecules. Nevertheless, it is likely that severalchemokines, in addition to MIP-1a and MIP-1b, have the capacityto regulate both the homing of T lymphocytes in the physiologicstate and the dramatically enhanced recruitment events that occurduring the development of an immune response. This notion isconsistent with the incomplete inhibition of T lymphocyte traffick-ing demonstrated in this contact hypersensitivity model.

Animals pretreated with a combination of anti-MIP-1a and anti-MIP-1b Abs before hapten application showed a modest but sig-nificant 19% inhibition of the contact hypersensitivity reaction

FIGURE 6. Chemokine inhibition reduces recruitment of T lympho-cytes in the lymph nodes. Four hours before sensitization with DNFB,experimental animals were pretreated by i.p. injection of 500mg of anti-MIP-1a and/or anti-MIP-1b neutralizing Abs. Control animals were in-jected with control goat IgG in doses comparable to those given the ex-perimental animals. Pretreatment with anti-MIP-1a and/or anti MIP-1bAbs significantly abrogated the recruitment of T lymphocytes into thedraining lymph nodes. Mice similarly treated with the maximum amount (1mg) of control goat IgG showed no inhibition of T cell accumulation afterDNFB sensitization. Each experimental and control group consisted of fouranimals. Results are presented as mean6 SD.

Table II. Effect of neutralizing anti-chemokine Abs administered before sensitzation on the subsequentelicitation of contact hypersensitivitya

Sensitization (%) Challenge (%)Treatment Before

SensitizationEar Thickness (mm),

Mean6 SD Reduction (%)

Acetone Acetone 606 0.2DNFB (0.2) 806 0.3

DNFB (0.5) DNFB (0.2) 3806 1.2DNFB (0.5) DNFB (0.2) Anti-MIP-1a and 1b 3056 0.3* 19.7DNFB (0.5) DNFB (0.2) Goat IgG 3656 0.2 NS 3.9DNFB (0.5) DNFB (0.2) Sodium chloride 3706 0.3 NS 2.6

a Five days after pretreatment with a combination of anti-MIP-1a and anti-MIP-1b or irrelevant Abs and sensitization withDNFB, mice were challenged by application of 10ml of 0.2% DNFB in acetone olive oil to one ear. Control mice were similarlypainted with the vehicle alone. The degree of ear swelling was measured before challenge and 24 h postchallenge by anindependent observer, using an engineer’s micrometers (Mitutoyo). Each ear lobe was measured twice and contact hypersen-sitivity determined as the amount of swelling of the DNFB-challenged ear compared with the thickness of the vehicle-treatedear expressed in micrometers. Mice that were challenged with the hapten without previous sensitization served as an additionalnegative control. Each experimental and control group consisted of four animals. Results are expressed as mean6 SD.

*p , 0.05 vs positive control; NS, not significant (Two-tailed Mann-WhitneyU test of the row data).

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elicited by ear lobe challenge (Table II). Therefore, pretreatment ofanimals with a combination of anti-MIP-1a and anti-MIP-1b be-fore hapten application not only significantly abrogates T cell re-cruitment to the draining nodes, but also partially blocks the sub-sequent delayed-type hypersensitivity reaction. This findingsuggests that mice injected with anti-MIP-1a and anti-MIP-1bAbs followed by hapten application became partially tolerant tothis hapten, perhaps as a result of decreased recruitment of hapten-primed T cells into the regional lymph nodes and thus a decreasein the expansion of hapten-specific T cells responsible for mount-ing a strong response upon subsequent challenge.

A striking finding in this work is the demonstration of mast cells asthe predominant cellular source of MIP-1b in the lymph nodes ofDNFB-treated mice. These cells were distinctively located in the sub-capsular and hilar regions of the nodes. Previous in vitro experimentshave described the expression of MIP-1b mRNA by mast cell lines(46, 47). However, the abundant expression of this chemokine bymast cells observed in this in vivo model of contact hypersensitivityis novel. The brisk appearance of mast cells in a distinctive location inthe subcapsular and hilar regions of the nodes, in association with thedecrease in their number in the affected skin shortly after repaintingwith DNFB, strongly suggests that mast cells travel from the skin viathe afferent lymphatic system. As expected, mast cell accumulation inthe draining nodes was unaffected by neutralization of MIP-1a andMIP-1b activity,4 indicating that movement of these cells into thenodes is independent of a concentration gradient of these chemokines.The novel observation of mast cells as the dominant source of MIP-1bindicated in this work clarifies the controversy regarding the role ofthese cells in contact hypersensitivity (48–51). In particular, mastcells are not only a source of histamine, serotonin, and other vasoac-tive amines that are believed to control vascular tone and permeability(52, 53), but also act as a key early regulator of T cell recruitment intodraining lymph nodes.

These findings provide the first direct in vivo evidence of che-mokine regulation of trafficking of T lymphocyte subpopulationsinto lymph nodes during the induction of an immune response.Mast cells accumulating in the nodes have been identified as theprincipal source of MIP-1b in this model of contact hypersensi-tivity. Further studies examining additional chemokines and othermodels of immune response are warranted, as interventions to ma-nipulate this chemokine-dependent T lymphocyte trafficking path-way may have profound influences on the outcome of host re-sponses to infection or autoimmune triggers.

AcknowledgmentsWe thank Ms. Angelina Enno for processing and cutting the histologicsections.

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