Leukocyte granule proteins mobilize innate host defenses and adaptive immune responses

68

Immunological Reviews 2000Vol. 177: 68–78Printed in Denmark. All rights reserved

Copyright © Munksgaard 2000

Immunological ReviewsISSN 0105-2896

Oleg ChertovDe YangO. M. Zack HowardJoost J. Oppenheim

Leukocyte granule proteins mobilize innate host defenses and adaptive immune responses

Authors’ addresses

Oleg Chertov1, De Yang2, O. M. Zack Howard1, Joost J. Oppenheim2,1Intramural Research Support Program, SAIC Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland, USA.2Laboratory of Molecular Immunoregulation, Division of Basic Sciences, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland, USA.

Correspondence to:

Joost J. OppenheimLaboratory of Molecular ImmunoregulationNational Cancer Institute-Frederick Cancer Research and Development CenterBldg 560, Rm 21-89Frederick MD 21702-1201USAFax: 1 301 846 7042 e-mail: [email protected]

Acknowledgements

This project has been funded in whole or in part with Federal funds from the National Cancer Institute, under Contract No. N01-CO-56000.The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organization imply endorsement by the U. S. Government.

Summary: “�It is likely that the leukocyte granulations are in fact secretory products,which the cell dissolves and spreads to the environment as needed”, Paul Ehrlich, 1900.Neutrophil granules have long been recognized as mediators of innate hostdefense. Newly discovered functions for individual granule proteins sug-gest that granule constituents may also participate in adaptive immuneresponses. Neutrophil granule-derived cathepsin G, azurocidin/CAP37and �-defensins have been shown to be chemotactic for mononuclear cellsand neutrophils. Analysis of the chemotactic activity of��-defensins showsthat they induce CD45RA+ and CD8 T-lymphocyte cell migration at con-centrations 10 to 100-fold below that required for direct bactericidal activ-ity. Additionally, � and � defensins form chemotactic gradients for imma-ture dendritic cells. Recruiting immature dendritic cells to sites of infec-tion is one way for neutrophil granule proteins to initiate adaptive immuneresponses. Granules found in other leukocytes such as mast cells also con-tain serine proteases, such as chymase, that are known to chemoattractneutrophils and mononuclear cells. Preliminary evidence suggests thatexocytosis of granule-derived products from a variety of leukocytes canmobilize inflammatory cells and immunocytes. Thus, leukocyte granule-derived proteins, more rapidly than chemokines, can mobilize cells thatmediate innate host defense and adaptive immunity.

Introduction

Neutrophils contribute to innate host defense by migrating

along chemoattractant gradients to sites of infection and

trauma, where they phagocytose opsonized foreign bodies and

release their granule contents in response to phagocytic stimuli

and activating factors such as interleukin (IL)-8, formylated

peptides, anaphylatoxin C5a, platelet-activating factor (PAF),

leukotriene B4 and activating cytokines (1). The four types of

neutrophil granules arise at different stages of maturation (2,

3). The primary or azurophil granule is formed first, with the

secondary or specific granules being formed shortly after. The

gelatinase granules and secretory vesicles are formed last and

found only in more mature segmented neutrophils (4, 5). The

generation and composition of the various granule types has

been recently reviewed by Borregaard & Cowland (5). The pro-

teins characteristic of azurophil granules include myeloperoxi-

dase (MPO), bactericidal permeability-increasing protein

Immunological Reviews 177/2000 69

Chertov et al · Neutrophil granule proteins in immunity

(BPI), defensins, azurocidin/CAP37, elastase, cathepsins and

proteinase-3. Some of the proteins contained in secondary

granules are vitamin B12-binding protein, lactoferrin, lysozyme

and properdin. Recent data has shown that while the mRNA

levels for some granule marker proteins are differentially regu-

lated during maturation, not all granule protein mRNA expres-

sion levels are modulated (6), suggesting that neutrophils func-

tion not only as static containers of phagocytosed, digested

material, but are specific regulatory effectors of immunity.

Examples relevant to this review are that myelocytes and meta-

myelocytes have the highest defensin (human neutrophil pep-

tide-3) mRNA levels. MPO, proteinase-3 and elastase mRNAs

are found in all neutrophil maturation states from myeloblast to

segmented bone marrow cells, with the highest message levels

being observed in the myeloblast and the least in the segmented

cell. In contrast the N-formyl-methionyl-leucyl-phenylalanine

(fMLP) high affinity receptor (FPR) mRNA is not expressed

until neutrophils are fully mature polymorphonuclear blood

cells (PMN) (6).

This review will focus on a number of proteins, present in

leukocyte granules, that are essential for innate host defense

and adaptive immunity. The products of azurophil (primary)

granules are known to act as innate immune effectors through

proteolytic or pore-mediated cytolytic activity We will discuss

the role of four neutrophil-derived proteins: cathepsin G,

azurocidin, defensins and properdin, in innate host defense

and examine the evidence that these proteins affect adaptive

immunity. We will also review the known effects of granule

proteins of other leukocytes (Fig. 1).

Cathepsin G

Background

Cathepsin G is a neutral serine proteinase primarily derived

from azurophilic granules, but is also expressed in a proteolyt-

ically active membrane-bound form. Cathepsin G is also found

in the granules of human monocytes and mast cells (7). It is

referred to as a chymotrypsin-like enzyme because it hydroly-

Fig. 1. Leukocyte granule proteins directly and indirectly mobilize immune cells. Mature polymorphonuclear neutrophils are stimulated by IL-8, fMLP, leukotriene B4, C5a, PAF, and TNF-� to release granule components. Direct chemoattractant activity is shown with a straight arrow. Individual cell types are labeled.

70 Immunological Reviews 177/2000


ses peptide bonds after leucine, methionine and phenylalanine

residues. However, cathepsin G is considered to be a rather inef-

ficient proteinase, degrading collagen and proteoglycan more

slowly than neutrophil elastase (8). Cathepsin G, cathepsin G-

like lymphocyte granzymes (B and H) and a/d chains of T-cell

receptor are closely linked on human chromosome 14q11.2

(9). Various physiological effects are ascribed to cathepsin G:

antimicrobial activity, degradation of extracellular matrix,

vasoregulation (8), activation of neutrophil elastase (10) and

processing of IL-8 (11).

Cathepsin G chemotactic activity

Subcutaneous injection of human IL-8 into SCID mice, which

were concomitantly injected intraperitoneally with human

peripheral blood lymphocytes, results in inflammatory res-

ponses that progress from an initial murine neutrophil infiltra-

tion at 4 h to subsequent infiltration of the subcutaneous injec-

tion site by considerable numbers of human T cells and a few

monocytes by 72 h (12). However, this in vivo chemotactic

effect of IL-8 on mononuclear cells appeared to be delayed and

murine neutrophil dependent. Since IL-8 induces degranula-

tion and release of neutrophil azurophilic and specific granule

markers (12), we hypothesized that granule proteins were

responsible for the later mononuclear cell accumulation.

Sequential biochemical purification of a chemotactic factor for

monocytes present in neutrophil granule fractions led to the

identification of cathepsin G (13).

The monocyte chemotactic activity of cathepsin G was

shown to be dose dependent with an optimal concentration of

0.5–1 mg/ml. Subsequently, cathepsin G was also found to be

an equally potent chemoattractant for neutrophils, but not for

lymphocytes. Cathepsin G is a more potent chemoattractant for

monocytes than either azurocidin or thrombin (13). To deter-

mine the relationship of the chemotactic activity of cathepsin G

to its enzymatic activity, cathepsin G was modified by diisopro-

pyl fluorophosphate or phenylmethanesulfonyl fluoride

(PMSF). The inhibition of proteolytic activity of cathepsin G

inactivated its monocyte chemotactic activity, suggesting that

the proteolytic activity of cathepsin G is essential for its chemo-

tactic activity. Using chemoattractant receptor-transfected cells

we found that chemotaxis of neutrophils and monocytes to

cathepsin G is mediated by the receptor for formyl peptides

(FPR) (O. Chertov, R. Ketteler, M. Potapnev, Q. Chen, D. Yang,

O. M. Z. Howard, J. J. Oppenheim, unpublished observation).

Subcutaneous injection of cathepsin G in mice induced

inflammatory responses despite the fact that human serum effi-

ciently inhibited the in vitro chemotactic effect of cathepsin G.

Presumably, the inhibitor(s) are not present in sufficient con-

centration in the tissue during the initial stages of inflammation

(14). There are two potential sources of proteinase inhibitors

in inflamed tissue: transudation and local synthesis in the tissue

as a result of stimulation by pro-inflammatory cytokines (15).

Apparently, it takes a finite time for the inhibitor to achieve

concentration sufficient to counterbalance the secreted protein-

ase. These observations suggest that it may be possible to inter-

fere with local inflammatory conditions with cathepsin G

inhibitors. Recent determination of cathepsin G three-dimen-

sional structure will provide a structural basis for the develop-

ment of novel highly efficient inhibitors (15).

Earlier studies of leukocyte chemotaxis revealed the

involvement of chymotrypsin-like protease in regulation of

leukocyte motility (16, 17). Lomas et al. (14) found that both

�-1-antichymotrypsin and antibodies to cathepsin G inhibit

fMLP and C5a-induced chemotaxis. These results suggested a

role for cathepsin G in regulation of leukocyte chemotaxis. The

physiological relevance of the involvement of this protease(s)

in chemotaxis remains unclear, since the cathepsin G knockout

mice are reported to have a normal chemotactic response to

fMLP.

Recently, the group of T. Ley (21) reported that neutrophils

from cathepsin G–/– mice have normal morphology and azuro-

phil granule formation, and display normal phagocytosis and

superoxide production. Neutrophils from cathepsin G–/– mice

also had normal chemotactic responses to C5a, fMLP and IL-8.

However, cathepsin G-deficient mice did have a decrease in

wound-breaking strength (22). Tissue sections of wounds

from mice lacking cathepsin G showed increased and pro-

longed neutrophil influx. Two factors may contribute to the

observed decrease in wound repair: 1) cathepsin G is a potent

chemoattractant for monocytes and macrophages, which are

known to play an important role in wound healing, and 2)

reduced attraction of monocytes, which in normal conditions

phagocytose neutrophils, could lead to prolonged neutrophil

infiltration of the wounds. These two conditions were observed

experimentally, indicating that while cathepsin G is not essen-

tial for mammalian development, it plays a role in neutrophil

trafficking.

Genetic studies of cathepsin G

Genetic defects affecting the cathepsin G content in neutrophil

granules have been described. A possible indication of an in vivo

functional role of cathepsin G comes from the study of leuko-

cytes of patients with rare neutrophil granule deficiency. Neu-

trophils of patients with Chediak–Higashi syndrome (CHS),

which are characterized by giant azurophil granules (18), do

not secrete cathepsin G, defensins or elastase efficiently. These



patients suffer from frequent severe bacterial infections and

often develop an atypical lymphoproliferative syndrome (19).

Further, Gallin et al. have described a human neutrophil gran-

ule disorder in a patient with recurrent infections (20). The

monocytes of this patient responded very well to fMLP in vitro

but appeared at significantly lower numbers in Rebuck (blister)

skin window sites. Moreover the patient’s PMN cells extract did

not manifest chemotactic activity for normal monocytes in vitro,

whereas the same extract from normal neutrophils was chemo-

tactic for monocytes. In view of our findings that cathepsin G

is a potent chemoattractant for monocytes, the immune defects

in these patients may be, at least partially, attributed to insuffi-

cient release of cathepsin G, which is necessary for proper

development of an inflammatory response. Consequently, the

deficiency observed in the patients with granule disorders may

be determined in part by reduced cathepsin G in neutrophils.

Cathepsin G in immunity

Recently, we observed that cathepsin G is a potent chemokinetic

stimulant for T lymphocytes (13). Based on these data and the

fact that human cathepsin G injection into murine skin induced

infiltration of neutrophils and mononuclear cells, we hypothe-

sized that cathepsin G may serve as a neutrophil-derived signal

that induces subsequent mononuclear cell and T-cell-depen-

dent immune responses. An in vivo model to evaluate the capac-

ity of cathepsin G to promote antibody responses to keyhole

limpet hemocyanin (KLH) in mice was developed. Cathepsin G

increased serum KLH-specific IgG1 and IgG2a subclasses (K.

Tani, O. Chertov, J. J. Oppenheim, unpublished data). Addition-

ally, we confirmed that human cathepsin G is mitogenic for

murine T cells, in agreement with previous reports that human

cathepsin G can increase DNA synthesis in both human T and

B cells and bind to human T cells, B cells and natural killer cells

(23, 24). Inactivation of cathepsin G enzymatic activity by

PMSF completely abolished its proliferative activity for murine

spleen cells. These results suggest that enzymatic activity of

cathepsin G is required for the stimulation of murine lympho-

cytes and possibly also antigen-specific Ig production (Table 1).

This agrees with previous reports showing that proteolytically

active cathepsin G is necessary for its stimulation of murine

B cells (25) and human lymphocytes (23, 24).

Co-administration of cathepsin G with an antigen (KLH) to

mice shows cathepsin G to have immunoadjuvant activity. In

vitro restimulation of lymph node cells from such KLH-immu-

nized mice showed that cathepsin G increased KLH-specific

lymphoproliferative responses and induced a marked increase

in interferon (IFN) production. IFN-� is presumably responsi-

ble for the cathepsin G-enhanced KLH-specific IgG2a response

of the immunized mice. Cathepsin G also increased KLH-spe-

cific production of the Th2 cytokine IL-4. This may account for

KLH-specific IgG1 production, because IL-4 is known to be

critical for the expansion of Th2 responses characterized by

increased synthesis of IgG1 (26, 27). Thus, cathepsin G

enhances both Th1 and Th2 limbs of the immune response.

Azurocidin

Azurocidin is a multifunctional heparin-binding microbicide

stored in primary neutrophil granules. Its known functions

range from protection of endothelial cells from inflammatory

cytokine-induced apoptosis (28) and modulation of monocyte

functions (29, 30) to serving as a marker for Alzheimer’s dis-

ease in the cerebral microvasculature (31). Azurocidin is a

potent monocyte chemoattractant that has also been shown to

prolong monocyte survival and enhance tumor necrosis factor

(TNF)-� production by lipopolysaccharide (LPS)-stimulated

monocytes (29, 30). Additionally, azurocidin is a weak

chemoattractant for human neutrophils (13). Competitive

binding studies suggest that azurocidin and fucoidan bind to a

common scavenger receptor on monocytes (30). Alternatively,

since both azurocidin and fucoidan interaction with human

monocytes is calcium dependent, these ligands may be inter-

�-Defensins Cathepsin G Azurocidin/CAP37

Chemotaxis T cells, monocytes (?) Monocytes, neutrophils T cells, monocytes, neutrophils

Local inflammatory response Moderate cell infiltration Moderate cell infiltration Mild cell infiltration

Spleen cell proliferation Co-mitogen for Con A response Polyclonal mitogen N.D.

Spleen cell cytokine production Enhanced IFN-� production Induces IFN-� production N.D.

Adjuvant effect (i.p. injection) IgG1, 2a, 2b IgG1, 2a Minimal

Adjuvant effect (intranasal delivery) IgG1, 2a, 2b, 3 N.D. N.D.

Immune spleen cells cytokine production IFN-�, IL-4, IL-5, IL-6, IL-10 IFN-� and IL-4 N.D.

Table 1. In vitro and in vivo effects of neutrophil granule signals

N.D.: not determined



acting with L- or P-selectins (30). In addition to its direct par-

ticipation in innate host defense, azurocidin is also chemotactic

for CD3+ lymphocytes (32), suggesting that azurocidin ampli-

fies the cellular immune response. However, pilot studies sug-

gest that azurocidin is only a weak in vivo immunoadjuvant (K.

Tani, J. J. Oppenheim, unpublished data).

Defensins

Defensins are cysteine-rich cationic proteins initially isolated as

endogenous peptide antibiotics from the azurophilic granules

of rabbit and human neutrophils (33, 34). Based on investiga-

tions over the last decade, it has become clear that defensins are

generated by plants (35, 36), insects (37, 38) and all vertebrate

species thus far examined (33, 34, 39–48). Vertebrate

defensins range from 18 to 42 amino acids in length and con-

tain six highly conserved cysteine residues. They are classified

into three subfamilies, namely �-, �-, and �-defensins, based on

patterns of their three intramolecular disulfide bridges

(46–48). The one �-defensin was isolated from monkey neu-

trophils, and it is cyclic with its six cysteines linked C1 to C6,

C2 to C5, and C3 to C4 (46). Both �- and �-defensins have sim-

ilar tertiary structures (49, 50): the three disulfide bonds of �-

defensins are paired C1 to C6, C2 to C4, and C3 to C5 (51),

while the cysteine pairs in �-defensins are C1 to C5, C2 to C4,

and C3 to C6 (52). The genes for �- and �-defensins reside in

the same gene cluster e.g. 8p21 to pter in humans (53, 54).

The cell source and biological activities of human

defensins are summarized in Table 2. In mammalian species, all

three classes of defensins are products of neutrophil granules

(33, 34, 39–41, 46). In a given species, however, not all three

classes of defensins co-exist in neutrophils. Human neutrophil

granules possess only four �-defensins, termed human neutro-

phil peptide (HNP) 1–4 (34, 55). Bovine neutrophil granules

have as many as 13 different �-defensins (41). Monkey neutro-

phil granules contain both �- and �-defensins (46, 56), while

mouse neutrophil granules contain neither. Defensins are also

produced by macrophages, skin keratinocytes, and mucosal

epithelial cells of the respiratory, digestive, urinary and repro-

ductive systems (42, 57–66). All characterized defensins share

the capacity to kill and/or to inactivate bacteria, fungi and

enveloped viruses in vitro (33, 34, 39–46). This direct antimi-

crobial activity is presumably executed in vivo and therefore

defensins are generally considered to contribute to host innate

immunity against microbial invasion (for details, please refer to

recent reviews (47, 48)). However, evidence has been accumu-

lating suggesting that defensins may contribute to both innate

host defense and adaptive immune responses of the host against

microbial invasion.

Contribution of defensins to innate host defense

In addition to direct killing of invading microbes, a number of

other defensin activities may indirectly contribute to innate

host defense. HNP1 enhances the phagocytosis of cultured

mouse peritoneal macrophages (67). Several guinea pig,

human, and rabbit �-defensins are able to degranulate rat peri-

toneal mast cells, resulting in the release of mast cell granule

products, including histamine (68, 69). Since mast cells modu-

late neutrophil influx and bacterial clearance at sites of infec-

tion, and histamine is known to increase the permeability of

small blood vessels (70, 71), defensin-induced release of mast

cell granule products would contribute to the recruitment of

Classification

Type Subtype Producing cells Biological activity relevant to immunity

� HNP1, HNP2, HNP3, HNP4 Neutrophils, macrophages 1. Antimicrobial effect

2. Chemotactic for CD4/CD45RA, and CD8 T cells

3. Chemotactic for immature dendritic cells

4. Degranulating mast cells

5. Inducing IL-8 production

6. Regulating complement activation

7. Inhibiting glucocorticoid production

8. Enhancing antigen-specific immunity

HD5, HD6 Paneth cells Antimicrobial effect

� HBD1 Epithelial cells 1. Antimicrobial effect

HBD2 Keratinocytes 2. Chemotactic for immature dendritic cells

3. Chemotactic for CD45RO T cells

Table 2. Classification, origin and activity of human defensins

HNP: human neutrophil peptide; HD: human defensin;BD: human �-defensin



more neutrophils to the inflammatory sites. Human neutrophil

defensins are reported to be capable of stimulating IL-8 produc-

tion by human primary bronchial epithelial cells and A549

lung epithelial cell line by augmenting IL-8 gene transcription

(72, 73). Since IL-8 is a potent neutrophil chemoattractant (74,

75), it is likely that defensin-stimulated production of IL-8 at a

site of infection would also aid in recruiting more neutrophils.

This is in accord with the report that in vivo administration of

human neutrophil defensins increase the ability of mice to

resist local infection by enhancing neutrophil recruitment to

infected tissues (76). Human neutrophil defensins are also

reported to bind to complement component 1q (77–79). This

raises the possibility that neutrophil-derived �-defensins at an

inflammatory site may participate in fine regulation of comple-

ment activation. The importance of neutrophils, macrophages

and the complement system in innate host defense supports the

hypothesis that neutrophil defensins contribute to innate host

defense against microbial invasion.

Contribution of defensins to adaptive immune responses

The earliest indication that defensins may also be involved in

adaptive immune response was the report showing that HNP1

and HNP2 are T-cell chemoattractants (32). Human neutrophil

defensins not only chemoattracted human peripheral blood

T cells in vitro, but also induced the recruitment of T cells to the

sites of in vivo administration within 4 h after subcutaneous

injection (32). Furthermore, when administered intranasally

or intraperitoneally simultaneously with antigens into mice,

human neutrophil �-defensins enhanced antigen-specific

humoral immune responses (80, 81). CD4+ T cells isolated

from mice immunized with antigen in the presence of �-

defensins displayed higher antigen-specific proliferative

responses and elevated production of IFN-�, IL-5, IL-6 and IL-

10 than T cells from mice immunized with antigen alone (80).

However, the capacity of neutrophil defensins to enhance sys-

temic antigen-specific immune responses is based on more

than their ability to recruit T cells to the site of co-administra-

tion of antigen and defensins.

The search for additional biological activities of defensins

led to the observation that both human �- and �-defensins are

selectively chemotactic for human immature, but not mature,

dendritic cells generated in vitro from either human CD34+ he-

matopoietic progenitor cells or peripheral blood monocytes

(82, 83). Although in vivo neutrophil defensins are stored in

azurophilic granules, they can be released into the extracellular

environment by neutrophil disruption or degranulation (32,

84). Production of �-defensins, especially murine �-defensin-2

and 3, are induced by bacterial LPS or pro-inflammatory cyto-

kines such as IL-1� and TNF-� (57, 60, 64, 65). Consequently,

both �- and �-defensins are presumably present at sites of in-

flammation, forming chemotactic gradients for immature den-

dritic cells. Since dendritic cells are crucial for the induction of

adaptive immune responses (85, 86), it is likely that the greater

the recruitment of immature dendritic cells to the site of anti-

gen deposition, the higher the induction of antigen-specific

immune responses.

HNP1 is also selectively chemotactic for CD4+/CD45RA+

and CD8+ human T cells (83). Human �-defensins, on the

other hand, are selectively chemotactic for CD4+/CD45RO+

human peripheral blood T cells (82). Therefore, defensins may

also facilitate the effector phase of adaptive immune response

by recruiting effector T cells to the sites of microbial intrusion.

It has also been reported that nanomolar concentrations of

some defensins can inhibit the production of immunosuppres-

sive adrenal steroid hormones (87–89). During systemic infec-

tions, defensin levels in plasma can reach up to 100 µg/ml (90,

91), a concentration that is sufficient for inhibiting the produc-

tion of adrenal glucocorticoids. Thus inhibition of adrenal ste-

roid production may be another way for defensins to enhance

adaptive immune response as well as innate inflammatory host

reactions.

The importance of defensins in host defense against micro-

bial invasion is indicated by experiments of nature, such as CHS

and specific granule deficiency, two disorders characterized by

a deficiency in neutrophil defensins that manifest increased

susceptibility to recurrent bacterial infection (19, 92, 93). Even

more direct support comes from the study showing that genetic

knockout of matrilysin gene, whose product is required for the

processing of murine �-defensins, rendered mice more suscep-

tible to Salmonella typhimurium (94).

Mode of action

The mechanism by which defensins execute their effects is far

from understood, except for their inhibitory effect on adrenal

steroid production (87, 88), which is achieved by blocking the

adrenocorticotropin receptor (89, 95). The microbicidal mec-

hanism of defensins has been extensively studied and is dis-

cussed in several recent reviews (38, 47, 48, 96). The widely

accepted view is that defensins bind to highly negatively

charged prokaryotic cell membranes and subsequently disrupt

the cell membrane in a non-receptor-dependent manner.

Analysis of the chemotactic activity of defensins revealed

several unique characteristics. First, defensins induce cell migra-

tion at concentrations that are 10 to 100-fold below those re-

quired for killing microbes (32, 82, 83). The chemotactic ef-

fect of defensins is not inhibited by the presence of 5~10% of



serum that can completely block their microbicidal effect (32).

In addition, the chemotactic effect of defensins has high selec-

tivity for particular subsets of T cells or immature dendritic

cells (82, 83). These characteristics suggested that both �- and

�-defensins may mediate their chemotactic effects through spe-

cific receptors. Furthermore, �- and �-defensin-induced chemo-

taxis is inhibitable by pretreatment of target cells with pertussis

toxin, suggesting Gi� protein-coupled receptors must be in-

volved (82, 83). Based on the characteristics of HBD’s chemo-

tactic effect (target cell spectrum, pertussis toxin sensitivity,

mutual cross-desensitization of HBDs and liver and activation

chemokine (LARC)), binding competition with LARC and

blockade of HBD’s effect by anti-CCR6 antibodies (Table 3), hu-

man CC chemokine receptor 6 (CCR6) was identified as a recep-

tor for human �-defensins (82). Although �-defensins also use

Gi� protein-coupled receptor(s) to mediate chemotaxis, it is

distinct from CC chemokine receptor 6.

The capacity of �-defensin to degranulate mast cells (68,

69) and enhance IL-8 production from airway epithelial cells

(72, 73) may also be receptor dependent. This is supported by

the following: 1) Defensin-induced histamine release from

mast cells is also inhibited by pertussis toxin, indicating that a

G protein-dependent mechanism is involved (38). 2) The mast

cell degranulating capacity of defensins differs from their

microbicidal capacity, indicating that the underlying mecha-

nisms are different (38). 3) Defensin-induced IL-8 production

is achieved by increasing IL-8 gene transcription (72), which

is, under most circumstances, the result of receptor-mediated

activation of transcription factor(s). 4) Treatment of defensins

with elastase or cathepsin G blocks their IL-8-inducing, but not

microbicidal, activity (73), suggesting that different domains

of defensins are needed for each function.

Although �-defensins’ chemotactic, degranulating and IL-

8-inducing effects are likely to be executed by receptor-medi-

ated mechanism(s), the receptor(s) for �-defensins are still

unidentified. Interestingly enough, Hs-AFP1, an antifungal

plant defensin, is shown to bind target fungal cells specifically

with high affinity, highly indicative of a ligand–receptor inter-

action (97). Is it plausible to propose that defensins kill pro-

karyotic cells in a non-receptor-mediated manner, while inter-

acting with eukaryotic cells in a receptor-mediated manner.

Once the receptor(s) is cloned, the role of �-defensins in

immunity, in particular in adaptive immune response, can be

more clearly defined.

Properdin

Properdin is stored in neutrophil secondary granules (98).

While this 53 kDa protein is not known to induce cell migra-

tion directly, it accelerates the complement pathway that pro-

duces C5a, thereby indirectly enhancing neutrophil, macroph-

age and mast cell migration (99). The importance of properdin

in host defense is demonstrated by individuals deficient in

properdin. They suffer from a great number of gram negative

bacterial infections with a high (75%) fatality rate (99).

Research into localization of dendritic cells has shown that

immature dendritic cells also respond chemotactically to C5a

(100), suggesting that properdin indirectly augments the

recruitment of immature dendritic cells to sites of infection by

generating C5a. Recently, Ottonello et al. demonstrated that

naive and memory B cells are also chemoattracted by C5a

(101). Thus, neutrophil granular proteins both directly (defen-

sins) and indirectly (properdin) induce the recruitment of sev-

eral immune cell subsets, dendritic cells, T cells (102) and B

cells that are responsible for the initiation of adaptive immu-

nity.

Granule proteins of other leukocytes

The above effects of neutrophil granule proteins suggested that

granule proteins from other leukocytes, such as monocytes,

mast cells, natural killers cells, cytotoxic T lymphocytes, baso-

phils and eosinophils, are likely to participate in cell-mediated

host defenses and adaptive immunity. The similarity between

the types of proteins found in leukocytes (2) and our prelimi-

nary evidence (D. Yang, H. F. Rosenborg, Q. Chen, J. J. Oppen-

heim, unpublished data) suggests that granule proteins found

in other leukocytes do participate in both innate host defense

and adaptive immune function. Furthermore, exocytosis of

leukocyte granule contents may provide a mechanism for more

rapid mobilization of innate host defenses and adaptive

immune responses than can be achieved by inducible cytokine

signals.

1. HBD is selectively chemotactic for human immature dendritic cells and CD45RO T cells, but not for mature dendritic cells and CD45RA T cells.

2. HBD is selectively chemotactic for CCR6/HEK293 cells, but not for CCR1/HEK293, CCR5/HEK293, CXCR4/HEK293 or parental HEK293 cells.

3. The chemotaxis of target cells by HBD is pertussis toxin sensitive.

4. HBD and LARC cross-desensitize each other’s chemotactic effect on CCR6/HEK293 cells.

5. HBD competes for binding of LARC to CCR6/HEK293 cells.

6. Anti-CCR6 antibodies block the migration of human immature DC in response to HBD.

Table 3. Human CCR6 acts as a receptor for human �-defensin: experimental evidence



Mast cells

The mast cell granule-derived serine proteases provide perhaps

the best support for the concept that granule proteins signal

both innate host defenses and adaptive immunity. Chymase, a

serine protease found in human mast cell granules, has been

shown to induce monocyte and neutrophil migration directly

(103). Murine mast cell protease-6 (mMCP-6) (also known as

tryptase) was shown to induce neutrophil extravasation indi-

rectly by enhancing endothelial cell IL-8 production (104).

However, mMCP-6 did not induce endothelial cells to express

general pro-inflammatory cytokines such as IL-1 and TNF-�� or

RANTES. The complexity of the mast cell granule proteases has

been underscored by the discovery that there are at least ten dif-

ferent serine proteases expressed by rat mast cells (105), and

that the protease expression patterns correlate with functional

subpopulations of mast cells (106). These data suggest that a

number of mast cell granule proteins enhance innate host

defense and have the potential of mediate adaptive immune

responses.

Eosinophils

Evaluation of current literature supports the role of eosinophil

granule proteins in both innate host defense and the adaptive

immune response. Eosinophil granules contain proteins with

strong bactericidal activity, major basic protein, eosinophil cat-

ionic protein (ECP) and BPI (107, 108). ECP and another basic

eosinophil protein, eosinophil-derived neurotoxin, are func-

tional ribonucleases (109). These data indicate that eosinophil

granule proteins are likely to be robust participants in host

defense against bacterial and viral infections. Recently, eosino-

phil granules were shown to also store RANTES (110), a CC

chemokine known to be chemotactic for monocytes, lympho-

cytes, basophils, dendritic cells and natural killer cells (111).

Earlier data demonstrated that eosinophil granules also contain

leukocyte growth factors like granulocyte-monocyte colony-

stimulating factor (112) and IL-4 (113). Therefore, eosinophil

granule proteins may mediate adaptive immune responses by

recruiting specific antigen-presenting cells like dendritic cells

and promoting TH2 T-cell development with growth factors.

Thus, eosinophil granule proteins may mobilize both innate

host defenses and adaptive immunity.

Cytotoxic T lymphocytes and natural killer cells

The granules of cytotoxic T lymphocytes and natural killer cells

are lysosomes that mediate host defense by releasing lytic pro-

teins (114). The pore-forming protein perforin and a family of

serine proteases called granzymes mediate the direct cytotoxic-

ity of these cells. Lymphocyte granzymes B and H are cathepsin

G-like serine proteases, suggesting that they may have chemo-

tactic activity for monocytes and neutrophils. Preliminary data

from our lab suggests that granzyme H is indeed chemotactic

for monocytes. Thus, the cytolytic granule proteins of cytotoxic

T lymphocytes and natural killer cells may contribute to host

defense, but their role in adaptive immunity remains to be fully

demonstrated.

Conclusion

In conclusion, neutrophils are not the only leukocytes that con-

tain granules, merely the most plentiful. Each cell type appears

to express a unique set of granule proteins that perform specific

functions in defense against either bacteria, virus and fungi or

recruitment and activation of immune effector cells. Regardless

of their cellular origin, granule proteins, due to their proximal

location and rapid release, are major participants in innate host

defense and play potentially pivotal roles in the initiation of

adaptive immune response.

References1. Boulay F, Naik N, Giannini E, Tardif M,

Brouchon L. Phagocyte chemoattractant receptors. Ann N Y Acad Sci 1997;832:69–84.

2. Gullberg U, Bengtsson N, Bulow E, Garwicz D, Lindmark A, Olsson I. Processing and targeting of granule proteins in human neutrophils. J Immunol Methods 1999;232:201–210.

3. Tsuruta T, Tani K, Hoshika A, Asano S. Alkaline phosphatase, defensin gene expression and effect of myeloid cell growth factors in normal and leukemic cells. Leuk Lymphoma 1999;32:237–247.

4. Borregaard N. Development of neutrophil granule diversity. Ann N Y Acad Sci 1997;832:62–68.

5. Borregaard N, Cowland JB. Granules of the human neutrophilic polymorphonuclear leukocyte. Blood 1997;89:3503–3521.

6. Cowland JB, Borregaard N. The individual regulation of granule protein mRNA levels during neutrophil maturation explains the heterogeneity of neutrophil granules. J Leukoc Biol 1999;66:989–995.

7. Salvesen G, Farley D, Shuman J, Przybyla A, Reilly C, Travis J. Molecular cloning of human cathepsin G: structural similarity to mast cell and cytotoxic T lymphocyte proteinases. Biochemistry 1987;26:2289–2293.

8. Travis J. Structure, function, and control of neutrophil proteinases. Am J Med 1988;84:37–42.

9. Caughey GH, et al. The human mast cell chymase gene (CMA1): mapping to the cathepsin G/granzyme gene cluster and lineage-restricted expression. Genomics 1993;15:614–620.



10. Kubes P, Smith R, Grisham MD, Granger DN. Neutrophil-mediated proteolysis. Differential roles for cathepsin G and elastase. Inflammation 1993;17:321–332.

11. Padrines M, Wolf M, Walz A, Baggiolini M. Interleukin-8 processing by neutrophil elastase, cathepsin G and proteinase-3. FEBS Lett 1994;352:231–235.

12. Taub DD, Anver M, Oppenheim JJ, Longo DL, Murphy WJ. T lymphocyte recruitment by interleukin-8 (IL-8). IL-8-induced degranulation of neutrophils releases potent chemoattractants for human T lymphocytes both in vitro and in vivo. J Clin Invest 1996;97:1931–1941.

13. Chertov O, et al. Identification of human neutrophil-derived cathepsin G and azurocidin/CAP37 as chemoattractants for mononuclear cells and neutrophils. J Exp Med 1997;186:739–747.

14. Lomas DA, et al. The control of neutrophil chemotaxis by inhibitors of cathepsin G and chymotrypsin. J Biol Chem 1995;270:23437–12443.

15. Kanemaru K, Meckelein B, Marshall DC, Sipe JD, Abraham CR. Synthesis and secretion of active �1-antichymotrypsin by murine primary astrocytes. Neurobiol Aging 1996;17:767–771.

16. Ward PA. Chemotoxis of mononuclear cells. J Exp Med 1968;128:1201–1221.

17. Ward PA, Becker EL. Biochemical demonstration of the activatable esterase of the rabbit netrophil involved in the chemotactic response. J Immunol 1970;105:1057–1067.

18. Kjeldsen L, Calafat J, Borregaard N. Giant granules of neutrophils in Chediak–Higashi syndrome are derived from azurophil granules but not from specific and gelatinase granules. J Leukoc Biol 1998;64:72–77.

19. Ganz T, Metcalf JA, Gallin JI, Boxer LA, Lehrer RI. Microbicidal/cytotoxic proteins of neutrophils are deficient in two disorders: Chediak–Higashi syndrome and “specific” granule deficiency. J Clin Invest 1988;85:552–556.

20. Gallin JI, Fletcher MP, Seligmann BE, Hoffstein S, Cehrs K, Mounessa N. Human neutrophil-specific granule deficiency: a model to assess the role of neutrophil-specific granules in the evolution of the inflammatory response. Blood 1982;59:1317–1329.

21. MacIvor DM, Shapiro SD, Pham CT, Belaaouaj A, Abraham SN, Ley TJ. Normal neutrophil function in cathepsin G-deficient mice. Blood 1999;94:4282–4293.

22. Abbott RE, Corral CJ, MacIvor DM, Lin X, Ley TJ, Mustoe TA. Augmented inflammatory responses and altered wound healing in cathepsin G-deficient mice. Arch Surg 1998;133:1002–1006.

23. Hase-Yamazaki T, Aoki Y. Stimulation of human lymphocytes by cathepsin G. Cell Immunol 1995;160:24–32.

24. Yamazaki T, Aoki Y. Cathepsin G binds to human lymphocytes. J Leukoc Biol 1997;61:73–79.

25. Hsieh CS, Heimberger AB, Gold JS, O’Garra A, Murphy KM. Differential regulation of T helper phenotype development by interleukins 4 and 10 in an �� T-cell-receptor transgenic system. Proc Natl Acad Sci USA 1992;89:6065–6069.

26. Le Gros G, Ben-Sasson SZ, Seder R, Finkelman FD, Paul WE. Generation of interleukin 4 (IL-4)-producing cells in vivo and in vitro: IL-2 and IL-4 are required for in vitro generation of IL-4- producing cells. J Exp Med 1990;172:921–929.

27. Vischer TL, Bretz U, Baggiolini M. In vitro stimulation of lymphocytes by neutral proteinases from human polymorphonuclear leukocyte granules. J Exp Med 1976;144:863–872.

28. Olofsson AM, et al. Heparin-binding protein targeted to mitochondrial compartments protects endothelial cells from apoptosis. J Clin Invest 1999;104:885–894.

29. Heinzelmann M, Platz A, Flodgaard H, Polk HC Jr, Miller FN. Endocytosis of heparin-binding protein (CAP37) is essential for the enhancement of lipopolysaccharide-induced TNF-� production in human monocytes. J Immunol 1999;162:4240–4245.

30. Heinzelmann M, Polk HC Jr, Miller FN. Modulation of lipopolysaccharide-induced monocyte activation by heparin-binding protein and fucoidan. Infect Immun 1998;66:5842–5847.

31. Pereira HA, Kumar P, Grammas P. Expression of CAP37, a novel inflammatory mediator, in Alzheimer’s disease. Neurobiol Aging 1996;17:753–759.

32. Chertov O, et al. Identification of defensin-1, defensin-2, and CAP37/azurocidin as T-cell chemoattractant proteins released from interleukin-8-stimulated neutrophils. J Biol Chem 1996;271:2935–3940.

33. Selsted ME, Szklarek D, Lehrer RI. Purification and antibacterial activities of antimicrobial peptides of rabbit granulocytes. Infect Immun 1984;45:150–154.

34. Ganz T, et al. Defensins: natural peptide antibiotics of human neutrophils. J Clin Invest 1985;76:1427–1435.

35. Colilla FJ, Rocher A, Mendez E. Gamma-purothionins: amino acid sequence of two polypeptides of a new family of thionins from wheat endosperm. FEBS Lett 1990;270:191–194.

36. Broekaert WF, Terras FRG, Cammue BPA, Osborn RW. Plant defensins: novel antimicrobial peptides as components of host defense system. Plant Physiol 1995;108:1353–1358.

37. Matsuyama K, Natori S. Purification of three antimicrobial proteins from the culture medium of NIH Sape-4, an embryonic cell line of Sarcophaga peregrina. J Biol Chem 1988;263:17112–17116.

38. Bulet P, Hetru C, Dimarcq J-L, Hoffmann D. Antimicrobial peptides in insects: structure and function. Dev Comp Immunol 1999;23:329–344.

39. Selsted ME, Harwig SSL. Purification, primary structure, and antimicrobial activities of a guinea pig neutrophil defensins. Infect Immun 1987;55:2281–2286.

40. Eisenhauer PB, Harwig SSL, Szklarek D, Ganz T, Selsted ME, Lehrer RI. Purification and antimicrobial properties of three defensins from rat neutrophils. Infect Immun 1989;57:2021–2027.

41. Selsted ME, et al. Purification, primary structures, and antimicrobial activities of �-defensins, a new family of antimicrobial peptides from bovine neutrophils. J Biol Chem 1993;268:6641–6648.

42. Ouellette AJ, Miller SI, Henschen AH, Selsted ME. Purification and primary structure of murine crytidin-1, a Paneth cell defensin. FEBS Lett 1992;304:146–148.

43. Harwig SSL, et al. Gallinacins: cysteine-rich antimicrobial peptides of chicken leukocytes. FEBS Lett 1994;342:281–285.

44. Mak P, Wojcik K, Thogersen B, Dubin A. Isolation, antimicrobial activities, and primary structures of hamster neutrophil defensins. Infect Immun 1996;64:4444–4449.

45. Zhang G, Wu H, Shi J, Ganz T, Ross CR, Blecha F. Molecular cloning and tissue expression of porcine �-defensin-1. FEBS Lett 1998;424:37–40.

46. Tang Y-Q, et al. A cyclic antimicrobial peptide produced in primate leukocytes by the ligation of two truncated �-defensins. Science 1999;286:498–502.

47. Ganz T, Lehrer RI. Antimicrobial peptides of vertebrates. Curr Opin Immunol 1998;10:41–44.

48. Lehrer RI, Ganz T. Antimicrobial peptides in mammalian and insect host defense. Curr Opin Immunol 1999;11:23–27.

49. Pardi A, Zhang X-L, Selsted ME, Skalicky JJ, Yip PF. NMR studies of defensin antimicrobial peptides: 2. Three-dimensional structures of rabbit NP-2 and human HNP-1. Biochemistry 1992;31:11357–11364.



50. Zimmermann GR, Legault P, Selsted ME, Pardi A. Solution structure of bovine neutrophil �-defensin-12: the peptide fold of the �-defensins is identical to that of the classical defensins. Biochemistry 1995;34:13663–13671.

51. Zhang X-L, Selsted ME, Pardi A. NMR studies of defensin antimicrobial peptides: 1. Resonance assignment and secondary structure determination of rabbit NP-2 and human HNP-1. Biochemistry 1992;31:11348–11356.

52. Tang Y-Q, Selsted ME. Characterization of the disulfide motif in BNBD-12, an antimicrobial �-defensin peptide from bovine neutrophils. J Biol Chem 1993;268:6649–6653.

53. Mars WM, Patmasiriwat P, Maity T, Huff V, Weil MM, Womack GF. Inheritance of unequal numbers of the genes encoding the human neutrophil defensins HP-1 and HP-3. J Biol Chem 1995;270:30371–30376.

54. Linzmeier R, Ho CH, Hoang BV, Ganz T. A 450-kb contig of defensin genes on human chromosome 8p23. Gene 1999;233:205–211.

55. Wilde CG, Griffith JE, Marra MN, Snable JL, Scott RW. Purification and characterization of human neutrophil peptide 4, a novel member of the defensin family. J Biol Chem 1989;264:11200–11203.

56. Tang Y-Q, Yuan J, Miller CJ, Selsted ME. Isolation, characterization, cDNA cloning, and antimicrobial properties of two distinct subfamilies of �-defensins from rhesus macaque leukocytes. Infect Immun 1999;67:6139–6144.

57. Schonwetter BS, Stolzenberg ED, Zasloff MA. Epithelial antibiotics induced at sites of inflammation. Science 1995;276:1645–1648.

58. Zhao C, Wang I, Lehrer RI. Widespread expression of �-defensin hBD-1 in human secretory glands and epithelial cells. FEBS Lett 1996;396:319–322.

59. Ouellette AJ, Selsted ME. Paneth cell defensins: endogenous peptide components of intestinal host defense. FASEB J 1996;10:1280–1289.

60. Harder J, Bartels J, Christophers E, Schroder JM. A peptide antibiotic from human skin. Nature 1997;387:861.

61. Svinarich DM, Wolf NA, Gomez R, Gonik B, Romero R. Detection of human defensin 5 in reproductive tissues. Am J Obstet Gynecol 1997;176:470–475.

62. Valore EV, Park CH, Quayle AJ, Wiles KR, McCrag PB Jr, Ganz T. Human �-defensin-1: an antimicrobial peptide of urogenital tissues. J Clin Invest 1998;101:1633–1642.

63. Krisanaprakornkit S, Weinberg A, Perez CN, Dale BA. Expression of the peptide antibiotic human �-defensin 1 in cultured gingival epithelial cells and gingival tissue.Infect Immun 1998;66:4222–4228.

64. Bals R, Wang X, Wu Z, Bafna V, Zasloff M, Wilson JM. Human �-defensin 2 is a salt-sensitive peptide antibiotic expressed in human lung. J Clin Invest 1998;102:874–880.

65. Bals R, et al. Mouse �-defensin 3 is an inducible antimicrobial peptide expressed in the epithelia of multiple organs.Infect Immun 1999;67:3542–3547.

66. Shirafuji Y, Oono T, Kanzaki H, Hirakawa S, Arata J. Detection of cryptidin in mouse skin. Clin Diagn Lab Immunol 1999;6:336–340.

67. Ichinose M, Asai M, Imai K, Sawada M. Enhancement of phagocytosis by corticostatin I (CSI) in cultured mouse peritoneal macrophages. Immunopharmacology 1996;35:103–109.

68. Yamashita T, Saito K. Purification, primary structure, and biological activity of guinea pig neutrophil cationic peptides. Infect Immun 1989;57:2405–2409.

69. Befus AD, Mowat C, Gilchrist M, Hu J, Solomon S, Bateman A. Neutrophil defensins induce histamine secretion from mast cells: mechanisms of action. J Immunol 1999;163:947–953.

70. Echtenacher B, Mannel DN, Hultner L. Critical protective role of mast cells in a model of acute septic peritonitis. Nature 1996;381:75–77.

71. Malaviya R, Ikeda T, Ross E, Abraham SN. Mast cell modulation of neutrophil influx and bacterial clearance at sites of infection through TNF-�. Nature 1996;381:77–80.

72. Van-Wetering S, Mannesse-Lazeroms SPG, Van-Sterkenburg MAJA, Daha MR, Dijkman JH, Hiemstra PS. Effect of defensins on interleukin-8 synthesis in airway epithelial cells. Am J Physiol 1997;272: L888–L96.

73. Van-Wetering S, Mannesse-Lazeroms SPG, Dijkman JH, Hiemstra PS. Effect of neutrophil serine proteinases and defensins on lung epithelial cells: modulation of cytotoxicity and IL-8 production. J Leukoc Biol 1997;62:217–226.

74. Oppenheim JJ, Zachariae CO, Mukaida N, Matsushima K. Properties of the novel proinflammatory supergene “intercrine” cytokine family. Annu Rev Immunol 1991;9:617–648.

75. Baggiolini MB, Dewalk D, Moser B. Interleukin-8 and related chemotactic cytokines: CXC and CC chemokines. Adv Immunol 1994;55:97–179.

76. Welling MM, et al. Antibacterial activity of human neutrophil defensins in experimental infections in mice is accompanied by increased leukocyte accumulation. J Clin Invest 1998;102:1583–1590.

77. Panyutich AV, Szold O, Poon PH, Tseng Y, Ganz T. Identification of defensin binding to C1 complement. FEBS Lett 1994;356:169–173.

78. Prohaszka Z, Nemet K, Csermely P, Hudecs F, Mezo G, Fust G. Defensins purified from human granulocytes bind C1q and activate the classical complement pathway like the transmembrane glycoprotein gp41 of HIV-1. Mol Immunol 1997;34:809–816.

79. Van-den-Berg RH, Faber-Krol MC, van-Wetering S, Hiemstra PS, Daha MR. Inhibition of activation of classical pathway of complement by human neutrophil defensins. Blood 1998;92:3898–3903.

80. Lillard JW Jr, Boyaka PN, Chertov O, Oppenheim JJ, McGhee JR. Mechanisms for induction of acquired host immunity by neutrophil peptide defensins. Proc Natl Acad Sci USA 1999;96:651–656.

81. Tani K, et al. Defensins act as potent adjuvants that promote cellular and humoral immune responses in mice to a lymphoma idiotype and carrier antigens. Int Immunol 2000;12:691–700.

82. Yang D, et al. �-Defensins: linking innate and adaptive immunity through dendritic and T cell CCR6. Science 1999;286:525–528.

83. Yang D, Chen Q, Chertov O, Oppenheim JJ. Human neutrophil defensins selectively chemoattract naïve T and immature dendritic cells. J Leukoc Biol (In press).

84. Ganz T. Extracellular release of antimicrobial defensins by human polymorphonuclear leukocytes. Infect Immun 1987;55:568–571.

85. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998;392:245–251.

86. Yang D, Howard OMZ, Chen Q, Oppenheim JJ. Immature dendritic cells generated from monocytes in the presence of TGF-�1 express functional C-C chemokine receptor 6. J Immunol 1999;163:1737–1741.

87. Zhu Q, Hu J, Mulay S, Esch F, Shimasaki S, Solomon S. Isolation and structure of corticostatin peptides from rabbit fetal and adult lung. Proc Natl Acad Sci USA 1988;85:592–596.



88. Tominaga T, Fukata J, Naito Y, Funakoshi S, Fujii N, Imura H. Effects of corticostatin-I on rat adrenal cells in vitro. J Endocrinol 1990;125:287–292.

89. Solomon S, et al. Corticostatic peptides. J Steroid Biochem Mol Biol 1991;40:391–398.

90. Panyutich AV, Panyutich EA, Krapivin VA, Baturevich EA, Ganz T. Plasma defensin concentrations are elevated in patients with septicemia or bacterial meningitis. J Lab Clin Med 1993;122:202–207.

91. Shiomi K, Nakazato M, Ihi T, Kangawa K, Matsuo H, Matsukura S. Establishment of radioimmunoassay for human neutrophil peptides and their increases in plasma and neutrophil in infection. Biochem Biophys Res Commun 1993;195:1336–1344.

92. Tamura A, et al. A marked decrease in defensin mRNA in the only case of congenital neutrophil-specific granule deficiency reported in Japan. Int J Hematol 1994;59:137–142.

93. Saxena RK, Saxena QB, Adler WH. Defective T-cell response in beige mutant mice. Nature 1982;295:240–241.

94. Wilson CL, et al. Regulation of intestinal �-defensin activation by the metalloproteinase matrilysin in innate host defense. Science 1999;286:113–117.

95. Zhu Q, Solomon S. Isolation and mode of action of rabbit corticostatic (antiadrenocorticotropin) peptides. Endocrinology 1992;130:1413–1423.

96. Gudmundsson GH, Agerberth B. Neutrophil antibacterial peptides, multifunctional effector molecules in the mammalian immune systen. J Immunol Methods 1999;232:45–54.

97. Thevissen K, Osborn RW, Acland DP, Broekaert WF. Specific, high affinity binding sites for an antifungal plant defensin on Neurospora crassa hyphae and microsomal membranes. J Biol Chem 1997;272:32176–32181.

98. Schwaeble W, et al. Properdin, a positive regulator of complement activation, is expressed in human T cell lines and peripheral blood T cells. J Immunol 1993;151:2521–2528.

99. Schwaeble WJ, Reid KB. Does properdin crosslink the cellular and the humoral immune response? Immunol Today 1999;20:17–21.

100. Sozzani S, et al. Migration of dendritic cells in response to formyl peptides, C5a, and a distinct set of chemokine. J Immunol 1995;155:3292–3295.

101. Ottonello L, et al. rC5a directs the in vitro migration of human memory and naive tonsillar B lymphocytes: implications for B cell trafficking in secondary lymphoid tissues. J Immunol 1999;162:6510–6517.

102. Nataf S, Davoust N, Ames RS, Barnum SR. Human T cells express the C5a receptor and are chemoattracted to C5a. J Immunol 1999;162:4018–4023.

103. Tani K, et al. Chymase is a potent chemoattractant for human monocytes and neutrophils. J Leukoc Biol (In press).

104. Huang C, et al. Induction of a selective and persistent extravasation of neutrophils into the peritoneal cavity by tryptase mouse mast cell protease 6. J Immunol 1998;160:1910–1919.

105. Lutzelschwab C, Pejler G, Aveskogh M, Hellman L. Secretory granule proteases in rat mast cells. Cloning of 10 different serine proteases and a carboxypeptidase A from various rat mast cell populations. J Exp Med 1997;185:13–29.

106. Whitaker-Menezes D, Schechter NM, Murphy GF. Serine proteinases are regionally segregated within mast cell granules. Lab Invest 1995;72:34–41.

107. Watanabe M, Nittoh T, Suzuki T, Kitoh A, Mue S, Ohuchi K. Isolation and partial characterization of eosinophil granule proteins in rats–eosinophil cationic protein and major basic protein. Int Arch Allergy Immunol 1995;108:11–18.

108. Calafat J, et al. The bactericidal/permeability-increasing protein (BPI) is present in specific granules of human eosinophils. Blood 1998;91:4770–4775.

109. Slifman NR, Loegering DA, McKean DJ, Gleich GJ. Ribonuclease activity associated with human eosinophil-derived neurotoxin and eosinophil cationic protein. J Immunol 1986;137:2913–2917.

110. Lacy P, et al. Rapid mobilization of intracellularly stored RANTES in response to interferon-� in human eosinophils. Blood 1999;94:23–32.

111. Zlotnick A, Morales J, Hedrick JA. Recent advances in chemokines and chemokine receptors.Crit Rev Immunol 1999;19:1–47.

112. Levi-Schaffer F, et al. Association of granulocyte-macrophage colony-stimulating factor with the crystalloid granules of human eosinophils. Blood 1995;85:2579–2586.

113. Moqbel R, et al. Identification of messenger RNA for IL-4 in human eosinophils with granule localization and release of the translated product. J Immunol 1995;155:4939–4947.

114. Page LJ, Darmon AJ, Uellner R, Griffiths GM. L is for lytic granules: lysosomes that kill. Biochim Biophys Acta 1998;1401:146–156.

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Leukocyte granule proteins mobilize innate host defenses and adaptive immune responses