Mechanisms of Neutrophil Defensins Induce Histamine

Neutrophil Defensins Induce Histamine Secretion from Mast Cells: Mechanisms of Action

This information is current as A. Dean Befus, Connie Mowat, Mark Gilchrist, Jing Hu, of September 28, 2021. Samuel Solomon and Andrew Bateman J Immunol 1999; 163:947-953; ; http://www.jimmunol.org/content/163/2/947 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Neutrophil Defensins Induce Histamine Secretion from Mast Cells: Mechanisms of Action1

A. Dean Befus,2* Connie Mowat,† Mark Gilchrist,* Jing Hu,‡ Samuel Solomon,‡ and Andrew Bateman‡

Defensins are endogenous antimicrobial peptides stored in neutrophil granules. Here we report that a panel of defensins from human, rat, guinea pig, and rabbit neutrophils all have histamine-releasing activity, degranulating rat peritoneal mast cells with

EC50 ranging from 70 to 2500 nM, and between 45 and 60% of the total histamine released. The EC50 for defensin-induced histamine secretion correlates with their net basic charge at neutral pH. There is no correlation between histamine release and antimicrobial potency. Degranulation induced by defensins has characteristics similar to those of activation by substance P. The maximum percent histamine release is achieved in <10 s, and it can be markedly inhibited by pertussis toxin (100 ng/ml) and by pretreatment of mast cells with neuraminidase. These properties differ from those for degranulation induced by IgE-dependent Downloaded from Ag stimulation and by the calcium ionophore A23187. GTPase activity, a measure of G protein activation, was induced in a membrane fraction from mast cells following treatment with defensin. Thus, neutrophil defensins are potent mast cell secretagogues that act in a manner similar to substance P and 48/80, through a rapid G protein-dependent response that is mechanis- tically distinct from Ag/IgE-dependent mast cell activation. Defensins may provide important pathways for communication between neutrophils and mast cells in defenses against microbial agents and in acute inﬂammatory responses. The Journal of

Immunology, 1999, 163: 947–953. http://www.jimmunol.org/

ctivation of mast cells by cross-linkage of surface IgE In studies largely with human basophils, a diverse group of sub- with specific Ag is widely recognized as a major path- stances that induce histamine secretion has been identified. These A way for the induction of inflammatory responses, par- are collectively referred to as histamine-releasing factors (HRF)3 ticularly immediate hypersensitivity reactions (1, 2). There are (3) and can be derived from T and B lymphocytes, alveolar mac- other pathways of mast cell activation, (2–4) but, unfortunately, rophages, platelets, mononuclear cells, fibroblasts, and neutrophils little is known about the significance of these in host defenses or (14). HRF have been found in nasal washings from atopic indi- in the pathogenesis of inflammatory diseases. For example, Aske- viduals (15), bronchoalveolar lavage fluids from individuals with by guest on September 28, 2021 nase and co-workers (5) have identified an Ag-specific, T cell- pulmonary fibrosis (16), tissue fluids from Ag-stimulated late derived factor that can sensitize mouse mast cells and together phase skin reactions (17), and PBMC of individuals with occupa- with Ag selectively induce serotonin release as opposed to hista- tional asthma (18). Several forms of HRF can be derived from mine secretion. Several other secretagogues can activate selected mononuclear cells that have been activated (14). An 8- to 10-kDa mast cell populations, including anaphylatoxins (C3a and C5a), form appears to be connective tissue-activating peptide, and its neuropeptides (substance P, neurotensin, vasoactive intestinal pep- closely related cleavage product is neutrophil-activating peptide tide, somatostatin, etc.), bee venom peptides, 48/80, polylysine, (19). Monocyte chemotactic and activating factor (20), monocyte and polymyxin (2–4, 6–10). Many of these secretagogues share chemotactic protein 1 (21), macrophage inflammatory protein-1␣ the characteristic of being polycations. Recent evidence suggests (22), RANTES (23), macrophage chemotactic protein-2 and –3 and fibroblast-induced cytokine (24), and IL-3 (25) also induce that they act by direct stimulation of G proteins of the Gi subtype following their binding to complementary cell surface sites, not to histamine secretion from human basophils. In addition, a novel classical receptors (7, 10–13). The pathway of activation involved IgE-dependent HRF has been recently defined by MacDonald and is distinct from that of IgE-dependent mast cell activation. colleagues as highly homologous with mouse p21 and human p23 (26). Neutrophil-derived HRF appears to be different from these, because it is constitutively produced, heat stable, and has an esti- mated molecular mass of 1400–2300 Da (27). Others have iden- *Pulmonary Research Group, Department of Medicine, University of Alberta, Ed- tified that HRF activity from neutrophils lies within cationic lyso- monton, Alberta, Canada; †Immunology Research Group, University of Calgary, Cal- somal proteins of about 5 kDa (28). gary, Alberta, Canada; and ‡Department of Medicine, Obstetrics and Gynecology, Endocrine Laboratory, Royal Victoria Hospital/McGill University, Montreal, Quebec, Recently, there has been an explosion of interest in defensins, a Canada family of highly cationic proteins of about 3500 Da derived from Received for publication July 2, 1998. Accepted for publication April 30, 1999. neutrophils and other cells (29–31). Defensins are variably cat- The costs of publication of this article were defrayed in part by the payment of page ionic, arginine-rich, nonglycosylated peptides that contain a char- charges. This article must therefore be hereby marked advertisement in accordance acteristic cysteine motif. They are best known for their antimicro- with 18 U.S.C. Section 1734 solely to indicate this fact. bial activity. Defensins have also been identified as corticostatins 1 This work was supported by operating grants from the Medical Research Council of because they bind specifically to the ACTH receptor and inhibit Canada (to A.D.B., A.B., and S.S.). 2 Address correspondence and reprint requests to Dr. Dean Befus, Glaxo-Heritage Asthma Research Laboratory, Pulmonary Research Group, Room 574, Heritage Med- ical Research Centre, University of Alberta, Edmonton, Alberta, T6G 2S2 Canada. 3 Abbreviations used in this paper: HRF, histamine-releasing factor; PMC, peritoneal E-mail address: [email protected] mast cells; HTB, HEPES-Tyrode’s buffer; MCDP, mast cell-degranulating peptide.

Table I. The nomenclature and primary structures of defensins tested for mast cell degranulating activity

Sequence

Consensus –C-CR—C—ER–G-C—G—–CC Rabbit NP3a/CSI GICACRRRF-CPNSERFSGYCRVNGARYVRCCSRR NP3b/CSII GRCVCRKQLLCSYRERRIGDCKIRGVRFPFCCPR NP1/CSIII VVCACRRAL-CLPRERRAGFCRIRGRIHPLCCRR Human HNP-1/HP1 ACYCRIPA-CIAGERRYGTCIYQGRLWAFCC HNP-4/HP4 VCSCRLVF-CRRTELRVGNCLIGGVSFTYCCTRV Guinea pig GPNP-1/GPCSI RRCICTTRTCRFPYRRLGTCIFQNRVYTFCC GPCSIII RRPRCFCRLHCRC

the actions of ACTH on the adrenal glands (32, 33). They are also lease of histamine was normally Ͻ2% of the total and was subtracted from chemotactic for human monocytes (34), and at least two guinea pig the percent release to give the speciﬁc secretion shown in the text, tables, defensins can induce histamine release from rat peritoneal mast and ﬁgures. From dose-response curves of various secretagogues, the con-

centrations that specifically release 50% of the cellular histamine were Downloaded from cells (PMC) (35). Given this single report about the ability of calculated (EC50). defensins to induce histamine secretion and the possibility that The secretagogues used in the studies included an extract of N. brasil- neutrophil-derived HRF activity involves defensins (27, 28), we iensis Ags (8), the neuropeptide substance P (10Ϫ5 M), the ionophore Ϫ6 characterized the ability of several defensins to induce histamine A23187 (5 ϫ 10 M), and a panel of defensins from humans, rabbits, and guinea pigs. Defensins were isolated from bone marrow for the rabbit (37) secretion and studied their mode of action. and guinea pig (38) peptides and from leukocytes obtained from the peritoneal exudate of patients with peritonitis for the human peptides (39, 40). Materials and Methods The peptides were purified by reverse phase and size exclusion chroma- http://www.jimmunol.org/ Animals tography as previously described (37–40). The identity, purity, and quan- tity of each peptide were determined by amino acid analysis. Purified pep- Outbred male Sprague Dawley rats (375–450 g) purchased from Charles tides were stored lyophilized at Ϫ20°C until use. The nomenclature and River Canada (St. Constant, Canada) were used. They were maintained in structure of the defensins used in this manuscript are summarized in Table an isolated room in filter-top cages to minimize unwanted infections. The I. The guinea pig peptide GPCSIII is more closely related to the protegrin animals were given food and water ad libitum and were maintained on a family (41) of cysteine-rich cationic neutrophil peptides than to the classic 12-h light (0700 h), 12-h dark (1900 h) cycle. No rats were used for ex- defensins and exists as an antiparallel homodimer (38). perimentation until a minimum of 1 wk following arrival in our facilities, To compare the time course of histamine secretion induced by various so as to reduce the effects of stress associated with transport, handling, and secretagogues, the secretory reaction was abruptly terminated at various new environmental conditions. All experimental procedures were approved times following mixing the secretagogue and cells by adding 1 ml of ice- by guest on September 28, 2021 by the University animal care committee and were performed in accor- cold HTB, placing the tubes immediately on ice, and then separating into dance with the guidelines of the Canadian Council on Animal Care. supernatant and pellet fractions by centrifugation at 180 ϫ g for 5 min at Either normal rats or rats infected 35–100 days previously with 3000 4°C. In several experiments, highly enriched mast cells were pretreated third stage larvae of the nematode, Nippostrongylus brasiliensis (to induce with pertussis toxin (List Biochemical Laboratory, Campbell, CA) for 2 h mast cell hyperplasia and IgE-dependent sensitization) were used to isolate at 37°C (42) or with type V neuraminidase from Clostridium perfringens PMC for study (8). (Sigma, St. Louis, MO) for1hat37°C (43). Cells were washed twice with 1 ml of HTB and were resuspended in HTB for subsequent studies of their Mast cell isolation and enrichment responses to secretagogues.

Normal rats or rats previously infected with N. brasiliensis were anesthe- Isolation of mast cell membrane fraction tized, and 15 ml of cold Tyrode’s solution containing 12 mM HEPES and 1% BSA, pH 7.3 (HTB), were injected into the peritoneal cavity. The A mast cell membrane fraction was isolated as described previously (44). abdomen was massaged, and the fluid was collected and centrifuged for 4 Briefly, purified PMC (30 ϫ 106) were resuspended in homogenization ϫ ␮ min at 180 g at 4°C. Peritoneal exudate cells were layered on a 30%/80% buffer (HEPES buffer with 2 mM MgCl2, 1 mM ATP, 100 g/ml PMSF, discontinuous gradient of Percoll (Pharmacia, Dorval, Canada) for 20 min and a protease inhibitor mixture consisting of 5 ␮g/ml each of leupeptin, at 220 ϫ g at 4°C. The cell pellet was washed twice and examined for mast aprotinin, and tosyl-arginine methyl ester (TAME)). Cells were ruptured by cell purity (Ն98%) and viability (Ն97%) by trypan blue exclusion (8). repeated passage (5–10 times) through a 12-␮m clearance in a ball-bearing Mast cell viability was also assessed using trypan blue exclusion following homogenizer (EMBL, Heidelberg, Germany), with resulting organelles re- treatment with each of the peptides tested as well as with pertussis toxin moved by centrifugation at 400 ϫ g for 10 min. The clear supernatant was and neuraminidase treatments. None of the treatments significantly altered then centrifuged at 100,000 ϫ g for1hat4°C. The membrane pellet was viability. resuspended in a small volume of 10 mM triethanolamine/HCl buffer and was stored at Ϫ70°C until use. Histamine secretion and its modulation by pertussis toxin and neuraminidase GTPase assay Ն For studies of histamine secretion, 50,000 highly enriched PMC ( 98%) GTPase activity was assayed by the hydrolysis of phosphatei from and the secretagogues to be used were preincubated separately at 37°C for [␥-32P]GTP as previously described (45). Briefly, a reaction mixture (100 ␮ 5 min, and then the secretagogues were added to the cells and incubated at L) containing 50 mM triethanolamine/HCl (pH 7.5), 2 mM MgCl2,1mM 37°C for 10 min. The reaction was terminated by the addition of 1 ml of DTT, 0.1 mM EGTA, 2 mg/ml BSA, 0.8 mM adenosine 5Ј-(␤,␥-imino)- cold HTB and was centrifuged at 4°C to separate the supernatant and cell triphosphate, 0.1 mM ATP, 0.4 mg/ml creatine kinase, 5 mM creatine pellet. After the pellets were brought to 1.5 ml using HTB, the samples phosphate, and 5 nM [␥-32P]GTP was prewarmed (5 min, 30°C). Reactions were boiled for 10 min to release cell-associated histamine and destroy were started by addition of membrane fraction (1.0–2.0 ␮g of protein) to histaminase activity. After TCA precipitation of proteins, histamine levels the prewarmed incubation mixture containing substance P or NP3a and were measured in both supernatant and pellet fractions by fluorometric allowed to proceed for 25 min at 30°C. The reaction was stopped by ad- assay (36) using a model LS-3B Perkin-Elmer fluorescence spectrometer dition of 700 ␮L of 10 mM sodium phosphate (pH 2.0) containing 5% (Norwalk, CT). Histamine release was expressed as a percentage of the (w/v) activated charcoal. The tubes were chilled for 30 min on ice, then total cellular histamine content as calculated by the formula: (histamine in centrifuged at 13,000 ϫ g for 10 min. A 500-␮L aliquot was removed, and supernatant/histamine in supernatant and pellet) ϫ 100. Spontaneous re- GTPase activity was evaluated by determination of Cherenkov radiation. The Journal of Immunology 949

Statistical analyses Data are given as the mean Ϯ SEM. Differences among treatment groups were examined for signiﬁcance by two-way ANOVA. Individual differences were assessed for statistical signiﬁcance ( p Ͻ 0.05) by paired or unpaired Student’s t test. Results

Dose response and EC50 for defensins The abilities of various concentrations of rabbit, guinea pig, and human defensins to induce histamine secretion from puriﬁed rat PMC were studied (Fig. 1). The rabbit defensins NP1, NP3a, and NP3b induced histamine secretion at low nanomolar concentrations and induced a maximum of 50–60% speciﬁc histamine release. The EC50 for these rabbit peptides varied from 70–620 nM (Table II). A similar pattern of histamine secretion occurred with guinea pig defensins, GPNP1 and GPCSIII (Fig. 1B and Table II). The human defensins HNP1 and HNP4, were less potent in induc- ing histamine secretion from rat mast cells. At 2.5 ␮M the maximum histamine secretion was 40–55% with EC50 for HNP1 and Downloaded from for HNP4 of 2.9 and 2.2 ␮M, respectively (Fig. 1C and Table II). Although the amounts of rat defensins available for study were limited, in a single experiment R2 (5 nM), R4 (0.5 nM), and R5 (0.5 nM) induced histamine secretion (15.2, 51.7, and 45.8%, respectively); R3 induced no histamine secretion.

The EC50 of the defensins studied generally correlated with the http://www.jimmunol.org/ nominal net charge of the peptides at pH 7.4 as calculated from their known sequences (Table II). These values correlate with their relative charge as determined by acid-urea gel electrophoresis. Rabbit NP3a was the most potent inducer of speciﬁc histamine release (EC50, 70 nM; Table II). Thus, for further studies of the mechanisms involved in histamine secretion with defensins we used NP3a as a representative peptide. For comparisons, the characteristics of histamine secretion induced by NP3a under different experimental conditions (time-course analysis and effects of per- by guest on September 28, 2021 tussis toxin and neuraminidase) were compared with those of substance P, Ag, and the calcium ionophore A23187. Interestingly, NP3a did not induce histamine secretion from mucosal mast cells isolated (8) from rat small intestinal mucosa (results not shown).

Time course analysis of histamine secretion Given that differences in the time course of histamine secretion occur with various secretagogues (polycations have a rapid time course, Յ10 s) and relate to the activation pathways involved, we studied the time course of histamine secretion induced by NP3a and other secretagogues. NP3a and the polycation substance P ex- hibited a rapid time course (Ͻ10 s required for maximum percent FIGURE 1. Specific histamine secretion induced by 10-min exposure of histamine release), whereas the time courses of Ag- and ionophore purified rat peritoneal mast cells to defensins from: A, rabbit (NP1, NP3a, (A23187)-induced histamine secretion were delayed and reached a and NP3b); B, guinea pig (GPCSI, GPCSIII); and human (HNP1 and Ϯ ϭ maximum after 20 or at least 60 s, respectively (Fig. 2). HNP4). Values are the mean SE (n 4 separate experiments for each defensin). The percent histamine secretion for each of the rabbit peptides Effects of pretreatment with pertussis toxin on histamine (A) is significantly different from the others at 50, 140, and 420 nM; for the secretion guinea pig peptides (B) at 30, 90, and 280 nM; and for the human peptides (C) at 280, 830, and 2500 nM (p Յ 0.05). Histamine secretion induced by polycations such as 48/80 and substance P is highly sensitive to pertussis toxin, whereas Ag- and ionophore-induced histamine release is not (10, 13, 46–48). Therefore, we investigated the effects of pertussis toxin on NP3a- Ag-induced histamine secretion. Inhibition was evident with as induced histamine secretion (Fig. 3). Purified PMC were preincu- little as 10 ng/ml pertussis toxin during a 2-h pretreatment. bated for 2 h with various concentrations (1–100 ng/ml) of pertussis toxin, washed, and then stimulated with various Effects of pretreatment with neuraminidase on histamine secretagogues for 10 min. Pertussis toxin (100 ng/ml) almost com- secretion pletely inhibited histamine secretion induced by substance P Histamine secretion induced by polycations, but not by A23187, is (94.5 Ϯ 3.8% inhibition) and NP3a (96.3 Ϯ 2.3% inhibition), also highly sensitive to inhibition by the removal of sialic acid whereas it was without effect on A23187-induced histamine se- residues from the cell surface using neuraminidase (10). Thus, to cretion (data not shown) and only partially inhibited (48 Ϯ 5.5%) characterize the sensitivity of NP3a-induced histamine secretion to 950 DEFENSINS INDUCE MAST CELL HISTAMINE SECRETION

Table II. Maximum histamine secretion, EC50, and nominal net charge at pH 7.4 of defensins tested for mast cell degranulating activity

Maximum Histamine Conc. EC50 Net Species Peptides Release (%) (nM) (nM) Charge

Guinea pig GPNPI 67.4 Ϯ 1.7 830 100 ϩ7 GPCSIII 68.8 Ϯ 1.7 2500 300 ϩ10

Human HP-1 43.1 Ϯ 3.2 2500 2870 ϩ3 HP-4 55.4 Ϯ 3.2 2500 2160 ϩ4

Rabbit NP3a 65.2 Ϯ 1.8 420 70 ϩ8 NP3b 55.0 Ϯ 1.5 1260 240 ϩ8 NP1 53.6 Ϯ 3.1 1260 620 ϩ9

neuraminidase, we pretreated purified PMC with various concen- tostatin promote IgE-independent histamine release from mast trations (0.01–0.1 U/ml) of neuraminidase for 1 h, washed them, cells. Rat intestinal mucosal mast cells often contact subepithelial and then challenged them with various secretagogues (Fig. 4). substance P neurons (49), suggesting a physiological role for sub- NP3a- and substance P-induced histamine secretion was highly stance P-induced mast cell degranulation. This observation re- Downloaded from sensitive to the removal of sialic acid residues (80.0 Ϯ 2.4% and ceived strong support when Janiszewski et al. (9), showed that 72.7 Ϯ 8.0% inhibition, respectively). By contrast, histamine se- concentrations as low as 5 pM substance P primed mast cells for cretion induced by Ag stimulation was partially (33.8 Ϯ 5.2%) subsequent activation by substance P and other secretagogues. inhibited by neuraminidase treatment, and there was no effect of However, peptide-induced histamine release also may be an un- neuraminidase on histamine secretion induced by A23187 (data wanted side effect in the development of pharmacologically useful not shown). peptide agents; for example, many luteinizing hormone-releasing hormone antagonists degranulate mast cells (50). Neutrophils elab- http://www.jimmunol.org/ Enhancement of GTPase activity by NP3a orate low molecular mass (Յ5 kDa) HRF whose identity is not Because the activation of histamine secretion from mast cells by known but which appears by biological and chromatographic cri- NP3a appeared to be similar to that of substance P and presumably teria to differ from other biochemically characterized histamine- other polycationic peptides, we studied whether NP3a could in- releasing polypeptides. Neutrophil-derived guinea pig defensins duce GTPase activity, a measure of activation of G proteins, in a activate histamine release from mast cells (35). We therefore ex- membrane fraction of PMC. An isolated membrane fraction from amined the histamine-releasing properties and mechanisms of ac- purified PMC induced a significant ( p Ͻ 0.05) GTPase response in tion of several defensins from different species as candidate

the presence of 140 nM NP3a (7.7 Ϯ 1.6 pmol/min/␮g), consistent neutrophil-derived HRF. by guest on September 28, 2021 with or greater than the positive response to 10Ϫ5 M substance P The ability to degranulate mast cells is common to all defensins ␮ (7.3 Ϯ 1.7 pmol/min/mg; Fig. 5). This was reproducible in mul- tested, with EC50 ranging from 70 nM to 2.9 M. Defensins re- tiple studies with a single membrane preparation as well as with semble wasp MCDP in terms of their cysteine-rich character and independent membrane preparations. their potencies on rat PMC: 20 nM for MCDP (51, 52) and 70 nM

for NP3a. On a molar basis the EC50 for the highly cationic de- Discussion fensins (net positive charge at pH 7.4 above 6) are lower or in the same range as those for anaphylatoxins C5a and C3a (53) on der- Several cationic peptides, including venom peptides such as mast mal mast cells and are orders of magnitude lower than those of cell-degranulating peptide (MCDP), and neuroendocrine peptides such as substance P, vasoactive intestinal polypeptide, and soma-

FIGURE 3. Effects of pretreatment with pertussis toxin (2 h, 37°C) on FIGURE 2. Time-course analysis of specific histamine secretion in- the inhibition of specific histamine secretion stimulated by substance P duced by substance P (10Ϫ5 M), NP3a (140 nM), A23187 (5 ϫ 10Ϫ6 M), (10Ϫ5 M), NP3a (140 nM), A23187 (5 ϫ 10Ϫ6 M), or N. brasiliensis (five or N. brasiliensis (five worm equivalents) Ag (mean Ϯ SE; n ϭ 3–6 ex- worm equivalents) Ag (mean Ϯ SE; n ϭ 3–6). NP3a and substance P are periments for each secretagogue). NP3a and substance P are significantly significantly different from Ag and A23187 at all doses of pertussis toxin .(p Յ0.05 ,ء) .(p Յ 0.05 ,ء) different from Ag and A23187 at 5 and 10 s The Journal of Immunology 951

to that of other polycationic peptides. This is also consistent with the lack of effect of NP3a on histamine secretion from intestinal mucosal mast cells, as this mast cell subset is unresponsive to other polycations. This unresponsiveness may protect these mucosal mast cells from spurious activation by defensin-like cryptdins thought to be constitutively released from Paneth cells in the intestinal crypts (56). Alternately, cryptdins may activate mucosal mast cells, whereas neutrophil-derived defensins do not. Peptide- induced histamine release is thought to involve receptor-independent activation of G proteins (13). The details of how such peptides activate G proteins remain unclear, but involve direct interaction of the peptide with the G protein. Charge and membrane penetrance are important; however, HP-1, which is membrane penetrating FIGURE 4. Effects of pretreatment with neuraminidase (1 h, 37°C) on (57), only weakly activates mast cell histamine secretion. In stud- the inhibition of specific histamine secretion stimulated by substance P ies on synthetic magainin analogues (amphipathic helical antimi- (10Ϫ5 M), NP3a (140 nM), A23187 (5 ϫ 10Ϫ6 M), or N. brasiliensis (five crobial peptides from frog skin) Cross et al. (58) were able to worm equivalents) Ag (mean Ϯ SE inhibition, n ϭ 4–5; n ϭ 2 for uncouple strong histamine-releasing activity from peptide- A23187). NP3a and substance P are significantly different from Ag and induced membrane perturbations. The amino acid configuration Downloaded from A23187 at neuraminidase concentrations of 0.025, 0.05, 0.075, and 0.1 may exert an important influence on peptide-induced mast cell .(p Յ 0.05 ,ء) U/ml degranulation, since L-toD-arginine substitutions in substance P analogues, which conserved overall charge, showed reduced mast cell activation (58). noncysteinyl neuroendocrine peptides (8) such as somatostatin There is growing evidence of important communication be- ϳ ␮ ϳ (EC50, 2.0 M), vasoactive intestinal polypeptide (EC50, 6.0 tween mast cells and neutrophils in inflammatory reactions. Neu- ␮ ϳ ␮ http://www.jimmunol.org/ M), and substance P (EC50, 40.0 M) on rat PMC. The range trophil recruitment to inflammatory sites is mast cell dependent of defensin EC50 as mast cell secretagogues shows a strong cor- (59), involves their production of TNF-␣ (60), and has important relation with the net charge of the peptide at pH 7.4; the more basic antimicrobial activities (61, 62). In addition, mast cell-derived defensins are more potent mast cell-degranulating agents. The po- tryptase has a pronounced effect on neutrophil recruitment (63, tency of mast cell degranulation also tends to follow the rank order 64). In turn, neutrophil-derived factors have long been implicated of these peptides as inhibitors of the ACTH receptor (corticostatic in histamine release at sites of inflammation, and defensin-induced activity), suggesting similarities in the underlying mechanisms of histamine release may contribute to this process. During infection, these two responses. HP-1 and HP-4, which have the weakest his- plasma defensin concentrations may greatly exceed the levels that

tamine-releasing activity are antimicrobial (54, 55), demonstrating by guest on September 28, 2021 that the structure-activity correlates that govern histamine secre- induce degranulation of mast cells in vitro. In sepsis, cumulative tion differ from those that determine microbicidal action. concentrations in plasma of the human defensins HNP1–3 rise In common with other cationic peptides, defensins degranulate from 40 ng/ml in healthy individuals to as high as 170 ng/ml (65, mast cells through a pertussis toxin-inhibitable mechanism, imply- 66). Highly elevated levels of HNP1–3 occur in sputum from cys- ing an action on G proteins. The time course of histamine release tic fibrosis patients (67) and in pleural effusions from patients with by defensins is identical with that of substance P, and both peptides empyema (68). Solid tissue infiltrated with neutrophils may have are inhibited by neuraminidase treatment of mast cells. Moreover, total HNP1–3 levels up to 25 nmol/g wet weight or roughly 90 ␮g as with substance P, NP3a induces GTPase activity. Thus, the (39). Defensins are prominent within the extracellular spaces of mechanism of defensin-induced mast cell degranulation is similar experimentally induced syphilitic lesions in rabbits (69). The plasma level of rabbit NP3a, the most active MCDP, increases from 8 ng/ml in uninfected animals to 234 ng/ml during experimental peritonitis (70). The release of defensins may arise both from the lysis of expended neutrophils and from cytokine-stimulated secretion. IL-8 (64, 68) or PMA (68) stimulates the release of HNP1–3 from human neutrophils, and at least in pleural effusions the local levels of IL-8 exceed the concentrations required to promote the secretion of neutrophil defensins. Since defensins are released from neutrophils in response to IL-8 and can be proinflammatory (71), these peptides may also be released by mast cell-derived TNF-␣ and tryptase and accumulate at sites of inflammation. Thus, it appears that this bidirectional communication between mast cells and neutrophils is important in acute inflammatory responses. It may involve important innate responses that help direct the kinds of immune and inflammatory responses that evolve following injury and Ag exposure and clearly requires further study. Regardless of the physiological or FIGURE 5. Increase in hydrolysis of phosphate from [␥-32P]GTP (GT- Pase activity) following treatment of membrane fraction isolated from mast pathological outcomes of defensin-induced histamine release and cells with substance P (10Ϫ5 M) or rabbit defensin, NP3a (140 nM), at inflammatory cascades, the design of novel antibiotics based on 30°C (results of three experiments from two independent membrane prep- defensin structures will have to take account of their propensity to .p Ͻ 0.05). degranulate mast cells ,ء :arations 952 DEFENSINS INDUCE MAST CELL HISTAMINE SECRETION

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