Porcine Polymorphonuclear Leukocytes Generate Extracellular

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(Filtron Technology Corp., Northborough, Mass.). Concentrated ... neutrophil elastase, MeOSucAlaAlaProVal-chloromethyl ketone (CMK; Sigma). Degranulation ...
INFECTION AND IMMUNITY, Mar. 1997, p. 978–985 0019-9567/97/$04.0010 Copyright q 1997, American Society for Microbiology

Vol. 65, No. 3

Porcine Polymorphonuclear Leukocytes Generate Extracellular Microbicidal Activity by Elastase-Mediated Activation of Secreted Proprotegrins ALEXANDER PANYUTICH, JISHU SHI, PAUL L. BOUTZ, CHENGQUAN ZHAO,

AND

TOMAS GANZ*

Will Rogers Institute Pulmonary Research Laboratory, Departments of Medicine and Pathology, School of Medicine, University of California at Los Angeles, Los Angeles, California 90095 Received 8 October 1996/Returned for modification 14 November 1996/Accepted 13 December 1996

Antimicrobial peptides of several structural classes have been found in phagocytes and epithelial cells of many animals. The broadly microbicidal protegrins (PG1, -2, and -3) were originally isolated as 16 to 18-amino-acid peptides from pig neutrophil lysates, but the corresponding cDNA sequences encoded much larger precursors that belonged to the cathelicidin family of antimicrobial peptides. We explored the storage, secretion, and microbicidal activation of protegrins in porcine neutrophils and in a model system consisting of recombinant proprotegrin 3 (pPG3) and various serine proteases and their inhibitors. Protegrins were stored in neutrophils as inactive proforms that were cleaved by neutrophil elastase to mature protegrins during the preparation of granule lysate and during phorbol myristate acetate-stimulated granule secretion from intact neutrophils. Recombinant pPG3 was efficiently cleaved by trace amounts of human neutrophil elastase or equivalent amounts of elastase activity from porcine neutrophils, but pPG3 was relatively resistant to porcine pancreatic elastase or human neutrophil cathepsin G. The recombinant pPG3 and neutrophil proprotegrins lacked microbicidal activity, but the mature protegrins generated in the elastase-mediated cleavage reaction were as active against Listeria monocytogenes as the chemically synthesized protegrin. The secretion and elastase-mediated activation of proprotegrins accounted for much of the stable microbicidal activity of porcine neutrophil secretions against L. monocytogenes. Secreted proprotegrins and trace amounts of elastase constitute a binary microbicidal system that is likely to contribute to the antimicrobial activity of porcine inflammatory fluids. and Bac7 (5), are stored in specialized (nonazurophil) granules and during phagocytosis undergo proteolytic maturation by neutrophil elastase that cleaves the cathelin motif from the C-terminal active peptide (21, 30, 31). The C-terminal peptides were thought to exert their microbicidal activity in phagocytic vacuoles (21). In contrast, two cathelicidins from rabbit neutrophils, p15a and p15b, are secreted and are active extracellularly without such proteolytic cleavage (11, 12, 27). Our study examined the processing and bactericidal activation of protegrins in porcine neutrophils and in systems reconstituted from purified components. We present evidence that, during secretion from activated porcine neutrophils, trace amounts of elastase cleave inactive protegrin precursors to active protegrins and that such neutrophils generate sufficient concentrations of protegrins to render extracellular fluids bactericidal.

Antimicrobial peptides have been found in the phagocytes and epithelial structures of many animals (10, 13). Protegrins PG1, PG2, and PG3 are broad-spectrum antibiotics purified from porcine neutrophil extracts as C-terminally amidated peptides (7) with sequences RGGRLCYCRRRFCVCVGRam, RGGRLCYCRRRFCICVam, and RGGGLCYCRRRFCVCV GRam, respectively (boldface indicates differences among the three peptides). To denote the C-terminal amidation of the natural peptides, they will be referred to as PG1am, PG2am, and PG3am. The corresponding protegrin cDNAs (23, 33) encoded a 149 (147 for PG2)-amino-acid protein with an Nterminal 29-amino-acid endoplasmic reticulum-targeting sequence, a 101-amino-acid cathelin motif, and a C-terminal 19-amino-acid mature protegrin sequence that terminated in a glycine presumably posttranslationally converted to the C-terminal amide (17). Two additional protegrin peptides, PG4 and PG5, were deduced from a bone marrow cDNA sequence and a gene sequence (32, 33), respectively, but have not yet been purified from natural sources. The presence of the cathelin motif in their precursors identified the protegrins as members of the recently described cathelin-related or cathelicidin family of microbicidal proteins and peptides (29). Most cathelicidin sequences were established by cDNA or gene cloning, and in some cases sequences were established by isolation of C-terminal peptides; however, the structures and posttranslational processing of cathelicidin proteins have not been extensively investigated (22). Two bovine members of this family, Bac5

MATERIALS AND METHODS Construction of pPG3 baculovirus. The overexpression of proprotegrin 3 (pPG3) in insect Spodoptera frugiperda (Sf21) cells was performed as described earlier for prodefensin (25). Briefly, PG3 cDNA (33) fragments, each of which contained translation start codon ATG, the entire coding region of pre-pPG3, a stop codon, and BamHI restriction sites at the 59 and 39 ends of the insert were cloned into the BamHI restriction site of the pBacPAK1 transfer vector and transformed into Escherichia coli XL-1 Blue. Selection of pPG3-containing clones was accomplished by Southern blotting with radiolabeled PG3 cDNA. The correct orientation of the PG3 insert was verified by restriction analysis of DNA minipreps with HindIII and NcoI. The BacPAK2-pPG3 transfer plasmid was purified by ultracentrifugation on cesium chloride gradients and was cotransfected with Bsu 361-digested BacPAK6 (Autographa californica nuclear polyhedrosis) viral DNA into Sf21 cells with Lipofectin (GIBCO-BRL, Grand Island, N.Y.). Recombinant baculovirus was harvested from culture media 72 h after cotransfection. Individual viral plaques were isolated from infected monolayers of Sf21 cells, and the eluted virus was used to infect Sf21 cells for virus amplification and test production of protein. After 72 h, the cells from infected test cultures were lysed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis

* Corresponding author. Mailing address: Department of Medicine, CHS 37-055, 10833 Le Conte Ave., UCLA School of Medicine, Los Angeles, CA 90095. Phone: (310) 825-6112. Fax: (310) 206-8766. Email: [email protected]. 978

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(SDS-PAGE) loading buffer and analyzed by acid-urea-PAGE (AU-PAGE) and Tricine-SDS-PAGE to identify pPG3-expressing viral clones. Purification of pPG3. Hi-Five (Trichoplusia ni) insect cells were infected with recombinant baculovirus from amplified stocks. Pilot experiments determined that these cells released pPG3 into the medium. At ;60 h after infection, cell debris was removed from the culture medium by centrifugation, and the culture medium was concentrated ;20-fold and diafiltered with 5% acetic acid by using a tangential-flow concentration apparatus with a 10-kDa Omega membrane (Filtron Technology Corp., Northborough, Mass.). Concentrated proteins were separated by a continuous-flow preparative gel electrophoresis apparatus (Model 491 Prep Cell; Bio-Rad Laboratories, Richmond, Calif.) in an AU–12% polyacrylamide gel. Fractions containing pPG3 were assayed by AU-PAGE and were further purified by reverse-phase high-pressure liquid chromatography (RPHPLC) on a Vydac C18 column (4.6 by 250 mm; Separation Group, Hesperia, Calif.) by using a 1%/min acetonitrile increment in 0.1% trifluoracetic acid. The amount of pPG3 was standardized by using the calculated A280 value of 0.36 for a 1 mg/ml solution. A determination of protein concentration by MicroBCA assay (Pierce, Rockford, Ill.) yielded a 10% lower estimate for the standard preparation. Mass spectrometry. Fast atom bombardment mass spectrometric analysis was performed by Kristine Swiderek at the Division of Immunology, Beckman Research Institute, City of Hope, Duarte, Calif., with a double-focusing JEOL HX100HF magnetic sector mass spectrometer as described before (7). Enzymatic cleavage of pPG3. The enzymatic activities of human neutrophil elastase (HNE), cathepsin G (CG), and porcine pancreatic elastase (PPE) were determined exactly according to manufacturer’s protocols (Elastin Products Company, Inc., Owensville, Mo.). For analytical enzymatic cleavage of pPG3, enzymes (for amounts see figure legends) in 1 ml of 0.01% bovine serum albumin in H2O were incubated with pPG3 (4.5 mg) in 5 ml of 0.1 M Tris–0.15 M NaCl, pH 7.5, (HNE) or 5 ml of 0.1 M Tris, pH 8.2, (CG) at 378C for 30 min or as described in the figure legends. The reaction was stopped by adding 5 ml of 5% acetic acid, and the products were analyzed by AU-PAGE. For the preparation of pPG3 fragments, 200 mg of pPG3 was digested with 1 mg of HNE for 10 min at 378C, the reaction was stopped by adding 90 ml of acetic acid, and the mixture was analyzed by RP-HPLC on a Vydac C18 column (4.6 by 250 mm) by using a 1%/min acetonitrile increment in 0.1% trifluoracetic acid. Two major peaks were resolved and individually analyzed by mass spectrometry. Preparation of granule extract and degranulation of porcine neutrophils. Porcine peripheral blood neutrophils were isolated from 8-to-10-week-old healthy pigs as described earlier (20). Briefly, 30 ml of EDTA-treated blood was mixed with 30 ml of 3% dextran and was incubated for 20 min at room temperature. The supernatant was carefully removed from the dextran-sedimented blood, was overlaid on a Percoll gradient solution which had been previously prepared by the underlay of 10 ml of a 75% Percoll solution under 10 ml of 62.5% Percoll solution, and was centrifuged at 300 3 g for 25 min at room temperature. Neutrophils (.95% of the cells) in the second band from the top were collected and then washed once in ice-cold Dulbecco’s phosphate-buffered saline (PBS), pH 7.4, and counted. Neutrophil granule extract was prepared as described earlier (4). Briefly, neutrophils were resuspended to 108/ml in ice-cold 0.34 M sucrose adjusted to pH 7.4 with sodium bicarbonate. The cell suspension was sonicated, and then unbroken cells and nuclei were removed by centrifugation at 200 3 g for 10 min. Granule pellets were collected by centrifugation at 13,000 3 g for 30 min at 48C. In some experiments, protease inhibitors were added as indicated. Granules were solubilized in 0.1 M sodium acetate, pH 4, in the presence or absence of the specified protease inhibitors (see Results). For degranulation experiments, freshly isolated neutrophils were resuspended at 108/ml in PBS containing 1 mM Ca21 and 1 mM Mg21. Neutrophils were then incubated with phorbol myristate acetate (PMA; 100 ng/ml) at 378C for 30 min in the presence or absence of a 0.5 mM concentration of the specific inhibitor for neutrophil elastase, MeOSucAlaAlaProVal-chloromethyl ketone (CMK; Sigma). Degranulation solutions were collected after 1 min of centrifugation at 13,000 3 g to remove the neutrophils. Western blot analysis. Monoclonal anti-PG1, -2, and -3 antibodies were produced as described earlier (16) with pPG3 as antigen. The resulting monoclonal antibodies were screened in an enzyme immunoassay against synthetic mature PG3 to identify monoclonal antibodies reactive against this moiety. The specificity of the antibodies was confirmed by dot blot analysis in which the antibodies recognized synthetic PG1, PG2, and PG3 (7) but reacted very weakly with synthetic PG4 and PG5 (data not shown). To detect protegrins, AU-PAGE gels of neutrophil granule extracts were electroblotted to an Immobilon-P membrane in 0.7% acetic acid and the blots were probed with a 1:5 dilution of the supernatant of monoclonal hybridoma cells (anti-PG1, -2, and -3) and a 1:1,000 dilution of rabbit anti-mouse immunoglobulin G alkaline phosphatase conjugate, then developed in 5-bromo-4-chloro-3-indolylphosphate–nitroblue tetrazolium solution. Recombinant pPG3 and synthetic PG3 were used as controls. Bacteria. Overnight cultures of Listeria monocytogenes EGD were subcultured in 3% Trypticase soy broth (TSB) and grown for 2.5 h to exponential phase in a shaking incubator at 378C. Bacterial concentrations were estimated photometrically (an optical density at 620 nm of 0.2 corresponds to ;5 3 107 bacteria/ml). Bacteria were washed and diluted to 5 3 106 cells/ml in PBS.

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FIG. 1. Small cationic proteins in porcine neutrophil granule lysate are generated by serine proteases. Shown is a Coomassie-stained AU-PAGE gel. Lanes: PG3am1pPG3, a mixture of PG3am (1 mg; lower band) and pPG3 (1 mg; upper band); PMN GE, granule lysate of 5 3 106 porcine neutrophils; DFP-PMN GE, granule lysate from 5 3 106 DFP-treated neutrophils. Antibacterial gel overlay assay. The gel overlay assay was performed as described previously (8). Briefly, proteins and peptides were separated by AUPAGE, and the gel was neutralized by washing for 5 min in saline with 10 mM sodium phosphate, pH 7.4, (0.01 M PBS) and 0.01 N NaOH, then by washing in 0.01 M PBS alone for 15 min. The gel was then placed on a plate (10 by 10 cm) containing a 10-ml solid layer of 1% agarose with 0.1% TSB and 106 L. monocytogenes cells and was incubated at 378C for 3 h to allow the proteins and peptides in the gel to diffuse into the bacterial layer. The gel was then removed, and the agarose was overlaid with a nutrient layer that contained 10 ml of 6% TSB in 1% agarose. After 18 h of incubation at 378C to allow visible bacterial growth, the agarose plate was stained with 0.001% Coomassie blue for 10 h. Antibacterial activity was indicated by a clear zone (no bacterial growth). CFU assay. Equal volumes of bacteria (106 cells/ml) and neutrophil degranulation solutions or other peptide solutions were mixed and incubated in a 378C shaking incubator for 30 min, then diluted 100-fold in PBS, and aliquots were plated in quadruplicate on TSB-agar plates. After incubation for 12 to 16 h at 378C, the colonies were counted and the numbers of CFU per milliliter were calculated.

RESULTS Protegrins PG1am, PG2am, and PG3am were originally isolated in their mature forms from neutrophil lysates, but it was not certain whether they were stored as such in granules or cleaved from larger precursors during their extraction and purification. To examine the cleavage of neutrophil granule proteins by serine proteases during granule isolation, we prepared resting neutrophils from pig peripheral blood, treated one-half of the neutrophil suspension with cell-permeant serine protease inhibitor diisopropylfluorophosphate (DFP; 0.5 mM), and processed the treated and untreated portions in parallel. A comparison of the compositions of granule lysates from untreated and DFP-treated granules in AU-PAGE gel (Fig. 1) identified a group of high-mobility bands in untreated granule lysates that were absent in DFP-treated granule lysates. These were tentatively identified as protegrins because of the positions of the bands relative to synthetic PG3am (7). Thus, peptides with the electrophoretic characteristics of protegrins (7)

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FIG. 2. Protegrins in porcine neutrophil granule lysates are generated by elastase-mediated cleavage of proprotegrins. Shown is a Western blot of an AU-PAGE gel with anti-PG1, -2, and -3 antibodies. Granule lysates from 5 3 106 neutrophils/20 ml of 0.1 M NaOAc (pH 4.0) were adjusted to pH 7.4 by the addition of 8 ml of 2 M Tris solution and were incubated for the time indicated (in minutes) in the presence (1) or absence (2) of 1 mM specific elastase inhibitor CMK. The reaction was stopped by adding an equal volume of 5% acetic acid, and the products were analyzed by AU-PAGE. After electrophoresis, proteins and peptides were electroblotted to an Immobilon-P membrane, probed with monoclonal anti-PG1, -2, and -3 antibodies, and developed as outlined in Materials and Methods. Marker proPGs indicates the position of proprotegrins, and marker PGs indicates the position of mature protegrins.

were generated by serine neutrophil proteases during granule isolation and lysis. The protegrin precursors encoded by the cDNAs contain valines immediately N-terminal to the mature protegrins (23, 33), suggesting that neutrophil elastase could cleave the precursors at this site to yield the mature protegrins. To determine whether such cleavage takes place during extraction from neutrophils, we compared granule lysates prepared with or without highly specific neutrophil elastase inhibitor CMK and identified protegrin forms by Western blotting with a monoclonal antibody to PG1, -2, and -3. The rapidly migrating protegrin bands seen in Coomassie-stained gels (data not shown) and Western blots of untreated neutrophil granule lysates were absent (Fig. 2) when CMK was included in the lysis solution, confirming that protegrins were stored in granules as larger precursors and cleaved by an elastase-like serine protease after the granules were lysed. In order to characterize the role of neutrophil elastase as the processing enzyme for protegrins we analyzed the effect of purified neutrophil proteases on pPG3. Because of past problems with trace contamination of the cathelin moiety of protegrin precursors (9, 19) by copurifying highly active protease inhibitors, we elected to biosynthesize pPG3 in preference to purifying the native porcine precursor. The pPG3 was prepared in recombinant baculovirus-infected insect cells and was characterized by mass spectrometry and SDS-PAGE. The purified precursor, which had an N-terminal glutamine-to-pyroglutamate modification as in the naturally occurring procathelicidins (19) but which retained the C-terminal glycine instead of the naturally occurring amide group (our insect cells apparently do not C-terminally amidate this protein), had a mass that matched the predicted mass of pPG3, assuming pPG3 to be a protein containing amino acid residues 30 to 149 encoded in the cDNA (13,383 Da measured versus 13,385 Da predicted). Since the proteases of pig neutrophils have not been systematically studied, we tested the processing activity of HNE

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FIG. 3. Time dependence of cleavage of pPG3 by HNE. Shown is a Coomassie-stained AU-PAGE gel. pPG3 (4.5 mg) was incubated with HNE (20 ng) for the times indicated, and the reactions were stopped by acidification with equal volumes of 5% acetic acid and subjected to AU-PAGE and Coomassie blue staining. Controls and markers: pPG3 (1 mg) and AA (pPG3 incubated with HNE for 120 min in the presence of 2.5% acetic acid). The arrows indicate the positions of the uncleaved pPG3 and the large cleavage fragment cathelin (C). Note that cathelin is less cationic than pPG3 and thus, despite its smaller size, migrates slower.

and CG. Trace amounts of HNE (20 ng) cleaved pPG3 (4.5 mg) in a time-dependent manner into two fragments with the electrophoretic characteristics of cathelin and mature PG3 (Fig. 3). As expected, the rate of cleavage increased with increasing concentration of HNE (Fig. 4). The cleavage was completely inhibited by specific HNE inhibitor CMK (Fig. 5A). In contrast, similar amounts of CG (20 ng) did not cleave pPG3 (data not shown). Very large amounts of CG (2 mg) cleaved pPG3 as rapidly as did 10 ng of HNE, but this activity was due to minor contamination of CG by HNE since the cleavage was completely inhibited by CMK (Fig. 5A). The mixture of CG and

FIG. 4. Dependence of pPG3 cleavage on the concentration of HNE. Shown is a Coomassie-stained AU-PAGE gel. pPG3 (4.5 mg) was incubated with the indicated amounts of HNE at 378C for 10 min, the reactions were stopped by adding equal volumes of 5% acetic acid, and the products were subjected to AU-PAGE and Coomassie blue staining. Control lane, PG3ac (0.5 mg). The arrows indicate the positions of the uncleaved pPG3 and the large cleavage fragment cathelin (C). Note that cathelin is less cationic than pPG3 and thus, despite its smaller size, migrates slower.

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FIG. 5. (A) Cleavage of pPG3 by HNE and CG. Shown is a Coomassie-stained AU-PAGE gel. pPG3 (4.5 mg) was incubated with HNE (20 ng) or CG (2,000 ng) in the presence or absence of 1 mM CMK at 378C for the times indicated (9, minutes). The reactions were stopped by adding equal volumes of 5% acetic acid, and the products were subjected to AU-PAGE and Coomassie blue staining. Control lane, PG3ac (1 mg). The arrows indicate the positions of PG3, the uncleaved pPG3, and the large cleavage fragment cathelin (C). Note that cathelin is less cationic than pPG3 and thus, despite its smaller size, migrates slower. (B) Cleavage of pPG3 by PPE. Shown is a Coomassie-stained AU-PAGE gel. pPG3 (4.5 mg) was incubated with HNE (20 ng) or PPE (20,200, or 2,000 ng) at 378C for 30 min. The reactions were stopped by adding equal volumes of 5% acetic acid, and the products were subjected to AU-PAGE and stained with Coomassie blue. Control lanes: PG3ac (0.5 mg) and PPE 2000 ng. The smaller arrow marks the position of the cationic fragment cleaved by PPE; the bigger arrow marks the position of synthetic PG3 and PG3 cleaved by HNE.

CMK retained proteolytic activity towards other proteins, as indicated by the lack of inhibition of proteolysis of bovine serum albumin used as a protein carrier in the incubation mixture. We confirmed the HNE cleavage site in pPG3 by separating the two proteolytic fragments of pPG3 by RP-HPLC, then subjecting them to mass spectrometry. The heavier fragment matched porcine cathelin with an N-terminal pyroglutamate modification (mass, 11,287.0 Da measured versus 11,288.7 Da predicted). The lighter fragment matched the 19-amino-acid mature PG3 with a C-terminal glycine (mass, 2,115.4 Da measured versus 2,114.5 Da predicted) and will be referred to as PG3. The error in measurement was similar to that for chemically synthesized internal standard PG3ac (C-terminal desgly acid form of PG3; mass, 2,058.2 Da measured versus 2,057.5 Da predicted). While the cleavage of pPG3 by HNE was both efficient and highly site specific, 10- to 100-fold more PPE was required to cleave pPG3 to the same extent in a 30-min incubation (Fig. 5B). As seen in the figure, the pancreatic enzyme generated proteolytic fragments distinct from those cleaved by HNE. To detect the porcine homolog of HNE, we assayed the porcine neutrophil granule lysate by a colorimetric assay with the small-peptide substrate MeOSuc-Ala-Ala-Pro-Val-pNA. Human and porcine neutrophil lysates contained similar amounts of elastase activity (data not shown). When aliquots of HNE and porcine granule extract were matched for elastase activity, they cleaved pPG3 to the same extent and yielded electrophoretically identical fragments and both reactions were completely inhibited by CMK (Fig. 6). Thus, the porcine enzyme that converts pPG3 to PG3 in granule lysates appears to be the functional homolog of HNE. We hypothesized that cleavage by porcine elastase converted the inactive proprotegrins to bactericidal mature protegrins. Indeed, recombinant pPG3 was not bactericidal by itself, but preincubation of pPG3 with HNE for 30 min followed by

the addition of CMK generated sufficient PG3 to kill 99.9% of the L. monocytogenes cells inoculated (Fig. 7). Compared to equimolar concentrations of chemically synthesized PG3ac or PG3am, cleaved pPG3 had equal bactericidal activity. When

FIG. 6. Cleavage of pPG3 by porcine neutrophil granule extract. Shown is a Coomassie-stained AU-PAGE gel. pPG3 (3 mg) was incubated with porcine neutrophil granule extract (equivalent to 2 ng of HNE) for 30 min at 378C in the presence or absence of neutrophil elastase inhibitor CMK. Controls: PG3ac, (0.5 mg) and GE alone (granule extract [103 cell equivalents]).

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FIG. 7. Bactericidal activity of pPG3 is dependent on its activation by neutrophil elastase. Shown are the results of a CFU assay. L. monocytogenes (50 ml of a 106-CFU/ml suspension) was incubated with an equal volume of indicated peptide solution for 30 min (except for T 5 0 point) at 378C. An aliquot (20 ml) was diluted, plated on TSB agar, and incubated overnight to allow colony development. PBS, T 5 0, number of CFU in the initial bacterial inoculum; PBS, T 5 30, number of CFU after 30 min of incubation in PBS. The remaining bars indicate the number of CFU after incubation with the indicated peptide mixture: pPG3 (17 mg/ml), (pPG31HNE)1CMK (17 mg of pPG3 per ml was preincubated with 300 ng of HNE per ml for 30 min, then CMK was added to a 1 mM final concentration, and the mixture was incubated with bacteria), PG3ac and PG3am (2.5 mg/ml), (CMK1HNE)1pPG3 (1 mM CMK was mixed with 300 ng of HNE per ml, pPG3 was added to a final concentration of 17 mg/ml, and the mixture was preincubated for 30 min; then the mixture was incubated with bacteria), and CMK1HNE (1 mM CMK was mixed with 300 ng of HNE per ml, preincubated for 30 min, then incubated with bacteria). The means plus standard errors of quadruplicate experiments are shown.

pPG3 was omitted from the incubation mixture or when CMK was added at the beginning of incubation before pPG3, no bactericidal activity was generated. Next we determined whether the elastase-dependent listericidal activity in cleaved pPG3 was due to the cathelin fragment, PG3, or both. We blotted AUPAGE gels of intact and HNE-cleaved pPG3 onto a lawn of L. monocytogenes (Fig. 8) to identify the bactericidal fragment(s). The zone of bacterial clearance overlaid the position of the PG3 band in the gel, with no activity seen in the area of cathelin or pPG3. If porcine neutrophils employed extracellular elastase activity to generate microbicidal fragments from inactive precursors, neutrophil secretions released into CMK-containing medium would be expected to be less microbicidal than they would be in the absence of this inhibitor. We prepared neutrophil secretions by stimulating porcine neutrophils with PMA, then removed the neutrophils by centrifugation and tested the medium for bactericidal activity in a CFU assay with L. monocytogenes. While unstimulated secretions were not bactericidal, the PMA-stimulated secretions killed more than 5 logs of bacteria. To detect the activating effect of neutrophil elastase on secreted proteins we added the inhibitor CMK either before PMA, to block elastase activity during and after secretion, or at the end of the 30-min incubation. Pretreatment with CMK made the secretions much less bactericidal than did posttreatment (Fig. 9). Neither CMK nor PMA affected L. monocytogenes CFU directly. Thus, porcine neutrophil elastase activity was required for the activation of bactericidal substances in neutrophil secretions. When the secretions were stored frozen for up to 1 month, their bactericidal activity remained stable. We surmised that the elastase-dependent bactericidal activity in neutrophil secretions was at least in part due to protegrins generated by the cleavage of secreted proprotegrins. To

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FIG. 8. PG3 is the bactericidal fragment generated by the elastase cleavage of pPG3. Shown is a gel-overlay assay. Peptides were subjected to AU-PAGE as described in Materials and Methods, and bactericidal bands were detected by overlay onto a bacterial lawn. After electrophoresis, the AU gel was washed in PBS (pH 7.4) for 30 min and was overlaid on 1% agarose gel with 0.1% TSB containing 106 L. monocytogenes cells per ml. This complex was incubated at 378C for 3 h to transfer protein bands onto the bacterial lawn. The AU gel was then removed, and 10 ml of 1% agarose gel with 6% TSB was poured on the bacterial lawn. The two-layer complex was then incubated at 378C overnight to develop microcolonies. Bactericidal activity was indicated by a clear zone. Lanes: PG3ac, 0.5 mg; pPG3, 3 mg; pPG31HNE, 3 mg of pPG3 plus 20 ng of HNE. The contents of each lane were preincubated for 30 min at 378C. The arrow indicates the position of pPG3 as detected by Coomassie blue staining.

test this possibility, we compared the bactericidal proteins in neutrophil secretions released in the presence of elastase inhibitor CMK with those in secretions released in the absence of CMK. In pilot experiments, the secreted proteins were concentrated either on a resin (Strataclean; Stratagene, La Jolla, Calif.) or by ultrafiltration on a 1,000-molecular-weight cutoff membrane before analysis by AU-PAGE and Western blotting with monoclonal antibodies to PG1, -2, and -3. Since the two methods yielded identical electrophoretic and Western blot patterns (data not shown), in subsequent experiments we concentrated the secreted proteins and peptides by the simpler and less expensive resin method. In a bactericidal gel overlay assay (Fig. 10), secretions generated in the presence of CMK substantially lacked the fast-migrating bactericidal bands that were prominent when CMK was added only 30 min after activation. These bands were readily visualized by Coomassie blue staining, and their identity as protegrins was confirmed by staining with monoclonal anti-protegrin antibody (Fig. 11). The Western blots also confirmed that the electrophoretic mobilities of PG1, -2, and -3 precursors from unstimulated porcine neutrophils were similar to that of recombinant pPG3, suggesting that the storage form of protegrins in granules is likely identical or nearly identical to the recombinant form. Inhibition of neutrophil elastase by CMK also resulted in an increase of total immunoreactive protegrin forms recovered from the medium, indicating either that mature protegrins generated by elastase from proprotegrin are lost from the medium by adsorption to cells and the container or that in the absence of protease inhibitors mature protegrins are eventually degraded by neutrophil elastase. A consistent slower-migrating listericidal band was equally prominent in neutrophil secretions obtained in the presence

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FIG. 9. Bactericidal activity of fluid conditioned by porcine neutrophils is dependent on the presence of active elastase during secretion. Shown are the results of a CFU assay. An L. monocytogenes suspension (50 ml of a 106-CFU/ml suspension) was incubated for 30 min at 378C with an equal volume of medium conditioned by secretions of a suspension of 108 porcine polymorphonuclear leukocytes (PMN) per ml. An aliquot (20 ml) of the above mixture was diluted, spread on TSB agar, and incubated overnight to develop colonies. Bars show the numbers of CFU in the following media: PMN, medium conditioned by unstimulated PMN; PMN1PMA, medium conditioned by PMN stimulated with PMA (100 ng/ml); (PMN1CMK)1PMA, medium conditioned by PMN stimulated with PMA (100 ng/ml) in the presence of 1 mM CMK; (PMN1PMA)1CMK, medium conditioned by PMN stimulated with PMA (100 ng/ml) for 30 min then adjusted to 1 mM CMK; CMK1PMA, medium containing CMK (1 mM) and PMA (100 ng/ml) but no neutrophils. The means plus standard errors of quadruplicate experiments are shown.

and absence of CMK and was variable but less prominent in secretions of unstimulated neutrophils (Fig. 10). It comigrated with lysozyme, as identified by gel overlay with dried Micrococcus lysodeikticus (data not shown). Under the conditions of the gel overlay assay (Dulbecco’s PBS [pH 7.4] in L. monocytogenes-containing agarose, supplemented by 6% TSB in the top layer) no other microbicidal bands were seen. DISCUSSION Neutrophils contain an impressive arsenal of antimicrobial peptides and proteins both in their phagocytic (primary, azurophil) granules and their secretory (secondary, specific) granules (10, 13). Some polypeptides are stored in granules in active form after posttranslational processing that ranges in complexity from mere removal of the signal sequence (p15 of rabbit neutrophils) (11, 12, 27) to multistep proteolytic maturation (defensins) (6, 24). Other antimicrobial proteins are stored in granules as inactive precursors that undergo activation by proteolytic cleavage during phagocytosis (Bac5 and Bac7 of bovine neutrophils) (21, 30, 31). The antimicrobial peptides of phagocytic granules can reach very high concentrations in the sequestered environment of the phagolysosome and there are likely to contribute to the killing of microbes, notwithstanding the known inhibitory effects of blood plasma. However, neutrophils can also confer antimicrobial properties on cell-free inflammatory fluids (26, 27). To function in the extracellular milieu, secreted antimicrobial proteins and peptides must retain their activity at much lower concentrations, even in the presence of plasma electrolytes. The retention of the antimicrobial activity of protegrins at 1- to 10-mg/ml concentrations in physiologic medium and 1 to 10% serum (7a, 17) suggested that these peptides could also function extracellularly. In this study, we have shown that stimulated porcine neutrophils release protegrin precursors that undergo proteo-

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FIG. 10. Release of bactericidal protegrins by porcine PMN requires active elastase. Shown is a gel overlay assay. A bactericidal overlay assay was performed as described in the legend for Fig. 8. Each lane contained the secretions of 107 porcine polymorphonuclear leukocytes (PMN). Lanes: Diluent, medium conditioned by PMN exposed to diluent only for 30 min; PMA, medium conditioned by PMN stimulated with PMA (100 ng/ml) for 30 min; CMK1PMA, medium conditioned by PMN in the presence of 1 mM CMK and PMA (100 ng/ml) for 30 min. Markers designate the locations of proprotegrins (proPGs), lysozyme (LZ), and mature protegrins (PGs).

lytic cleavage to generate microbicidal protegrins active against L. monocytogenes. In these experiments we employed a high density of porcine neutrophils (108/ml) to facilitate the biochemical detection of secreted microbicidal proteins and peptides. Such a concentration may occur in vivo in abscesses and other acute inflammatory conditions, but it is also possible that over longer periods of time much lower densities of neutrophils are sufficient to render fluids microbicidal or microbistatic, especially if the neutrophils turn over rapidly. Experimental studies of infected pigs will be required to determine whether and under what circumstances protegrins contribute to host defense. Trace amounts of the porcine homolog of HNE are both necessary and sufficient for extracellular conversion of proprotegrins to active protegrins. The release of such small amounts of elastase is likely to occur during phagocytosis (3) even if (as is the case for human neutrophils) elastase is stored in the nonsecretory granule population. Preliminary experiments indicated that the proteolytic activation of secreted protegrins by stimulated porcine neutrophils is resistant to the inhibitory effect of a-1 proteinase inhibitor (a-1 antitrypsin; a1PI). The potential mechanisms that could negate the inhibitory effects of a1PI include its destruction by the products of respiratory burst or metalloproteinases (2, 18, 28), the relative inaccessibility of the cell surface-bound neutrophil elastase to this inhibitor (15), and the high efficiency of proprotegrin cleavage by neutrophil elastase. The presence of the approximately 100-amino-acid cathelin motif is the common feature of precursors of an increasing number of antimicrobial peptides with presumed host defense function, members of the cathelicidin family (29). The remark-

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lular microbicidal activity may be another common characteristic of cathelicidins. Although their contribution to host defense in the infected animal remains to be delineated, the capacity of these peptides to kill bacteria in extracellular fluids is likely to make them attractive candidates in the search for new classes of antimicrobial compounds. ACKNOWLEDGMENTS We thank Chris Carangi and John Robert for their excellent technical support and Robert Lehrer for many helpful discussions and the sharing of unpublished results. This work was supported by grant HL46809 from the National Institutes of Health. REFERENCES

FIG. 11. Release of protegrins by porcine polymorphonuclear leukocytes (PMN) requires active elastase. Shown are an AU-PAGE gel and a Western blot. Porcine peripheral blood neutrophils were incubated with or without PMA (100 ng/ml) and/or CMK (1 mM) for 30 min at 378C. The conditioned media were subjected to AU-PAGE and Western blot analysis with monoclonal anti-PG1, -2, and -3 antibodies. (Left panel) Coomassie-stained AU-PAGE gel. Lanes: Diluent, medium conditioned by PMN in the presence of diluent; CMK1PMA, medium conditioned by PMN in the presence of 1 mM CMK and PMA (100 ng/ml); PMA, medium conditioned by PMN in the presence of PMA (100 ng/ml); PG3am1pPG3, standards (1 mg each). (Right panel) Western blot analysis. A sister gel of the one in the left panel was electroblotted and probed with antiPG1, -2, and -3 monoclonal antibodies. Rapidly migrating bands reactive with anti-PG1, -2, and -3 antibodies are mature protegrins (labeled PGs).

able attribute of this family is the extreme diversity of the C-terminal peptides, which range in structure from a-helical to b-sheet to proline rich, while the cathelin motif in the precursors is highly conserved. The cathelin motif in turn is significantly similar to the cystatin motif found in various cystatins, naturally occurring inhibitors of thiol proteases, and in serum kininogen, which flanks the vasodilator and pain-inducing nonapeptide bradykinin. In the case of kininogen and bradykinin, the potential adverse consequences of inadvertent activation are quite obvious. It is tempting to speculate that the cathelin motif, like the cystatin motif, serves to increase the specificity of the interaction with the activating protease, neutrophil elastase, and to interfere with the activity of other proteases. In the case of protegrins Bac5 and Bac7 (21), the adjacent cathelin motif neutralizes the microbicidal activity of the C-terminal peptide, but this is not a universal feature of all cathelin-like motifs since the p15 peptides of rabbit neutrophils are microbicidal with the cathelin motif on the active peptide form (10, 27). It is notable that several members of the cathelicidin family have either been localized to the secretory granules of neutrophils, i.e., the specific (secondary) granules in human and murine neutrophils (1, 14) and the large granules in bovine neutrophils (30) or have been detected in active concentrations in biological fluids (27), suggesting that participation in extracel-

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