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The Journal of Immunology, 2002, 168: 3024-3032.
Copyright © 2002 by The American Association of Immunologists

Antimicrobial Peptides Initiate IL-1{beta} Posttranslational Processing: A Novel Role Beyond Innate Immunity

David G. Perregaux, Kanan Bhavsar, Len Contillo, Jishu Shi and Christopher A. Gabel1

Department of Antibacterials, Immunology, and Inflammation, Pfizer Global Research and Development, Pfizer, Inc., Groton, CT 06340


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human monocytes stimulated with LPS produce large quantities of prointerleukin-1{beta}, but little of this cytokine product is released extracellularly as the mature biologically active species. To demonstrate efficient proteolytic cleavage and export, cytokine-producing cells require a secondary effector stimulus. In an attempt to identify agents that may serve as initiators of IL-1{beta} posttranslational processing in vivo, LPS-activated human monocytes were treated with several individual antimicrobial peptides. Two peptides derived from porcine neutrophils, protegrin (PTG)-1 and PTG-3, promoted rapid and efficient release of mature IL-1{beta}. The PTG-mediated response engaged a mechanism similar to that initiated by extracellular ATP acting via the P2X7 receptor. Thus, both processes were disrupted by a caspase inhibitor, both were sensitive to ethacrynic acid and CP-424,174, two pharmacological agents that suppress posttranslational processing, and both were negated by elevation of extracellular potassium. Moreover, the PTGs, like ATP, promoted a dramatic change in monocyte morphology and a loss of membrane latency. The PTG response was concentration dependent and was influenced profoundly by components within the culture medium. In contrast, porcine neutrophil antimicrobial peptides PR-26 and PR-39 did not initiate IL-1{beta} posttranslational processing. The human defensin HNP-1 and the frog peptide magainin 1 elicited export of 17-kDa IL-1{beta}, but these agents were less efficient than PTGs. As a result of this ability to promote release of potent proinflammatory cytokines such as IL-1{beta}, select antimicrobial peptides may possess important immunomodulatory functions that extend beyond innate immunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Production of the inflammatory cytokine IL-1 by monocytes and macrophages involves a number of highly regulated cellular processes (1). Like other cytokines, IL-1 appears to act as an intercellular messenger. High-affinity receptors for this cytokine exist on many cells (2, 3), and when engaged by their polypeptide ligands these receptors activate signaling cascades leading to proinflammatory responses (4, 5). No high-affinity intracellular binding sites for IL-1 have been identified, although intracellular functions have been proposed (6, 7, 8). To function as an extracellular effector, however, IL-1 must be released by producing cells so that it can engage target cell surface receptors. Although such a requirement is common to all cytokine mediators, IL-1 faces a challenge not encountered by most other secreted polypeptides. Eukaryotic proteins destined to be secreted typically are synthesized in the rough endoplasmic reticulum (ER)2 (9). This localization is achieved as a result of a signal peptide being associated with the nascent polypeptide chain that directs the translational complex composed of ribosomal subunits and messenger RNA to specific sites on the ER membrane. The ER-bound complex then facilitates passage of the nascent polypeptide across the membrane, resulting in the release of the completed polypeptide chain within the lumen of the ER. From here, newly synthesized secretory proteins are packaged into transport vesicles for delivery to the Golgi apparatus and ultimately to the cell surface (10). Newly synthesized IL-1, in contrast, does not contain a signal peptide and, as a result, is not sequestered to the ER (11, 12). Rather, IL-1 polypeptides accumulate within the cytoplasm of activated monocytes and macrophages (13, 14).

The mechanism by which IL-1, both the {alpha} and {beta} species, gains access to the extracellular environment is not well understood. Both cytokine species are synthesized as 31-kDa propolypeptides that must be processed by proteases to generate their mature 17-kDa species; this processing further complicates the export process. In the case of IL-1{beta}, proteolytic processing is required for binding to the IL-1R (15). The enzyme responsible for cleavage of proIL-1{beta} is caspase-1 (16, 17). In contrast, proIL-1{alpha} can bind to IL-1R and elicit a biologic response (15); nonetheless, proIL-1{alpha} may be cleaved to a 17-kDa species by a non-caspase-1-mediated process (18). Within LPS-activated monocytes and macrophages, the procytokines are readily detected but the proteolytically processed forms are difficult to find (19, 20). Based on this, the suggestion has been put forward that cleavage is closely associated temporally with release of cytokine to the medium (20). Indeed, evidence has been presented to suggest that caspase-1 may be a component of the secretory apparatus (21).

In vitro studies have demonstrated that secretion of mature IL-1{beta} from LPS-activated monocytes and macrophages is not a constitutive process (22, 23, 24, 25, 26). Rather, to display efficient IL-1 export, these cytokine-producing cells must encounter a secondary stimulus that specifically activates the posttranslational processing events. To date, agents that have demonstrated an ability to initiate IL-1{beta} posttranslational processing from cultured human monocytes include ATP, nigericin, hypotonic stress, and bacterial toxins (24, 25, 27, 28, 29, 30, 31, 32). These agents all share an ability to promote major changes to the intracellular ionic environment, and K+ efflux appears to be a necessary component of the cytokine export process (27, 28, 31, 32). Moreover, all of these treatments lead to death of the cytokine-producing cell; whether this death is achieved via an apoptotic or necrotic response pathway is unclear, as hallmarks of both processes have been observed (24, 27, 33).

The need for a separate secretion stimulus does not appear to be an artificial requirement resulting from the use of isolated cells in culture. Indeed, when LPS is injected into the peritoneal cavity of mice, resident macrophages generate large quantities of cell-associated proIL-1{beta}, but little cytokine is recovered in the peritoneal lavage fluid. However, following a subsequent injection of ATP, large quantities of cell-dissociated 17-kDa IL-1{beta} are generated (34). Likewise, cell-free IL-1 can be detected in plasma following LPS activation of human whole blood ex vivo, but cytokine levels are increased dramatically by coadministration of ATP (35). Therefore, efficient generation of biologically active IL-1 appears to require two separate stimuli: 1) a priming stimulus to promote synthesis of the procytokine and 2) a secretion stimulus to initiate posttranslational processing and release. Currently, stimuli that function to promote IL-1 secretion in vivo remain to be identified. ATP, acting via the P2X7 receptor, may operate in this capacity (36), and in the context of an infection bacterially derived toxins could function as secretion stimuli (32). A search for other physiologically relevant effectors that may function in vivo to promote IL-1{beta} posttranslational processing led us to ask whether neutrophil-derived antimicrobial peptides of the defensin superfamily possess this type of activity. This selection was based on the well characterized ability of many of these peptides to promote ion movements across biological membranes. In this report, we demonstrate that protegrins (PTGs) are effective inducers of human monocyte IL-1{beta} posttranslational processing. These mediators of innate immunity may therefore possess additional functions that impact the acquired immune response as a result of their ability to facilitate release of leaderless peptides such as IL-1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Synthesis and source of antimicrobial peptides

PTG-1 and -3 were synthesized at Pfizer (Groton, CT) using modified cycles on a 433a peptide synthesizer (Applied Biosystems, Foster City, CA) with F-moc chemistry. Formation of the disulfide bonds was achieved as follows. Five milligrams of the linear peptide (PTG-1 or PTG-3) were solubilized in 100 ml of 100 mM Tris (pH 7.8), containing 52 mg cystine-2HCl, 13.5 mg acetyl-cysteine-2H2O, and 164 mg methionine. The mixtures were stirred for 2 h at 4°C, after which the folded peptides were isolated by HPLC. The final peptide products were analyzed by mass spectrometry and found to possess masses of 2155.0 and 2056.51 Da, respectively, for PTG-1 and PTG-3. These values are in complete agreement with theoretical values. Synthesis of the proline- and arginine-rich peptides PR-26 and PR-39 was performed by American Peptide Company (Sunnyvale, CA); mass of the synthetic products was confirmed by mass spectrometry. Magainins I and II and {beta}-defensin-1, {beta}-defensin-2, and {alpha}-defensin-1 (HNP-1) all were purchased from Peptide Institute (Osaka, Japan).

Human monocyte IL-1{beta} posttranslational processing assay: ELISA format

Blood collected from normal volunteers in the presence of heparin was fractionated using lymphocyte separation medium (ICN Pharmaceuticals, Aurora, OH). The region of the resulting gradient containing mononuclear cells was harvested and diluted with an equal volume of maintenance medium (RPMI 1640 medium, 5% FBS, 25 mM HEPES (pH 7.2), and 1% penicillin/streptomycin) and the cells were collected by centrifugation. The resulting cell pellet was suspended in 10 ml of maintenance medium and a cell count was performed. Each well of a 96-well tissue culture plate then was seeded with 2 x 105 cells (in a total volume of 0.1 ml of maintenance medium). Monocytes were allowed to adhere for 2 h, after which medium supernatants were discarded. Attached cells were rinsed twice with maintenance medium and then incubated in 0.1 ml of maintenance medium overnight at 37°C in a 5% CO2 environment. The following morning, LPS (Escherichia coli serotype 055:B5; Sigma-Aldrich, St. Louis, MO) was introduced to some wells to achieve a final concentration of 10 ng/ml and the cultures were activated for 2 h at 37°C. Media then were removed and 0.1 ml of fresh medium (RPMI 1640 containing 1% FBS, 20 mM HEPES (pH 6.9), 5 mM NaHCO3) was added to each well. Where indicated, a compound to be tested as an inhibitor of posttranslational processing also was added to this medium. An antimicrobial peptide then was introduced, and the cultures were incubated for an additional 3 h at 37°C. The 96-well plates subsequently were centrifuged and the resulting clarified medium supernatants were harvested. IL-1{beta} content of these supernatants was determined by ELISA (R&D Systems, Minneapolis, MN).

Human monocyte IL-1{beta} posttranslational processing assay: metabolic format

Human mononuclear cells were prepared as described above, and 1 x 107 cells (in 2 ml of maintenance medium) were seeded into each well of six-well multiplates. After 2 h of adherence, media and nonadherent cells were removed and 2 ml of fresh maintenance medium was added; the cultures were incubated overnight at 37°C. LPS was added to each well the following morning (final concentration of 10 ng/ml) and the cultures were incubated for an additional 2 h. Media then were removed and replaced with 1 ml of methionine-free RPMI 1640 medium containing 1% dialyzed FBS, 25 mM HEPES (pH 7.2), and 83 µCi/ml [35S]methionine (1000 Ci/mmol; Amersham, Arlington Heights, IL); the cells were labeled for 60 min. Pulse media then were removed, the adherent cells were rinsed once with 2 ml of RPMI 1640 containing 1% FBS, 25 mM HEPES (pH 6.9), and 5 mM NaHCO3, and 1 ml of the same medium, with or without an effector molecule, was added to each well. Where indicated, ATP was added (from a 100 mM stock solution (pH 7)) to achieve a final concentration of 2 mM. The cultures were incubated at 37°C for 3 h after which their media were harvested and clarified by centrifugation; these samples were adjusted to 1% Triton X-100, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM iodoacetic acid, 1 µg/ml leupeptin, and 1 µg/ml pepstatin by the addition of a concentrated stock solution of these reagents. Adherent monocytes were solubilized by addition of 1 ml of 25 mM HEPES (pH 7), 1% Triton X-100, 150 mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM iodoacetic acid, 1 µg/ml pepstatin, 1 µg/ml leupeptin, and 1 mg/ml OVA. After a 30-min incubation on ice, both the media and cell extracts were clarified by centrifugation at 45,000 rpm for 30 min in a tabletop ultracentrifuge using a TLA 45 rotor (Beckman Coulter, Fullerton, CA).

IL-1{beta} subsequently was recovered from the soluble fraction of the media and cell-associated samples by immunoprecipitation as described previously (27). Resulting immunoprecipitates were analyzed by SDS-PAGE and autoradiography. In some cases, the amount of radioactivity associated with a specific polypeptide species was determined by phosphor imager analysis.

Other reagents

Ethacrynic acid was obtained from Sigma-Aldrich and KN-62 was obtained from Research Biochemicals International (Natick, MA) The caspase inhibitor YVAD-cmk was obtained from Bachem (Torrance, CA). CP-424,174 was synthesized at Pfizer. In some experiments, monocytes were incubated in an isotonic medium composed of 137 mM NaCl, 0.9 mM CaCl2, 0.5 mM MgCl2, 1.5 mM KH2PO4, 5 mM glucose, 5 mM KHCO3, 2.7 mM KCl (pH 6.9); where indicated, 50 mM KCl was added to this medium and the NaCl concentration was reduced to 87 mM. Lactate dehydrogenase (LDH) was measured as previously detailed (27).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PTGs promote IL-1{beta} posttranslational processing

To determine whether antimicrobial peptides are capable of stimulating human monocyte IL-1 posttranslational processing, a staged assay was used. Cells were stimulated with LPS to initiate proIL-1{beta} synthesis and then treated with an effector peptide to promote cytokine posttranslational processing and release. Initially, peptides corresponding to two different subtypes of the cathelicidin family were analyzed (37). Porcine antimicrobial peptides PTG-1 and PTG-3 are arginine- and cysteine-rich peptides that contain two intramolecular disulfide bonds (Fig. 1GoA). PTG-1 and PTG-3 each contain 18 amino acids and differ only in the replacement of an arginine residue found in PTG-1 with a glycine residue in PTG-3 (38, 39). In contrast, proline- and arginine-rich antimicrobial peptides PR-26 and PR-39 contain no intrachain disulfide bonds (Fig. 1Go). LPS-activated human monocytes incubated with increasing concentrations of the individual PR-rich peptides <=100 µg/ml released no significant IL-1{beta} to the medium (Fig. 1GoB). In contrast, LPS-activated monocyte cultures treated with PTG-1 or PTG-3 released large quantities of cytokine to the medium (Fig. 1GoB). For both PTG peptides a similar bell-shaped dose response curve was observed, with 12.5 µg/ml being the optimal concentration.



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  		FIGURE 1. PTGs selectively initiate IL-1 release from LPS-activated human monocytes. A, Amino acid sequence of the synthetic PTGs and proline- and arginine-rich peptides used in these studies. In the case of PTGs, disulfide bond pairs are indicated. B, LPS-activated human monocytes were treated with the indicated concentration of peptide for 3 h, after which media were removed and their content of IL-1{beta} was determined by ELISA. Each point is the mean of triplicate determinations. In this and all subsequent figures, PTG-1 and PTG-3 are designated as PG1 and PG3, respectively.

 
All four of the individual synthetic peptides were assessed for their ability to kill E. coli. Using a standard assay format (40), each of the synthetic peptides demonstrated antimicrobial activity (data not shown), with a minimal inhibitory concentration of 2–10 µg/ml. Thus, the ability to promote release of IL-1 from LPS-activated monocytes did not correlate with antimicrobial activity.

PTG treatment leads to formation of 17-kDa IL-1{beta}

The ELISA used in the above experiment is reported by the manufacturer to preferentially recognize mature IL1{beta}, but this assay also detects the procytokine species. Therefore, to determine whether PTGs actually elicit formation and release of biologically active 17-kDa IL-1{beta} a metabolic assay format was used. Human monocytes were activated with LPS, labeled with [35S]methionine for 60 min, and then placed in isotope-free medium in the absence or presence of an initiator of IL-1 posttranslational processing. Following a 3-h treatment, media were harvested and IL-1{beta} was recovered by immunoprecipitation. In the absence of an effector, no radiolabeled IL-1{beta} was released extracellularly (Fig. 2GoB), but large quantities of proIL-1{beta} were recovered from extracts of the cells (Fig. 2GoA). In contrast, in the presence of ATP, a known initiator of IL-1 posttranslational processing (24, 27, 28, 29), large amounts of 17-kDa IL-1{beta} and smaller quantities of 31-kDa proIL-1{beta} and a 28-kDa species were recovered from the medium; the latter species may represent an alternate caspase-1 cleavage product (41). Cytokine that remained cell associated following ATP treatment persisted as the 31-kDa procytokine species (Fig. 2GoA). Treatment with 10 µg/ml PTG-1 led to the appearance of 17-kDa IL-1{beta} in the media (Fig. 2GoB). Relative to ATP-treated cultures, PTG-1-treated cells released more 31-kDa proIL-1{beta}. Increasing the PTG-1 concentration to 25 µg/ml resulted in less 17-kDa IL-1{beta} and greater quantities of the procytokine species relative to the lower peptide concentration. This suggests that the efficiency of proteolytic processing was reduced at the higher PTG-1 concentration. Similarly, PTG-3-treated monocytes externalized large quantities of 17-kDa IL-1{beta} (Fig. 2GoB). As with PTG-1, the efficiency at which the released cytokine was processed to the 17-kDa species was reduced at 25 µg/ml relative to that achieved at 10 µg/ml (Fig. 2GoB). With both the PTG-1- and PTG-3-treated cultures, IL-1 that remained cell-associated persisted as the 31-kDa procytokine species (Fig. 2GoA).



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  		FIGURE 2. PTGs promote formation and release of mature IL-1{beta}. LPS-activated, [35S]methionine-labeled human monocytes were treated with the indicated effector for 3 h. Media and cell-associated fractions were harvested separately and IL-1{beta} was recovered from each by immunoprecipitation. The resulting immunoprecipitates were analyzed by SDS-PAGE and autoradiography. The autoradiograms correspond to the cell-associated (A) and media (B) samples. The migration positions of the 17-kDa mature and 31-kDa proform of IL-1{beta} are indicated. Each condition was performed in duplicate.

 
ATP-induced IL-1{beta} posttranslational processing is accompanied by cell death and loss of plasma membrane latency (24, 27). To determine whether PTG-initiated IL-1 posttranslational processing also altered cell viability, distribution of the cytoplasmic enzyme LDH was assessed. From the cultures described above, 7, 25, 37, 72, 27, and 69% of the total culture-associated LDH was recovered in the medium following 3 h of treatment with no effector, ATP, 10 µg/ml PTG-1, 25 µg/ml PTG-1, 10 µg/ml PTG-3, or 25 µg/ml PTG-3, respectively.

PTG-1 induces a dramatic change in monocyte morphology

Loss of cell viability caused by initiators of IL-1{beta} posttranslational processing such as ATP and nigericin is accompanied by a dramatic morphology change (27). To ascertain whether PTG-induced processing demonstrated a similar type of response, LPS-activated human monocytes were treated with PTG-1 and analyzed by light microscopy. Cultures of LPS-activated monocytes were heterogeneous in appearance, with some cells demonstrating tight adherence to the plastic surface and others remaining less adherent and round. Exposure to PTG-1 for 2.5 min did not produce a noticeable change in monocyte morphology (Fig. 3Go). However, after 5 min of treatment, loss of the adherent phenotype was observed and some cells demonstrated cytoplasmic extensions. By 10 min, the cytoplasm of many cells appeared devoid of contrast and the nucleus became very pronounced. With longer PTG-1 exposures, the majority of cells showed loss of cytoplasmic contrast and achieved a distended, swollen appearance (Fig. 3Go). These changes in morphology are comparable to those observed in the presence of ATP or nigericin (27).



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  		FIGURE 3. PTG-1 causes a change in cell morphology. LPS-activated human monocytes were exposed to PTG-1 (12.5 µg/ml) for the times indicated at 37°C in RPMI 1640 medium, after which individual cultures were examined by light microscopy. Photographs were taken using a x40 objective.

 
Pharmacological sensitivity of the PTG response

ATP-induced IL-1{beta} posttranslational processing is disrupted by a number of pharmacological agents (17, 42, 43, 44, 45, 46, 47). To determine whether the PTG-1-mediated response was similarly affected, monocytes were exposed to the peptide in the presence of several pharmacological agents. For example, the caspase-1 inhibitor YVAD-cmk blocked production of 17-kDa IL-1{beta} by ATP-treated/LPS-activated human monocytes (Fig. 4Go). In the presence of this agent, ATP-treated cells continued to release proIL-1{beta}, but quantities of the 17-kDa species were greatly reduced relative to that generated in the absence of the caspase inhibitor. Similarly, the caspase-1 inhibitor blocked PTG-1-mediated production of 17-kDa IL-1{beta}; the YVAD-cmk-treated cells continued to release 31-kDa proIL-1{beta} (Fig. 4Go).



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  		FIGURE 4. PTG-1 mediated release of mature IL-1{beta} is blocked by a caspase inhibitor. LPS-activated, [35S]methionine-labeled human monocytes were treated with 2 mM ATP or 10 µg/ml PTG-1, in the absence and presence of 50 µM YVAD-cmk, for 3 h. Media fractions subsequently were harvested and IL-1{beta} was recovered by immunoprecipitation; resulting immunoprecipitates were analyzed by SDS-PAGE and autoradiography. Migration positions of the 17-kDa mature and 31-kDa proforms of IL-1{beta} are indicated. Each condition was performed in duplicate.

 
Nonselective inhibitors of anion transport block ATP-induced IL-1{beta} posttranslational processing (44). Thus, ethacrynic acid inhibited ATP-induced IL-1{beta} release from LPS-activated human monocytes (Fig. 5GoA). Likewise, ethacrynic acid inhibited PTG-1-mediated IL-1 production (Fig. 5GoA), and the inhibitor demonstrated a similar dose response relationship against the ATP- and PTG-1-mediated responses. ATP mediates IL-1{beta} posttranslational processing by activating the P2X7 receptor, a ligand-gated ion channel (36). KN-62 is an antagonist of the P2X7 receptor (46), and this agent blocked ATP-induced IL-1 production in a dose-dependent manner (Fig. 5GoB). In contrast, KN-62 did not inhibit PTG-1 mediated IL-1 production (Fig. 5GoB).



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  		FIGURE 5. ATP- and PTG-1-mediated responses are sensitive to pharmacological intervention. LPS-activated human monocytes were treated with 10 µg/ml PTG-1 or 2 mM ATP in the absence or presence of the indicated concentration of ethacrynic acid (A) or KN-62 (B). After a 3-h treatment, media were harvested and assessed for IL-1{beta} content by ELISA. The amount of cytokine produced, expressed as a percentage relative to non-pharmacophore-treated cultures, is indicated as a function of test agent concentration.

 
A novel class of IL-1 posttranslational processing inhibitors exemplified by CP-424,174 recently was identified (47). These agents block cytokine processing induced by ATP, cytolytic T cells, and hypotonic stress, suggesting that they disrupt a step in the overall cellular response pathway downstream of the initiating stimulus. Moreover, these cytokine release inhibitory drugs (CRIDs) arrest the cytokine-producing cells in a state that preserves membrane latency and maintains proIL-1{beta} intracellularly (47); the CRID molecular target remains to be identified. [35S]methionine-labeled/LPS-activated monocytes were treated with PTG-1 in the absence or presence of CP-424,174, after which cytokine released to the medium was recovered by immunoprecipitation and analyzed by SDS-PAGE/autoradiography. PTG-1 again initiated generation and release of 17-kDa IL-1{beta} (Fig. 6Go). Monocytes treated with the antimicrobial peptide also released smaller amounts of proIL-1{beta} (Fig. 6Go). Addition of CP-424,174 to the cultures caused a dose-dependent inhibition in the production of 17-kDa IL-1{beta} but did not affect externalization of the procytokine species (Fig. 6Go); formation of the 17-kDa cytokines species was inhibited with an IC50 value of 100 nM (Fig. 6GoB).



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  		FIGURE 6. The CRID CP-424,174 blocks PTG-1-mediated processing. LPS-activated, [35S]methionine-labeled human monocytes were treated with 10 µg/ml PTG-1, in the absence and presence of the indicated concentration of CP-424,174 for 3 h. Control cultures received neither PTG-1 nor CP-424,174. Media fractions subsequently were harvested and IL-1{beta} was recovered by immunoprecipitation. A, The resulting immunoprecipitates were analyzed by SDS-PAGE and autoradiography. Migration positions of the 17-kDa mature and 31-kDa proforms of IL-1{beta} are indicated. Each condition was performed in duplicate. B, The amount of radioactivity associated with the 17-kDa species was determined by phosphor imager analysis and is indicated, as a percentage of the control, as a function of CP-424,174 concentration.

 
Medium constituents influence PTG-1-mediated IL-1 processing

Activity of defensin-like peptides is known to be affected by the composition of the medium in which cells are exposed to these agents. Likewise, the ability of PTG-1 to mediate IL-1 posttranslational processing was dependent on media composition. In RPMI 1640 medium, PTG-1 treatment of [35S]methionine-labeled/LPS-activated monocytes led to production of large quantities of extracellular IL-1{beta} (Fig. 7Go). However, the same concentration of PTG-1 was unable to promote mature IL-1{beta} production from monocytes maintained in MEM (Fig. 7Go). When the monocytes were treated with PTG-1 in an isotonic NaCl-based medium, robust production of 17-kDa IL-1{beta} was observed in the absence of divalent cations (Ca2+ and Mg2+), but the addition of these cations diminished yield of the mature cytokine product (Fig. 7Go). In contrast, in an isotonic medium containing 50 mM KCl, no 17-kDa IL-1{beta} was generated in the presence of PTG-1; under these conditions the peptide did induce release of proIL-1{beta}, particularly in the absence of divalent cations (Fig. 7Go).



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  		FIGURE 7. Media composition affects PTG-1-mediated IL-1 posttranslational processing. LPS-activated, [35S]methionine-labeled human monocytes were treated with 10 µg/ml PTG-1 in the indicated medium for 3 h. IL-1{beta} released extracellularly was recovered by immunoprecipitation and the resulting immunoprecipitates were analyzed by SDS-PAGE and autoradiography. Migration positions of the 17-kDa mature and 31-kDa proforms of IL-1{beta} are indicated. Each condition was performed in duplicate. Where indicated, 50 mM KCl was substituted for 50 mM NaCl and/or CaCl2 and MgCl2 were removed (w/o).

 
Search for other antimicrobial peptides that promote IL-1 posttranslational processing

A large number of antimicrobial peptides from a host of different organisms have been identified (37, 38, 39, 48, 49). To determine whether other members of this superfamily can initiate IL-1 posttranslational processing, [35S]methionine-labeled/LPS-activated human monocytes were treated with several commercially available peptides. Magainin-1 is an antimicrobial peptide isolated from frog skin (50). Under the used experimental conditions, magainin-1 (100 µg/ml) elicited some mature IL-1{beta} production relative to that released from cells in the absence of an effector (Fig. 8Go). However, relative to PTG-1-treated cells, the amount of the 17-kDa cytokine generated was much reduced (Fig. 8Go). Likewise, the human {alpha}-defensin HNP-1 caused formation and release of 17-kDa IL-1{beta}; increasing the peptide concentration from 10 to 100 µg/ml enhanced production of the mature cytokine product, but the level generated at the highest HNP-1 concentration was again reduced relative to that generated by PTG-1 (Fig. 8Go).



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  		FIGURE 8. Characterization of other antimicrobial peptides as initiators of IL-1 posttranslational processing. LPS-activated, [35S]methionine-labeled human monocytes were treated with the indicated effector in RPMI 1640 medium for 3 h; controls were maintained in the absence of an effector. IL-1{beta} released extracellularly was recovered by immunoprecipitation and the resulting immunoprecipitates were analyzed by SDS-PAGE and autoradiography. Migration positions of the 17-kDa mature and 31-kDa proforms of IL-1{beta} are indicated. Each condition was performed in duplicate.

 
Several other antimicrobial peptides were tested in the IL-1{beta} posttranslational processing assay and found to be totally inactive, including the human {beta}-defensins 1 and 2 and magainin-2 (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Agents that promote human monocyte IL-1{beta} posttranslational processing in vitro include ATP, the potassium ionophore nigericin, and hypotonic stress (24, 25, 27, 28, 29, 30, 31). These diverse agents share an ability to mobilize intracellular K+, and their activity as initiators of IL-1 posttranslational processing is negated by raising the concentration of K+ within the medium (27, 28, 31). Remarkably, Berlin and Wood (51) reported that elevated media K+ concentrations inhibited release of endogenous pyrogen from rabbit leukocytes long before this factor had been purified and identified as IL-1. Therefore, K+ efflux appears to be a necessary element of the IL-1 posttranslational processing mechanism. A recent report noted that normal intracellular K+ ion concentrations inhibit cytochrome c-dependent formation of the apoptosome, a protein assembly required for activation of effector procaspases (52). Although the mechanism of procaspase-1 activation is not understood, perhaps intracellular K+ levels also regulate activation of this protease and/or its cellular distribution (21). Given this ionic requirement, we initiated a search for other physiologically relevant molecules that had a potential to elicit K+ efflux from LPS-activated monocytes and, in turn, to initiate IL-1{beta} posttranslational processing. Results presented in this report demonstrate that antimicrobial peptides, particularly PTGs, can promote IL-1{beta} posttranslational processing. Identification of this novel activity suggests that antimicrobial peptides may possess biological functions extending beyond the innate immune response.

The superfamily of antimicrobial peptides encompasses a wide variety of structures and sequences; the reason for this complex diversity remains unclear. Peptides of this sort are considered to be integral components of an animal’s innate immune response because they can directly kill bacteria. Nonetheless, a number of additional activities have been reported for some members of the family, suggesting functions that extend beyond the prototypical antibacterial role (53, 54). For example, PR-39 is a potent antimicrobial peptide but also can inhibit NADPH oxidase activity associated with neutrophils and elicit a chemotactic response (55). Likewise, cytolytic T cells produce granulysin, a peptide that demonstrates broad spectrum antimicrobial activity, but also induces apoptosis in mammalian cells (56). Diversity also is observed within the mechanisms by which the antimicrobial peptides kill their bacterial targets. Peptides such as the PTGs form channels within the bacterial cell membrane that lead to disruption of normal ionic homeostasis (57, 58). Other peptides, for example PR-39, appear to kill bacteria via non-channel-mediated processes (59).

Despite a wide and diverse membership, the antimicrobial peptide superfamily can be categorized based on shared structural elements. For example, all PTGs are derived by proteolysis of cathelicidin precursor molecules (37, 38, 39). These propolypeptides consist of a conserved amino terminus containing ~100 amino acids attached to variable carboxyl-terminal domains. The latter variable regions correspond to the antimicrobial peptides, and proteolysis is required to release these segments. The liberated peptides subtype-dependently form {alpha}-helical, {beta}-sheet, or nonordered conformations. Five distinct porcine PTGs have been identified, and each is composed of a {beta}-sheet structure with two intrachain disulfide bonds. Proline- and arginine-rich antimicrobial peptides such as porcine PR-39 and PR-26 also are derived from cleavage of a cathelicidin precursor; these peptides, however, lack cysteine residues and are thus disulfide bond-free (37). PR-26 and PR-39 also lack {alpha}-helical and/or {beta}-sheet structural features. Defensins are derived not from cathelicidins but from precursor polypeptides (60). Following cleavage, the resulting mature defensins are comprised of amphiphilic {beta}-sheet structures containing three intrachain disulfide bonds (49). The defensin family is further subdivided into {alpha} and {beta} members, based in part on disulfide bond configuration (60).

Antimicrobial peptides appear to bind to their bacterial targets via non-receptor-mediated processes; this initial binding may result from an interaction of the peptides with anionic phospholipids within the bacterial cell membrane (39, 61). In some cases, the antimicrobial peptide may function to kill only a limited target population; for example, PR-39 kills Gram- organisms but not Gram+ species (40). In contrast, PTG-like peptides target a broad range of bacteria (38, 39). Despite their efficiency as antimicrobial agents, these innate mediators of immunity are not overtly toxic to mammalian cells (62). Nonetheless, mammalian cell toxicities have been observed. For example, human defensins HNP-1, -2, and -3 are reported to have toxic effects on murine tumor cells (63) and human lymphocytes (64). Likewise, members of the cathelicidin family, including PTGs, are reported to kill some types of mammalian cells (38, 65); the mechanism by which these agents achieve cell-type selective killing remains to be determined.

The effect of PTGs on LPS-activated human monocytes is dramatic. After just 10 min of exposure of monocyte cultures to PTG-1, the cells had responded, as evidenced by the dramatic change in their morphology. This change, like that induced by nigericin or ATP, is characterized by clearing of cytoplasmic contents and swelling of the plasma membrane. These features are not typical of an apoptotic process and are more consistent with oncosis, an osmotically driven process (66). In addition to inducing similar changes in morphology, ATP- and PTG-mediated IL-1 posttranslational processing display similar sensitivity to pharmacological intervention. Thus, PTG-1- and ATP-treated LPS-activated human monocytes released 17-kDa IL-1{beta} extracellularly, and formation of the mature cytokine was blocked by the caspase-1 inhibitor YVAD-cmk. This inhibition implies that PTG-1 and ATP activate caspase-1 which, in turn, cleaves proIL-1{beta}. In contrast, YVAD-cmk did not prevent release of proIL-1{beta} in response to either ATP or PTG-1 challenge, suggesting that YVAD-cmk-sensitive caspase activity is not required for cell death; similar conclusions have been reported previously (67). Moreover, PTG-1 mediated release of 17-kDa IL-1{beta} was prevented by increasing the concentration of extracellular K+; as with an ATP stimulus, therefore, an efflux of K+ appears to be a necessary element of the PTG-1-mediated response. Whether PTG-1 actually forms channels within monocytes through which K+ is effluxed remains to be established. The PTG-1-mediated response also was blocked by a nonselective inhibitor of anion transport, ethacrynic acid, and a selective inhibitor of IL-1 posttranslational processing, CP-424,174.

Given the striking similarities between the ATP- and PTG-1-mediated responses, we considered the possibility that PTGs activated IL-1 posttranslational processing indirectly by facilitating release of ATP from monocytes which then engaged the P2X7 receptor. If this type of mechanism occurred, then the PTG-mediated response would be sensitive to an antagonist of this receptor, KN-62 (46). Although KN-62 effectively inhibited ATP-induced IL-1 posttranslational processing, this agent had no effect on PTG-1-mediated events. Thus, PTG-1 does not act through the P2X7 receptor.

The ability of PTG-1 to mediate human monocyte IL-1{beta} posttranslational processing was dose dependent and was influenced by the medium in which the cells were exposed to the antimicrobial peptide. Maximal production of 17-kDa IL-1{beta} was achieved at PTG concentrations near 10 µg/ml. Similar concentrations were required to kill E. coli, and studies have suggested that concentrations of this magnitude can be achieved in vivo (68). Monocytes treated with 100 µg/ml of the PTGs continued to release IL-1{beta}, but the efficiency at which the released cytokine was processed to the 17-kDa species was reduced. This decrease in processing is assumed to account for the bell-shaped dose response curve observed by ELISA (the kit is reported to prefer the mature species). High concentrations of the peptides may elicit a massive, rapid ionic flux that bypasses a required sequence of ionic changes leading to caspase-1 activation; detergents such as saponin that disrupt the plasma membrane also promote release of non-caspase-processed proIL-1{beta} (25). The media dependence of the PTG-mediated response was striking. Thus, in RPMI 1640 medium PTG-1 was a robust initiator of cytokine processing, but in MEM this same peptide was ineffective. The reason for this media dependence is not clear. PTG-1 effectively mediated IL-1{beta} posttranslational processing when monocytes were treated in a basal isotonic medium, although the addition of divalent cations dramatically reduced activity. Thus, special additives present in cell culture medium are not required for the PTG-1-mediated response. Rather, MEM may contain elements that inactivate PTG-1. For example, the relative abundance of divalent cations (Ca2+ and Mg2+) is 3-fold higher in MEM (2.62 mM) than in RPMI (0.83 mM). Alternatively, intrachain disulfide bonds necessary for PTG-1-mediated antimicrobial activity (69) are susceptible to reduction, and MEM possesses reduced cysteine and ascorbic acid, not present in RPMI 1640, that may facilitate reduction of these critical linkages. Additional work will be required to understand how environmental factors affect the antimicrobial peptide-initiated response.

To date, we have characterized only a few of the known antimicrobial peptides as initiators of IL-1 posttranslational processing. The two PTGs tested were very efficient inducers of cytokine posttranslational processing. The {alpha}-defensin HNP-1 demonstrated some activity, as did magainin-1. In contrast, the proline- and arginine-rich peptides PR-26 and PR-39 and the {beta}-defensins 1 and 2 were totally inactive. Therefore, the ability to promote IL-1{beta} posttranslational processing is not shared by all antimicrobial peptides. In view of the profound effects that media components had on the activity of PTG-1, we cannot rule out the possibility that peptides observed to be inactive may become active when tested under a different set of experimental conditions. As noted above, PTGs form ion channels in target cell membranes (39, 57, 58), and this type of ionophoretic-like activity may be responsible for initiating IL-1 posttranslational processing. Consistent with this hypothesis, PR-26 and PR-39 are not thought to form ion channels, and they failed to initiate the cytokine response. In contrast, defensins are capable of forming ion-conducting channels, yet they failed to activate processing (49, 58). Defensin-induced channel activity, however, is most optimal under low ionic conditions (64); PTGs, in contrast, form channels under normal physiological saline concentrations (69). Further work is needed to clarify the mechanism by which PTGs selectively engage human monocyte IL-1 posttranslational processing.

The ability of some antimicrobial peptides to serve as initiators of IL-1{beta} posttranslational processing suggests an entirely new function for these mediators of innate immunity. Within the context of an inflammatory lesion, monocytes and/or macrophages may become primed and begin to synthesize proIL-1{beta}. In the absence of an appropriate initiator of IL-1 posttranslational processing, however, these cytokine-producing cells will not release their cytokine product. Should the activated monocyte/macrophage move into an environment where an appropriate antimicrobial peptide has been released, in contrast, then IL-1{beta} posttranslational processing would be engaged rapidly. Many cell types produce antimicrobial peptides, including epithelial cells at sites of inflammation (70), and the selective release of these effector peptides within a local environment may provide a mechanism whereby a potent mediator of inflammation like IL-1 can be externalized on a need-only, tightly regulated basis. Initiation of IL-1 posttranslational processing, therefore, may represent yet another mechanism by which antimicrobial peptides contribute to the acquired immune response.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Christopher A. Gabel, Department of Antibacterials, Immunology, and Inflammation, PGRD, Pfizer, Inc., Groton, CT 06340. E-mail address: christopher_a_gabel{at}groton.pfizer.com Back

2 Abbreviations used in this paper: ER, endoplasmic reticulum; LDH, lactate dehydrogenase; PTG, protegrin; CRID, cytokine release inhibitory drug. Back

Received for publication November 13, 2001. Accepted for publication January 17, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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