Species-specific NLRP3 regulation and its role in CNS autoinflammatory diseases.

The NLRP3 inflammasome is essential for caspase-1 activation and the release of interleukin (IL)-1ß, IL-18, and gasdermin-D in myeloid cells. However, research on species-specific NLRP3's physiological impact is limited. We engineer mice with the human NLRP3 gene, driven by either the human or mouse promoter, via syntenic replacement at the mouse Nlrp3 locus. Both promoters facilitate hNLRP3 expression in myeloid cells, but the mouse promoter responds more robustly to LPS. Investigating the disease impact of differential NLRP3 regulation, we introduce the D305N gain-of-function mutation into both humanized lines. Chronic inflammation is evident with both promoters; however, CNS outcomes vary significantly. Despite poor response to LPS, the human promoter results in D305N-associated aseptic meningitis, mirroring human pathology. The mouse promoter, although leading to increased CNS expression post-LPS, does not induce meningitis in D305N mutants. Therefore, human-like NLRP3 expression may be crucial for accurate modeling of its role in disease pathogenesis.

Target Cell Activation of a Structurally Novel NOD-Like Receptor Pyrin Domain-Containing Protein 3 Inhibitor NT-0796 Enhances Potency.

The NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome is a central regulator of innate immunity, essential for processing and release of interleukin-1ß and pyroptotic cell death. As endogenous NLRP3 activating triggers are hallmarks of many human chronic inflammatory diseases, inhibition of NLRP3 has emerged as a therapeutic target. Here we identify NDT-19795 as a novel carboxylic acid-containing NLRP3 activation inhibitor in both human and mouse monocytes and macrophages. Remarkably, conversion of the carboxylate to an isopropyl-ester (NT-0796) greatly enhances NLRP3 inhibitory potency in human monocytes. This increase is attributed to the ester-containing pharmacophore being more cell-penetrant than the acid species and, once internalized, the ester being metabolized to NDT-19795 by carboxylesterase-1 (CES-1). Mouse macrophages do not express CES-1, and NT-0796 is ineffective in these cells. Mice also contain plasma esterase (Ces1c) activity which is absent in humans. To create a more human-like model, we generated a mouse line in which the genome was modified, removing Ces1c and replacing this segment of DNA with the human CES-1 gene driven by a mononuclear phagocyte-specific promoter. We show human CES-1 presence in monocytes/macrophages increases the ability of NT-0796 to inhibit NLRP3 activation both in vitro and in vivo. As NLRP3 is widely expressed by monocytes/macrophages, the co-existence of CES-1 in these same cells affords a unique opportunity to direct ester-containing NLRP3 inhibitors precisely to target cells of interest. Profiling NT-0796 in mice humanized with respect to CES-1 biology enables critical modeling of the pharmacokinetics and pharmacodynamics of this novel therapeutic candidate. SIGNIFICANCE STATEMENT: Inhibition of NLRP3 represents a desirable therapeutic strategy for the treatment of multiple human disorders. In this study pharmacological properties of a structurally-novel, ester-containing NLRP3 inhibitor NT-0796 are characterized. To study pharmacodynamics of NT-0796 in vivo, a mouse line was engineered possessing more human-like traits with respect to carboxylesterase biology. In the context of these hCES-1 mice, NT-0796 serves as a more effective inhibitor of NLRP3 activation than the corresponding acid, highlighting the full translational potential of the ester strategy.

Discovery of Clinical Candidate NT-0796, a Brain-Penetrant and Highly Potent NLRP3 Inflammasome Inhibitor for Neuroinflammatory Disorders.

The NLRP3 inflammasome is a component of the innate immune system involved in the production of proinflammatory cytokines. Neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, multiple sclerosis, and amyotrophic lateral sclerosis, have been shown to have a component driven by NLRP3 inflammasome activation. Diseases such as these with large unmet medical needs have resulted in an interest in inhibiting the NLRP3 inflammasome as a potential pharmacological treatment, but to date, no marketed drugs specifically targeting NLRP3 have been approved. Furthermore, the requirement for CNS-penetrant molecules adds additional complexity to the search for NLRP3 inflammasome inhibitors suitable for clinical investigation of neuroinflammatory disorders. We designed a series of ester-substituted carbamate compounds as selective NLRP3 inflammasome inhibitors, leading to NT-0796, an isopropyl ester that undergoes intracellular conversion to NDT-19795, the carboxylic acid active species. NT-0796 was shown to be a potent and selective NLRP3 inflammasome inhibitor with demonstrated in vivo brain penetration.

Discovery and Optimization of Triazolopyrimidinone Derivatives as Selective NLRP3 Inflammasome Inhibitors.

The NLRP3 inflammasome is a multiprotein complex that facilitates activation and release of the proinflammatory cytokines interleukin-1ß (IL-1ß) and IL-18 in response to infection or endogenous stimuli. It can be inappropriately activated by a range of danger signals resulting in chronic, low-grade inflammation underlying a multitude of diseases, such as Alzheimer's disease, Parkinson's disease, osteoarthritis, and gout. The discovery of potent and specific NLRP3 inhibitors could reduce the burden of several common morbidities. In this study, we identified a weakly potent triazolopyrimidone hit (1) following an in silico modeling exercise. This was optimized to furnish potent and selective small molecule NLRP3 inflammasome inhibitors. Compounds such as NDT-30805 could be useful tool molecules for a scaffold-hopping or pharmacophore generation project or used as leads toward the development of clinical candidates.

Discovery of a series of ester-substituted NLRP3 inflammasome inhibitors.

The NLRP3 inflammasome is a component of the innate immune system involved in the production of proinflammatory cytokines. Aberrant activation by a wide range of exogenous and endogenous signals can lead to chronic, low-grade inflammation. It has attracted a great deal of interest as a drug target due to the association with diseases of large unmet medical need such as Alzheimer's disease, Parkinson's disease, arthritis, and cancer. To date, no drugs specifically targeting inhibition of the NLRP3 inflammasome have been approved. In this work, we used the known NLRP3 inflammasome inhibitor CP-456,773 (aka CRID3 or MCC 950) as our starting point and undertook a Structure-Activity Relationship (SAR) analysis and subsequent scaffold-hopping exercise. This resulted in the rational design of a series of novel ester-substituted urea compounds that are highly potent and selective NLRP3 inflammasome inhibitors, as exemplified by compounds 44 and 45. It is hypothesized that the ester moiety acts as a highly permeable delivery vehicle and is subsequently hydrolyzed to the carboxylic acid active species by carboxylesterase enzymes. These molecules are greatly differentiated from the state-of-the-art and offer potential in the treatment of NLRP3-driven diseases, particularly where tissue penetration is required.

Novel protein therapeutic joint retention strategy based on collagen-binding Avimers.

Designing drugs to treat diseases associated with articular joints, particularly those targeting chondrocytes, is challenging due to unique local environmental constraints including the avascular nature of cartilage, the absence of a closed joint compartment, and a highly cross-linked extracellular matrix. In an effort to address these challenges, we developed a novel strategy to prolong residence time of intra-articularly administered protein therapeutics. Avimer domains are naturally found in membrane polypeptides and mediate diverse protein-protein interactions. Screening of a phage Avimer domain library led to identification of several low affinity type II collagen-binding Avimers. Following several rounds of mutagenesis and reselection, these initial hits were transformed to high affinity, selective type II collagen-binding Avimers. One such Avimer (M26) persisted in rat knees for at least 1 month following intra-articular administration. Fusion of this Avimer to a candidate therapeutic payload, IL-1Ra, yielded a protein construct which simultaneously bound to type II collagen and to IL-1 receptor. In vitro, IL-1Ra_M26 bound selectively to cartilage explants and remained associated even after extensive washing. Binding appeared to occur preferentially to pericellular regions surrounding chondrocytes. An acute intra-articular IL-1-induced IL-6 challenge rat model was employed to assess in vivo pharmacodynamics. Whereas both IL-1Ra_M26 and native IL-1Ra inhibited IL-6 output when co-administered with the IL-1 challenge, only IL-1Ra_M26 inhibited when administered 1 week prior to IL-1 challenge. Collagen-binding Avimers thus represent a promising strategy for enhancing cartilage residence time of protein therapeutics. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1238-1247, 2018.

Interleukin-36 (IL-36) ligands require processing for full agonist (IL-36α, IL-36ß, and IL-36γ) or antagonist (IL-36Ra) activity.

IL-36α, IL-36ß, and IL-36γ (formerly IL-1F6, IL-1F8, and IL-1F9) are IL-1 family members that signal through the IL-1 receptor family members IL-1Rrp2 (IL-1RL2) and IL-1RAcP. IL-36Ra (formerly IL-1F5) has been reported to antagonize IL-36γ. However, our previous attempts to demonstrate IL-36Ra antagonism were unsuccessful. Here, we demonstrate that IL-36Ra antagonist activity is dependent upon removal of its N-terminal methionine. IL-36Ra starting at Val-2 is fully active and capable of inhibiting not only IL-36γ but also IL-36α and IL-36ß. Val-2 of IL-36Ra lies 9 amino acids N-terminal to an A-X-Asp motif conserved in all IL-1 family members. In further experiments, we show that truncation of IL-36α, IL-36ß, and IL-36γ to this same point increased their specific activity by ∼10(3)-10(4)-fold (from EC(50) 1 µg/ml to EC(50) 1 ng/ml). Inhibition of truncated IL-36ß activity required ∼10(2)-10(3)-fold excess IL-36Ra, similar to the ratio required for IL-1Ra to inhibit IL-1ß. Chimeric receptor experiments demonstrated that the extracellular (but not cytoplasmic) domain of IL-1Rrp2 or IL-1R1 is required for inhibition by their respective natural antagonists. IL-36Ra bound to IL-1Rrp2, and pretreatment of IL-1Rrp2-expressing cells with IL-36Ra prevented IL-36ß-mediated co-immunoprecipitation of IL-1Rrp2 with IL-1RAcP. Taken together, these results suggest that the mechanism of IL-36Ra antagonism is analogous to that of IL-1Ra, such that IL-36Ra binds to IL-1Rrp2 and prevents IL-1RAcP recruitment and the formation of a functional signaling complex. In addition, truncation of IL-36α, IL-36ß, and IL-36γ dramatically enhances their activity, suggesting that post-translational processing is required for full activity.

Discovery, synthesis and SAR of azinyl- and azolylbenzamides antagonists of the P2X7 receptor.

The discovery, of a series of 2-Cl-5-heteroaryl-benzamide antagonists of the P2X(7) receptor via parallel medicinal chemistry is described. Initial analogs suffered from poor metabolic stability and low Vd(ss). Multi parametric optimization led to identification of pyrazole 39 as a viable lead with excellent potency and oral bioavailability. Further attempts to improve the low Vd(ss) of 39 via introduction of amines led to analogs 40 and 41 which maintained the favorable pharmacology profile of 39 and improved Vd(ss) after iv dosing. But these analogs suffered from poor oral absorption, probably driven by poor permeability.

Optimization of the physicochemical and pharmacokinetic attributes in a 6-azauracil series of P2X7 receptor antagonists leading to the discovery of the clinical candidate CE-224,535.

High throughput screening (HTS) of our compound file provided an attractive lead compound with modest P2X(7) receptor antagonist potency and high selectivity against a panel of receptors and channels, but also with high human plasma protein binding and a predicted short half-life in humans. Multi-parameter optimization was used to address the potency, physicochemical and pharmacokinetic properties which led to potent P2X(7)R antagonists with good disposition properties. Compound 33 (CE-224,535) was advanced to clinical studies for the treatment of rheumatoid arthritis.

Externalization of the leaderless cytokine IL-1F6 occurs in response to lipopolysaccharide/ATP activation of transduced bone marrow macrophages.

An interesting trait shared by many members of the IL-1 cytokine family is the absence of a signal sequence that can direct the newly synthesized polypeptides to the endoplasmic reticulum. As a result, these cytokines accumulate intracellularly. Recent studies investigating IL-1beta export established that its release is facilitated via activation of an intracellular multiprotein complex termed the inflammasome. The purpose of the current study was to explore the mechanism by which murine IL-1F6 is released from bone marrow-derived macrophages (BMDMs) and to compare this mechanism to that used by IL-1beta. BMDMs were engineered to overexpress IL-1F6 by retroviral transduction; cells overexpressing GFP also were generated to provide a noncytokine comparator. The transduced cells constitutively expressed IL-1F6 and GFP, but they did not constitutively release these polypeptides to the medium. Enhanced release of IL-1F6 was achieved by treating with LPS followed by ATP-induced activation of the P2X(7) receptor; GFP also was released under these conditions. No obvious proteolytic cleavage of IL-1F6 was noted following P2X(7) receptor-induced release. Stimulus-induced release of IL-1F6 and GFP demonstrated comparable susceptibility to pharmacological modulation. Therefore, transduced IL-1F6 is released in parallel with endogenous mature IL-1beta from LPS/ATP-treated BMDMs, but this externalization process is not selective for cytokines as a noncytokine (GFP) shows similar behavior. These findings suggest that IL-1F6 can be externalized via a stimulus-coupled mechanism comparable to that used by IL-1beta, and they provide additional insight into the complex cellular processes controlling posttranslational processing of the IL-1 cytokine family.

P2 purinergic receptor modulation of cytokine production.

Cytokines serve important functions in controlling host immunity. Cells involved in the synthesis of these polypeptide mediators have evolved highly regulated processes to ensure that production is carefully balanced. In inflammatory and immune disorders, however, mis-regulation of the production and/or activity of cytokines is recognized as a major contributor to the disease process, and therapeutics that target individual cytokines are providing very effective treatment options in the clinic. Leukocytes are the principle producers of a number of key cytokines, and these cells also express numerous members of the purinergic P2 receptor family. Studies in several cellular systems have provided evidence that P2 receptor modulation can affect cytokine production, and mechanistic features of this regulation have emerged. This review highlights three separate examples corresponding to (1) P2Y6 receptor mediated impact on interleukin (IL)-8 production, (2) P2Y11 receptor-mediated affects on IL-12/23 output, and (3) P2X7 receptor mediated IL-1ß posttranslational processing. These examples demonstrate important roles of purinergic receptors in the modulation of cytokine production. Extension of these cellular observations to in vivo situations may lead to new therapeutic strategies for treating cytokine-mediated diseases.

Structure-activity relationships of triazolopyridine oxazole p38 inhibitors: identification of candidates for clinical development.

The synthesis, structure-activity relationship, in vivo activity, and metabolic profile for a series of triazolopyridine-oxazole based p38 inhibitors are described. The deficiencies of the lead structure in the series, CP-808844, were overcome by changes to the C4 aryl group and the triazole side-chain culminating in the identification of several potential clinical candidates.

Theoretical and experimental design of atypical kinase inhibitors: application to p38 MAP kinase.

Mimics of the benzimidazolone nucleus found in inhibitors of p38 kinase are proposed, and their theoretical potential as bioisosteres is described. A set of calculated descriptors relevant to the anticipated binding interaction for the fragments 1-methyl-1H-benzotriazole 5, 3-methyl-benzo[d]isoxazole 3, and 3-methyl-[1,2,4]triazolo[4,3-a]pyridine 4, pyridine 1, and 1,3-dimethyl-1,3-dihydro-benzoimidazol-2-one 2 are reported. The design considerations and synthesis of p38 inhibitors based on these H-bond acceptor fragments is detailed. Comparative evaluation of the pyridine-, benzimidazolone-, benzotriazole-, and triazolopyridine-based inhibitors shows the triazoles 20 and 25 to be significantly more potent experimentally than the benzimidazolone after which they were modeled. An X-ray crystal structure of 25 bound to the active site shows that the triazole group serves as the H-bond acceptor but unexpectedly as a dual acceptor, inducing movement of the crossover connection of p38alpha. The computed descriptors for the hydrophobic and pi-pi interaction capacities were the most useful in ranking potency.

Role of p38 MAPK in UVB-induced inflammatory responses in the skin of SKH-1 hairless mice.

The p38 mitogen-activated protein kinase (MAPK) signaling pathway is activated by numerous inflammatory mediators and environmental stresses. We assessed the effects of ultraviolet B (UVB) on the p38 MAPK pathway and determined whether cyclooxygenase (COX)-2 expression is downstream of this kinase in the skin of UVB-irradiated SKH-1 mice. SKH-1 mice were irradiated with a single dose of UVB (360 mJ per cm2), and activation of the epidermal p38 MAPK pathway was assessed. UVB-induced phosphorylation of p38 MAPK occurred in a time-dependent manner. Phosphorylation of MAPK-activated protein kinase-2 (MAPKAPK-2) also was detected and correlated with an increase in its kinase activity. Phosphorylation of heat shock protein 27 (HSP27), a substrate for MAPKAPK-2, also was detected post-irradiation. Oral administration of the p38 inhibitor, SB242235, prior to UVB irradiation, blocked activation of the p38 MAPK cascade, and abolished MAPKAPK-2 kinase activity and phosphorylation of HSP27. Moreover, SB242235 inhibited expression of the pro-inflammatory cytokines interleukin (IL)-6 and KC (murine IL-8) and COX-2. Our data demonstrate that UVB irradiation of murine skin activates epidermal p38 MAPK signaling and induces a local pro-inflammatory response. Blockade of the p38 MAPK pathway may offer an effective approach to reducing or preventing skin damage resulting from acute solar radiation.

Correlation of polymorphic variation in the promoter region of the interleukin-1 beta gene with secretion of interleukin-1 beta protein.

OBJECTIVE: Significant variation in interleukin-1 beta (IL-1 beta) protein secretion between subjects has been observed when using a lipopolysaccharide (LPS)/ATP-mediated ex vivo blood stimulation assay. To explore the potential relationships between genetic polymorphisms in the IL1B cytokine gene and cellular responses to inflammatory stimuli such as LPS, we investigated the hypothesis that polymorphisms within the promoter and exon 5 of the IL1B gene contribute to the observed differences in IL-1 beta protein secretion. METHODS: The IL1B gene polymorphisms C-511T, T-31C, and C3954T were tested for association with LPS-induced secretion of IL-1 beta protein as measured by an ex vivo blood stimulation assay. Samples from 2 independent study populations (n = 31 and n = 25) were available for use in the ex vivo assay after consent was obtained to analyze the DNA. RESULTS: A specific haplotype, composed of the T allele at -511 and the C allele at -31, was significantly associated with a 2-3-fold increase in LPS-induced IL-1 beta protein secretion. This association was observed in both of the independent study populations (P = 0.0084 and P = 0.0017). CONCLUSION: These data suggest that polymorphisms within the promoter region of the IL1B gene contribute to observed differences in LPS-induced IL-1 beta protein secretion.

Benzimidazolone p38 inhibitors.

The synthesis and in vitro p38 alpha activity of a novel series of benzimidazolone inhibitors is described. The p38 alpha SAR is consistent with a mode of binding wherein the benzimidazolone carbonyl serves as the H-bond acceptor to Met109 of p38 alpha in a manner analogous to the pyridine nitrogen of prototypical pyridylimidazole p38 inhibitors. Potent p38 alpha activity comparable to that of several previously reported p38 inhibitors is observed for this novel chemotype.

Deletion of the P2X7 nucleotide receptor reveals its regulatory roles in bone formation and resorption.

The P2X7 nucleotide receptor is an ATP-gated ion channel expressed widely in cells of hematopoietic origin. Our purpose was to explore the involvement of the P2X7 receptor in bone development and remodeling by characterizing the phenotype of mice genetically modified to disrupt the P2X7 receptor [knockout (KO)]. Femoral length did not differ between KO and wild-type (WT) littermates at 2 or 9 months of age, indicating that the P2X7 receptor does not regulate longitudinal bone growth. However, KO mice displayed significant reduction in total and cortical bone content and periosteal circumference in femurs, and reduced periosteal bone formation and increased trabecular bone resorption in tibias. Patch clamp recording confirmed expression of functional P2X7 receptors in osteoclasts from WT but not KO mice. Osteoclasts were present in vivo and formed in cultures of bone marrow from KO mice, indicating that this receptor is not essential for fusion of osteoclast precursors. Functional P2X7 receptors were also found in osteoblasts from WT but not KO mice, suggesting a direct role in bone formation. P2X7 receptor KO mice demonstrate a unique skeletal phenotype that involves deficient periosteal bone formation together with excessive trabecular bone resorption. Thus, the P2X7 receptor represents a novel therapeutic target for the management of skeletal disorders such as osteoporosis.

Glutathione s-transferase omega 1-1 is a target of cytokine release inhibitory drugs and may be responsible for their effect on interleukin-1beta posttranslational processing.

Stimulus-induced posttranslational processing of human monocyte interleukin-1beta (IL-1beta) is accompanied by major changes to the intracellular ionic environment, activation of caspase-1, and cell death. Certain diarylsulfonylureas inhibit this response, and are designated cytokine release inhibitory drugs (CRIDs). CRIDs arrest activated monocytes so that caspase-1 remains inactive and plasma membrane latency is preserved. Affinity labeling with [(14)C]CRIDs and affinity chromatography on immobilized CRID were used in seeking potential protein targets of their action. Following treatment of intact human monocytes with an epoxide-bearing [(14)C]CRID, glutathione S-transferase (GST) Omega 1-1 was identified as a preferred target. Moreover, labeling of this polypeptide correlated with irreversible inhibition of ATP-induced IL-1beta posttranslational processing. When extracts of human monocytic cells were chromatographed on a CRID affinity column, GST Omega 1-1 bound selectively to the affinity matrix and was eluted by soluble CRID. Recombinant GST Omega 1-1 readily incorporated [(14)C]CRID epoxides, but labeling was negated by co-incubation with S-substituted glutathiones or by mutagenesis of the catalytic center Cys(32) to alanine. Peptide mapping by high performance liquid chromatography-mass spectrometry also demonstrated that Cys(32) was the site of modification. Although S-alkylglutathiones did not arrest ATP-induced IL-1beta posttranslational processing or inhibit [(14)C]CRID incorporation into cell-associated GST Omega 1-1, a glutathione-CRID adduct effectively demonstrated these attributes. Therefore, the ability of CRIDs to arrest stimulus-induced IL-1beta posttranslational processing may be attributable to their interaction with GST Omega 1-1.

Absence of the P2X7 receptor alters leukocyte function and attenuates an inflammatory response.

When challenged with extracellular ATP, leukocytes respond and activate processes attributed to the P2X(7) receptor (P2X(7)R), an unusual ligand-gated ion channel. To prove P2X(7)R involvement, blood samples from P2X(7)R-deficient mice were characterized. Monocytes and lymphocytes associated with wild-type blood responded to ATP and underwent volume/shape changes and shed L-selectin. In contrast, leukocytes from P2X(7)R-deficient animals demonstrated no change in physical properties or L-selectin expression following ATP challenge. Blood stimulated with LPS or ATP individually generated minimal quantities of the leaderless polypeptide IL-1 beta, but sequential treatment of wild-type, but not P2X(7)R-deficient, blood with LPS and ATP yielded large amounts of cell-free cytokine. Based on these differences, wild-type and P2X(7)R-deficient animals were compared following induction of monoclonal anti-collagen-induced arthritis. Ab-treated wild-type animals subsequently challenged with LPS developed inflamed, swollen paws; their joint cartilage demonstrated lesions, loss of proteoglycan content, and the presence of collagen degradation products. P2X(7)R-deficient animals subjected to the same challenge were markedly less affected; both the incidence and severity of disease were reduced. These data indicate that ATP does act via the P2X(7)R to affect leukocyte function and that the P2X(7)R can serve as an important component of an in vivo inflammatory response.

Antimicrobial peptides initiate IL-1 beta posttranslational processing: a novel role beyond innate immunity.

Human monocytes stimulated with LPS produce large quantities of prointerleukin-1beta, 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-1beta 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-1beta. The PTG-mediated response engaged a mechanism similar to that initiated by extracellular ATP acting via the P2X(7) 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-1beta posttranslational processing. The human defensin HNP-1 and the frog peptide magainin 1 elicited export of 17-kDa IL-1beta, but these agents were less efficient than PTGs. As a result of this ability to promote release of potent proinflammatory cytokines such as IL-1beta, select antimicrobial peptides may possess important immunomodulatory functions that extend beyond innate immunity.