Clifford D. Wright

Conversion of proepithelin to epithelins: roles of SLPI and elastase in host defense and wound repair.

Increased leukocyte elastase activity in mice lacking secretory leukocyte protease inhibitor (SLPI) leads to impaired wound healing due to enhanced activity of TGFbeta and perhaps additional mechanisms. Proepithelin (PEPI), an epithelial growth factor, can be converted to epithelins (EPIs) in vivo by unknown mechanisms with unknown consequences. We found that PEPI and EPIs exert opposing activities. EPIs inhibit the growth of epithelial cells but induce them to secrete the neutrophil attractant IL-8, while PEPI blocks neutrophil activation by tumor necrosis factor, preventing release of oxidants and proteases. SLPI and PEPI form complexes, preventing elastase from converting PEPI to EPIs. Supplying PEPI corrects the wound-healing defect in SLPI null mice. Thus, SLPI/elastase act via PEPI/EPIs to operate a switch at the interface between innate immunity and wound healing.

Inhibition of allergen-induced pulmonary responses by the selective tryptase inhibitor 1,5-bis-{4-[(3-carbamimidoyl-benzenesulfonylamino)-methyl]-phenoxy}-pentane (AMG-126737)

Emerging evidence suggests that mast cell tryptase is a therapeutic target for the treatment of asthma. The effects of this serine protease are associated with both pathophysiologic pulmonary responses and pathologic changes of the asthmatic airway. In this study, the tryptase inhibitor 1,5-bis-[4-[(3-carbamimidoyl-benzenesulfonylamino)-methyl]-p henoxy]-pentane (AMG-126737) was evaluated for its pharmacologic effects against allergen-induced airway responses. AMG-126737 is a potent inhibitor of human lung mast cell tryptase (Ki = 90 nM), with greater than 10- to 200-fold selectivity versus other serine proteases. Intratracheal administration of AMG-126737 inhibited the development of airway hyperresponsiveness in allergen-challenged guinea pigs with an ED50 of 0.015 mg/kg. In addition, the compound exhibited oral activity in the guinea pig model. The in vivo activity of AMG-126737 was confirmed in a sheep model of allergen-induced airway responses, where the compound inhibited early and late phase bronchoconstriction responses and the development of airway hyperresponsiveness. These results support the proposed role of tryptase in the pathology of asthma and suggest that AMG-126737 has potential therapeutic utility in this pulmonary disorder.

Secretory leukocyte protease inhibitor, an inhibitor of neutrophil activation, is elevated in serum in human sepsis and experimental endotoxemia.

Objectives: To document changes in serum secretory leukocyte protease inhibitor (SLPI) in human sepsis and in experimental endotoxemia in vivo. To compare changes in serum SLPI in human sepsis with changes in interleukin (IL)‐6, IL‐10, and tumor necrosis factor (TNF)‐α. To determine whether or not changes in SLPI correlate with the severity of multiple organ dysfunction syndrome as measured by the maximal multiple organ dysfunction score. Finally, because neutrophils have been implicated in tissue injury associated with organ dysfunction, to determine whether recombinant human SLPI blocks activation of isolated human neutrophils. Design: Case‐control study and ex‐vivo cellular assay. Setting: Surgical intensive care unit and clinical research center of university hospitals; laboratory of a medical school. Interventions: None. Measurements and Main Results: There was a significant dose‐dependent elevation (50.2 ± 4.0 ng/mL, p = .01) in plasma SLPI 12 hrs after administration of lipopolysaccharide to seven healthy adults (36.4 ± 2.3 ng/mL). Further, serum concentrations of SLPI (132 ± 15 ng/mL) were elevated in septic surgical patients compared with healthy controls (43 ± 2 ng/mL, p < .01) and nonseptic surgical controls (69 ± 10 ng/mL, p = .01). Serum SLPI concentrations correlated (r2 = .71, p < .01) better with organ dysfunction as measured by maximal multiple organ dysfunction score than did serum IL‐6 (r2 = .49, p < .01), IL‐10 (r2 = .05, p = .22), or TNF‐α (r2 = .02, p = .44). We found that recombinant human SLPI in vitro inhibits TNF‐α‐induced hydrogen peroxide production by human neutrophils (ID50 = 1‐2 μg/mL). Conclusions: Serum SLPI is elevated in human sepsis and experimental endotoxemia. Maximal concentrations of serum SLPI correlate significantly with maximal multiple organ dysfunction scores in patients with sepsis. Secretory leukocyte protease inhibitor may function to limit ongoing neutrophil‐mediated tissue injury associated with organ dysfunction.

Anti-Inflammatory Effects of Mutant Forms of Secretory Leukocyte Protease Inhibitor

The secretory leukocyte protease inhibitor (SLPI) is found in a variety of secreted fluids in mammals and is a known inhibitor of serine proteases. Wild-type (WT) SLPI has recently been shown to block nuclear factor kappaB (NF-kappaB) activation in rat lungs and to interfere with the ensuing inflammatory response and recruitment of neutrophils after an intrapulmonary deposition of IgG immune complexes. In this study, WT SLPI and SLPI mutants with various degrees of protease-inhibitory capacity (for trypsin, chymotrypsin, and elastase) were evaluated for their ability to suppress the lung-vascular leak, neutrophil accumulation, and NF-kappaB activation in the lung inflammatory model. The SLPI mutant with Gly(72) (replacing Leu(72) ) lost its ability to block in vivo activation of NF-kappaB, as well as its ability to suppress the lung vascular leak and neutrophil recruitment. The Phe(72) and Gly(20) mutants were as effective as the WT SLPI in suppressing NF-kappaB activation and neutrophil recruitment. The Lys(72) mutant had the most suppressive effects of the lung vascular leak and for neutrophil recruitment into the lung. The in vivo suppressive effects of SLPI mutants on lung vascular permeability, neutrophil recruitment, and NF-kappaB activation appear to be most closely related to their trypsin-inhibiting activity. These data suggest that the suppressive effects of SLPI on the intrapulmonary activation of NF-kappaB and neutrophil recruitment into the lung may be linked to their antiprotease activity, directed, perhaps, at the intracellular proteases.

Proteinase-activated receptor-2 mediates hyperresponsiveness in isolated guinea pig bronchi.

The mast cell serine protease tryptase has been implicated as a critical mediator of airway hyperresponsiveness in vitro and in vivo. We have previously demonstrated that tryptase promotes hyperresponsiveness in isolated guinea pig bronchi. In this study, we have investigated the potential role of tryptase-mediated activation of proteinase-activated receptor-2 (PAR-2) in promoting airway hyperresponsiveness. Ex vivo exposure of guinea pig bronchi to the PAR-2 agonists H(2)N-Ser-Leu-Ile-Gly-Arg-Leu-CONH(2) (SLIGRL) and t-cinnamoyl-H(2)N-Leu-Ile-Gly-Arg-Leu-O-CONH(2) (t-c-LIGRLO) (0.1-10 microM) induced a concentration-dependent increase of contractile response to histamine. Treatment with 10 microM SLIGRL or t-c LIGRLO for 45 min increased subsequent responsiveness to histamine (0.3mM) by 54+/-3% and 69+/-5%, respectively (P<0.05 vs. control). In contrast, the PAR-1 agonist peptide H(2)N-Ser-Phe-Leu-Leu-Arg-Asn-CONH(2) (SFLLRN) did not promote significant changes in the airway. Effects of the peptides were observed following at least a 30-min preincubation with the tissue. Coincubation with indomethacin or removal of epithelial cells is required for PAR-2-mediated hyperreactivity. The inactive analogue H(2)N-Leu-Ser-Ile-Gly-Arg-Leu-CONH(2) (LISGRL; 10 microM) failed to promote hyperresponsiveness. Neuropeptide antagonists blocked the effect of the PAR-2 agonists. Selective antagonists of NK1 (L-703,606), NK2 (L-659,877), and CGRP (alphaCGRP 8-37) provided additive inhibition of PAR-2-mediated hyperreactivity. Pretreatment of bronchi with capsaicin (0.8 microM) also prevented the effects of SLIGRL. These results demonstrate the potential involvement of tryptase-mediated activation of PAR-2 in promoting airway hyperresponsiveness. These results further demonstrate that the PAR-2-mediated response involves a neurogenic mechanism involving neuropeptide release.

TRYPTASE MEDIATES HYPERRESPONSIVENESS IN ISOLATED GUINEA PIG BRONCHI

Hyperresponsiveness of airway smooth muscle to allergens and environmental factors has long been associated with the pathophysiology of asthma. Tryptase, a serine protease of lung mast cells, has been implicated as one of the mediators involved in the induction of hyperresponsiveness. As a consequence, tryptase inhibitors have become the subject of study as potential novel therapeutic agents for asthma. Secretory leukocyte protease inhibitor (SLPI) is a naturally occurring protein of human airways which exhibits anti-tryptase activity. To assess the potential therapeutic utility of SLPI in asthma, its effects were evaluated using in vitro and ex vivo models of airway hyperresponsiveness and compared with the effects of the small molecule tryptase inhibitor APC-366. Our results demonstrate that SLPI inhibits tryptase-mediated hyperresponsiveness in vitro and attenuates the hyperresponsiveness observed in airway smooth muscle from antigen-sensitized animals subjected to antigen exposure. The small molecule tryptase inhibitor APC-366 has a similar inhibitory effect. Thus, tryptase appears to be a significant contributor to the development of hyperresponsiveness in these models. To the extent that tryptase contributes to the development and progression of asthma, SLPI may possess therapeutic potential in this disease setting.

Salicylanilides: Selective inhibitors of interleukin-12p40 production

Interleukin (IL)-12p40, a subunit component of both IL-12 and IL-23, is being widely studied for its role in inflammatory disease. As part of an effort to profile cellular signaling pathways across different cell types, we report salicylanilide inhibitors of IL-12p40 production in stimulated dendritic cells. Based on a hypothesis that a desirable therapeutic profile is one that could block IL-12p40 but not IL-6 production, we engaged in directed analoging. This resulted in salicylanilides with similar IL-12p40 related potency but enhanced selectivity relative to IL-6 production.

Regulation of polymorphonuclear leukocyte membrane fluidity: effect of cytoskeletal modification

We previously demonstrated that the f‐actin cytoskeleton modulates oxygen radical production associated with polymorphonuclear leukocyte (PMN) oxidative burst activity. Given the close association of the actin and microtubule cytoskeletons with the plasma membrane and the transmembrane location of the PMN NADPH oxidase, it is likely cytoskeletal change may affect PMN membrane responses, such as cellular anisotropy. Changes in PMN membrane fluidity were therefore examined after PMN activation by the chemoattractant N‐formyl‐1‐methionyl‐1‐leucyl‐1‐phenylalanine (fMLP) in the presence or absence of phalloidin or cytochalasin B (CB), agents that stabilize and disrupt f‐actin, or taxol and vincristine, which stabilize and disrupt microtubules, respectively. Phalloidin and taxol treatment of PMN significantly decreased whereas CB and vincristine significantly increased membrane fluidity. Activation of PMN by fMLP (10‐6 M) resulted in a significant increase in membrane fluidity that was attenuated by PMN pretreatment with phalloidin or taxol. CB and vincristine pretreatment of PMN did not alter the fMLP response. These data suggest that stabilization of the f‐actin or microtubule cytoskdeton may prevent increases in cellular membrane fluidity associated with PMN activation.—J. Leukoc. Biol. 56: 192–199; 1994.

Selective regulation of human neutrophil functions by the cell activation inhibitor CI-959.

The cell activation inhibitor CI‐959 [5‐methoxy‐3‐(1‐methylethoxy)‐N‐1H‐tetrazol‐5‐ylbenzo[b]thiophene‐2‐carboxamide, monosodium salt] was evaluated for its effects on human neutrophil functions. CI‐959 inhibited spontaneous migration and chemotaxis toward N‐formyl–methionyl‐l‐leucyl‐l‐phenylalanine (fMLP) with 50% inhibition (IC50) values of 3.6 and 3.1 μM, respectively. CI‐959 also inhibited superoxide anion generation in response to C5a, fMLP, serum‐opsonized zymosan (SOZ), concanavalin A (Con A), and calcium ionophore A23187 with IC50 values of 2.5, 4.7, 14.5, 5.4, and 14.8 μM, respectively. In comparison, CI‐959 inhibited myeloperoxidase release in response to C5a, fMLP, SOZ, and Con A with IC50 values of 11.6, 16.1, 7.5, and <1.0 μM, respectively, while inhibiting the response to A23187 by only 5.5% at 100 μM. At concentrations up to 100 μM, CI‐959 had no effect on the respiratory burst or degranulation in response to L‐α‐1,2‐dioctanoylglycerol (DiC8) or phorbol 12‐myristate 13‐acetate (PMA). In addition, the compound inhibited leukotriene B4 release stimulated by fMLP and SOZ (IC50 values 4.0 and 2.5 μM, respectively), while having less activity against the A23187‐stimulated response (IC50>100 μM). These results demonstrate that CI‐959 inhibits cellular responses to stimuli that mobilize intracellular calcium. For cellular responses to ionophore‐mediated calcium influx, only oxygen radical production was inhibited by CI‐959. CI‐959 was further evaluated for its effects on neutrophil stimulus‐response coupling. At 100 μM, CI‐959 had no effect on human neutrophil phospholipase C or protein kinase C. CI‐959 inhibited fMLP‐stimulated intracellular calcium mobilization and calcium influx with IC50 values of 16.7 and 3.1 μM, respectively, and exhibited less potent calmodulin antagonist activity (IC50 = 90.5 μM). These results indicate that CI‐959 may exert its stimulus‐ and response‐specific inhibitory effects on neutrophil functions, in part, through inhibition of calcium‐regulated signalling mechanisms. J. Leukoc. Biol. 55: 443–451; 1994.

Identification of 14−3−3ζ by Chemical Affinity with Salicylanilide Inhibitors of Interleukin-12p40 Production

Salicylanilides were found as selective inhibitors of interleukin-12p40 production in stimulated dendritic cells. The conversion of one of these bioactive salicylanilides into a comparably bioactive, chemically labeled derivative was achieved using a facile and systematic functional group derivatization strategy. This resulted in a tool reagent that was then employed in an affinity chromatography approach that resulted in the identification of the protein 14-3-3zeta as having selective affinity for the chromatography matrix that was derivatized with a salicylanilide that inhibited IL-12p40 production.