Klotho and smoking--An interplay influencing the skeletal muscle function deficits that occur in COPD.

BACKGROUND: Klotho is an 'anti-ageing' hormone and transmembrane protein; Klotho deficient mice develop a similar ageing phenotype to smokers including emphysema and muscle wasting. The objective of this study was to evaluate skeletal muscle and circulating Klotho protein in smokers and COPD patients and to relate Klotho levels to relevant skeletal muscle parameters. We sought to validate our findings by undertaking complimentary murine studies. METHODS: Fat free mass, quadriceps strength and spirometry were measured in 87 participants (61 COPD, 13 'healthy smokers' and 13 never smoking controls) in whom serum and quadriceps Klotho protein levels were also measured. Immunohistochemistry was performed to demonstrate the location of Klotho protein in human skeletal muscle and in mouse skeletal muscle in which regeneration was occurring following injury induced by electroporation. In a separate study, gastrocnemius Klotho protein was measured in mice exposed to 77 weeks of smoke or sham air. RESULTS: Quadriceps Klotho levels were lower in those currently smoking (p = 0.01), irrespective of spirometry, but were not lower in patients with COPD. A regression analysis identified current smoking status as the only independent variable associated with human quadriceps Klotho levels, an observation supported by the finding that smoke exposed mice had lower gastrocnemius Klotho levels than sham exposed mice (p = 0.005). Quadriceps Klotho levels related to local oxidative stress but were paradoxically higher in patients with established muscle wasting or weakness; the unexpected relationship with low fat free mass was the only independent association. Within locomotor muscle, Klotho localized to the plasma membrane and to centralized nuclei in humans and in mice with induced muscle damage. Serum Klotho had an independent association with quadriceps strength but did not relate to quadriceps Klotho levels or to spirometric parameters. CONCLUSIONS: Klotho is expressed in skeletal muscle and levels are reduced by smoking. Despite this, quadriceps Klotho protein expression in those with established disease appears complex as levels were paradoxically elevated in COPD patients with established muscle wasting. Whilst serum Klotho levels were not reduced in smokers or COPD patients and were not associated with quadriceps Klotho protein, they did relate to quadriceps strength.

CD23 shedding: requirements for substrate recognition and inhibition by dipeptide hydroxamic acids.

CD23 (low affinity IgE receptor, FcepsilonRII) is expressed as a Type II extracellular protein on a variety of cells such as B cells, monocytes and macrophages and is cleaved from the cell surface to generate several distinct fragments. The expression of CD23 on the cell surface as well as the generation of soluble fragments of CD23 has been shown to be involved in regulation of IgE synthesis. CD23 is released from the cell surface by a metalloprotease, analogous to the cleavage of other cell surface molecules such as TNF-alpha. This activity has been extensively studied with respect to biochemical characterization and ability to cleave specific mutants of CD23. Both local sequence and distal domains have been shown to affect cleavage of CD23. Selective dipeptide hydroxamic acid inhibitors of CD23 processing have been identified and demonstrated to very potently and selectively inhibit CD23 processing.

Modulation of human monocyte activities by tranilast, SB 252218, a compound demonstrating efficacy in restenosis.

Tranilast (SB 252218) is a compound initially identified as an anti-atopic agent. Recently the compound has demonstrated clear beneficial effects in animal models of restenosis. Here we confirm tranilast has broad and profound effects on human monocytes, which could contribute to the vascular antifibrotic activity. Tranilast exhibited significant immunomodulatory activity inhibiting endotoxin-induced prostaglandin E(2) (PGE(2); IC(50) = approximately 1-20 microM), thromboxane B(2) (IC(50) = approximately 10-50 microM), transforming growth factor-beta1 (TGF-beta1; IC(50) = approximately 100-200 microM), and interleukin-8 (IC(50) = approximately 100 microM) formation, but had no effect on tumor necrosis factor-alpha. Interleukin-12 and -18-induced interferon-gamma formation by monocytes was also attenuated by tranilast. A23187-induced monocyte leukotriene C(4) or PGE(2) formation was inhibited by tranilast at IC(50) values of 10-40 microM and 2-20 microM, respectively, incubated with or without exogenous arachidonic acid. Interestingly, tranilast (up to 1000 microM) had no direct effects on cyclooxygenase I or II activity, nor did it have significant effects on human type IIA 14 kDa or type IV 85 kDa phospholipase A(2) activity. Furthermore, tranilast had no effect on endotoxin-induced cyclooxygenase II protein expression, suggesting tranilast modulates eicosanoid production and release by an as yet unidentified mechanism. Alternatively, the expression of TGF-beta1 was inhibited by tranilast but found to be due in part to inhibition of PGE(2) because exogenous PGE(2) could abrogate tranilast-mediated inhibition of TGF-beta1. Taken together, although a reported direct inhibitor of fibroblast proliferation, we show tranilast also attenuates the proinflammatory activity of human monocytes, adding to its potential efficacy as a therapeutic agent in restenosis.

Inhibition of CD23 processing correlates with inhibition of IL-4-stimulated IgE production in human PBL and hu-PBL-reconstituted SCID mice.

BACKGROUND: CD23, the low affinity serum immunoglobulin E (IgE) receptor, is upregulated on B cells following interleukin (IL)-4 stimulation and is concomitantly cleaved to generate soluble CD23 (sCD23) fragments with cytokine-like activity. OBJECTIVE: Compounds that selectively inhibit the proteolytic release of CD23 to generate sCD23 were assessed for their ability to inhibit IgE production in order to evaluate the contribution of sCD23 in the production of human IgE as well as the ability of such compounds to block IgE production. METHODS: IgE production was measured in IL-4-stimulated human peripheral blood lymphocytes (PBL) and PBL-reconstituted SCID mice in the presence of a broad-spectrum matrix metalloprotease (MMP) inhibitor, a compound selective for inhibition of CD23 processing over MMPs and an anti-CD23 mAb, MHM6. RESULTS: The two compounds were equipotent in inhibiting IgE production without inhibition of IgG production by IL-4/anti-CD40-stimulated PBL. Soluble CD23 release was also shown to precede IgE accumulation in the cell-free medium. Addition of compound at later times other than day 0 in the 14 day assay resulted in progressively less inhibition of both IgE and sCD23, and exactly paralleled the effect of an anti-CD23 mAb, MHM6 on IgE levels. Both compounds also inhibited the release of CD23 from human RPMI 8866 cells adoptively transferred i. p. to mice. Doses required for inhibition of CD23 correlated well with the doses required for inhibition of IgE production in IL-4-challenged hu-PBL-SCID mice. IgE was selectively inhibited over total IgG in the SCID mice as well. CONCLUSIONS: Inhibition of CD23 processing alone is sufficient to inhibit IL-4-stimulated IgE production both in vitro and in vivo.

Selective inhibition of low affinity IgE receptor (CD23) processing: P1' bicyclomethyl substituents.

Using a variety of alpha-hydroxy hydroxamic acid derivatives, the size and shape of the S1' pocket for the CD23 processing metalloprotease has been explored. It has been demonstrated that a P1' 2-naphthylmethyl group occupies most of the available space and gives excellent selectivity against fibroblast collagenase (matrix metalloproteinase-1, MMP-1) and other MMPs.

Anti-human RP105 sera induces lymphocyte proliferation.

Cellular environment dictates whether antigen binding to the B lymphocyte receptor together with co-stimulatory molecules will result in proliferation, anergy, or apoptosis. Murine RP105 is a member of the leucine-rich repeat family of proteins, which is specifically expressed on mature B cells. Monoclonal antibodies to the murine RP105 induce proliferation and protect B cells from apoptosis, suggesting an important regulatory role in murine B lymphocyte function. We identified a human RP105 homolog and mapped the gene to chromosome 5q12.3-13.1. Tissue distribution analysis shows that the transcript is found predominately in lymphoid tissues including spleen, tonsils, appendix, and peripheral blood leukocytes. Polymerase chain reaction analysis of isolated primary human cell populations confirms that mRNA exists in spleen B lymphocytes and monocytes but not T lymphocytes. Western blot analysis demonstrates specific expression of human RP105 in human B lymphocytes. Murine anti-human RP105 sera was generated using DNA immunization. The antisera contained antibodies that recognized and bound to human B lymphocytes from both spleen and peripheral blood as assessed by flow cytometry. Assessment of biological function showed that human peripheral blood leukocytes incubated with anti-RP105 sera were induced to proliferate as measured by tritiated thymidine incorporation. Moreover, anti-CD40 and interleukin-4-treated cells but not those exposed to anti-RP105 sera produced soluble CD23, suggesting distinct functional roles. This is the first demonstration of both the existence of RP105 protein on human B lymphocytes and its role in the regulation of B lymphocyte activation.

Respective roles of the 14 kDa and 85 kDa phospholipase A2 enzymes in human monocyte eicosanoid formation.

Human monocytes possess both the cytosolic 85 kDa phospholipase (PLA) A2 and a 14 kDa PLA2 and are capable of simultaneously producing prostanoids (PG), leukotrienes (LT) and platelet activating factor (PAF). As the exact roles of the two enzymes in monocyte lipid mediator formation was unclear, both selective PLA2 inhibitors and antisense were used to elucidate their respective roles. Reduction in 85 kDa PLA2 cellular protein levels by initiation site-directed antisense (SK 7111) or exposure to the 85 kDa PLA2 inhibitor, arachidonyl trifluormethyl ketone (AACOCF3), prevented A23187 or zymosan-stimulated monocytes prostanoid formation but not LTC4 or PAF production. This confirmed the important role of the 85 kDa PLA2 in prostanoid formation but indicated a less significant role in LT or PAF biosynthesis. Alternatively, treatment of monocytes with the selective, active-site-directed 14 kDa PLA2 inhibitor, SB 203347, totally inhibited LT and PAF formation, while prostanoid formation was not altered. Addition of 20 uM exogenous arachidonic acid (AA) to monocytes exposed to SB 203347 did not alter A23187-induced LTC4 generation, indicating that SB 203347 had no effect on downstream AA metabolizing enzymes in this setting. Taken together, these results provide evidence that the 14 kDa PLA2 provides substrate for monocyte LT and PAF formation, while the 85 kDa PLA2 plays a more significant role in the formation of PG.

Inhibitors of the p38 mitogen-activated kinase modulate IL-4 induction of low affinity IgE receptor (CD23) in human monocytes.

CD23, the low affinity IgE receptor, is up-regulated on the surface of IL-4-treated B cells and monocytes and is immediately proteolytically processed, releasing soluble fragments of CD23. Here, we report that inhibitors of the p38 mitogen-activated kinase (p38 MAPK), SK&F 86002 or the more selective inhibitor, SB 203580, reduce the levels of soluble CD23 formed by IL-4-stimulated human monocytes or the human monocytic cell line, U937. In contrast to compounds such as the metalloprotease inhibitor batimastat ([4-(N-hydroxyamino)-2-(R)-isobutyl-3-(S)-(2-thiophenethiomethyl)s uccinyl]-(S)-phenylalanine-N-methylamide, sodium salt), p38 MAPK inhibitors do not directly inhibit proteolytic processing of CD23. Further, evaluation of surface intact CD23 (iCD23) by flow cytometry demonstrated that SK&F 86002 and SB 203580 reduced the surface expression of iCD23 in a concentration-dependent fashion, while batimastat increased the surface expression of iCD23. The decrease in surface iCD23 was accompanied by a decrease in total cell-associated CD23 protein levels but not CD23 mRNA. IL-4 induced a late (>4-h) increase in p38 MAPK activity and corresponding activation of its substrate MAPKAPK-2. This activation was blocked by addition of SB 203580 before IL-4 induction, in parallel with the inhibition of CD23 expression. Modulation of CD23 by antibodies has been shown to alleviate the symptoms of murine collagen-induced arthritis, implicating CD23 as an important proinflammatory agent. These data show that in addition to the known cytokine inhibitory actions of SK&F 86002 and SB 203580, they also confer an additional potential anti-inflammatory activity through modulation of CD23 expression.

The role of platelet activating factor and other lipid mediators in inflammatory angiogenesis.

Chronic inflammatory diseases are often accompanied by intense angiogenesis. A model of inflammatory angiogenesis is the murine air pouch granuloma which has a hyperangiogenic component. Proinflammatory lipid mediator generation is also a hallmark of chronic inflammation and the role of endogenous production of these mediators in angiogenesis is not known. The 14 kDa phospholipase A2 (PLA2) deacylates phospholipid, liberating arachidonic acid, which is used for leukotriene production, and lysophospholipid, which can drive the production of platelet-activating factor (PAF). Therefore, SB 203347, an inhibitor of the 14 kDa PLA2, zileuton, an inhibitor of 5-lipoxygenase, and Ro 24-4736 a PAF receptor antagonist were evaluated for their effects in the murine air pouch granuloma. SB 203347 reduced both LTB4 and PAF, but not PGD2 levels measured in the day 6 granuloma. This correlated with a significant reduction in angiogenesis. Zileuton reduced LTB4 levels as expected, but did not significantly inhibit angiogenesis, whereas Ro 24-4736 potently reduced angiogenesis. These data support the hypothesis that PAF, and to a lesser extent leukotrienes contribute to the angiogenic phenotype in chronic inflammation.

Pharmacological effects of SB 220025, a selective inhibitor of P38 mitogen-activated protein kinase, in angiogenesis and chronic inflammatory disease models.

Chronic inflammatory diseases often are accompanied by intense angiogenesis, supporting the destructive proliferation of inflammatory tissues. A model of inflammatory angiogenesis is the murine air pouch granuloma, which has a hyperangiogenic component. In this model, we explored the regulation of inflammatory angiogenesis using SB 220025, a specific inhibitor of human p38 mitogen-activated protein (MAP) kinase, with an IC50 value of 60 nM and 50- to 1000-fold selectivity vs. other kinases tested. In vivo, this compound reduced the lipopolysaccharide-induced production of tumor necrosis factor at an ED50 value of 7.5 mg/kg. In the inflammatory angiogenesis model, over the course of granuloma development, we observed elevated levels of interleukin-1beta and tumor necrosis factor-alpha during the chronic inflammatory phase when intense angiogenesis occurs. SB 220025 at 30 mg/kg b.i.d. p.o. was able to greatly reduce the expression of these cytokines and inhibit angiogenesis by approximately 40%. To further study the effects of p38/CSBP MAP kinase inhibition in angiogenesis-dependent chronic inflammatory disease, SB 220025 was tested in murine collagen-induced arthritis. In this model, SB 220025 was able to prevent the progression of established arthritis. Thus, this p38/CSBP MAP kinase inhibitor, which can reduce inflammatory cytokine production and inhibit angiogenesis, is an effective treatment for chronic proliferative inflammatory disease.

Hydroxamate-based inhibitors of low affinity IgE receptor (CD23) processing.

A series of hydroxamic acids related to the non-selective matrix metalloprotease inhibitor Batimastat is described, which inhibits the proteolytic cleavage of the low affinity IgE receptor from cell membrane preparations. Limited SAR studies suggest that the structural requirements for effective inhibition are distinct from those required for the inhibition of collagenase.

Selective inhibition of low affinity IgE receptor (CD23) processing.

A series of hydroxamic acids related to the non-selective matrix metalloproteinase inhibitor Batimastat has been prepared, some members of which are potent inhibitors of the processing of the low affinity IgE receptor (CD 23). Increased activity is obtained by appropriate substitution at the alpha-position, whilst selectivity is gained by use of a P1' benzyl group in conjunction with a C-terminal primary amide.

Evidence that 85 kDa phospholipase A2 is not linked to CoA-independent transacylase-mediated production of platelet-activating factor in human monocytes.

Platelet-activating factor (PAF) production is carefully controlled in inflammatory cells. The specific removal of arachidonate (AA) from 1-O-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine (GPC), thought to be mediated by CoA-independent transacylase (CoA-IT), is required to generate the PAF precursor 1-O-alkyl-2-lyso-GPC in human neutrophils. Exposure of A23187-stimulated human monocytes to the CoA-IT inhibitors SK&F 98625 and SK&F 45905 inhibited PAF formation (IC50s of 10 and 12 microM, respectively), indicating that these cells also need CoA-IT activity for PAF production. Because CoA-IT activity transfers arachidonate to a 2-lyso phospholipid substrate, its activity is obligated to an sn-2 acyl hydrolase to form the 2-lyso phospholipid substrate. SB 203347, an inhibitor of 14 kDa phospholipase A2 (PLA2), and AACOCF3, an inhibitor of 85 kDa PLA2, both inhibited AA release from A23187-stimulated human monocytes. However, AACOCF3 had no effect on A23187-induced PAF formation at concentrations as high as 3 microM. Further, depletion of 85 kDa PLA2 using antisense (SB 7111, 1 microM) had no effect on PAF production, indicating a lack of a role of 85 kDa PLA2 in PAF biosynthesis. Both SB 203347 and the 14 kDa PLA2 inhibitor scalaradial blocked PAF synthesis in monocytes (IC50s of 2 and 0.5 microM, respectively), suggesting a key role of 14 kDa PLA2 in this process. Further, A23187-stimulated monocytes produced two forms of PAF: 80% 1-O-alkyl-2-acetyl-GPC and 20% 1-acyl-2-acetyl-GPC, which were both equally inhibited by SB 203347. In contrast, inhibition of CoA-IT using SK&F 45905 (20 microM) had a greater effect on the production of 1-O-alkyl (-80%) than of 1-acyl (-14%) acetylated material. Finally, treatment of U937 cell membranes with exogenous human recombinant (rh) type II 14 kDa PLA2, but not rh 85 kDa PLA2, induced PAF production. Elimination of membrane CoA-IT activity by heat treatment impaired the ability of 14 kDa PLA2 to induce PAF formation. Taken together, these results suggest that a 14 kDa PLA2-like activity, and not 85 kDa PLA2, is coupled to monocyte CoA-IT-induced PAF production.

Depletion of human monocyte 85-kDa phospholipase A2 does not alter leukotriene formation.

Human monocytes possess several acylhydrolase activities and are capable of producing both prostanoids (PG) and leukotriene (LT) products upon acute stimulation with calcium ionophore, A23187 or phagocytosis of zymosan particles. The cytosolic 85-kDa phospholipase (PLA) A2 co-exists with the 14-kDa PLA2 in the human monocyte, but their respective roles in LT production are not well understood. Reduction in 85-kDa PLA2 cellular protein levels by initiation site-directed antisense (SK 7111) or exposure to the 85-kDa PLA2 inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3), prevented A23187 or zymosan-stimulated monocyte prostanoid formation. In contrast, neither treatment altered stimulated LTC4 production. This confirmed the important role of the 85-kDa PLA2 in prostanoid formation but suggests that it has less of a role in LT biosynthesis. Alternatively, treatment of monocytes with the selective, active site-directed 14-kDa PLA2 inhibitor, SB 203347, prior to stimulation had no effect on prostanoid formation at concentrations that totally inhibited LT formation. Addition of 20 microM exogenous arachidonic acid to monocytes exposed to SK 7111 or SB 203347 did not alter A23187-induced PGE2 or LTC4 generation, respectively, indicating that these agents had no effect on downstream arachidonic acid-metabolizing enzymes in this setting. Taken together, these results provide evidence that the 85-kDa PLA2 may play a more significant role in the formation of PG than LT. Further, utilization of SB 203347 provides intriguing data to form the hypothesis that a non-85-kDa PLA2 sn-2 acyl hydrolase, possibly the 14-kDa PLA2, may provide substrate for LT formation.

Quantitation of human tissue and immune cell type II 14 kDa phospholipase A(2) by enzyme immunoassay.

The metabolism of arachidonic acid into inflammatory mediators (e.g. prostaglandin, leukotrienes) is dependent upon the rate-limiting enzyme phospholipase A(2). Localization and quantification of type II 14 kDa phospholipase A(2) (PLA(2)) in cells or tissue preparations has historically been accomplished through activity measurements, a process that can provide variable results due to interference by exogenous substances with hydrolysis assessment. Others have reported on the use of sandwich enzyme immunoassays (EIA) to measure 14 kDa PLA(2) by mass in serum and exudate fluids, e.g. synovial fluid. Herein, we report the utilization of a human recombinant type II 14 kDa PLA(2) sandwich EIA to directly measure cell or tissue-residing 14 kDa PLA(2). It is known that type II 14 kDa PLA(2) resists acid treatment, and this technique was applied to cell fractions which liberated the enzyme from cellular membrane components prior to quantitation by EIA. Two human immune cell populations were assessed and shown to contain measurable levels of 14 kDa PLA(2). Neutrophil or monocyte cytosolic fractions contained no measurable levels whereas the respective 100 000g particulate fractions contained 2.6+/-0.8 pg (neutrophil) and 2.1+/-0.6 pg (monocyte) 14 kDa PLA(2)/mug protein. Human placenta cytosolic fractions contained no measurable levels while 100 000g particulate contained approximately 25 ng 14 kDa PLA(2)/mg protein. This EIA, in conjunction with acid extraction, provides an easy and reproducible assay to identify and quantify this enzyme in cells and whole tissues, expanding our ability to study the relationship of this enzyme to inflammatory processes.

IgE secretion is attenuated by an inhibitor of proteolytic processing of CD23 (Fc epsilonRII).

CD23, the low-affinity IgE receptor, is up-regulated on interleukin (IL)-4-stimulated B cells and monocytes, with a concomitant increase in the release of soluble fragments of CD23 (sCD23) into the medium by proteolytic processing of the surface-bound intact CD23. The effect of inhibition of the processing of CD23 on IgE production in human and mouse cells and in a mouse model in vivo was evaluated. CD23 processing to sCD23 from RPMI 8866 (a human Epstein-Barr virus-transformed B cell line) cell membranes was inhibited by a broad-spectrum matrix-metalloprotease inhibitor, batimastat, with an IC50 of 0.15 microM. Batimastat also inhibited CD23 processing in whole RPMI 8866 cells as well as in IL-4-stimulated purified human monocytes with similar IC50. Batimastat inhibited IgE production from IL-4/anti-CD40-stimulated human tonsil B cells as well as mouse splenic B cells in a manner consistent with inhibition of CD23 processing. Release of soluble fragments of CD23 in the cell supernatants of tonsil B cells was inhibited over the concentration range of 1-10 microM batimastat and intact cell surface CD23 was increased on mouse splenic B cells in the presence of these concentrations of batimastat. IgE production of IL-4-stimulated human peripheral blood mononuclear cells was also blocked by 1-10 microM batimastat, again with comparable inhibition of sCD23 release over the same concentration range. Finally, in a mouse model of IgE production, batimastat inhibited IgE production in response to ovalbumin challenge as determined by serum IgE levels. Taken together, the data support a role of CD23 in IgE production and point to CD23 processing to sCD23 as a therapeutically relevant control point in the regulation of IgE synthesis.

Prostaglandin E2 requirement for transforming growth factor beta 1 inhibition of elicited macrophage 14 kDa phospholipase A2 release.

1. Cultured elicited-peritoneal macrophages release a soluble type II 14 kDa phospholipase A2 (PLA2) over time, reaching a plateau by 20-24 h of incubation and maintaining these levels over 72 h. Prostaglandin E2 (PGE2) is also produced but does not plateau until 48-72 h. 2. Transforming growth factor beta 1 (TGF beta 1) reduces cellular 14 kDa PLA2 and its subsequent release by approximately half, but does not alter PGE2 production. Co-incubation of TGF beta 1 with indomethacin interfered, in a concentration-dependent manner, with the ability of TGF beta 1 to reduce cellular 14 kDa PLA2 and its subsequent release over 24 h. The regulation of TGF beta 1 was not specific to indomethacin since other non-steroidal anti-inflammatory drugs had the same effect. This suggested that cyclooxygenase activity was essential for TGF beta 1 to exert its effect and indeed, the addition of exogenous PGE2 restored the TGF beta 1 action. 3. PGE2 alone exerted a concentration-dependent negative feedback action on elicited-macrophage 14 kDa PLA2 release. The inhibitory concentration (IC50 = approximately 180 ng PGE2 ml-1) approximated the PGE2 levels measured in the 24 h macrophage conditioned media (85-140 ng PGE2 ml-1) where PLA2 release began to plateau. Further, incubation of cells with indomethacin over 48 h resulted in the enhancement of 14 kDa PLA2 activity compared to that released from untreated cells. Forskolin failed to inhibit 14 kDa PLA2 release, suggesting PGE2 was not acting through an increase in adenylate cyclase. 4. Taken together, the data are consistent with the immunosuppressive aspects reported for both mediators during inflammation and demonstrates the requirement of PGE2 for TGF beta 1 action on the elicited macrophage.

SB 203347, an inhibitor of 14 kDa phospholipase A2, alters human neutrophil arachidonic acid release and metabolism and prolongs survival in murine endotoxin shock.

Phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 fatty acyl group [predominantly arachidonic acid (AA)] of membrane phospholipids, the products of which are further metabolized, forming a variety of eicosanoids and/or platelet-activating factor. PLA2 activity is significantly enhanced during inflammation and therefore offers an intriguing target in designing anti-inflammatory drugs. SB 203347 (2-[2-[3,5-bis (trifluoromethyl) sulfonamido]-4- trifluoromethylphenoxy] benzoic acid) potently inhibits rh type II 14 kDa PLA2 (IC50 = 0.5 microM) but exhibits a 40-fold weaker inhibition of 85 kDa PLA2 (IC50 = 20 microM) using [3H]-AA E. coli as substrate. A specific interaction with rh type II 14 kDa PLA2 was confirmed both by observing the pH dependence of its IC50 and by demonstrating linear inhibition in a "scooting" kinetic model using radiolabeled phospholipid reporter substrate in a 1,2-dimyristoyl phosphatidylmethanol vesicle. Before evaluating the effect of SB 203347 on AA metabolism in intact human neutrophil, we showed that it fully inhibits PLA2 activity in acid extracted-intact human neutrophil homogenate (IC50 = 4.7 microM). SB 203347 inhibited A23187-induced intact human neutrophil AA mass release in a concentration-dependent manner (IC50 = 1 microM), which coincided with reductions in the biosynthesis of platelet-activating factor (IC50 = 1.5 microM) and leukotriene B4 (IC50 = 2.3 microM). Finally, SB 203347 prolonged survival in a mouse model of endotoxin shock delivered i.p. Taken together, the data support a role of cellular 14 kDa PLA2 in the formation of AA-derived proinflammatory lipid mediator.(ABSTRACT TRUNCATED AT 250 WORDS)