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.

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.

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.

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.