Fibroblast activation protein in remodeling tissues.

Tissue remodeling is critical during development and wound healing. It also characterizes a number of pathologic conditions, including chronic inflammation, fibrosis and cancer. It is well appreciated that reactive stromal cells play critical roles in these settings. However, understanding of the mechanisms involved in the differentiation of reactive stromal cells and their biologic activities has been hampered by the fact that they are generated from diverse progenitors, and by their phenotypic and function heterogeneity. Furthermore, molecular markers that are expressed by all reactive stromal cells and that distinguish them from all other cell types have been lacking. Fibroblast activation protein (FAP) is a serine protease that was originally discovered as a cell surface protein expressed on astrocytomas and sarcomas. Over the last two decades, FAP has attracted increasing attention as a selective marker of carcinoma-associated fibroblasts (CAFs) and more broadly, of activated fibroblasts in tissues undergoing remodeling of their extracellular matrix (ECM) due to chronic inflammation, fibrosis or wound healing. Herein we review the evidence that FAP is indeed a robust and selective marker for reactive mesenchymal stromal cells associated with pathophysiologic tissue remodeling. We also review recent insights obtained using FAP as a tool to define the relationship between subpopulations of reactive stromal cells in various settings of tissue remodeling. Furthermore, we review recent genetic and pharmacologic data indicating that FAP and FAP-expressing cells play important roles in such conditions. Finally, we discuss the potential risks and therapeutic benefits of targeting FAP and FAP-expressing cells, as well as approaches to do so.

The adhesion receptor CD44 promotes atherosclerosis by mediating inflammatory cell recruitment and vascular cell activation.

Atherosclerosis causes most acute coronary syndromes and strokes. The pathogenesis of atherosclerosis includes recruitment of inflammatory cells to the vessel wall and activation of vascular cells. CD44 is an adhesion protein expressed on inflammatory and vascular cells. CD44 supports the adhesion of activated lymphocytes to endothelium and smooth muscle cells. Furthermore, ligation of CD44 induces activation of both inflammatory and vascular cells. To assess the potential contribution of CD44 to atherosclerosis, we bred CD44-null mice to atherosclerosis-prone apoE-deficient mice. We found a 50-70% reduction in aortic lesions in CD44-null mice compared with CD44 heterozygote and wild-type littermates. We demonstrate that CD44 promotes the recruitment of macrophages to atherosclerotic lesions. Furthermore, we show that CD44 is required for phenotypic dedifferentiation of medial smooth muscle cells to the "synthetic" state as measured by expression of VCAM-1. Finally, we demonstrate that hyaluronan, the principal ligand for CD44, is upregulated in atherosclerotic lesions of apoE-deficient mice and that the low-molecular-weight proinflammatory forms of hyaluronan stimulate VCAM-1 expression and proliferation of cultured primary aortic smooth muscle cells, whereas high-molecular-weight forms of hyaluronan inhibit smooth muscle cell proliferation. We conclude that CD44 plays a critical role in the progression of atherosclerosis through multiple mechanisms.

Effects of coexpression of the LDL receptor and apoE on cholesterol metabolism and atherosclerosis in LDL receptor-deficient mice.

The low density lipoprotein receptor (LDLR) plays a major role in regulation of plasma cholesterol levels as a ligand for apolipoprotein B-100 and apolipoprotein E (apoE). Consequently, LDLR-deficient mice fed a Western-type diet develop significant hypercholesterolemia and atherosclerosis. ApoE not only mediates uptake of atherogenic lipoproteins via the LDLR and other cell-surface receptors, but also directly inhibits atherosclerosis. In this study, we examined the hypothesis that coexpression of the LDLR and apoE would have greater effects than either one alone on plasma cholesterol levels and the development of atherosclerosis in LDLR-deficient mice. LDLR-deficient mice fed a Western-type diet for 10 weeks were injected with recombinant adenoviral vectors encoding the genes for human LDLR, human apoE3, both LDLR and apoE3, or lacZ (control). Plasma lipids were analyzed at several time points after vector injection. Six weeks after injection, mice were analyzed for extent of atherosclerosis by two independent methods. As expected, LDLR expression alone induced a significant reduction in plasma cholesterol due to reduced VLDL and LDL cholesterol levels, whereas overexpression of apoE alone did not reduce plasma cholesterol levels. When the LDLR and apoE were coexpressed in this model, the effects on plasma cholesterol levels were no greater than with expression of the LDLR alone. However, coexpression did result in a substantial increase in large apoE-rich HDL particles. In addition, although the combination of cholesterol reduction and apoE expression significantly reduced atherosclerosis, its effects were no greater than with expression of the LDLR or apoE alone. In summary, in this LDLR-deficient mouse model fed a Western-type diet, there was no evidence of an additive effect of expression of the LDLR and apoE on cholesterol reduction or atherosclerosis.

A crucial role for CD44 in inflammation.

Current therapies for chronic inflammatory diseases typically act through the nonspecific downregulation of immune cell activation. However, it is becoming increasingly evident that parenchymal cells are also active participants in the inflammatory process. Future prospects for the treatment of inflammation should therefore include the targeting of specific inflammatory pathways in both immune cells and parenchymal cells. CD44, a cell-adhesion molecule that is ubiquitously expressed on leukocytes and parenchymal cells, has been implicated, together with its ligand hyaluronan (HA), in several inflammatory diseases. The mechanisms of action of CD44-HA interactions in inflammation might provide potential targets for therapy.

A role for CD44 in the production of IFN-gamma and immunopathology during infection with Toxoplasma gondii.

The interaction of activated CD44 with its ligand, low m.w. hyaluronan, is involved in inflammation, but no role has been identified for this interaction in the regulation of an immune response to infection. In these studies, infection of C57BL/6 mice with Toxoplasma gondii resulted in increased expression of CD44 on T cells, B cells, NK cells, and macrophages, and a small percentage of CD4(+) T cells express an activated form of CD44. Administration of anti-CD44 to infected mice prevented the development of a CD4(+) T cell-dependent, infection-induced inflammatory response in the small intestine characterized by the overproduction of IFN-gamma. The protective effect of anti-CD44 treatment was associated with reduced production of IFN-gamma, but not IL-12, in vivo and in vitro. Furthermore, the addition of low m.w. hyaluronan to cultures of splenocytes or purified CD4(+) T cells from infected mice resulted in the production of high levels of IFN-gamma, which was dependent on IL-12 and TCR stimulation. Together, these results identify a novel role for CD44 in the regulation of IFN-gamma production by CD4(+) T cells during infection and demonstrate a role for CD44 in the regulation of infection-induced immune pathology.

Reduction of isoprostanes and regression of advanced atherosclerosis by apolipoprotein E.

Apolipoprotein E is a multifunctional protein synthesized by hepatocytes and macrophages. Plasma apoE is largely liver-derived and known to regulate lipoprotein metabolism. Macrophage-derived apoE has been shown to reduce the progression of atherosclerosis in mice. We tested the hypothesis that liver-derived apoE could directly induce regression of pre-existing advanced atherosclerotic lesions without reducing plasma cholesterol levels. Aged low density lipoprotein (LDL) receptor-deficient (LDLR(-/-)) mice were fed a western-type diet for 14 weeks to induce advanced atherosclerotic lesions. One group of mice was sacrificed for evaluation of atherosclerosis at base line, and two other groups were injected with a second generation adenoviruses encoding human apoE3 or a control empty virus. Hepatic apoE gene transfer increased plasma apoE levels by 4-fold at 1 week, and apoE levels remained at least 2-fold higher than controls at 6 weeks. There were no significant changes in plasma total cholesterol levels or lipoprotein composition induced by expression of apoE. The liver-derived human apoE gained access to and was retained in arterial wall. Compared with base-line mice, the control group demonstrated progression of atherosclerosis; in contrast, hepatic apoE expression induced highly significant regression of advanced atherosclerotic lesions. Regression of lesions was accompanied by the loss of macrophage-derived foam cells and a trend toward increase in extracellular matrix of lesions. As an index of in vivo oxidant stress, we quantitated the isoprostane iPF(2 alpha)-VI and found that expression of apoE markedly reduced urinary, LDL-associated, and arterial wall iPF(2 alpha)-VI levels. In summary, these results demonstrate that liver-derived apoE directly induced regression of advanced atherosclerosis and has anti-oxidant properties in vivo that may contribute to its anti-atherogenic effects.

Deficiency in inducible nitric oxide synthase results in reduced atherosclerosis in apolipoprotein E-deficient mice.

Inducible NO synthase (iNOS) present in human atherosclerotic plaques could contribute to the inflammatory process of plaque development. The role of iNOS in atherosclerosis was tested directly by evaluating the development of lesions in atherosclerosis-susceptible apolipoprotein E (apoE)-/- mice that were also deficient in iNOS. ApoE-/- and iNOS-/- mice were cross-bred to produce apoE-/-/iNOS-/- mice and apoE-/-/iNOS+/+ controls. Males and females were placed on a high fat diet at the time of weaning, and atherosclerosis was evaluated at two time points by different methods. The deficiency in iNOS had no effect on plasma cholesterol, triglyceride, or nitrate levels. Morphometric measurement of lesion area in the aortic root at 16 wk showed a 30-50% reduction in apoE-/-/iNOS-/- mice compared with apoE-/-/iNOS+/+ mice. Although the size of the lesions in apoE-/-/iNOS-/- mice was reduced, the lesions maintained a ratio of fibrotic:foam cell-rich:necrotic areas that was similar to controls. Biochemical measurements of aortic cholesterol in additional groups of mice at 22 wk revealed significant 45-70% reductions in both male and female apoE-/-/iNOS-/- mice compared with control mice. The results indicate that iNOS contributes to the size of atherosclerotic lesions in apoE-deficient mice, perhaps through a direct effect at the site of the lesion.

Hepatic expression of apolipoprotein E inhibits progression of atherosclerosis without reducing cholesterol levels in LDL receptor-deficient mice.

Apolipoprotein E (apoE) is a multifunctional protein synthesized by the liver and by tissue macrophages. Plasma apoE (derived primarily from the liver) regulates plasma lipoprotein metabolism, but macrophage-derived apoE was shown to slow the progression of atherosclerosis independent of plasma lipid levels. We utilized liver-directed gene transfer to test the hypothesis that hepatic expression of human apoE would inhibit atherogenesis even in a model in which apoE expression has little effect on plasma lipoproteins. LDL receptor-deficient mice fed a western-type diet for 5 weeks were injected with a second-generation recombinant adenovirus encoding human apoE3 or control virus. Plasma cholesterol levels were not significantly different in the two groups of mice after virus injection. Four weeks after injection, atherosclerosis was examined using three independent assays. Expression of apoE was associated with significantly reduced atherosclerosis compared with control mice in both the aortic arch (decreased by 43%) and the aortic root (decreased by 59%). In summary, hepatic overexpression of apoE inhibited progression of atherosclerosis in LDL receptor-deficient mice without reducing plasma cholesterol levels. This finding indicates that liver-derived plasma apoE can influence early atherogenesis through mechanisms other than modulation of lipoprotein metabolism and that liver-directed gene transfer and overexpression of apoE may be a therapeutic approach to atherosclerosis.

Oncostatin M and transforming growth factor-beta 1 induce post-translational modification and hyaluronan binding to CD44 in lung-derived epithelial tumor cells.

CD44, a receptor for hyaluronan (HA), has been implicated in tumor growth and metastasis. Most CD44-positive cells fail to exhibit constitutive HA receptor function but CD44-mediated HA binding on hematopoetic cells can be induced by antibody cross-linking of the receptor and by physiologic stimuli, including cytokines. We now demonstrate that oncostatin M (OSM) and transforming growth factor-beta1, cytokines known to regulate the growth of tumor cells, stimulate HA binding in lung epithelial-derived tumor cells. In lung epithelial-derived tumor cells, cytokine-induced binding resulted from post-translational modification of the receptor. OSM-induced HA binding was associated with a reduction in N-linked carbohydrate content of CD44. In addition, OSM induced HA binding via a novel mechanism requiring sulfation of chondroitin sulfate chains linked to CD44. The mechanism underlying transforming growth factor-beta1 induced HA binding was distinct from the effects of OSM. The data presented indicate that modulation of the glycosylation and sulfation of CD44 by cytokines provides mechanisms for regulating cell adhesion during tumor growth and metastasis.

Regression of atherosclerosis induced by liver-directed gene transfer of apolipoprotein A-I in mice.

BACKGROUND: The ability of apolipoprotein (apo)A-I to induce regression of preexisting atherosclerotic lesions has not been determined, and a mouse model of atherosclerosis regression has not yet been reported. METHODS AND RESULTS: LDL receptor-deficient mice were fed a western-type diet for 5 weeks to induce atherosclerotic lesions. A second-generation recombinant adenovirus encoding human apoA-I or a control adenovirus were injected intravenously in order to express apoA-I in the liver. Three days after injection, total apoA-I levels in mice injected with the apoA-I-expressing adenovirus were 216+/-16.0 mg/dL, compared with 68.0+/-3.0 mg/dL in control virus-injected mice (P<0.001). HDL cholesterol levels in mice injected with the AdhapoA-I vector 7 days after injection were 189+/-21.0 mg/dL, compared with 123+/-8.0 mg/dL in control virus-injected mice (P<0.02). Total and non-HDL cholesterol levels did not differ between the 2 groups. Atherosclerotic lesion area was quantified by en face analysis of the aorta and cross-sectional analysis of the aortic root. Compared with baseline mice, atherosclerosis progressed in mice injected with the control adenovirus. In contrast, in mice expressing apoA-I compared with baseline mice, total en face aortic lesion area was reduced by 70% and aortic root lesion was reduced by 46%. Expression of apoA-I was associated with a significant reduction in the fraction of lesions occupied by macrophages and macrophage-derived foam cells. CONCLUSIONS: Liver-directed gene transfer of human apoA-I resulted in significant regression of preexisting atherosclerotic lesions in LDL receptor-deficient mice as assessed by 2 independent methods.

Rapid regression of atherosclerosis induced by liver-directed gene transfer of ApoE in ApoE-deficient mice.

Apolipoprotein E (apoE) is a multifunctional protein synthesized by the liver and tissue macrophages. ApoE-deficient mice have severe hyperlipidemia and develop accelerated atherosclerosis on a chow diet. Both liver-derived and macrophage-derived apoEs have been shown to reduce plasma lipoprotein levels and slow the progression of atherosclerosis in apoE-deficient mice, but regression of atherosclerosis has not been demonstrated in this model. We utilized second-generation adenoviruses to achieve hepatic expression of human apoE in chow-fed, apoE-deficient mice with established atherosclerotic lesions of different stages. As expected, hepatic expression of human apoE3 significantly reduced plasma cholesterol levels. Liver-derived apoE also accumulated substantially within preexisting atherosclerotic lesions, indicating that plasma apoE gained access to the arterial intima. Hepatic expression of human apoE3 for 6 weeks resulted in significant quantitative regression of both early fatty streak lesions as well as advanced, complex lesions in both the aortic root and the aortic arch. In addition, hepatic expression of apoE induced substantial morphological changes in lesions, including decreased foam cells and increased smooth muscle cells and extracellular matrix content. In parallel, human apoE4 and apoE2 were also expressed in the liver by using recombinant adenoviruses. ApoE4 reduced cholesterol levels to the same extent as did apoE3 and also prevented progression but did not induce significant regression of preexisting lesions. ApoE2 reduced cholesterol levels to a lesser degree than did apoE3 and apoE4 and lesion progression was reduced, but regression was not induced. In summary, (1) regression of preexisting atherosclerotic lesions in apoE-deficient mice can be rapidly induced by hepatic expression of apoE, despite the absence of macrophage-derived apoE; (2) the morphological changes seen in this model of regression resemble those in other animal models, induced over longer periods of time; (3) liver-derived apoE gained access to and was retained by intimal atherosclerotic lesions; and (4) apoE4 was less effective in inducing regression, despite its effects on plasma lipoproteins that were similar to those of apoE3. The rapid regression of preexisiting atherosclerotic lesions induced by apoE gene transfer in apoE-deficient mice could provide a convenient murine model for investigation of the molecular events associated with atherosclerosis regression.

The tyrosine kinases Syk and Lyn exert opposing effects on the activation of protein kinase Akt/PKB in B lymphocytes.

The protein kinase Akt/PKB is a crucial regulator of cell survival in response to mitogenic signals. The increased kinase activity of v-akt, an oncogenic form of Akt/PKB, causes mouse T cell lymphoma, and overexpression of Akt/PKB is associated with progression of several tumor types in human. In this study, we demonstrate that ligation of B cell antigen receptor (BCR) leads to activation of Akt/PKB in B lymphocytes. BCR-induced activation of Akt/PKB required the tyrosine kinase Syk, which was not previously known to regulate Akt/PKB. In contrast, BCR crosslinking of Lyn-deficient B cells resulted in markedly enhanced hyperphosphorylation and activation of Akt/PKB compared with wild-type B cells, indicating that this Src-family kinase acts as an endogenous antagonist of BCR-induced Akt/PKB activation. Lyn inhibited Akt/PKB additively with an okadaic acid-sensitive endogenous phosphatase(s). Expression of exogenous Lyn in mutant cells restored normal BCR-induced phosphorylation of Akt/PKB. Negative regulation of Akt/PKB by Lyn was not dependent on the protein phosphatases SHP-1, SHP-2, or SHIP. Our results show that Lyn provides a mechanism for negative regulation and opposes the effect of Syk on BCR-mediated activation of Akt/PKB. Deregulation of Akt/PKB correlates with the hyperresponsiveness of B cells from Lyn-deficient mice stimulated by BCR crosslinking and may contribute to the autoimmune syndrome that develops in Lyn-deficient animals.

Characterization of anergic anti-DNA B cells: B cell anergy is a T cell-independent and potentially reversible process.

Anti-single stranded DNA (ssDNA) and anti-double stranded DNA (dsDNA) B cells are regulated in non-autoimmune mice. In this report we show that while both anti-ssDNA and anti-dsDNA B cells are blocked in their ability to differentiate into antibody-secreting cells, other phenotypic and functional characteristics distinguish them from one another. Splenic anti-ssDNA B cells are found distributed throughout the B cell follicle, and are phenotypically mature and long-lived. On the other hand, splenic anti-dsDNA B cells are short-lived, exhibit an immature and antigen-experienced phenotype, and localize to the T-B interface of the splenic follicle. Functionally, anti-ssDNA B cells proliferate, albeit suboptimally, in response to anti-IgM, lipopolysaccharide (LPS) and CD40L/IL-4 + anti-IgM stimulation, and tyrosine phosphorylate intracellular proteins upon mIgM cross-linking. Anti-dsDNA B cells, on the other hand, are functionally unresponsive to anti-IgM and LPS stimulation, and do not phosphorylate intracellular proteins, including Syk, upon mIg stimulation. Importantly, anti-DNA B cell anergy is maintained in the absence of T cells since both anti-ssDNA and anti-dsDNA B cells are as efficiently regulated in RAG2(-/-) mice as in their RAG2(+/+) counterparts. Interestingly, the severely anergic state of anti-dsDNA B cells is partially reversible upon stimulation with CD40 ligand and IL-4. In response to these signals, anti-dsDNA B cells remain viable, up-regulate cell surface expression of B7-2 and IgM, and restore their ability to proliferate and phosphorylate Syk upon mIg cross-linking. Collectively, these data suggest that anti-DNA B cell anergy encompasses distinct phenotypes which, even in its most severe form, may be reversible upon stimulation with T cell-derived factors.

Distinct roles of prostaglandin H synthases 1 and 2 in T-cell development.

Prostaglandin G and H synthases, or cyclooxygenases (COXs), catalyze the formation of prostaglandins (PGs). Whereas COX-1 is diffusely expressed in lymphoid cells in embryonic day 15.5 thymus, COX-2 expression is sparse, apparently limited to stromal cells. By contrast, COX-2 is predominant in a subset of medullary stromal cells in three- to five-week-old mice. The isozymes also differ in their contributions to lymphocyte development. Thus, experiments with selective COX-1 inhibitors in thymic lobes from normal and recombinase-activating gene-1 knockout mice support a role for this isoform in the transition from CD4(-)CD8(-) double-negative (DN) to CD4(+)CD8(+) double-positive (DP). Concordant data were obtained in COX-1 knockouts. Pharmacological inhibition and genetic deletion of COX-2, by contrast, support its role during early thymocyte proliferation and differentiation and, later, during maturation of the CD4 helper T-cell lineage. PGE2, but not other PGs, can rescue the effects of inhibition of either isoform, although it acts through distinct EP receptor subtypes. COX-dependent PG generation may represent a mechanism of thymic stromal support for T-cell development.

Suboptimal cross-linking of antigen receptor induces Syk-dependent activation of p70S6 kinase through protein kinase C and phosphoinositol 3-kinase.

Ligation of the B cell antigen receptor (BCR) induces a cascade of signaling pathways that lead to clonal expansion, differentiation, or abortive activation-induced apoptosis of B lymphocytes. BCR-mediated cross-linking induces the rapid phosphorylation of protein tyrosine kinases. However, the pathways leading to the activation of downstream serine/threonine kinases such as mitogen-activated protein kinase, p90(Rsk), and p70S6 kinase (p70(S6k)) that mediate reorganization of the actin cytoskeleton, cell cycle progression, gene transcription, and protein synthesis have not been delineated. We recently demonstrated that cross-linking of BCR leads to activation of p70(S6k) in B lymphocytes. In this report, we demonstrate that multiple protein tyrosine kinase-dependent signal transduction pathways induced by BCR lead to the activation of p70(S6k). These distinct pathways exhibit different thresholds with respect to the extent of receptor cross-linking required for their activation. Activation of p70(S6k) by suboptimal doses of anti-Ig is Syk-dependent and is mediated by protein kinase C and phosphoinositol 3-kinase. Moreover, the activation of p70(S6k) results in phosphorylation of S6 protein which is important for ribosomal protein synthesis and may be coupled to BCR-induced protein and DNA synthesis in primary murine B cells.

Hyaluronan fragments synergize with interferon-gamma to induce the C-X-C chemokines mig and interferon-inducible protein-10 in mouse macrophages.

Hallmarks of chronic inflammation and tissue fibrosis are increased influx of activated inflammatory cells, mediator release, and increased turnover and production of the extracellular matrix (ECM). Recent evidence has suggested that fragments of the ECM component hyaluronan play a role in chronic inflammation by inducing macrophage expression of chemokines. Interferon-gamma (IFN-gamma), an important regulator of macrophage functions, has been shown to induce the C-X-C chemokines Mig and IP-10. These chemokines affect T-cell recruitment and inhibit angiogenesis. The purpose of this investigation was to determine the effect of hyaluronan (HA) on IFN-gamma-induced Mig and IP-10 expression in mouse macrophages. We found a marked synergy between HA and IFN-gamma on Mig and IP-10 mRNA and protein expression in mouse macrophages. This was most significant with Mig, which was not induced by HA alone. The synergy was specific for HA, was not dependent on new protein synthesis, was not mediated by tumor necrosis factor-alpha, was selective for Mig and IP-10, and occurred at the level of gene transcription. These data suggest that the ECM component HA may influence chronic inflammatory states by working in concert with IFN-gamma to alter macrophage chemokine expression.

Inhibition of interferon gamma induced interleukin 12 production: a potential mechanism for the anti-inflammatory activities of tumor necrosis factor.

Inflammation is associated with production of cytokines and chemokines that recruit and activate inflammatory cells. Interleukin (IL) 12 produced by macrophages in response to various stimuli is a potent inducer of interferon (IFN) gamma production. IFN-gamma, in turn, markedly enhances IL-12 production. Although the immune response is typically self-limiting, the mechanisms involved are unclear. We demonstrate that IFN-gamma inhibits production of chemokines (macrophage inflammatory proteins MIP-1alpha and MIP-1beta). Furthermore, pre-exposure to tumor necrosis factor (TNF) inhibited IFN-gamma priming for production of high levels of IL-12 by macrophages in vitro. Inhibition of IL-12 by TNF can be mediated by both IL-10-dependent and IL-10-independent mechanisms. To determine whether TNF inhibition of IFN-gamma-induced IL-12 production contributed to the resolution of an inflammatory response in vivo, the response of TNF+/+ and TNF-/- mice injected with Corynebacterium parvum were compared. TNF-/- mice developed a delayed, but vigorous, inflammatory response leading to death, whereas TNF+/+ mice exhibited a prompt response that resolved. Serum IL-12 levels were elevated 3-fold in C. parvum-treated TNF-/- mice compared with TNF+/+ mice. Treatment with a neutralizing anti-IL-12 antibody led to resolution of the response to C. parvum in TNF-/- mice. We conclude that the role of TNF in limiting the extent and duration of inflammatory responses in vivo involves its capacity to regulate macrophage IL-12 production. IFN-gamma inhibition of chemokine production and inhibition of IFN-gamma-induced IL-12 production by TNF provide potential mechanisms by which these cytokines can exert anti-inflammatory/repair function(s).

CD40-mediated signal transduction in human airway smooth muscle.

CD40 is a member of the TNF receptor family that was initially described on the surface of B cells. Recently, CD40 has also been described on mesenchymal cells, such as endothelial cells and fibroblasts, where engagement by its ligand CD40 ligand can lead to up-regulation of costimulatory and cell adhesion molecules, as well as secretion of proinflammatory cytokines. Since airway inflammation potentially involves cell-cell interactions of T cells and eosinophils (which express CD40 ligand) with airway smooth muscle (ASM) cells, we postulated that ASM may express CD40 and that engagement of ASM CD40 may modulate smooth muscle cell function. We demonstrate that CD40 is expressed on cultured human ASM and that expression can be increased by treatment with TNF-alpha or IFN-gamma. Cross-linking CD40 on ASM resulted in enhanced IL-6 secretion and an increase in intracellular calcium concentrations, which were dependent on calcium influx. We show that CD40-mediated signaling events include protein tyrosine phosphorylation and activation of NF-kappaB. Pretreatment of ASM with the tyrosine kinase inhibitors genistein or herbimycin inhibited the rapid mobilization of calcium induced via CD40, suggesting that calcium mobilization was coupled to activation of protein tyrosine kinases. In addition, inhibition of calcium influx inhibited both CD40-mediated NF-kappaB activation and enhancement of IL-6 secretion. These results delineate a potentially important CD40-mediated signal-transduction pathway in ASM, involving protein tyrosine kinase-dependent calcium mobilization, NF-kappaB activation, and IL-6 production. Together, these results suggest a mechanism whereby T cell/smooth muscle cell interactions may potentiate airway inflammation.

Regulation of the type II oncostatin M receptor expression in lung-derived epithelial cells.

Oncostatin M (OSM) is a potent modulator of human lung-derived epithelial cell function. This cytokine binds two distinct receptor complexes: type I OSM receptor which is also a functional receptor for leukemia inhibitory factor (LIF), and type II OSM-specific receptor. The role of these two distinct receptors in mediating the response of individual cell types to OSM has not been delineated. In contrast to LIF, OSM induces synthesis of alpha1-antichymotrypsin and alpha1-antiproteinase inhibitor in lung-derived epithelial cells. The differential responsiveness to LIF and OSM suggested that the response of lung epithelial cells to OSM may be mediated by the OSM-specific receptor. Therefore, we characterized lung-derived epithelial cells for the expression of type II OSM receptor mRNAs, and the regulation of the mRNAs encoding the components of the OSM-specific receptor by cytokines and dexamethasone.