Our results confirm the overexpression of IL-6 in dcSSc and support the potential of IL-6 as a surrogate marker for clinical outcome in this disease. The data also provide rationale for clinical studies targeting IL-6 trans-signalling as a potential antifibrotic therapy for SSc.

Objective. Connective tissue growth factor (CTGF; CCN2) is overexpressed in systemic sclerosis (SSc) and has been hypothesized to be a key mediator of the pulmonary fibrosis frequently observed in this disease. CTGF is induced by transforming growth factor ␤ (TGF␤) and is a mediator of some profibrotic effects of TGF␤ in vitro. This study was undertaken to investigate the role of CTGF in enhanced expression of type I collagen in bleomycin-induced lung fibrosis, and to delineate the mechanisms of action underlying the effects of CTGF on Col1a2 (collagen gene type I ␣2) in this mouse model and in human pulmonary fibroblasts.Methods. Transgenic mice that were carrying luciferase and ␤-galactosidase reporter genes driven by the Col1a2 enhancer/promoter and the CTGF promoter, respectively, were injected with bleomycin to induce lung fibrosis (or saline as control), and the extracted pulmonary fibroblasts were incubated with CTGF blocking agents. In vitro, transient transfection, promoter/ reporter constructs, and electrophoretic mobility shift assays were used to determine the mechanisms of action of CTGF in pulmonary fibroblasts.Results. In the mouse lung tissue, CTGF expression and promoter activity peaked 1 week after bleomycin challenge, whereas type I collagen expression and Col1a2 promoter activity peaked 2 weeks postchallenge. Fibroblasts isolated from the mouse lungs 14 days after bleomycin treatment retained a profibrotic expression pattern, characterized by greatly elevated levels of type I collagen and CTGF protein and increased promoter activity. In vitro, inhibition of CTGF by specific small interfering RNA and neutralizing antibodies reduced the collagen protein expression and Col1a2 promoter activity. Moreover, in vivo, anti-CTGF antibodies applied after bleomycin challenge significantly reduced the Col1a2 promoter activity by ϳ25%. The enhanced Col1a2 promoter activity in fibroblasts from bleomycintreated lungs was partly dependent on Smad signaling, whereas CTGF acted on the Col1a2 promoter by a mechanism that was independent of the Smad binding site, but was, instead, dependent on the ERK-1/2 and JNK MAPK pathways. The CTGF effect was mapped to the proximal promoter region surrounding the inverted CCAAT box, possibly involving CREB and c-Jun. In human lung fibroblasts, the human COL1A2 promoter responded in a similar manner, and the mechanisms of action also involved ERK-1/2 and JNK signaling.Conclusion. Our results clearly define a direct profibrotic effect of CTGF and demonstrate its contribution to lung fibrosis through transcriptional activation

Monocytes and macrophages participate in a wide variety of host defense mechanisms. Annexin II, a fibrinolytic receptor, binds plasminogen and tissue plasminogen activator (t-PA) independently at the cell surface, thereby enhancing the catalytic efficiency of plasmin production. We demonstrated previously that annexin II on the surface of both cultured monocytoid cells and monocyte-derived macrophages promotes their ability to remodel extracellular matrix. Here, we demonstrate that human peripheral blood monocytes represent the major circulating annexin II-expressing cell. Annexin II supported t-PA-dependent generation of cell surface plasmin and the matrixpenetrating activity of human monocytes. Compared to polymorphonuclear leukocytes, monocytes supported a 12.9-fold greater rate of plasmin generation in the presence of exogenous t-PA, and this activity was largely attributable to annexin II. Likewise, anti-annexin II IgG directed against the t-PA-binding tail domain inhibited plasminogen-dependent, cytokine-directed monocyte migration through extracellular matrix. On differentiation of monocytes to macrophages, there was a 2.4-fold increase in annexin II-specific mRNA, and a 7.9-fold increase in surface annexin II. Thioglycolate-elicited peritoneal macrophages, furthermore, displayed an additional 3.8-fold increase in annexin II surface expression compared with resident cells. Thus, annexin II-mediated assembly of plasminogen and t-PA on monocyte/macrophages contributes to plasmin generation, matrix remodeling, and directed migration. IntroductionAnnexin II belongs to a family of widely distributed, phospholipidbinding, calcium-regulated, peripheral membrane proteins known as the annexins. 1 Annexin II is richly expressed in the epithelial cells of intestine and lung and is also found on the cell surface of vascular endothelial cells, myelomonocytic leukemia cells, 2 and cells of several monocyte-like lines. 3 Through the expression of independent binding sites for both plasminogen and tissue plasminogen activator (t-PA), annexin II assembles these 2 proteins on the cell surface, thereby accelerating the production of plasmin. 4 Kinetic studies indicate that annexin II enhances the catalytic efficiency (k cat /K m ) of t-PA-dependent plasminogen activation by 60-fold, 5 suggesting that endothelial cell surface generation of plasmin likely contributes to fibrinolytic surveillance, thereby promoting blood vessel patency.Monocytes and macrophages are large, motile, phagocytic cells that have a wide range of biosynthetic and secretory activities depending on their exposure to local stimuli and mediators. 6 They participate routinely in host defense mechanisms, including antigen presentation and clearance of debris, and contribute critically to acute and chronic inflammatory responses, pathogen clearance, and wound healing. 7,8 In many pathologic processes, such as atherosclerosis or disorders of lipid metabolism, furthermore, lipid-laden macrophages accumulate in tissues. 9 The monocyte/macrophage, moreover, ma...

COX-2-dependent prostaglandin (PG) E 2 synthesis regulates macrophage MMP expression, which is thought to destabilize atherosclerotic plaques. However, the administration of selective COX-2 inhibitors paradoxically increases the frequency of adverse cardiovascular events potentially through the loss of anti-inflammatory prostanoids and/or disturbance in the balance of pro-and anti-thrombotic prostanoids. To avoid these collateral effects of COX-2 inhibition, a strategy to identify and block specific prostanoid-receptor interactions may be required. We previously reported that macrophage engagement of vascular extracellular matrix (ECM) triggers proteinase expression through a MAPK erk1/2 -dependent increase in COX-2 expression and PGE 2 synthesis. Here we demonstrate that elicited macrophages express the PGE 2 receptors EP1-4. When plated on ECM, their expression of EP2 and EP4, receptors linked to PGE 2 -induced activation of adenylyl cyclase, is strongly stimulated. Forskolin and dibutryl cyclic-AMP stimulate macrophage matrix metalloproteinase (MMP)-9 expression in a dose-dependent manner. However, an EP2 agonist (butaprost) has no effect on MMP-9 expression, and macrophages from EP2 null mice exhibited enhanced COX-2 and MMP-9 expression when plated on ECM. In contrast, the EP4 agonist (PGE 1 -OH) stimulated macrophage MMP-9 expression, which was inhibited by the EP4 antagonist ONO-AE3-208. When compared with COX-2 silencing by small interfering RNA or inhibition by celecoxib, the EP4 antagonist was as effective in inhibiting ECM-induced proteinase expression. In addition, ECM-induced MMP-9 expression was blocked in macrophages in which EP4 was silenced by small interfering RNA. Thus, COX-2-dependent ECM-induced proteinase expression is effectively blocked by selective inhibition of EP4, a member of the PGE 2 family of receptors.Atherosclerosis is a chronic inflammatory disease characterized by lipid accumulation, macrophage recruitment, smooth muscle proliferation, and fibrosis (1, 2). Macrophage proteinase expression compromises the structural integrity of atherosclerotic lesions by degrading components of the extracellular matrix (ECM), 2 which contributes to lesion ulceration or rupture and subsequent sequelae of thrombosis, myocardial infarction, and stroke (3-6). A substantial body of evidence has identified cyclooxygenase (COX)-2 as a targetable component of the signaling pathway responsible for increased proteinase expression by macrophages in atherosclerotic lesions. COX metabolizes arachidonic acid to an unstable endoperoxide, which is then converted to the principal prostaglandins (PG) by specific synthases (7,8). COX-2 expression is elevated in atherosclerotic lesions (9 -14). PGE 2 , an important mediator of the inflammatory response, stimulates proteinase expression by a variety of cells including macrophages (15-18). Both PGE synthase and matrix metalloproteinase (MMP) activities are elevated in regions of symptomatic plaques rich in macrophages and susceptible to rupture (13,19). Moreover, tr...

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