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Objective: To investigate the role of macrophage migration inhibitory factor (MIF) in the pathogenesis of ankylosing spondylitis (AS). Methods: Patients who met the modified New York criteria for AS were recruited for the study. Healthy volunteers, rheumatoid arthritis patients, and osteoarthritis patients were included as controls. Based on the annual rate of increase in modified Stoke AS Spine Score (mSASSS), AS patients were classified as progressors or nonprogressors. MIF levels in serum and synovial fluid were quantitated by enzyme-linked immunosorbent assay. Predictors of AS progression were evaluated using logistic regression analysis. Immunohistochemical analysis of ileal tissue was performed to identify MIF-producing cells. Flow cytometry was used to identify MIF-producing subsets, expression patterns of the MIF receptor (CD74), and MIF-induced tumor necrosis factor (TNF) production in the peripheral blood. MIF-induced mineralization of osteoblast cells (SaOS-2) was analyzed by alizarin red S staining, and Western blotting was used to quantify active β-catenin levels. Results: Baseline serum MIF levels were significantly elevated in AS patients compared to healthy controls and were found to independently predict AS progression. MIF levels were higher in the synovial fluid of AS patients, and MIF-producing macrophages and Paneth cells were enriched in their gut. MIF induced TNF production in monocytes, activated β-catenin in osteoblasts, and promoted the mineralization of osteoblasts. Conclusion: Our findings indicate an unexplored pathogenic role of MIF in AS and a link between inflammation and new bone formation.

Inflammation is a multistep process triggered when innate immune cells - for example, DCs - sense a pathogen or injured cell or tissue. Edema formation is one of the first steps in the inflammatory response; it is fundamental for the local accumulation of inflammatory mediators. Injection of LPS into the skin provides a model for studying the mechanisms of inflammation and edema formation. While it is known that innate immune recognition of LPS leads to activation of numerous transcriptional activators, including nuclear factor of activated T cells (NFAT) isoforms, the molecular pathways that lead to edema formation have not been determined. As PGE2 regulates many proinflammatory processes, including swelling and pain, and it is induced by LPS, we hypothesized that PGE2 mediates the local generation of edema following LPS exposure. Here, we show that tissue-resident DCs are the main source of PGE2 and the main controllers of tissue edema formation in a mouse model of LPS-induced inflammation. LPS exposure induced expression of microsomal PGE synthase-1 (mPGES-1), a key enzyme in PGE2 biosynthesis. mPGES-1 activation, PGE2 production, and edema formation required CD14 (a component of the LPS receptor) and NFAT. Therefore, tissue edema formation induced by LPS is DC and CD14/NFAT dependent. Moreover, DCs can regulate free antigen arrival at the draining lymph nodes by controlling edema formation and interstitial fluid pressure in the presence of LPS. We therefore suggest that the CD14/NFAT/mPGES-1 pathway represents a possible target for antiinflammatory therapies.

5-Lipoxygenase (5-LO) is a potential target for pharmacological intervention with various inflammatory and allergic diseases. Starting from the natural dual 5-LO/microsomal prostaglandin E2 synthase (mPGES)-1 inhibitor embelin (2,5-dihydroxy-3-undecyl-1,4-benzoquinone, 2) that suppresses 5-LO activity in human primary leukocytes with IC50 Combining double low line 0.8-21/4M, we synthesized 48 systematically modified derivatives of 2. We modified the 1,4-quinone to 1,2-quinone, mono- or bimethylated the hydroxyl groups, and varied the C11-n-alkyl residue (C4- to C16-n-alkyl or prenyl) of 2. Biological evaluation yields potent analogues being superior over 2 and obvious structure-activity relationships (SAR) for inhibition of 5-LO. Interestingly, conversion to 1,2-benzoquinone and bimethylation of the hydroxyl moieties strongly improves 5-LO inhibition in polymorphonuclear leukocytes versus 2 up to 60-fold, exemplified by the C12-n-alkyl derivative 22c (4,5-dimethoxy-3-dodecyl-1,2-benzoquinone) with IC50 Combining double low line 29 nM. Regarding inhibition of mPGES-1, none of the novel benzoquinones could outperform the parental compound 2 (IC50 Combining double low line 0.21 1/4M), and only modest suppressive effects on 12- and 15-LOs were evident. Together, our detailed SAR study reveals 22c as highly potent 5-LO-selective lead compound in intact cells that warrants further preclinical evaluation as anti-inflammatory agent. © 2015 Published by Elsevier Masson SAS.

Caveolae, the flask-shaped membrane invaginations abundant in endothelial cells, have acquired a prominent role in signal transduction. Evidence, that events occurring in caveolae participate in cell survival and angiogenesis, has been recently substantiated by the identification of two novel caveolar constituents: prostacyclin synthase (PGIS) and the cellular form of prion protein (PrPc). We have shown that PGIS, previously described as an endoplasmic reticulum component, is bound to caveolin-1 (cav-1) and localized in caveolae in human endothelial cells. By generating prostacyclin, PGIS is involved in angiogenesis. Previous observations regarding the localization of PrPc in caveolae-like membrane domains (CLDs) have been recently confirmed and extended. It has been demonstrated that PrPc is bound to cav-1 and, by recruiting Fyn kinase, can participate in signal transduction events connected to cell survival and differentiation. The new entries of PGIS and PrPc in caveolar components place caveolae and CLDs at the centre of a network, where cells decide whether to proliferate or differentiate and whether to survive or to suicide by apoptosis. © 2002 Elsevier Science Inc. All rights reserved.

A variety of evidence suggests that endothelial cell functions are impaired in altered gravity conditions. Nevertheless, the effects of hypergravity on endothelial cell physiology remain unclear. In this study we cultured primary human endothelial cells under mild hypergravity conditions for 24-48 h, then we evaluated the changes in cell cycle progression, caveolin1 gene expression and in the caveolae status by confocal microscopy. Moreover, we analyzed the activity of enzymes known to be resident in caveolae such as endothelial nitric oxide synthase (eNOS), cycloxygenase 2 (COX-2), and prostacyclin synthase (PGIS). Finally, we performed a three-dimensional in vitro collagen gel test to evaluate the modification of the angiogenic responses. Results indicate that hypergravity shifts endothelial cells to G 0/G1 phase of cell cycle, reducing S phase, increasing caveolin1 gene expression and causing an increased distribution of caveolae in the cell interior. Hypergravity also increases COX-2 expression, nitric oxide (NO) and prostacyclin (PG12) production, and inhibits angiogenesis as evaluated by 3-D collagen gel test, through a pathway not involving apoptosis. Thus, endothelial cell caveolae may be responsible for adaptation of endothelium to hypergravity and the mechanism of adaptation involves an increased caveolin1 gene expression coupled to upregulation of vasodilators as NO and PG12. © 2003 Wiley-Liss, Inc.