The chemokine CXC ligand 8 (CXCL8)͞IL-8 and related agonists recruit and activate polymorphonuclear cells by binding the CXC chemokine receptor 1 (CXCR1) and CXCR2. Here we characterize the unique mode of action of a small-molecule inhibitor (Repertaxin) of CXCR1 and CXCR2. Structural and biochemical data are consistent with a noncompetitive allosteric mode of interaction between CXCR1 and Repertaxin, which, by locking CXCR1 in an inactive conformation, prevents signaling. Repertaxin is an effective inhibitor of polymorphonuclear cell recruitment in vivo and protects organs against reperfusion injury. Targeting the Repertaxin interaction site of CXCR1 represents a general strategy to modulate the activity of chemoattractant receptors. L eukocyte trafficking into tissue sites of inflammation is directed by chemokines. Chemokines are grouped into four families based on a cysteine motif in the amino terminus of the protein (1, 2). Human CXC ligand 8 (CXCL8)͞IL-8 and related molecules are polymorphonuclear cells (PMN) chemoattractants. Two high-affinity human CXCL8 receptors are known, CXC chemokine receptor 1 (CXCR1) and CXC chemokine receptor 2 (CXCR2). Only one corresponding receptor has been identified in the mouse, and this is recognized by ligands that act as neutrophil attractant, although a mouse orthologue of CXCL8 has not been identified. By recruiting and activating PMN, CXCL8 and related rodent molecules have been implicated in a wide range of disease states characterized by PMN infiltration in organs, including reperfusion injury (RI) (3).G protein-coupled receptors (GPCR) are a prime target for the development of new strategies to control diverse pathologies (4-6). Antichemokine strategies include antibodies, N-terminal modified chemokines, and small-molecule antagonists (7-9). Here we describe a class of GPCR inhibitors that specifically block the inflammatory CXCL8 chemokine receptors CXCR1 and CXCR2 by means of an allosteric noncompetitive mode of interaction and protection against RI. Materials and MethodsReagents. Repertaxin (R)(Ϫ)-2-(4-isobutylphenyl)propionyl methansulfonamide) salified with L-lysine was dissolved in saline. Chemokines were from PeproTech (London). Chemicals, cell culture reagents, and protease inhibitors were from Sigma.Migration. Cell migration of human PMN and monocytes and rodent peritoneal PMN were evaluated in a 48-well microchemotaxis chamber with or without Repertaxin. Agonists (1 nM CXCL8, 10 nM N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), 10 nM CXCL1, 2.5 nM CCL2, 1 nM C5a, 5 nM rat and mouse CXCL1, and 2.5 nM rat and mouse CXCL2) were seeded in the lower compartment. The chemotaxis chamber was incubated for 45 min (human PMN), 1 h (rodent PMN), or 2 h (monocytes). L1.2 migration was evaluated by using 5-m pore-size Transwell filters (Costar) (10). Mutation Analysis of CXCR1 and Signaling. The human CXCR1 ORF was PCR amplified from a CXCR1͞pCEP4 plasmid (kindly provided by P. M. Murphy, National Institutes of Health, Bethesda). Receptor mutants and chimeric re...

Objective Systemic juvenile idiopathic arthritis (JIA) is associated with high levels of interleukin‐6 (IL‐6) in the serum and synovial fluid, and impairment of natural killer (NK) cell function is often observed. This study was undertaken to evaluate a possible link between these 2 biologic findings and whether they may be associated with the development of macrophage activation syndrome, a condition frequently observed in systemic JIA. Methods Splenocytes from wild‐type (WT) or IL‐6–transgenic (Tg) mice were evaluated for NK cell cytotoxicity using a 51Cr‐release assay. Numbers of NK cells and expression of perforin, granzyme B, CD69, and CD107a were evaluated by flow cytometry. Human peripheral blood mononuclear cells (PBMCs) isolated from healthy donors were treated with IL‐6 and cultured in the presence or absence of tocilizumab (TCZ), an IL‐6 receptor blocker. Human polyclonal NK cells from healthy donor PBMCs were evaluated for cell cytotoxicity and expression of perforin, granzyme B, and CD107a. PBMCs harvested from patients with systemic JIA during periods of active or inactive disease were left untreated or treated with IL‐6 in combination with soluble IL‐6 receptor and analyzed for the expression of perforin and granzyme B. Results Splenic NK cell cytotoxicity was reduced in IL‐6–Tg mice compared to WT mice. Levels of CD69 and CD107a showed no significant differences, whereas expression of perforin and granzyme B was impaired in NK cells from IL‐6–Tg mice. Exposure of human peripheral blood NK cells to IL‐6 led to reduced expression of perforin and granzyme B. Culturing human polyclonal NK cells in the presence of TCZ significantly increased cell cytotoxicity, and also increased expression of perforin and granzyme B. In patients with systemic JIA, a reduction in IL‐6 plasma levels during disease remission correlated with the rescue of perforin and granzyme B expression in NK cells from these patients. Conclusion In both mice and humans, IL‐6 down‐modulated the cytotoxic activity of NK cells. This decrease was associated with reduced perforin and granzyme B levels in the absence of altered granule exocytosis.

Peritoneal dialysis is a form of renal replacement alternative to the hemodialysis. During this treatment, the peritoneal membrane acts as a permeable barrier for exchange of solutes and water. Continual exposure to dialysis solutions, as well as episodes of peritonitis and hemoperitoneum, can cause acute/chronic inflammation and injury to the peritoneal membrane, which undergoes progressive fibrosis, angiogenesis, and vasculopathy, eventually leading to discontinuation of the peritoneal dialysis. Among the different events controlling this pathological process, epithelial to mesenchymal transition of mesothelial cells plays a main role in the induction of fibrosis and in subsequent functional deterioration of the peritoneal membrane. Here, the main extracellular inducers and cellular players are described. Moreover, signaling pathways acting during this process are elucidated, with emphasis on signals delivered by TGF-β family members and by Toll-like/IL-1β receptors. The understanding of molecular mechanisms underlying fibrosis of the peritoneal membrane has both a basic and a translational relevance, since it may be useful for setup of therapies aimed at counteracting the deterioration as well as restoring the homeostasis of the peritoneal membrane.

SUMMARYEpithelial-to-mesenchymal transition (EMT) occurs in fibrotic diseases affecting the kidney, liver and lung, and in the peritoneum of patients undergoing peritoneal dialysis. EMT in the peritoneum is linked to peritoneal membrane dysfunction, and its establishment limits the effectiveness of peritoneal dialysis. The molecular regulation of EMT in the peritoneum is thus of interest from basic and clinical perspectives. Treatment of primary human mesothelial cells (MCs) with effluent from patients undergoing peritoneal dialysis induced a genuine EMT, characterized by downregulated E-cadherin and cytokeratin expression, cell scattering, and spindle-like morphology. This EMT was replicated by co-stimulation with transforming growth factor (TGF)-β1 and interleukin (IL)-1β. Retroviral overexpression of a mutant inhibitor of kappaB (IκB) demonstrated that NF-κB activation is required for E-cadherin and cytokeratin downregulation during EMT. Pre-treatment with the MAP kinase kinase (MEK)-1/2 inhibitor U0126 showed that cytokinetriggered NF-κB nuclear translocation and transcriptional activity are mediated by activation of extracellular regulated kinase (ERK). Cytokine-mediated induction of mRNA expression of the transcription factor Snail1, a repressor of E-cadherin expression and a potent inducer of EMT, was prevented by blockade of ERK or NF-κB. Finally, blockade of ERK/NF-κB signaling in ex vivo MCs that were cultured from peritoneal dialysis effluents reverted cells to an epithelioid morphology, upregulated E-cadherin and cytokeratin expression, and downregulated Snail1 expression. Modulation of the ERK/NF-κB/Snail1 pathway may provide a means of counteracting the progressive structural and functional deterioration of the peritoneal membrane during peritoneal dialysis.

SummaryThe transcriptional regulator YAP orchestrates many cellular functions, including tissue homeostasis, organ growth control, and tumorigenesis. Mechanical stimuli are a key input to YAP activity, but the mechanisms controlling this regulation remain largely uncharacterized. We show that CAV1 positively modulates the YAP mechanoresponse to substrate stiffness through actin-cytoskeleton-dependent and Hippo-kinase-independent mechanisms. RHO activity is necessary, but not sufficient, for CAV1-dependent mechanoregulation of YAP activity. Systematic quantitative interactomic studies and image-based small interfering RNA (siRNA) screens provide evidence that this actin-dependent regulation is determined by YAP interaction with the 14-3-3 protein YWHAH. Constitutive YAP activation rescued phenotypes associated with CAV1 loss, including defective extracellular matrix (ECM) remodeling. CAV1-mediated control of YAP activity was validated in vivo in a model of pancreatitis-driven acinar-to-ductal metaplasia. We propose that this CAV1-YAP mechanotransduction system controls a significant share of cell programs linked to these two pivotal regulators, with potentially broad physiological and pathological implications.

The MAP kinase (MAPK) p38 plays a key role in regulating inflammatory responses. Here, we demonstrate that beta1 integrin ligation on human NK cells results in the activation of the p38 MAPK signaling pathway, which is required for integrin-triggered IL-8 production. In addition, we identified some of the upstream events accompanying the beta1 integrin-mediated p38 MAPK activation, namely, the activation of the Rac guanine nucleotide exchange factor (GEF) p95 Vav, the small G protein Rac1, and the cytoplasmic kinases Pak1 and MKK3. Finally, we provide direct evidence that p95 Vav and Rac control the activation of p38 MAPK triggered by beta1 integrins.

Objective. To investigate whether prolonged exposure to interleukin-6 (IL-6) affects the inflammatory response induced by Toll-like receptor (TLR) ligands.Methods. IL-6-transgenic mice and wild-type mice were stimulated with different TLR ligands; survival rates, blood cell counts, and biochemical parameters were analyzed. Murine splenic mononuclear cells and peritoneal macrophages were stimulated with lipopolysaccharide (LPS), lipoteichoic acid, poly(I-C), or CpG. Human macrophages were cultured for 4 days in the presence of IL-6 and then stimulated with LPS. Inflammatory cytokine expression was measured by enzyme-linked immunosorbent assay or reverse transcription-polymerase chain reaction. Activation of STAT-3, ERK-1/2 (MAPK), and p65 NF-B was evaluated by Western blotting or confocal analysis.Results. Treatment of IL-6-transgenic mice with TLR ligands led to an increased fatality rate and elevated levels of IL-1␤, tumor necrosis factor ␣ (TNF␣), IL-6, and IL-18. Macrophages from IL-6-transgenic mice produced increased levels of inflammatory cytokines, which were associated with increased phosphorylation of STAT-3 and ERK-1/2 and with increased NF-B nuclear translocation. Human macrophages treated with IL-6 and then stimulated with LPS showed elevated levels of cytokines and similarly elevated signaling pathway activation. After LPS administration, IL-6-transgenic mice showed an increase in ferritin and soluble CD25 levels, as well as a decrease in platelet and neutrophil counts and in hemoglobin levels compared to wild-type mice.Conclusion. Our findings indicate that prolonged exposure to IL-6 in vivo and in vitro leads to an exaggerated inflammatory response to TLR ligands. Hematologic and biochemical abnormalities in IL-6-transgenic mice treated with LPS show striking similarities to macrophage activation syndrome.

No abstract