Objective. To explore the potential involvement of the chemokine system in synoviocyte-mediated tissue destruction in rheumatoid arthritis (RA), we studied the expression profile of chemokine receptors and their function in the migration, proliferation, and matrix metalloproteinase (MMP) production of cultured fibroblast-like synoviocytes (FLS) from RA patients.Methods. The presence of CC and CXC chemokine receptors on cultured FLS was studied at the messenger RNA (mRNA) level by reverse transcriptasepolymerase chain reaction and at the cell surface expression level by flow cytometry. Variations in cytosolic calcium influx induced by chemokine stimulation were assessed by flow cytometry on Fura Red-preloaded FLS. Two-compartment transwell chambers were used for FLS chemotaxis assays. Cell growth was measured by a fluorescence-based proliferation assay. Gelatinase and collagenase activities were determined by a fibril degradation assay and zymography.Results. FLS constitutively expressed the receptors CCR2, CCR5, CXCR3, and CXCR4, both at the cell surface and mRNA levels, but failed to express CCR3 and CCR6. Significant intracytosolic calcium influx was observed on FLS challenged with monocyte chemotactic protein 1 (MCP-1), stromal cell-derived factor 1␣ (SDF-1␣), and interferon-inducible protein 10 (IP-10). Stimulation with MCP-1, SDF-1␣, IP-10, and monokine induced by interferon-␥ enhanced the migration and proliferation of FLS. These chemokines, in addition to RANTES, increased in a dose-and time-dependent manner the gelatinase and collagenase activities in cell-free supernatants of cultured FLS. Interestingly, the chemokine-mediated up-regulation of MMP activities was significantly abrogated by the presence of anti-interleukin-1␤, but not anti-tumor necrosis factor ␣, blocking antibodies.Conclusion. These data suggest that through modulation of the migration, proliferation, and MMP production by FLS, the chemokine system may play a more direct role in the destructive phase of RA than is currently suspected, and thus emphasize the relevance of chemokines and their receptors as potential therapeutic targets in this disease.

A disintegrin and metalloproteinase domain (ADAM) proteins are a family of transmembrane glycoproteins with heterogeneous expression profiles and proteolytic, cell-adhesion, -fusion, and -signaling properties. One of its members, ADAM-8, is expressed by several cell types including neurons, osteoclasts, and leukocytes and, although it has been implicated in osteoclastogenesis and neurodegenerative processes, little is known about its role in immune cells. In this study, we show that ADAM-8 is constitutively present both on the cell surface and in intracellular granules of human neutrophils. Upon in vitro neutrophil activation, ADAM-8 was mobilized from the granules to the plasma membrane, where it was released through a metalloproteinase-dependent shedding mechanism. Adhesion of resting neutrophils to human endothelial cells also led to up-regulation of ADAM-8 surface expression. Neutrophils isolated from the synovial fluid of patients with active rheumatoid arthritis expressed higher amounts of ADAM-8 than neutrophils isolated from peripheral blood and the concentration of soluble ADAM-8 in synovial fluid directly correlated with the degree of joint inflammation. Remarkably, the presence of ADAM-8 both on the cell surface and in suspension increased the ectodomain shedding of membrane-bound L-selectin in mammalian cells. All these data support a potential relevant role for ADAM-8 in the function of neutrophils during inflammatory response.

BackgroundB cells exert their pathogenic action in rheumatoid arthritis (RA) locally in the synovium. This study was undertaken to elucidate the chemokines responsible for the recruitment of B cells in the inflamed synovium, taking into account that the rich chemokine milieu present in the synovial tissue can fine-tune modulate discrete chemokine receptors.MethodsExpression levels of chemokine receptors from the CC and CXC family, as well as CD27, were assessed by flow cytometry in CD20+ mononuclear cells isolated from the peripheral blood (PB) and synovial fluid (SF) of RA and psoriatic arthritis patients. Transwell experiments were used to study migration of B cells in response to a chemokine or in the presence of multiple chemokines.ResultsB cells from the SF of arthritis patients showed a significant increase in the surface expression of CCR1, CCR2, CCR4, CCR5 and CXCR4 with respect to PB. Conversely, SF B cells expressed consistently lower amounts of CXCR5, CXCR7 and CCR6, independent of CD27 expression. Analysis of permeabilized B cells suggested internalization of CXCR5 and CCR6 in SF B cells. In Transwell experiments, CCL20 and CXCL13, ligands of CCR6 and CXCR5, respectively, caused a significantly higher migration of B cells from PB than of those from SF of RA patients. Together, these two chemokines synergistically increased B-cell migration from PB, but not from SF.ConclusionsThese results suggest that CXCL13 and CCL20 might play major roles in RA pathogenesis by acting singly on their selective receptors and synergistically in the accumulation of B cells within the inflamed synovium.Electronic supplementary materialThe online version of this article (10.1186/s13075-018-1611-2) contains supplementary material, which is available to authorized users.

The P-selectin glycoprotein ligand-1 (PSGL-1) is involved in the initial contact of leukocytes with activated endothelium, and its adhesive function is regulated through its proteolytic processing. We have found that the metalloprotease ADAM8 is both associated with PSGL-1 through the ezrin-radixin-moesin actin-binding proteins and able to cause the proteolytic cleavage of this adhesion receptor. Accordingly, ADAM8 knockdown increases PSGL-1 expression, and functional assays show that ADAM8 is able to reduce leukocyte rolling on P-selectin and hence on activated endothelial cells. We conclude that ADAM8 modulates the expression and function of PSGL-1.Key words: ADAM8 . Adhesion . Cell migration . ERM . Neutrophil . PSGL-1 See accompanying Commentary by Zarbock and RossaintIntroduction ADAM family is a group of transmembrane glycoproteins that possess proteolytic and signaling properties and are implicated in both cell adhesion and cell fusion processes [1]. ADAMs are usually activated by furin or other convertases as well as autocatalytically, in the case of ADAM8 [2]. It has been described that ADAM8 cleaves important cell surface proteins [3,4], cytokines and growth factors [5]. ADAM8 is overexpressed under several pathological conditions involving inflammation and remodeling of the extracellular matrix, including malignant diseases and asthma [6][7][8].P-selectin glycoprotein ligand-1 (PSGL-1), through its interaction with P-, E-and L-selectins, mediates the tethering and rolling of leukocytes on endothelial cells prior to their extravasation [9,10], triggers the activation of transcription factors like cFos [11] in leukocytes and induces the generation of tolerogenic DCs which promote the differentiation of Treg cells [12]. Although it was described that PSGL-1 was a substrate of the proteases BACE1 and ADAM10 [13], neither the physiological context of cleavage nor the mechanism responsible for its shedding have been identified so far. In this work, we demonstrate that in leukocytes SHORT COMMUNICATION Ã These authors contributed equally to this work. 3436Frontline ADAM8 associates with PSGL-1 through ezrin-radixin-moesin (ERM) proteins, and that this interaction modulates the expression and function of this adhesion receptor. Results and discussionAssociation of PSGL-1 with ADAM8To identify intracellular molecules able to associate with PSGL-1, we performed a proteomic analysis of HL-60 cell lysates pulled down with the cytoplasmic tail of PSGL-1 fused to GST (GST-PSGL-1cyt) [11]. This analysis revealed the presence of a 98-kDa protein that corresponded to ADAM8 (data not shown). Additional pull-down experiments performed with fragments of the cytoplasmic tail of PSGL-1 fused to GST, detected an additional protein of 75-80 kDa, which likely corresponded to a cleaved form of ADAM8 (Fig. 1A, left panel). To map the region of PSGL-1 involved in this interaction, pull-down experiments from lysates of HL-60 cells were performed with different fragments of the cytoplasmic tail of PSGL-1 fused to GST. We found th...

Adrenergic receptors are expressed on the surface of inflammation-mediating cells, but their potential role in the regulation of the inflammatory response is still poorly understood. The objectives of this work were to study the effects of α2-adrenergic agonists on the inflammatory response in vivo and to determine their mechanism of action. In two mouse models of inflammation, zymosan air pouch and thioglycolate-induced peritonitis models, the i.m. treatment with xylazine or UK14304, two α2-adrenergic agonists, reduced neutrophil migration by 60%. The α2-adrenergic antagonist RX821002 abrogated this effect. In flow cytometry experiments, the basal surface expression of L-selectin and CD11b was modified neither in murine nor in human neutrophils upon α2-agonist treatment. Similar experiments in HUVEC showed that UK14304 prevented the activation-dependent upregulation of ICAM-1. In contrast, UK14304 augmented electrical resistance and reduced macromolecular transport through a confluent HUVEC monolayer. In flow chamber experiments, under postcapillary venule-like flow conditions, the pretreatment of HUVECs, but not neutrophils, with α2-agonists decreased transendothelial migration, without affecting neutrophil rolling. Interestingly, α2-agonists prevented the TNF-α–mediated decrease in expression of the adherens junctional molecules, VE-cadherin, β-catenin, and plakoglobin, and reduced the ICAM-1–mediated phosphorylation of VE-cadherin by immunofluorescence and confocal analysis and Western blot analysis, respectively. These findings indicate that α2-adrenoceptors trigger signals that protect the integrity of endothelial adherens junctions during the inflammatory response, thus pointing at the vascular endothelium as a therapeutic target for the management of inflammatory processes in humans.

Diphenylamine-based nonsteroidal antiinflammatory drugs (NSAIDs) are able to cause in vitro the shedding of L-selectin. The aim of this work was to determine the physiologic relevance of L-selectin shedding in the antiinflammatory effect exerted by NSAIDs in vivo. Chemical compounds structurally related to NSAIDs -including diphenylamine, N-phenylanthranilic acid (N-Ph), diphenylacetic acid -as well as the traditional NSAID indomethacin were studied using the zymosan air-pouch mouse model. Animals intramuscularly pretreated with indomethacin or N-Ph, but not with diphenylamine or diphenylacetic acid, showed a significant dose-dependent reduction in the number of neutrophils compared with untreated animals (N-Ph, IC50 = 6.7 mg/kg). Except for indomethacin, none of these compounds caused any significant reduction in cyclooxygenase-1 activity in vivo. In flow chamber experiments, N-Ph reduced the capability of human neutrophils to pass across the endothelial barrier by interfering with leukocyte rolling step on HUVEC. N-Ph, but not diphenylacetic acid, induced activationindependent L-selectin shedding in mouse neutrophils. Interestingly, N-Ph exerted an antiinflammatory effect similar to that of the anti-L-selectin blocking antibody Mel-14, although no additive action was observed when both compounds were combined. These data suggest that the L-selectin shedding induced by NSAIDs may be involved in the antiinflammatory action exerted by these compounds in clinical settings. Keywords:Air-pouch mouse model r L-selectin r Nonsteroidal antiinflammatory drugs r N-phenylanthranilic acid See accompanying Commentary by Zarbock and RossaintCorrespondence: Prof. Federico Díaz-González e-mail: federico.diaz.gonzalez@gmail.com IntroductionThe recruitment of leukocytes into tissues during the inflammatory response is preceded by a highly coordinated sequence of adhesive events between flowing leukocytes and endothelial cells, a process known as the adhesion cascade. Members of three major C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 56Ada Herrera-García et al. Eur. J. Immunol. 2013. 43: 55-64 adhesion receptor families have been implicated in this cascade: selectins, integrins, and the immunoglobulin superfamily [1,2]. In the field of inflammation, much effort is currently focused on developing antagonists of adhesion receptors, an approach known as antiadhesive therapy. This strategy is based on the assumption that if any one of the sequential steps of the adhesion cascade is inhibited, the inflammatory response is consequently suppressed or, at least, ameliorated [3,4]. Although antiadhesive therapies targeting the major leukocyte integrins LFA-1, Mac-1, and VLA-4 have proven to be relatively successful in several human inflammatory disorders [4], the inhibition of selectins and their ligands has only proven beneficial in certain animal models of inflammation [5][6][7][8], with apparently only limited clinical effects on human inflammatory conditions [9]. Nonsteroidal antiinflammatory drugs (NSAIDs...

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Neutrophils destroy invading microorganisms by phagocytosis by bringing them into contact with bactericidal substances, among which ROS are the most important. However, ROS also function as important physiological regulators of cellular signaling pathways. Here, we addressed the involvement of oxygen derivatives in the regulation of human neutrophil rolling, an essential component of the inflammatory response. Flow experiments using dihydroethidium‐preloaded human neutrophils showed that these cells initiate an early production of intracellular ROS during the rolling phase of the adhesion cascade, a phenomenon that required cell rolling, and the interaction of the chemokine receptor CXCR2 with their ligand CXCL8. Flow cytometry experiments demonstrated that L‐selectin shedding in neutrophils is triggered by ROS through an autocrine–paracrine mechanism. Preincubation of neutrophils with the NADPH oxidase complex inhibitor diphenyleniodonium chloride significantly increased the number of rolling neutrophils on endothelial cells. Interestingly, the same effect was observed when CXCL8 signaling was interfered using either a blocking monoclonal antibody or an inhibitor of its receptor. These findings indicate that, in response to CXCL8, neutrophils initiate ROS production during the rolling phase of the inflammatory response. This very early ROS production might participate in the modulation of the inflammatory response by inducing L‐selectin shedding in neutrophils.