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Background B 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. Methods Expression 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. Results B 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. Conclusions These 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 material The online version of this article (10.1186/s13075-018-1611-2) contains supplementary material, which is available to authorized users.

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., This work was supported by grants from the Fondo de Investigaciones Sanitarias of Spain Grants PI12/02499 and PI15/01810 (to. F.D.‐G.), co‐funded by the European Regional Development Fund. This work was also supported by Asociación para la Ayuda a la Investigación en Reumatología del Hospital Universitario de Canarias (REUNINVES) and by the Spanish Society of Rheumatology.

Primary Sjögren’s syndrome (pSS) is an autoimmune exocrinopathy in which the role that the immune response plays in reducing exocrine gland function, including the glandular microenvironment of cytokines, has not been fully understood. Epithelial cells from biopsies of human parotid gland (HPG) were used to establish a model of human salivary gland in vitro. In this model, the functional consequences of several proinflammatory soluble factors present in the pSS glandular microenvironment were assessed. Stimulation with isoproterenol and calcium produced a significant increase in the basal activity of amylase in the HPG cell supernatants. Under these conditions, the presence of TNF-α and CXCL12 increased amylase mRNA cellular abundance, but reduced the amylase activity in the cell-free supernatant in a dose-dependent manner. IL-1β and IFN-γ, but not TGF-β, also diminished amylase secretion by HPG cells. These results suggest that the glandular microenvironment of cytokine, by acting post-transcriptionally, may be responsible, at least in part, for the reduced exocrine function observed in pSS patients. These data may help to a better understanding of the pathogenesis of SS, which in turn would facilitate the identification of new therapeutic targets for this disorder.

Background: The precise mechanism linking systemic inflammation with insulin resistance (IR) in rheumatoid arthritis (RA) remains elusive. In the present study, we determined whether the incretin-insulin axis and incretin effect are disrupted in patients with RA and if they are related to the IR found in these patients. Methods: We conducted a cross-sectional study that encompassed 361 subjects without diabetes, 151 patients with RA, and 210 sex-matched control subjects. Insulin, C-peptide, glucagon-like peptide-1 (GLP-1), gastric inhibitory polypeptide (GIP), dipeptidyl peptidase 4 (DPP-4) soluble form, and IR indexes by homeostatic model assessment (HOMA2) were assessed. A multivariable analysis adjusted for IR-related factors was performed. Additionally, ten patients and ten control subjects underwent a 566-kcal meal test so that we could further study the postprandial differences of these molecules between patients and control subjects. Results: Insulin, C-peptide, and HOMA2-IR indexes were higher in patients than in control subjects. This was also the case for GLP-1 (0.49 ± 1.28 vs. 0.71 ± 0.22 ng/ml, p = 0.000) and GIP (0.37 ± 0.40 vs. 1.78 ± 0.51 ng/ml, p = 0.000). These differences remained significant after multivariable adjustment including glucocorticoid intake. Disease Activity Score in 28 joints with erythrocyte sedimentation rate (? coefficient 46, 95% CI 6?87, p = 0.026) and Clinical Disease Activity Index (? coefficient 7.74, 95% CI 1.29?14.20, p = 0.019) were associated with DPP-4 serum levels. GLP-1 positively correlated with ?-cell function (HOMA2 of ?-cell production calculated with C-peptide) in patients but not in control subjects (interaction p = 0.003). The meal test in patients with RA revealed a higher total and late response AUC for glucose response, a later maximal response of C-peptide, and a flatter curve in GIP response. Conclusions: The incretin-insulin axis, both during fasting and postprandial, is impaired in patients with RA. This work was supported by grants from the Spanish Ministry of Health, Subdirección General de Evaluación y Fomento de la Investigación, Plan Estatal de Investigación Científica y Técnica y de Innovación 2013–2016 Instituto de Salud Carlos III [ISCIII] PI14/00394) and by the Fondo Europeo de Desarrollo Regional (FEDER) (to IFA). The research of MAGG was supported by European Union FEDER funds and by the “Fondo de Investigación Sanitaria” (grants PI06/0024, PS09/00748, PI12/00060, and PI15/00525) of the Instituto de Salud Carlos III (ISCIII; Spanish Health Ministry). The research of MAGG was also partially supported by RETICS Programs RD12/0009 (RIER) and RD12/0009/0013 from the ISCIII (Spanish Health Ministry).

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.

Non-steroidal anti-inflammatory drugs (NSAIDs) induce the shedding of L-selectin in human neutrophils through a mechanism still not well understood. In this work we studied both the functional effect of NSAIDs on the neutrophils/endothelial cells dynamic interaction, and the potential involvement of reactive oxygen species (ROS) in the NSAIDs-mediated down-regulation of L-selectin. When human neutrophils were incubated with diclofenac, a significant reduction in the number of cells that rolled on activated endothelial cells was observed. Different NSAIDs (flufenamic acid, meclofenamic acid, diclofenac, indomethacin, nimesulide, flurbiprofen, meloxicam, phenylbutazone, piroxicam, ketoprofen and aspirin) caused variable increase in neutrophil intracellular ROS concentration, which was inversely proportional to the change produced in L-selectin surface expression. Pre-incubation of neutrophils with superoxide dismutase, but not with catalase, showed both a significant protective effect on the L-selectin down-regulation induced by several NSAIDs and a diminished effect of diclofenac on neutrophil rolling. Interestingly, diclofenac and flufenamic acid but not piroxicam significantly increased the extracellular superoxide anion production by neutrophils, and inhibition of nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase activity with diphenyleneiodonium prevented the down-regulation of L-selectin by diclofenac. In accordance with these results, neutrophils from patients with chronic granulomatous disease, a hereditary disease in which neutrophils show a reduced capacity to form superoxide radicals, exhibited a lower down-regulation of L-selectin (IC50: 15.3 μg/ml) compared to normal controls (IC50: 5.6 μg/ml) in response to diclofenac. Conclusion A group of NSAIDs is capable of interfering with the ability of neutrophils to interact with endothelial cells by triggering L-selectin-shedding through the NADPH-oxidase-dependent generation of superoxide anion at the plasma membrane.

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 physio-logic relevance of L-selectin shedding in the antiinflammatory effect exerted by NSAIDs in vivo. Chemical compounds structurally related to NSAIDs - including diphenyl-amine, 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 diphenyl-amine 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 activation-independent 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.

Objective.To study the qualitative and quantitative phenotypic changes that occur in molecules involved in antigen presentation and costimulation in synovial B cells from rheumatoid arthritis (RA) and psoriatic arthritis (PsA).Methods.The presence of HLA-DR, CD86, and CD40 in CD20+ cells was studied in RA synovium biopsies using immunohistochemistry and immunofluorescence. Expression was assessed by flow cytometry of the Class II molecules CD40, CD86, CD23, and CD27 on B cells from the synovial fluid (SF), with respect to peripheral blood, from 13 patients with RA and 15 patients with PsA. Expression of interferon-induced protein with tetratricopeptide repeats 4 (IFIT4) in immune-selected CD20+ cells from patients with RA was assessed by quantitative realtime PCR.Results.Infiltrating synovial RA, B cells expressed HLA-DR, CD40, and CD86. Increased expression of CD86, HLA-DR, and HLA-DQ in B cells from SF was found in patients with RA and PsA. HLA-DP was also elevated in rheumatoid SF B cells; conversely, a significantly lower expression was observed in SF from patients with PsA. CD40 expression was increased in SF B cells from PsA, but not in patients with RA. Interestingly, CD20 surface expression level was significantly lower in SF B cells (CD19+, CD138−) from RA, but not in patients with PsA. CD27 upregulation and CD23 downregulation were observed in synovial B cells in both pathologies. Finally, a 4-fold increase in IFIT4 mRNA content was shown in B cells from SF in patients with RA.Conclusion.Synovial B cells from patients with RA and patients with PsA express different antigen-presenting cell phenotypes, suggesting that this cell type plays a dissimilar role in the pathogenesis of each disease.

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 transcriptase–polymerase 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 1α (SDF-1α), and interferon-inducible protein 10 (IP-10). Stimulation with MCP-1, SDF-1α, IP-10, and monokine induced by interferon-γ 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-1β, but not anti–tumor necrosis factor α, 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.

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. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim., This work was supported by grants from the Spanish Ministry of Health (Fondo de Investigaciones Sanitarias) to A. Urzainqui (FIS-PI080894) and to F. Díaz González (FIS 09/02209), by grants from Ministerio de Ciencia e Innovación to F. S. M. (SAF2008-02635) and to F. M. (SAF2008-02251) and by the Redes RIER, RTICC (RD06/0020/1037) and RECAVA del Instituto de Salud Carlos III and RTICCR.

Objectives Rheumatoid Arthritis (RA) patients show alterations in cholesterol and lypoproteins levels. These disturbances seem to be related to the inflammation that takes place in the disease, but the pathological mechanisms that leed to it are unknown. Cholesterol ester transfer protein (CETP) is an enzyme that allows transference of cholesterol ester from HDL to LDL-cholesterol particles. CETP has recently gained interest as therapeutic goal of new treatments developed to treat dyslipidemia. The aim of this study is to describe the role of this enzyme in a model of chronic inflammation associated dyslipidemia as it occurs in rheumatoid arthritis. Methods 101 RA patients and 115 sex and age-matched controls were included. Serum levels of CEPT were measured by an enzimelinked inmunoassay and the enzymatic activity by a specific kit (Roar CEPT activity assay kit). In both patients and controls classical lipidic profile (cholesterol, triglycerides, HDL and LDL cholesterol, apolipoprotein A1, apolipoprotein B and lipoprotein A) was defined. Also ESR and CRP were determined. In RA patients disease activity indexes like DAS28 and HAQ were collected, as well as, sociodemographic variables, comorbidility and anthropometric variables. Multivariate analysis was performed to compare results between patients and controls adjusting for corticosteroids intake and for classical dyslipidemia risk factors. Results Serum protein levels were correlated with the enzimatic activity (r=0,5, p=0,00), showing that serum levels could be enough to express the activity of this enzime. Patients show lower CETP activity levels after adjusting for sex and age (beta coefficient -0,52 pmol/l, CI95% -0,87–0,18, p=0,01). In patients, CRP levels show a negative correlation with CETP activity (r=-0.34, p=0,03); in controls this correlation did not achieve the stadistic significance. Disease activity scores like DAS28 did not show association with CETP. Conclusions CETP is underexpressed in RA patients. Differencial characteristics of dyslipIdemia in RA could be partly explained by this fact. Disclosure of Interest None Declared

Objectives It has been suggested that resistance to insulin action is a feature that accompanies rheumatoid arthritis (RA), a disease where chronic inflammation predominates and classical triggers for insulin resistance (IR) like obesity and diabetes are absents. However, data regarding characterization of RA features associated with this insulin resistance (IR) are lacking. The aim of this study was to investigate how sensitivity to insulin and markers of beta cell dysfunction (proinsulin processing metabolites) are expressed in RA. Methods 101 non-diabetic RA patients and 99 non-diabetic sex and age-matched controls were included in this study. Insulin sensitivity function through homeostatic model assessment (HOMA2), and beta cell secretion through insulin, split and intact proinsulin, and C-peptide were assessed in both groups. We performed multiple regression analysis to compare IR between groups and to explore the relation between RA features and IR. Data were adjusted for glucocorticoids intake and for IR classical risk factors. Results RA patients compared to controls show higher HOMA-IR ( log HOMA-IR, beta coefficient, 0.40 [95% CI 0.20-0.59], p=0.00). When this data was adjusted for glucocorticoids intake, non-on corticosteroid patients maintained a higher IR index (beta coef. 0.14 [95% CI 0.05-0.24], p=0.00). Current prednisone treatment was not associated with higher IR in the patients’ intragroup comparison (beta coef. 0.56 [1.13-1.19], p=0.22). Insulin processing signaling in RA patients showed impaired features via an elevated intact proinsulin levels (beta coef. 3.13 [0.81-5.44] pMol/L, p=0.03 for the comparison between patients and controls). Split proinsulin levels were also higher in RA patients (beta coef. 13.7 pmol/L [3.57-9.40], p=0.00) even adjusting for prednisone intake (beta coef. 2.66 pmol/L [95%CI 1.62-5.95] for non steroids RA patients when compared to controls). In multiple regression analysis, RA features (disease duration, rheumatoid factor, erythrocyte sedimentation rate, C-reactive protein, disease activity through HAQ and DAS28 scores, and current non-biologic disease modifying antirheumatic drug), when adjusted for sex, age and body mass index, were not associated with IR or insulin propeptides. Conclusions Beta cell signaling is impaired in non-diabetic and non-corticoids RA patients. Disclosure of Interest None Declared

Objectives To explore the influence of body composition and abdominal adiposity on endothelial dysfunction and radiological damage in Rheumatoid Arthritis (RA) patients. Methods A total of 100 patients, 54 RA patients and 46 controls, adjusted for sex, body mass index (BMI), age and comorbidity, were included. Total body composition was measured by dual energy X-ray absorptiometry; total and regional lean mass, fat mass, fat free mass index (fat free mass/m2) and fat mass index (fat mass/m2) were established. Quantification of visceral and parietal abdominal fat area was determined using magnetic resonance imaging, as well as visceral/parietal fat index. Endothelial dysfunction was assessed by brachial artery flow-mediated dilatation (FMD) as the dilator response to 5 minutes distal cuff occlusion and after sublingual nitroglycerine administration, and the Sharp Score defined radiological damage. The 28-joint DAS (DAS-28) and disability using HAQ (Health Assessment Questionnaire) scores, erythrocyte sedimentation rate (ESR) and C-reactive protein were collected. Multivariate analysis was performed to compare body composition between controls and patients and the relationship between those and endothelial dysfunction and radiological damage, everything adjusted for demographic and comorbidity variables. Results In the univariate analysis RA patients showed less flow-mediated dilatation (5.9 vs 9.8 mm, p=0.03). Similarly, fat free mass index was higher in patients than in controls (beta coefficient 0,94 (CI95% 0,08-1,80), P=0,03) after adjusting for BMI, sex and age; on the other hand fat mass index tended to be higher in patients than in controls (beta coefficient 2,51 (CI95% -0,82-5,84), p=0.13). Parietal and visceral abdominal fat values did not show differences between controls and patients. Fat mass, lean mass and abdominal fat did not correlate with DAS-28, HAQ, ESR or CRP. Parietal abdominal fat through resonance imaging in RA patients showed a negative relation with flow-mediated dilatation (beta coefficient -0.045 mm FMD/cm2 parietal area, p=0.02) after adjusting for BMI. This relation was not found with visceral abdominal fat. Radiological damage was negatively correlated with total bone mass (beta coefficient -1,13, CI95% -2,05-0,22, p=0.02) and showed a negative trend to do it also with total lean mass (beta coefficient -1.11 CI95% 2,42-0,21, p=0,09). Conclusions Changes in body composition take place in RA patients. These changes can be related to the endothelial dysfunction and radiological damage that occurs in this disease. Disclosure of Interest None Declared

Objectives The retinol binding protein 4 (RBP4) has recently been described as a protein highly related with insulin resistance (IR) states like obesity and diabetes. The chronic inflammatory states, like in rheumatoid arthritis (RA), are linked also to insulin resistance by mechanisms that are unknown. Therefore, RA is considered as an insulin resistance model related with a chronic inflammatory state, where diabetes and obesity, classical factors for IR, are absent. The objective of this study is to estimate RBP4 expression in RA patients. Methods 101 RA patients and 115 age and sex-matched controls were included. Pancreatic beta cell function was estimated by classical insulinresistance indexes like HOMA (homeostatic model assessment 2). RBP4, C peptide and insuline levels were measured in patients and controls by a specific enzyme-linked immunosorbent assay (ELISA). Multivariate analysis was performed to compare results between patients and controls and the data were adjusted for glucocorticoids intake and for IR classical risk factors. Results RBP4 levels did not show differences between controls and patients, after adjusting for sex, age, body mass index (BMI) and waist circumference (lnRBP4 2,83 mcg/dl in patients vs. 2,70 mcg/dl, p=0,33). On going steroids patients showed higher RBP4 levels (lnRBP4 3,03 vs 2,61 mcg/dl, p=0.00), after adjusting for age, sex, BMI and waist circumference. Similarly, in the univariate analysis, RBP4 levels tended to correlate with steroid average dose (r=0,20, p=0,14). In our serie, RBP4 levels were not related with the classical IR characteristics, like abdominal waist and BMI, both in controls and patients. Nor these levels showed relation with ESR, CRP, insuline levels or disease activity indexes. Conclusions The molecular mechanisms that lead to IR in RA patients appear to be different from those that ocurr in obesity and diabetes status. RBP4 does not seem to play a role in IR in patients with RA. Disclosure of Interest None Declared

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.