María Jesús Domínguez-Luis

Expression and regulation of the metalloproteinase ADAM-8 during human neutrophil pathophysiological activation and its catalytic activity on L-selectin shedding.

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.

The metalloprotease ADAM8 is associated with and regulates the function of the adhesion receptor PSGL-1 through ERM proteins

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.

Superoxide anion mediates the L-selectin down-regulation induced by non-steroidal anti-inflammatory drugs in human neutrophils.

UNLABELLED 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.

In vivo modulation of the inflammatory response by nonsteroidal antiinflammatory drug-related compounds that trigger L-selectin shedding

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.

Differential Antigen-presenting B Cell Phenotypes from Synovial Microenvironment of Patients with Rheumatoid and Psoriatic Arthritis

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.

Role of CXCL13 and CCL20 in the recruitment of B cells to inflammatory foci in chronic arthritis

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.

Functional effects of proinflammatory factors present in Sjögren’s syndrome salivary microenvironment in an in vitro model of human salivary gland

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.

THU0056 Impaired beta cell signaling is present in non diabetic rheumatoid arthritis patients

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 (logHOMA-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