CD93 is rapidly shed from the surface of human myeloid cells and the soluble form is detected in human plasma

CD93 is a highly glycosylated transmembrane protein expressed on monocytes, neutrophils, endothelial cells, and stem cells. Antibodies directed at CD93 modulate phagocytosis, and CD93-deficient mice are defective in the clearance of apoptotic cells from the inflamed peritoneum. In this study we observe that CD93, expressed on human monocytes and neutrophils, is susceptible to phorbol dibutyrate-induced protein ectodomain shedding in a time- and dose-dependent manner. The soluble fragment found in culture supernatant retains the N-terminal carbohydrate recognition domain and the epidermal growth factor repeats after ectodomain cleavage. Importantly, a soluble form of the CD93 ectodomain was detected in human plasma, demonstrating that shedding is a physiologically relevant process. Inhibition of metalloproteinases with 1,10-phenanthroline inhibited shedding, but shedding was independent of TNF-alpha-converting enzyme (a disintegrin and metalloproteinase 17). Phorbol dibutyrate-induced CD93 shedding on monocytes was accompanied by decreased surface expression, whereas neutrophils displayed an increase in surface expression, suggesting that CD93 shed from the neutrophil surface was rapidly replaced by CD93 from intracellular stores. Cross-linking CD93 on human monocytes with immobilized anti-CD93 mAbs triggered shedding, as demonstrated by a decrease in cell-associated, full-length CD93 concomitant with an increase in CD93 intracellular domain-containing cleavage products. In addition, the inflammatory mediators, TNF-alpha and LPS, stimulated ectodomain cleavage of CD93 from monocytes. These data demonstrate that CD93 is susceptible to ectodomain shedding, identify multiple stimuli that trigger shedding, and identify both a soluble form of CD93 in human plasma and intracellular domain containing cleavage products within cells that may contribute to the physiologic role of CD93.     

ADAM-15 inhibits wound healing in human intestinal epithelial cell monolayers

The disintegrin metalloproteases (or ADAMs) are membrane-anchored glycoproteins that have been implicated in cell-cell or cell-matrix interactions and in proteolysis of molecules on the cell surface. The expression and/or the pathophysiological implications of ADAMs are not known in intestinal epithelial cells. Therefore, our aim was to investigate the expression and the role of ADAMs in intestinal epithelial cells. Expression of ADAMs was assessed by RT-PCR, Western blot analysis, and immunufluorescence experiments. Wound-healing experiments were performed by using the electric cell substrate impedence sensing technology. Our results showed that ADAMs-10, -12, and -15 mRNA are expressed in the colonic human cell lines Caco2-BBE and HT29-Cl.19A. An ADAM-15 complementary DNA cloned from Caco2-BBE poly(A)+ RNA, and encompassing the entire coding region, was found to be shorter and to present a different region encoding the cytoplasmic tail compared with ADAM-15 sequence deposited in the database. In Caco2-BBE cells and colonic epithelial cells, ADAM-15 protein was found in the apical, basolateral, and intracellular compartments. We also showed that the overexpression of ADAM-15 reduced cell migration in a wound-healing assay in Caco2-BBE monolayers. Our data show that 1) ADAM-15 is expressed in human intestinal epithelia, 2) a new variant of ADAM-15 is expressed in a human intestinal epithelial cell line, and 3) ADAM-15 is involved in intestinal epithelial cells wound-healing processes. Together, these results suggest that ADAM-15 may have important pathophysiological roles in intestinal cells.     

Tumor necrosis factor-alpha-converting enzyme controls surface expression of c-Kit and survival of embryonic stem cell-derived mast cells

Transmembrane metalloproteinases of the disintegrin and metalloproteinase (ADAM) family control cell signaling interactions via hydrolysis of protein extracellular domains. Prior work has shown that the receptor tyrosine kinase, c-Kit (CD117), is essential for mast cell survival and that serum levels of c-Kit increase in proliferative mast cell disorders, suggesting the existence of c-Kit shedding pathways in mast cells. In the present work, we report that tumor necrosis factor alpha-converting enzyme (TACE; ADAM-17) mediates shedding of c-Kit. Stimulation of transfected cells with phorbol 12-myristate 13-acetate (PMA) induced metalloproteinase-mediated release of c-Kit ectodomain, which increased further upon TACE overexpression. By contrast, TACE-deficient fibroblasts did not demonstrate inducible release, thus identifying TACE as the metalloproteinase primarily responsible for PMA-induced c-Kit shedding. Surface expression of c-Kit by the human mast cell-1 line decreased upon phorbol-induced shedding, which involved metalloproteinase activity susceptible to inhibition by tissue inhibitor of metalloproteinase (TIMP)-3. To further explore the role of TACE in shedding of c-Kit from mast cells, we compared the behavior of mast cells derived from murine embryonic stem cells. In these studies, PMA decreased surface c-Kit levels on mast cells expressing wild-type (+/+) TACE but not on those expressing an inactive mutant (DeltaZn/DeltaZn), confirming the role of TACE in PMA-induced c-Kit shedding. Compared with TACE(+/+) cells, TACE(DeltaZn/DeltaZn) mast cells also demonstrated decreased constitutive shedding and increased basal surface expression of c-Kit, with diminished apoptosis in response to c-Kit ligand deprivation. These data suggest that TACE controls mast cell survival by regulating shedding and surface expression of c-Kit.     

Synovial tissue macrophage as a source of the chemotactic cytokine IL-8

Cells of the synovial microenvironment may recruit neutrophils (PMN) and lymphocytes into synovial fluid, as well as lymphocytes into the synovial tissues, of arthritic patients. We have investigated the production of the chemotactic cytokine IL-8 by using sera, synovial fluid, synovial tissue, and macrophages and fibroblasts isolated from synovial tissues from 75 arthritic patients. IL-8 levels were higher in synovial fluid from rheumatoid (RA) patients (mean +/- SE, 14.37 +/- 5.8 ng/ml), compared with synovial fluid from osteoarthritis patients (0.135 +/- 17 ng/ml) (p less than 0.05) or from patients with other arthritides (5.52 +/- 5.11 ng/ml). IL-8 from RA sera was 8.44 +/- 2.33 ng/ml, compared with nondetectable levels found in normal sera. IL-8 levels from RA sera and synovial fluid were strongly positively correlated (r = 0.96, p less than 0.05). Moreover, RA synovial fluid chemotactic activity for PMN in these fluids was inhibited 40 +/- 5% upon incubation with neutralizing polyclonal antibody to IL-8. Synovial tissue fibroblasts released only small amounts of constitutive IL-8 but could be induced to produce IL-8 by stimulation with either IL-1 beta, TNF-alpha, or LPS. In contrast, unlike normal PBMC or alveolar macrophages, macrophages isolated from RA synovial tissue constitutively expressed both IL-8 mRNA and antigenic IL-8. RA synovial macrophage IL-8 expression was not augmented by incubation with either LPS, TNF-alpha, or IL-1 beta. Immunohistochemical analysis of synovial tissue showed that a greater percentage of RA macrophages than osteoarthritis macrophages reacted with anti-IL-8. Whereas macrophages were the predominant cell for immunolocalization of IL-8, less than 5% of synovial tissue fibroblasts were positive for immunolocalized IL-8. These results suggest that macrophage-derived IL-8 may play an important role in the recruitment of PMN in synovial inflammation associated with RA.     

Rab27a regulates exocytosis of tertiary and specific granules in human neutrophils

The correct mobilization of cytoplasmic granules is essential for the proper functioning of human neutrophils in host defense and inflammation. In this study, we have found that human peripheral blood neutrophils expressed high levels of Rab27a, whereas Rab27b expression was much lower. This indicates that Rab27a is the predominant Rab27 isoform present in human neutrophils. Rab27a was up-regulated during neutrophil differentiation of HL-60 cells. Subcellular fractionation and immunoelectron microscopy studies of resting human neutrophils showed that Rab27a was mainly located in the membranes of specific and gelatinase-enriched tertiary granules, with a minor localization in azurophil granules. Rab27a was largely absent from CD35-enriched secretory vesicles. Tertiary and specific granule-located Rab27a population was translocated to the cell surface upon neutrophil activation with PMA that induced exocytosis of both tertiary and specific granules. Specific Abs against Rab27a inhibited Ca(2+) and GTP-gamma-S activation and PMA-induced exocytosis of CD66b-enriched tertiary and specific granules in electropermeabilized neutrophils, whereas secretion of CD63-enriched azurophil granules was scarcely affected. Human neutrophils lacked or expressed low levels of most Slp/Slac2 proteins, putative Rab27 effectors, suggesting that additional proteins should act as Rab27a effectors in human neutrophils. Our data indicate that Rab27a is a major component of the exocytic machinery of human neutrophils, modulating the secretion of tertiary and specific granules that are readily mobilized upon neutrophil activation.     

Sulfhydryl regulation of L-selectin shedding: phenylarsine oxide promotes activation-independent L-selectin shedding from leukocytes

The L-selectin adhesion molecule mediates leukocyte recruitment to inflammatory sites and lymphocyte trafficking through the peripheral lymph nodes. In response to leukocyte activation, L-selectin is proteolytically released from the cell surface, disabling leukocytes from the subsequent L-selectin-dependent interactions. We have found that L-selectin shedding is sensitive to sulfhydryl chemistry; it is promoted by thiol-oxidizing or -blocking reagents and inhibited by reducing reagents. Phenylarsine oxide (PAO), a trivalent arsenical that interacts with vicinal dithiols, is most potent in inducing rapid shedding of L-selectin from isolated neutrophils, eosinophils, and lymphocytes as well as from neutrophils in whole blood. PAO does not cause cell activation, nor does it interfere with integrin function or alter the expression of several other cell surface molecules at the low concentrations that induce L-selectin shedding. PAO is not required to enter the cell to induce L-selectin shedding. TAPI-2 ((N-(D,L-[2-(hydroxyaminocarbonyl)-methyl]-4-methylpentanoyl)-L-3-(tert-butyl)-alanyl-l -alanine, 2-aminoethyl amide), which has previously been shown to inhibit the activation-dependent L-selectin shedding, is also capable of inhibiting PAO-induced L-selectin shedding. We hypothesize that PAO-induced L-selectin shedding involves a regulatory molecule, such as protein disulfide isomerase (PDI), an enzyme that plays a role in the formation and rearrangement of disulfide bonds, contains PAO-binding, vicinal dithiol-active sites, and is expressed on the neutrophil surface. Cell surface expression of PDI, L-selectin shedding induced by PDI-blocking Abs and by bacitracin, a known inhibitor of PDI activity, and direct binding of PDI to PAO, provide supporting evidence for this hypothesis.     

Actin polymerization induces shedding of FcgammaRIIIb (CD16) from human neutrophils

FcgammaRIIIb (CD16) is a glycosyl phosphatidylinositol (GPI)-anchored low-affinity IgG receptor, exclusively expressed on human neutrophils. FcgammaRIIIb associates with complement receptor 3 (CR3, Mac-1, CD11b/CD18), which may indirectly link FcgammaRIIIb to the actin cytoskeleton. Upon neutrophil activation, apoptosis, or chemotaxis, FcgammaRIIIb is shed from the cell surface. In all of these events, actin rearrangements play an important role. To establish a role for the actin cytoskeleton in the control of FcgammaRIIIb shedding, we treated human neutrophils with jasplakinolide, an actin-polymerizing peptide. We show that enhanced actin polymerization induces time- and dose-dependent shedding of FcgammaRIIIb. This effect was not restricted to FcgammaRIIIb, because the cell surface expression of CD43, CD44, and L-selectin was also downregulated after induction of actin polymerization. This actin-dependent pathway is staurosporine sensitive but does not appear to involve activation of PKC or CR3. These data show that the actin cytoskeleton can regulate protein ectodomain shedding from human neutrophils.     

TNF-Induced shedding of TNF receptors in human polymorphonuclear leukocytes: role of the 55-kDa TNF receptor and involvement of a membrane-bound and non-matrix metalloproteinase

A down-modulation of both the 55-kDa (TNF-R55) and the 75-kDa (TNF-R75) TNF receptors is observed in neutrophils exposed to a variety of stimuli. Proteolytic cleavage of the extracellular region of both receptors (shedding) and, with TNF, internalization of TNF-R55 and shedding of TNF-R75 are the proposed mechanisms. We have characterized the TNF-induced shedding of TNF receptors in neutrophils and determined the nature of the involved proteinase. Neutrophils exposed to TNF release both TNF receptors. A release of TNF receptors comparable to that observed with TNF was induced with TNF-R55-specific reagents (mAbs and a mutant of TNF) but not with the corresponding TNF-R75-specific reagents. A hydroxamic acid compound (KB8301) almost completely inhibited shedding of TNF-R55 and to a lesser degree shedding of TNF-R75. KB8301 also inhibited FMLP-induced shedding to a similar extent. Shedding was also inhibited by 1,10-phenanthroline, but this effect was considered nonspecific as the compound, at variance with KB8301, almost completely inhibited TNF and FMLP-induced PMN activation. Diisopropylfluorophosphate partially inhibited shedding of TNF-R75, suggesting the contribution of a serine proteinase to the release of this receptor. Shedding activity was not affected by matrix metalloproteinases inhibitors nor was it released in the supernatants of FMLP-stimulated neutrophils. These results suggest that TNF induces release of its receptors, that such a release is mediated via TNF-R55, and that a membrane-bound and non-matrix metalloproteinase is involved in the process. The possibility that ADAM-17, which we show to be expressed in neutrophils, might be the involved proteinase is discussed.     

FHL2 interacts with both ADAM-17 and the cytoskeleton and regulates ADAM-17 localization and activity

ADAM-17 is a metalloprotease-disintegrin responsible for the ectodomain shedding of several transmembrane proteins. Using the yeast two-hybrid system, we showed that ADAM-17 interacts with the Four and Half LIM domain 2 protein (FHL2), a LIM domain protein that is involved in multiple protein-protein interaction. We demonstrated that this interaction involved the amino-acid sequence of ADAM-17 from position 721 to739. In the cardiomyoblast cells H9C2, ADAM-17 and FHL2 colocalize with the actin-based cytoskeleton and we showed that FHL2 binds both ADAM-17 and the actin-based cytoskeleton. We found that mainly the mature form of ADAM-17 associates with the cytoskeleton, although the maturation of ADAM-17 by furin is not necessary for its binding to the cytoskeleton. Interestingly, less ADAM-17 was detected at the surface of wild-type mouse macrophages compared to FHL2 deficient macrophages. However, wild-type cells have a higher ability to release ADAM-17 substrates under PMA stimulation. Altogether, these results demonstrate a physical and functional interaction between ADAM-17 and FHL2 that implies that FHL2 has a role in the regulation of ADAM-17.     

TRAIL-R2 (DR5) mediates apoptosis of synovial fibroblasts in rheumatoid arthritis

TRAIL has been proposed as an anti-inflammatory cytokine in animal models of rheumatoid arthritis (RA). Using two agonistic mAbs specific for TRAIL-R1 (DR4) and TRAIL-R2 (DR5), we examined the expression and function of these death receptors in RA synovial fibroblast cells. The synovial tissues and primary synovial fibroblast cells isolated from patients with RA, but not those isolated from patients with osteoarthritis, selectively expressed high levels of cell surface DR5 and were highly susceptible to anti-DR5 Ab (TRA-8)-mediated apoptosis. In contrast, RA synoviocytes did not show increased expression of TRAIL-R1 (DR4), nor was there any difference in expression of Fas between RA and osteoarthritis synovial cells. In vitro TRA-8 induced apoptosis of RA synovial cells and inhibited production of matrix metalloproteinases induced by pro-inflammatory cytokines. In vivo TRA-8 effectively inhibited hypercellularity of a SV40-transformed RA synovial cell line and completely prevented bone erosion and cartilage destruction induced by these cells. These results indicate that increased DR5 expression and susceptibility to DR5-mediated apoptosis are characteristic of the proliferating synovial cells in RA. As highly proliferative transformed-appearing RA synovial cells play a crucial role in bone erosion and cartilage destruction in RA, the specific targeting of DR5 on RA synovial cells with an agonistic anti-DR5 Ab may be a potential therapy for RA.     

The migratory and phagocytic activity of polymorphonuclear leukocytes in rheumatoid arthritis and osteoarthritis patients

Polymorphonuclear neutrophils (PMN) play a central role in the elimination of most extracellular pathogenic microorganisms and any impairment of their functions therefore predisposes to defect immune defence. We investigated the migratory and phagocytic functions of the PMNs isolated from peripheral blood and synovial fluid of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). The results suggest that in RA the number and the migratory but not phagocytic capacity of synovial fluid (SF) neutrophils were enhanced, while in OA they were significantly decreased in synovial fluid cells comparatively with peripheral blood (PB). The migratory function of both PB and SF cells from RA patients was increased comparatively with that of the cells from OA patients. We found the different abnormal functions in synovial fluid neutrophils from RA and OA patients. These results may help to elucidate the underlying mechanism which leads to severe joint destruction and different susceptibility to infectious diseases in patients with rheumatic disorders.     

Role of ADAM-9 disintegrin-cysteine-rich domains in human keratinocyte migration

ADAM-9 belongs to a family of transmembrane, disintegrin-containing metalloproteinases involved in protein ectodomain shedding and cell-cell and cell-matrix interactions. The aim of this study was to analyze the expression of ADAM-9 in skin and to assess the role of this proteolytic/adhesive protein in skin physiology. In normal skin, ADAM-9 expression was detected in both the epidermis and dermis and in vitro in keratinocytes and fibroblasts. Here we report that ADAM-9 functions as a cell adhesion molecule via its disintegrin-cysteine-rich domain. Using solid phase binding assays and antibody inhibition experiments, we demonstrated that the recombinant disintegrin-cysteine-rich domain of ADAM-9 specifically interacts with the beta1 integrin subunit on keratinocytes. This was corroborated by co-immunoprecipitation. In addition, engagement of integrin receptors by the disintegrin-cysteine-rich domain resulted in ERK phosphorylation and increased MMP-9 synthesis. Treatment with the ERK inhibitor PD98059 inhibited MMP-9 induction. Furthermore, the presence of the soluble disintegrin-cysteine-rich domain did not interfere with cell migration on different substrates. However, keratinocytes adhering to the immobilized disintegrin-cysteine-rich domain showed increased motility, which was partially due to the induction of MMP-9 secretion. In summary, our results indicate that the ADAM-9 adhesive domain plays a role in regulating the motility of cells by interaction with beta1 integrins and modulates MMP synthesis.     

The disintegrin-like and cysteine-rich domains of ADAM-9 mediate interactions between melanoma cells and fibroblasts

A characteristic of malignant cells is their capacity to invade their surrounding and to metastasize to distant organs. During these processes, proteolytic activities of tumor and stromal cells modify the extracellular matrix to produce a microenvironment suitable for their growth and migration. In recent years the family of ADAM proteases has been ascribed important roles in these processes. ADAM-9 is expressed in human melanoma at the tumor-stroma border where direct or indirect interactions between tumor cells and fibroblasts occur. To analyze the role of ADAM-9 for the interaction between melanoma cells and stromal fibroblasts, we produced the recombinant disintegrin-like and cysteine-rich domain of ADAM-9 (DC-9). Melanoma cells and human fibroblasts adhered to immobilized DC-9 in a Mn(2+)-dependent fashion suggesting an integrin-mediated process. Inhibition studies showed that adhesion of fibroblasts was mediated by several β1 integrin receptors independent of the RGD and ECD recognition motif. Furthermore, interaction of fibroblasts and high invasive melanoma cells with soluble recombinant DC-9 resulted in enhanced expression of MMP-1 and MMP-2. Silencing of ADAM-9 in melanoma cells significantly reduced cell adhesion to fibroblasts. Ablation of ADAM-9 in fibroblasts almost completely abolished these cellular interactions and melanoma cell invasion in vitro. In summary, these results suggest that ADAM-9 expression plays an important role in mediating cell-cell contacts between fibroblasts and melanoma cells and that these interactions contribute to proteolytic activities required during invasion of melanoma cells.     

Evidence for cadherin-11 cleavage in the synovium and partial characterization of its mechanism

IntroductionEngagement of the homotypic cell-to-cell adhesion molecule cadherin-11 on rheumatoid arthritis (RA) synovial fibroblasts with a chimeric molecule containing the cadherin-11 extracellular binding domain stimulated cytokine, chemokine, and matrix metalloproteinases (MMP) release, implicating cadherin-11 signaling in RA pathogenesis. The objective of this study was to determine if cadherin-11 extracellular domain fragments are found inside the joint and if a physiologic synovial fibroblast cleavage pathway releases those fragments.MethodsCadherin-11 cleavage fragments were detected by western blot in cell media or lysates. Cleavage was interrupted using chemical inhibitors or short-interfering RNA (siRNA) gene silencing. The amount of cadherin-11 fragments in synovial fluid was measured by western blot and ELISA.ResultsSoluble cadherin-11 extracellular fragments were detected in human synovial fluid at significantly higher levels in RA samples compared to osteoarthritis (OA) samples. A cadherin-11 N-terminal extracellular binding domain fragment was shed from synovial fibroblasts after ionomycin stimulation, followed by presenilin 1 (PSN1)-dependent regulated intramembrane proteolysis of the retained membrane-bound C-terminal fragments. In addition to ionomycin-induced calcium flux, tumor necrosis factor (TNF)-α also stimulated cleavage in both two- and three-dimensional fibroblast cultures. Although cadherin-11 extracellular domains were shed by a disintegrin and metalloproteinase (ADAM) 10 in several cell types, a novel ADAM- and metalloproteinase-independent activity mediated shedding in primary human fibroblasts.ConclusionsCadherin-11 undergoes ectodomain shedding followed by regulated intramembrane proteolysis in synovial fibroblasts, triggered by a novel sheddase that generates extracelluar cadherin-11 fragments. Cadherin-11 fragments were enriched in RA synovial fluid, suggesting they may be a marker of synovial burden and may function to modify cadherin-11 interactions between synovial fibroblasts.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0647-9) contains supplementary material, which is available to authorized users.     

Human Synovial Lubricin Expresses Sialyl Lewis x Determinant and Has L-selectin Ligand Activity

Lubricin (or proteoglycan 4 (PRG4)) is an abundant mucin-like glycoprotein in synovial fluid (SF) and a major component responsible for joint lubrication. In this study, it was shown that O-linked core 2 oligosaccharides (Galβ1–3(GlcNAcβ1–6)GalNAcα1-Thr/Ser) on lubricin isolated from rheumatoid arthritis SF contained both sulfate and fucose residues, and SF lubricin was capable of binding to recombinant L-selectin in a glycosylation-dependent manner. Using resting human polymorphonuclear granulocytes (PMN) from peripheral blood, confocal microscopy showed that lubricin coated circulating PMN and that it partly co-localized with L-selectin expressed by these cells. In agreement with this, activation-induced shedding of L-selectin also mediated decreased lubricin binding to PMN. It was also found that PMN recruited to inflamed synovial area and fluid in rheumatoid arthritis patients kept a coat of lubricin. These observations suggest that lubricin is able to bind to PMN via an L-selectin-dependent and -independent manner and may play a role in PMN-mediated inflammation.     

Differential expression of CD148 on leukocyte subsets in inflammatory arthritis

Introduction

Monocytic cells play a central role in the aetiology of rheumatoid arthritis, and manipulation of the activation of these cells is an approach currently under investigation to discover new therapies for this and associated diseases. CD148 is a transmembrane tyrosine phosphatase that is highly expressed in monocytes and macrophages and, since this family of molecules plays an important role in the regulation of cell activity, CD148 is a potential target for the manipulation of macrophage activation. For any molecule to be considered a therapeutic target, it is important for it to be increased in activity or expression during disease.

Methods

We have investigated the expression of CD148 in two murine models of arthritis and in joints from rheumatoid arthritis (RA) patients using real-time PCR, immunohistochemistry, and studied the effects of proinflammatory stimuli on CD148 activity using biochemical assays.

Results

We report that CD148 mRNA is upregulated in diseased joints of mice with collagen-induced arthritis. Furthermore, we report that in mice CD148 protein is highly expressed in infiltrating monocytes of diseased joints, with a small fraction of T cells also expressing CD148. In human arthritic joints both T cells and monocytes expressed high levels of CD148, however, we show differential expression of CD148 in T cells and monocytes from normal human peripheral blood compared to peripheral blood from RA and both normal and RA synovial fluid. Finally, we show that synovial fluid from rheumatoid arthritis patients suppresses CD148 phosphatase activity.

Conclusions

CD148 is upregulated in macrophages and T cells in human RA samples, and its activity is enhanced by treatment with tumour necrosis factor alpha (TNFα), and reduced by synovial fluid or oxidising conditions. A greater understanding of the role of CD148 in chronic inflammation may lead to alternative therapeutic approaches to these diseases.     

Differential expression of plasminogen activator inhibitor-1, tumor necrosis factor-alpha,TNF-alpha converting enzyme and ADAMTS family members in murine fat territories

Our objective was to investigate expression of A disintegrin and metalloproteinase (ADAM) and ADAM proteins with a thrombospondin (TS) motif (ADAMTS) family members in adipose tissue of lean and obese mice. Five-week-old male mice were kept on standard chow (SFD) or on high fat diet (HFD) for 15 weeks, and subcutaneous (SC) and gonadal (GON) adipose tissue, as well as mature adipocytes and stromal-vascular (S-V) cells were harvested. mRNA levels of plasminogen activator inhibitor-1 (PAI-1), tumor necrosis factor-alpha (TNF-alpha), ADAM-17 (TACE or TNF-alpha converting enzyme), ADAMTS-1 and ADAMTS-8 were quantified in isolated adipose tissues and cell fractions, and during differentiation of murine preadipocytes. The HFD resulted in a significantly enhanced weight of isolated SC and GON fat pads, and in enhanced blood levels of glucose, cholesterol and PAI-1. ADAM-17, TNF-alpha, PAI-1, ADAMTS-1 and ADAMTS-8 mRNA were detected in both SC and GON adipose tissue of lean mice (SFD). In SC adipose tissue of obese mice (HFD), the expression of ADAM-17 and PAI-1 was enhanced and that of ADAMTS-1 reduced, whereas in GON adipose tissue expression of TNF-alpha was enhanced and that of ADAMTS-8 reduced. In lean and obese mice, expression of ADAM-17, ADAMTS-1 and ADAMTS-8 was higher in the S-V cell fraction than in mature adipocytes. During differentiation of murine 3T3-F442A preadipocytes, expression of ADAM-17 and ADAMTS-1 remained virtually unaltered, whereas that of ADAMTS-8 decreased as adipocytes matured. Several ADAM and ADAMTS family members are expressed in adipose tissue and during differentiation of preadipocytes. Modulation of their expression upon development of obesity is adipose tissue-dependent.     

Interleukin-1 induced gene expression of neutrophil activating protein (interleukin-8) and monocyte chemotactic peptide in human synovial cells

We report here that human synovial cells stimulated by interleukin-1 alpha and interleukin-1 beta express mRNA for both IL-8 (neutrophil chemotactic peptide) and monocyte chemotactic protein. IL-1 stimulated synovial cells from both osteoarthritis and rheumatoid arthritis patients exhibited similar mRNA expression of interleukin-8 and monocyte chemotactic protein. A capacity to produce factors selectively chemotactic for neutrophils, lymphocytes and monocytes provides a mechanism whereby synovial cells can facilitate inflammatory arthritis.