Scratching triggers skin flares in atopic dermatitis (AD). We demonstrate that scratching of human skin, and tape stripping of mouse skin, causes neutrophil influx. This influx in mice was largely dependent on the generation of leukotriene B4 (LTB4) by neutrophils and their expression of the LTB4 receptor BLT1. Allergic skin inflammation in response to epicutaneous (EC) application of ovalbumin to tape-stripped skin was severely impaired in Ltb4r1−/− mice, and required expression of BLT1 on both T cells and non-T cells. Co-transfer of WT neutrophils, but not neutrophils deficient in BLT1 or the LTB4 synthesizing enzyme LTA4H, restored the ability of WT CD4+ effector T cells to transfer allergic skin inflammation to Ltb4r1−/− recipients. Pharmacologic blockade of LTB4 synthesis inhibited allergic skin inflammation elicited by cutaneous antigen challenge in previously EC-sensitized mice. Our results demonstrate that a neutrophil-T cell axis reliant on LTB4-BLT1 interaction is required for allergic skin inflammation.
To define the expression pattern of cadherin-11 in destructive pannus tissue of patients with rheumatoid arthritis and to determine if cadherin-11 expression in fibroblast-like synoviocytes controls their invasive capacity.
Cadherin-11 expression in rheumatoid synovial tissue was evaluated using immunohistochemistry. To examine the role of cadherin-11 in regulating the invasive behavior of fibroblast-like synoviocytes, we generated L-cell clones expressing wild-type cadherin-11, mutant cadherin-11, and empty vector transfected controls. The invasive capacity of L-cell transfectants and cultured fibroblast-like synoviocytes treated with a blocking cadherin-11-Fc protein or control immunoglobulin was determined in Matrigel invasion assays.
Immunohistochemistry revealed that cadherin-11 is abundantly expressed in cells at the cartilage-pannus junction in rheumatoid synovitis. Invasion assays demonstrate a twofold increased invasive capacity of cadherin-11 transfected L-cells compared to L-cells transfected with E-cadherin or control vector. The invasive behavior of the L-cells stably transfected with a cadherin-11 construct that lacked the juxta-membrane cytoplasmic domain (cadherin-11 ΔJMD) was diminished to the level of vector control L-cells. Further, treatment with the cadherin-11-Fc fusion protein diminished the invasive capacity of fibroblast-like synoviocytes.
These in vitro studies implicate a role for cadherin-11 in promoting cell invasion and contribute insight into the invasive nature of fibroblast-like synoviocytes in chronic synovitis and rheumatoid arthritis.
Cadherin-11; Fibroblast-like Synoviocytes; Cell Invasion
Osteopontin (OPN) is a pro-inflammatory cytokine important in rheumatoid arthritis (RA). OPN can be cleaved by thrombin, leading to OPN-Arg (OPN-R) and exposing the cryptic C-terminal α4β1 and α9β1 integrin-binding motif (SVVYGLR). Thrombin-activatable carboxypeptidase B (CPB), also termed thrombin-activatable fibrinolysis inhibitor (TAFI), removes the C-terminal arginine from OPN-R, generating OPN-Leu (OPN-L) and abrogating its enhanced cell binding. We investigated the roles of OPN-R and OPN-L in: (i) synoviocyte adhesion, which contributes to formation of invasive pannus, and (ii) neutrophil survival, which affects inflammatory infiltrates, in RA.
Methods and Results
We developed ELISAs specific for OPN-R and OPN-L, and demonstrate elevations of OPN-R and OPN-L in RA, but not in osteoarthritis or psoriatic arthritis, synovial fluid samples. OPN-R and OPN-L levels correlated with multiple inflammatory cytokines including TNFα and IL-6. Immunohistochemical analyses demonstrated robust expression of OPN-FL, but minimal OPN-R, in RA synovium, suggesting that cleaved OPN is released into the synovial fluid. In cellular assays, OPN-FL, and to a lesser extent OPN-R and OPN-L, had an anti-apoptotic effect on neutrophils. OPN-R, but not OPN-L, augmented RA fibroblast-like synoviocyte binding mediated by SVVYGLR binding to α4β1.
Thrombin activation of OPN (OPN-R) and its subsequent inactivation by thrombin-activatable CPB (OPN-L) occurs locally within inflamed joints in RA. Our data suggest that thrombin-activatable CPB plays a central homeostatic role in RA, by regulating neutrophil viability and reducing synoviocyte adhesion.
osteopontin; thrombin-activatable carboxypeptidase B; thrombin-activatable fibrinolysis inhibitor; rheumatoid arthritis
Mechanisms by which mesenchymal-derived tissue lineages participate in amplifying and perpetuating synovial inflammation in arthritis have been relatively underinvestigated and are therefore poorly understood. Elucidating these processes is likely to provide new insights into the pathogenesis of multiple diseases. Leukotriene B4 (LTB4) is a potent proinflammatory lipid mediator that initiates and amplifies synovial inflammation in the K/BxN model of arthritis. We sought to elucidate mechanisms by which mesenchymal-derived fibroblast-like synoviocytes (FLSs) perpetuate synovial inflammation. We focused on the abilities of FLSs to contribute to LTB4 synthesis and to respond to LTB4 within the joint. Using a series of bone marrow chimeras generated from 5-lipoxygenase–/– and leukotriene A4 (LTA4) hydrolase–/– mice, we demonstrate that FLSs generate sufficient levels of LTB4 production through transcellular metabolism in K/BxN serum-induced arthritis to drive inflammatory arthritis. FLSs—which comprise the predominant lineage populating the synovial lining—are competent to metabolize exogenous LTA4 into LTB4 ex vivo. Stimulation of FLSs with TNF increased their capacity to generate LTB4 3-fold without inducing the expression of LTA4 hydrolase protein. Moreover, LTB4 (acting via LTB4 receptor 1) was found to modulate the migratory and invasive activity of FLSs in vitro and also promote joint erosion by pannus tissue in vivo. Our results identify novel roles for FLSs and LTB4 in joints, placing LTB4 regulation of FLS biology at the center of a previously unrecognized amplification loop for synovial inflammation and tissue pathology.
Human (h) CD7 is a 40 kDa single chain Ig superfamily molecule that is expressed on thymocytes, a major subunit of peripheral T cells, and most natural killer cells. Ligands for hCD7 include the epithelial cell-produced molecule, K-12, and galectin. Mice deficient in CD7 have been shown to be resistant to LPS-induced endotoxic shock syndromes. However, monoclonal antibodies (MAb) to mouse (m) CD7 have yet to be produced, nor is the distribution of mCD7 protein in mice known. We have raised a panel of three rat MAbs to mCD7 by immunizing rats with recombinant mCD7 protein. However, using Western blot and immunoprecipitation of tissue extracts from mouse thymus, spleen, liver, brain, lymph node and skin, these anti-mouse CD7 MAbs bound only to murine heat shock protein 60 (HSP-60) present both in wild-type (CD7+/+) and CD7-deficient (CD7−/−) mice. Epitope mapping of the sites on HSP-60 and recombinant mCD7 recognized by mCD7 MAbs demonstrated non-homologous amino acid sequence epitopes recognized by anti-CD7 MAbs on both proteins. These data demonstrated molecular mimicry of mCD7 with HSP-60, and leave open the question of surface expression of mCD7.
Immunoassay-based techniques, routinely used to measure serum estradiol (E2), are known to have reduced specificity, especially at lower concentrations, when compared with the gold standard technique of mass spectrometry (MS). Different measurement techniques may be responsible for the conflicting results of associations between serum E2 and clinical phenotypes in men.
Our objective was to compare immunoassay and MS measurements of E2 levels in men and evaluate associations with clinical phenotypes.
Design and Setting:
Middle-aged and older male subjects participating in the population-based Osteoporotic Fractures in Men (MrOS) Sweden study (n = 2599), MrOS US (n = 688), and the European Male Aging Study (n = 2908) were included.
Main Outcome Measures:
Immunoassay and MS measurements of serum E2 were compared and related to bone mineral density (BMD; measured by dual energy x-ray absorptiometry) and ankle-brachial index.
Within each cohort, serum E2 levels obtained by immunoassay and MS correlated moderately (Spearman rank correlation coefficient rS 0.53–0.76). Serum C-reactive protein (CRP) levels associated significantly (albeit to a low extent, rS = 0.29) with immunoassay E2 but not with MS E2 levels. Similar associations of immunoassay E2 and MS E2 were seen with lumbar spine and total hip BMD, independent of serum CRP. However, immunoassay E2, but not MS E2, associated inversely with ankle-brachial index, and this correlation was lost after adjustment for CRP.
Our findings suggest interference in the immunoassay E2 analyses, possibly by CRP or a CRP-associated factor. Although associations with BMD remain unaffected, this might imply for a reevaluation of previous association studies between immunoassay E2 levels and inflammation-related outcomes.
Immunoglobulins, antigens and complement can assemble to form immune complexes (IC). ICs can be detrimental as they propagate inflammation in autoimmune diseases. Like ICs, submicron extracellular vesicles termed microparticles (MP) are present in the synovial fluid from patients affected with autoimmune arthritis. We examined MPs in rheumatoid arthritis (RA) using high sensitivity flow cytometry and electron microscopy. We find that the MPs in RA synovial fluid are highly heterogeneous in size. The observed larger MPs were in fact MP-containing ICs (mpICs) and account for the majority of the detectable ICs. These mpICs frequently express the integrin CD41, consistent with platelet origin. Despite expression of the Fc receptor FcγRIIa by platelet-derived MPs, we find that the mpICs form independently of this receptor. Rather, mpICs display autoantigens vimentin and fibrinogen, and recognition of these targets by anti-citrullinated peptide antibodies contributes to the production of mpICs. Functionally, platelet mpICs are highly pro-inflammatory, eliciting leukotriene production by neutrophils. Taken together, our data suggest a unique role for platelet MPs as autoantigen-expressing elements capable of perpetuating formation of inflammatory ICs.
arthritis; autoantigens; immune complexes; microparticles; platelets
Rheumatoid arthritis culminates in joint destruction that in mouse models of disease, is supported by innate immune molecules including FcγRs and complement. However, the results may not predict outcomes in humans given the structural differences between murine and human activating FcγRs on neutrophils, a prominent component of joint exudates. In this study, we examined the role of the human neutrophil FcγRIIA in the development of arthritis and probed the underlying mechanism by which FcγRIIA initiated disease.
K/BxN serum transfer-induced arthritis was examined in mice that express FcγRIIA on neutrophils but lack their own activating FcγRs (γ-chain-deficient). The role of mast cells, complement (C3 and C5a) and CD18 integrins in FcγRIIA-initiated disease was examined using cell reconstitution approaches, inhibitors, and functional blocking antibodies respectively. Cross-talk between C5aR and FcγRIIA on neutrophils was evaluated in vitro.
Neutrophil FcγRIIA expression was sufficient to restore susceptibility to K/BxN serum induced neutrophil recruitment, synovitis and bone destruction in γ-chain-deficient mice. Joint inflammation was robust and proceeded even in the absence of mast cells and vascular permeability, shown to contribute to disease in wild-type mice. Neutrophil recruitment was dependent on CD18 integrin LFA-1 and C5aR. C5aR in addition significantly enhanced FcγRIIA mediated phagocytosis and oxidative burst in vitro.
Human and murine activating FcγRs on neutrophils are not functionally equivalent, and in humans may play a primary role in arthritis. Cross-talk between neutrophil C5aR and FcγRIIA is essential for disease thus highlighting a new aspect of complement during the effector phase of inflammatory arthritis.
Traumatic and degenerative meniscal tears have different anatomic features and different proposed etiologies, yet both are associated with development or progression of osteoarthritis (OA). In established OA, synovitis is associated with pain and progression, but a relationship between synovitis and symptoms in isolated meniscal disease has not been reported. Accordingly, we sought to characterize synovial pathology in patients with traumatic meniscal injuries and determine the relationships between inflammation, meniscal and cartilage pathology, and symptoms.
Thirty-three patients without evidence of OA undergoing arthroscopic meniscectomy for meniscal injuries were recruited. Pain and function were assessed preoperatively; meniscal and cartilage abnormalities were documented at the time of surgery. Inflammation in synovial biopsies was scored and associations between inflammation and clinical outcomes determined. Microarray analysis of synovial tissue was performed and gene expression patterns in patients with or without inflammation compared.
Synovial inflammation was present in 43% of patients and was associated with worse pre-operative pain and function scores, independent of age, gender, or cartilage pathology. Microarray analysis and real-time PCR revealed a chemokine signature in synovial biopsies with increased inflammation scores.
In patients with traumatic meniscal injury undergoing arthroscopic meniscectomy without clinical or radiographic evidence of OA, synovial inflammation occurs frequently and is associated with increased pain and dysfunction. Synovia with increased inflammation scores exhibit a unique chemokine signature. Chemokines may contribute to the development of synovial inflammation in patients with meniscal pathology; they also represent potential therapeutic targets for reducing inflammatory symptoms.
Meniscectomy; meniscal injury; inflammation; synovium; synovitis
In addition to the well-described role of platelets in thrombosis, a growing body of evidence implicates platelets in diverse inflammatory responses. We recently showed platelets can contribute to the pathophysiology of inflammatory arthritis via IL-1–containing microparticles. In this study, we demonstrate that platelets, and not platelet microparticles, actively contribute to synovitis via production of proinflammatory prostacyclin in an autoimmune arthritis model. Using both genetic and pharmacologic approaches, we establish that paracrine production of prostacyclin proceeds in the absence of cyclooxygenase-2. Furthermore, we also demonstrate that prostacyclin generation can arise via transcellular collaboration between platelets and fibroblast-like synoviocytes. In addition to shedding light on an unappreciated pathway of lipid synthesis in arthritis, we further delineate a novel effector activity by which platelets can contribute to inflammatory disease.
Mast cells are tissue-resident immune sentinels implicated in the pathogenesis of inflammatory joint disease. We hypothesized that complement fragments could be key activators of synovial mast cells in autoimmune arthritis.
In vivo studies employed murine K/BxN arthritis, a distal symmetric polyarthritis mediated by IgG immune complexes. Expression of C5aR on synovial mast cells was determined by immunohistochemical and functional studies. C5aR−/− and control mast cells were engrafted into mast cell-deficient W/Wv mice to examine the requirement for this receptor in arthritis. C5aR-dependent activation of mast cells was investigated in C5aR−/− animals and in murine and human mast cell cultures.
Murine synovial mast cells express functional C5aR. Unlike their wild-type counterparts, C5aR−/− mast cells adoptively transferred into W/Wv mice were incompetent to restore arthritis, despite equivalent synovial engraftment. Activation of C5aR−/− mast cells by K/BxN serum in vivo remained intact, indicating that C5aR is dispensable for normal IgG-mediated triggering. Consistent with this result, cultured mast cells treated with C5a failed to modulate expression of Fc γ receptors (FcγR) or otherwise alter activation threshold. In human mast cells, C5a promoted production of the neutrophil chemotaxin interleukin 8, and recruitment of neutrophils at 24h after serum administration was impaired in C5aR−/− mice, suggesting that enhanced neutrophil chemoattractant production underlies the requirement for C5aR on mast cells in arthritis.
Stimulation via C5aR is required to unleash the pro-inflammatory activity of synovial mast cells in immune complex arthritis, albeit via a mechanism distinct from C5a-modulated FcγR expression.
Galectin-1 (Gal-1), a β-galactoside–binding lectin, plays a profound role in modulating adaptive immune responses by altering the phenotype and fate of T cells. Experimental data showing recombinant Gal-1 (rGal-1) efficacy on T cell viability and cytokine production, nevertheless, is controversial due to the necessity of using stabilizing chemicals to help retain Gal-1 structure and function. To address this drawback, we developed a mouse Gal-1 human Ig chimera (Gal-1hFc) that did not need chemical stabilization for Gal-1 ligand recognition, apoptosis induction, and cytokine modulation in a variety of leukocyte models. At high concentrations, Gal-1hFc induced apoptosis in Gal-1 ligand+ Th1 and Th17 cells, leukemic cells, and granulocytes from synovial fluids of patients with rheumatoid arthritis. Importantly, at low, more physiologic concentrations, Gal-1hFc retained its homodimeric form without losing functionality. Not only did Gal-1hFc–binding trigger IL-10 and Th2 cytokine expression in activated T cells, but members of the CD28 family and several other immunomodulatory molecules were upregulated. In a mouse model of contact hypersensitivity, we found that a non-Fc receptor-binding isoform of Gal-1hFc, Gal-1hFc2, alleviated T cell-dependent inflammation by increasing IL-4+, IL-10+, TGF-β +, and CD25high/FoxP3+ T cells, and by decreasing IFN-γ + and IL-17+ T cells. Moreover, in human skin-resident T cell cultures, Gal-1hFc diminished IL-17+ T cells and increased IL-4+ and IL-10+ T cells. Gal-1hFc will not only be a useful new tool for investigating the role of Gal-1 ligands in leukocyte death and cytokine stimulation, but for studying how Gal-1–Gal-1 ligand binding shapes the intensity of immune responses.
We previously reported association of co-occurrence of HLA-DRB1 shared epitope (SE) and RANKL SNPs with younger age of RA onset in 182 rheumatoid factor positive (RF) European American (EA) early RA patients. Here, we fine-mapped the 48 kb RANKL region in the extended 210 EA RF-positive early RA cohort, sought replication of RA-associated SNPs in additional 501 EA and 298 African-Americans (AA) RA cohorts, and explored functional consequences of RA-associated SNPs.
SNP genotyping was conducted using pyrosequencing or TaqMan PCR assays. Associations of rs7984870 with RANKL expression in plasma, PBMC and isolated T cells were quantified using ELISA and RT-PCR. Site-directed mutagenesis of rs7984870 within the 2kb RANKL promoter was performed to drive the luciferase reporter gene in osteoblast and stromal cell lines. Interaction of DNA and protein was determined by electrophoretic mobility shift assay.
A single promoter SNP rs7984870 was consistently significantly associated with earlier age of RA onset in 3 independent seropositive (RF or anti-cyclic citrullinated peptide antibody positive) RA cohorts but not in seronegative RA patients. The risk C allele of rs7984870 conferred 2-fold higher plasma RANKL levels in RF-positive RA patients, significantly elevated RANKL mRNA expression in activated normal T cells, and increased promoter activity after stimulation in vitro via differential binding to transcription factor SOX5.
The RANKL promoter allele that increased transcriptional levels upon stimulation might promote interaction between activated T cells and dendritic cells, predisposing to younger RA onset in seropositive EA and/or AA individuals.
The immune and coagulation systems are both implicated in the pathogenesis of rheumatoid arthritis (RA). Plasma carboxypeptidase B (CPB), which is activated by the thrombin/thrombomodulin complex, plays a procoagulant role during fibrin clot formation. However, an antiinflammatory role for CPB is suggested by the recent observation that CPB can cleave proinflammatory mediators, such as C5a, bradykinin, and osteopontin. Here, we show that CPB plays a central role in downregulating C5a-mediated inflammatory responses in autoimmune arthritis. CPB deficiency exacerbated inflammatory arthritis in a mouse model of RA, and cleavage of C5a by CPB suppressed the ability of C5a to recruit immune cells in vivo. In human patients with RA, genotyping of nonsynonymous SNPs in the CPB-encoding gene revealed that the allele encoding a CPB variant with longer half-life was associated with a lower risk of developing radiographically severe RA. Functionally, this CPB variant was more effective at abrogating the proinflammatory properties of C5a. Additionally, expression of both CPB and C5a in synovial fluid was higher in patients with RA than in those with osteoarthritis. These findings suggest that CPB plays a critical role in dampening local, C5a-mediated inflammation and represents a molecular link between inflammation and coagulation in autoimmune arthritis.
The aim of this study was to determine the association of hormone levels with the occurrence of musculoskeletal pain. Men ages 40 to 79 years were recruited from population registers in 8 European centres. Subjects were asked to complete a postal questionnaire, which enquired about lifestyle and the occurrence of musculoskeletal pain over the past month. Total testosterone (T), oestradiol (E2), luteinising hormone (LH), and follicle-stimulating hormone (FSH) were assayed from a fasting blood sample. The association between pain status and hormone levels was assessed using multinomial logistic regression with results expressed as relative risk ratios (RRR) and 95% confidence intervals (CI). A total of 3206 men had complete data on pain status. Of these, 8.7% reported chronic widespread pain (CWP), whereas 50% had some pain although not CWP and were classified as having some pain. T and E2 were not associated with musculoskeletal pain, whereas significant differences in LH and FSH levels were found between pain groups. After adjustment for age and other possible confounders, the association between pain status and both LH and FSH persisted. Compared with those in the lowest tertile of LH, those in the highest tertile were more likely to report some pain (vs no pain, RRR = 1.28; 95% CI 1.09 to 1.50) and also CWP (vs no pain, RRR = 1.51; 95% CI 1.10 to 2.07). Similar results were found for FSH. Gonadotrophins, but not sex steroid hormone levels, are associated with musculoskeletal pain in men.
Higher levels of gonadotrophins but not androgens were significantly associated with musculoskeletal pain in men. Alterations in hypothalamic–pituitary–testicular feedback mechanisms may play a role in the onset of chronic widespread pain.
Musculoskeletal pain; Reproductive hormones; American College of Rheumatology; European Male Ageing Study; Epidemiology
Neutrophils serve as a vanguard of the acute innate immune response to invading pathogens. Neutrophils are also abundant at sites of autoimmune inflammation, such as the rheumatoid joint, although their pathophysiologic role is incompletely defined and relevant effector functions remain obscure. Using genetic and pharmacologic approaches in the K/BxN serum transfer model of arthritis, we find that autoantibody-driven erosive synovitis is critically reliant on the generation of leukotrienes, and more specifically on leukotriene B4 (LTB4), for disease induction as well as perpetuation. Pursuing the cellular source for this mediator, we find via reconstitution experiments that mast cells are a dispensable source of leukotrienes, whereas arthritis susceptibility can be restored to leukotriene-deficient mice by intravenous administration of wild-type neutrophils. These experiments demonstrate a nonredundant role for LTB4 in inflammatory arthritis and define a neutrophil mediator involved in orchestrating the synovial eruption.
Chemokine receptors are G-protein coupled receptors (GPCRs) phosphorylated by G-protein receptor kinases (GRKs) after ligand-mediated activation. We hypothesized that GRK subtypes differentially regulate granulocyte chemotaxis and clinical disease expression in the K/BxN model.
Clinical, histologic, and cytokine responses in GRK6−/−, GRK5−/−,GRK2+/−, and wildtype mice were evaluated using K/BxN serum transfer. Granulocyte chemotaxis was analyzed by transendothelial migration assays.
Both GRK6−/− and GRK2+/− mice had increased arthritis disease severity (p<0.001); whereas GRK5−/− was not different from controls. Acute weight loss was enhanced in GRK6−/− and GRK2+/− mice (p<0.001, days 3–10). However, GRK6−/− mice uniquely had more weight loss (>10%), elevated serum IL-6, and enhanced migration toward LTB4 and C5a in vitro.
GRK6 and - 2, but not GRK5,are involved in the pathogenesis of acute arthritis in the K/BxN model. In particular, GRK6 may dampen inflammatory responses by regulating granulocyte trafficking toward chemoattractants.
Phospholipase A2 (PLA2) catalyzes the release of arachidonic acid for generation of lipid mediators of inflammation and is crucial in diverse inflammatory processes. The functions of the secretory PLA2 enzymes (sPLA2), numbering 9 members in humans, are poorly understood, though they have been shown to participate in lipid mediator generation and the associated inflammation. To further understand the roles of sPLA2 in disease, we quantified the expression of these enzymes in the synovial fluid in rheumatoid arthritis and used gene-deleted mice to examine their contribution in a mouse model of autoimmune erosive inflammatory arthritis. Contrary to expectation, we find that the group V sPLA2 isoform plays a novel anti-inflammatory role that opposes the proinflammatory activity of group IIA sPLA2. Mechanistically, group V sPLA2 counter-regulation includes promotion of immune complex clearance by regulating cysteinyl leukotriene synthesis. These observations identify a novel anti-inflammatory function for a PLA2 and identify group V sPLA2 as a potential biotherapeutic for treatment of immune-complex-mediated inflammation.
secreted phospholipase A2; arthritis; autoimmunity; inflammation
Neutrophils represent a prominent component of inflammatory joint effusions and are required for synovial inflammation in mouse models, but mechanisms are poorly understood. We developed a system to test the importance of production of specific factors by neutrophils in a mouse model of arthritis.
Neutrophil-deficient Gfi-1−/− mice were sub-lethally irradiated, then engrafted with donor bone-marrow cells (BMC), which resulted in production of mature neutrophils within two weeks. By reconstituting with BMC from mice lacking selected pro-inflammatory factors, mice specifically lacking these factors on neutrophils were generated. Arthritis was initiated by transfer of K/BxN serum to identify the role of defined neutrophil factors on arthritis incidence and severity.
Neutrophils lacking the signaling chain of stimulatory Fc receptors (FcRγ −/−) were unable to elicit arthritis, but neutrophils lacking Fcγ RIII still did so. Neutrophils lacking the chemotactic or adhesion receptors C5aR or CD11a/LFA-1 also failed to initiate arthritis but could enter joints in which inflammation had been initiated by wild-type neutrophils. Neutrophils unable to produce interleukin-1 α and β (IL-1αβ −/−) or leukotrienes (5-LO−/−) produced arthritis of intermediate severity. Inability of neutrophils to make tumor necrosis factor (TNF), or to express receptors for TNF or IL-1, had no effect on arthritis.
A novel transfer system was developed to identify neutrophil production of FcRγ , C5aR, and CD11a/LFA-1 as critical components of autoantibody-mediated arthritis. Neutrophil production of IL-1 and leukotriene B4 likely contributes to inflammation but is not essential. Molecular requirements for neutrophil influx into joints become more permissive after inflammation is initiated.
arthritis; neutrophils; mouse model; inflammation; autoantibodies
Although the innate immune function of mast cells in the acute phase of parasitic and bacterial infections is well established, their participation in chronic immune responses to indolent infection remains incompletely understood. In parasitic infection with Trichinella spiralis, the immune response incorporates both lymphocyte and mast cell-dependent effector functions for pathogen eradication. Among the mechanistic insights still unresolved in the reaction to T. spiralis are the means by which mast cells respond to parasites and the mast cell effector functions that contribute to the immunologic response to this pathogen. We hypothesized that mast cell elaboration of tryptase may comprise an important effector component in this response. Indeed, we find that mice deficient in the tryptase mouse mast cell protease-6 (mMCP-6) display a significant difference in their response to T. spiralis larvae in chronically infected skeletal muscle tissue. Mechanistically, this is associated with a profound inability to recruit eosinophils to larvae in mMCP-6-deficient mice. Analysis of IgE-deficient mice demonstrates an identical defect in eosinophil recruitment. These findings establish that mast cell secretion of the tryptase mMCP-6, a function directed by the activity of the adaptive immune system, contributes to eosinophil recruitment to the site of larval infection, thereby comprising an integral link in the chronic immune response to parasitic infection.
In addition to their pivotal role in thrombosis and wound repair, platelets participate in inflammatory responses. We investigated the role of platelets in the autoimmune disease rheumatoid arthritis. We identified platelet microparticles—submicrometer vesicles elaborated by activated platelets—in joint fluid from patients with rheumatoid arthritis and other forms of inflammatory arthritis, but not in joint fluid from patients with osteoarthritis. Platelet microparticles were proinflammatory, eliciting cytokine responses from synovial fibroblasts via interleukin-1. Consistent with these findings, depletion of platelets attenuated murine inflammatory arthritis. Using both pharmacologic and genetic approaches, we identified the collagen receptor glycoprotein VI as a key trigger for platelet microparticle generation in arthritis pathophysiology. Thus, these findings demonstrate a previously unappreciated role for platelets and their activation-induced microparticles in inflammatory joint diseases.
Phospholipase A2 (PLA2) catalyses the release of arachidonic acid for generation of lipid mediators of inflammation and is crucial in diverse inflammatory processes. The functions of the secretory PLA2 enzymes (sPLA2), numbering nine members in humans, are poorly understood, though they have been shown to participate in lipid mediator generation and the associated inflammation. To further understand the roles of sPLA2 in disease, we quantified the expression of these enzymes in the synovial fluid in rheumatoid arthritis and used gene-deleted mice to examine their contribution in a mouse model of autoimmune erosive inflammatory arthritis. Contrary to expectation, we find that the group V sPLA2 isoform plays a novel anti-inflammatory role that opposes the pro-inflammatory activity of group IIA sPLA2. Mechanistically, group V sPLA2 counter-regulation includes promotion of immune complex clearance by regulating cysteinyl leukotriene synthesis. These observations identify a novel anti-inflammatory function for a PLA2 and identify group V sPLA2 as a potential biotherapeutic for treatment of immune-complex-mediated inflammation.
arthritis; autoimmunity; inflammation; secreted phospholipase A2
Induction of macrophage necrosis is an important strategy used by virulent Mycobacterium tuberculosis (Mtb) to avoid innate host defense. In contrast, attenuated Mtb causes apoptosis, which limits bacterial replication and promotes T cell cross priming by antigen presenting cells. Here we demonstrated that Mtb infection causes plasma membrane microdisruptions. Resealing of these lesions—a process crucial for preventing necrosis and promoting apoptosis—required the translocation of lysosome and Golgi apparatus-derived vesicles to the plasma membrane. Plasma membrane repair depended on prostaglandin E2 (PGE2), which regulates synaptotagmin 7, the Ca++ sensor involved in the lysosome-mediated repair mechanism. By inducing production of lipoxin A4 (LXA4), which blocks PGE2 biosynthesis, virulent Mtb prevented membrane repair and induced necrosis. Thus, virulent Mtb impairs macrophage plasma membrane repair to evade host defenses.
Although mast cells (MCs) often are abundant in the synovial tissues of patients with rheumatoid arthritis (RA), MC’s contribution to joint inflammation and cartilage loss remains poorly understood. MC-restricted tryptase•heparin complexes have pro-inflammatory activity, and significant amounts of hTryptase-β are present in RA synovial fluid. Mouse MC protease-6 (mMCP-6) is the ortholog of hTryptase-β, and this serine protease is abundant in the synovium of arthritic mice. We now report that C57BL/6 (B6) mice lacking their tryptase•heparin complexes have attenuated arthritic responses, with mMCP-6 as the dominant tryptase responsible for augmenting neutrophil infiltration in the K/B×N mouse serum-transfer arthritis model. While inflammation in this experimental arthritis model was not dependent on protease activated receptor-2, it was dependent on the chemokine receptor CXCR2. In support of the latter data, exposure of synovial fibroblasts to hTryptase-β•heparin or mMCP-6•heparin complexes resulted in expression of the neutrophil chemotactic factors CXCL1/KC, CXCL5/LIX, and CXCL8/IL-8. Our proteomics, histochemistry, and immunohistochemistry data also revealed substantial loss of cartilage-derived aggrecan proteoglycans in the arthritic joints of wild-type B6 mice but not mMCP-6-null B6 mice. These observations demonstrate the functional contribution of MC-restricted tryptase•heparin complexes in the K/B×N mouse arthritis model and connect our mouse findings with RA pathophysiology.
mast cell; rheumatoid arthritis; inflammation; chemokines; transgenic/knockout mice
To determine the glycaemic index (GI) of various staple carbohydrate-rich foods in the UK diet, and to consider the factors influencing the GI of foods.
Subjects were served with 25 or 50 g portions of glucose on three occasions, followed by a selection of test foods providing an equal amount of available carbohydrate, in random order. Each test food was consumed by 10 subjects. Capillary blood glucose levels were measured in the fasted state and over the 120 minutes following commencement consumption of the foods.
The study was carried out in a research institute (MRC Human Nutrition Research, Cambridge, UK).
42 healthy adult volunteers were studied.
The GI values of 33 foods were measured according to the WHO/FAO recommended methodology. These foods included various breads, breakfast cereals, pasta, rice and potatoes, all of which were commercially available in the UK.
The results illustrate a number of factors which are important in influencing the GI of a food, highlighting the importance of measuring the GI of a food, rather than assuming a previously published value for a similar food. This is useful both to researchers analysing dietary surveys or planning intervention studies, and also to health professionals advising individuals on their diets.
Glycaemic index; Carbohydrate; UK diet