Marianne Engström

Immunological changes in human skeletal muscle and blood after eccentric exercise and multiple biopsies.

1 A role of the immune system in muscular adaptation to physical exercise has been suggested but data from controlled human studies are scarce. The present study investigated immunological events in human blood and skeletal muscle by immunohistochemistry and flow cytometry after eccentric cycling exercise and multiple biopsies. 2 Immunohistochemical detection of neutrophil‐ (CD11b, CD15), macrophage‐ (CD163), satellite cell‐ (CD56) and IL‐1β‐specific antigens increased similarly in human skeletal muscle after eccentric cycling exercise together with multiple muscle biopsies, or multiple biopsies only. 3 Changes in immunological variables in blood and muscle were related, and monocytes and natural killer (NK) cells appeared to have governing functions over immunological events in human skeletal muscle. 4 Delayed onset muscle soreness, serum creatine kinase activity and C‐reactive protein concentration were not related to leukocyte infiltration in human skeletal muscle. 5 Eccentric cycling and/or muscle biopsies did not result in T cell infiltration in human skeletal muscle. Modes of stress other than eccentric cycling should therefore be evaluated as a myositis model in human. 6 Based on results from the present study, and in the light of previously published data, it appears plausible that muscular adaptation to physical exercise occurs without preceding muscle inflammation. Nevertheless, leukocytes seem important for repair, regeneration and adaptation of human skeletal muscle.

Systemic anti-tumor necrosis factor alpha therapy in rheumatoid arthritis down-regulates synovial tumor necrosis factor alpha synthesis.

OBJECTIVE To investigate the hypothesis that tumor necrosis factor alpha (TNFalpha) blockade in rheumatoid arthritis (RA) diminishes synovial synthesis of TNFalpha, interleukin-1alpha (IL-1alpha), and IL-1beta. METHODS Patients with active RA received a single 10 mg/kg infusion of infliximab. Multiple synovial biopsy specimens were obtained from a knee the day before infusion and 14 days later. A modified immunohistochemical method detecting cytokine-producing rather than cytokine-binding cells was applied to determine synthesis of TNFalpha, IL-1alpha, and IL-1beta in fixed, cryopreserved sections. Computerized image analysis using two different methodologies was performed by independent observers blinded to the identity of samples. RESULTS All 8 patients met the American College of Rheumatology 20% improvement response criteria (ACR 20) at 2 weeks, and half of these patients met the ACR 50. With a few exceptions, there was concordance between both image analysis methodologies regarding the direction of change in immunopositive area fraction for all cytokines analyzed. TNFalpha synthesis was significantly reduced after treatment (P = 0.05 at the Karolinska Institute, Stockholm, Sweden; P = 0.008 at the Kennedy Institute, London, UK). Patients meeting the ACR 50 were those with the highest baseline levels of TNFalpha synthesis. There was a significant correlation between baseline levels of TNFalpha expression and change in TNFalpha levels in response to therapy. Both IL-1alpha and IL-1beta synthesis were reduced in 3 patients; IL-1alpha synthesis alone was reduced in 2 patients and IL-1beta synthesis alone was reduced in 2 patients. In 1 patient, neither IL-1alpha nor IL-1beta synthesis was reduced. CONCLUSION Analysis of synovial tissue by means of immunomorphology and image analysis in a clinical trial setting may allow the drawing of biologically meaningful conclusions. Synovial TNFalpha synthesis was reduced 2 weeks after infliximab treatment. Reductions in IL-1alpha and IL-1beta synthesis were demonstrated in a subgroup of patients. High levels of synovial TNFalpha production prior to treatment may predict responsiveness to therapy.

Monoclonal IgG antibodies generated from joint-derived B cells of RA patients have a strong bias toward citrullinated autoantigen recognition

Synovial IgG-expressing B cells from patients with rheumatoid arthritis show specificity for citrullinated autoantigens.

Structural Changes and Antibody Enrichment in the Lungs Are Early Features of Anti–Citrullinated Protein Antibody–Positive Rheumatoid Arthritis

It has been suggested that immunologic events in the lungs may be involved in triggering immunity, in particular production of anti–citrullinated protein antibodies (ACPAs) during early phases of rheumatoid arthritis (RA). The aim of this study was to investigate the structural and immunologic features of the lungs in incident cases of early RA in relation to ACPA presence and smoking status.

Identification of a novel chemokine-dependent molecular mechanism underlying rheumatoid arthritis-associated autoantibody-mediated bone loss

Objectives Rheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide antibodies (ACPAs) appear before disease onset and are associated with bone destruction. We aimed to dissect the role of ACPAs in osteoclast (OC) activation and to identify key cellular mediators in this process. Methods Polyclonal ACPA were isolated from the synovial fluid (SF) and peripheral blood of patients with RA. Monoclonal ACPAs were isolated from single SF B-cells of patients with RA. OCs were developed from blood cell precursors with or without ACPAs. We analysed expression of citrullinated targets and peptidylarginine deiminases (PAD) enzymes by immunohistochemistry and cell supernatants by cytometric bead array. The effect of an anti-interleukin (IL)-8 neutralising antibody and a pan-PAD inhibitor was tested in the OC cultures. Monoclonal ACPAs were injected into mice and bone structure was analysed by micro-CT before and after CXCR1/2 blocking with reparixin. Results Protein citrullination by PADs is essential for OC differentiation. Polyclonal ACPAs enhance OC differentiation through a PAD-dependent IL-8-mediated autocrine loop that is completely abolished by IL-8 neutralisation. Some, but not all, human monoclonal ACPAs derived from single SF B-cells of patients with RA and exhibiting distinct epitope specificities promote OC differentiation in cell cultures. Transfer of the monoclonal ACPAs into mice induced bone loss that was completely reversed by the IL-8 antagonist reparixin. Conclusions We provide novel insights into the key role of citrullination and PAD enzymes during OC differentiation and ACPA-induced OC activation. Our findings suggest that IL8-dependent OC activation may constitute an early event in the initiation of the joint specific inflammation in ACPA-positive RA.

Cell Specific Synovial Expression of Nicotinic Alpha 7 Acetylcholine Receptor in Rheumatoid Arthritis and Psoriatic Arthritis

Neuroimmune interactions are known to influence several chronic inflammatory and rheumatic diseases, but the underlying mechanisms have been insufficiently elucidated. The cholinergic anti‐inflammatory pathway is characterized by neural regulation of systemic inflammation, mediated by the vagus nerve and specific cholinergic stimulation of the nicotinic α‐7 acetylcholine receptor (α7nAChR) on immune cells. Moreover, α7nAChR has been shown important for immune regulation also in the absence of nerves, but little is known about these mechanisms in chronic joint inflammation. The expression and localization of α7nAChR in synovial biopsies from patients with rheumatoid arthritis and psoriatic arthritis was investigated by immunohistochemistry using monoclonal antibody against α7nAChR. Surface staining of α7nAChR was observed in synovial tissue of all arthritis patients investigated and could also to a lesser extent be detected in the synovium of healthy individuals. α7nAChR positive cells were detected in mainly synovial lining cells and vessels. The α7nAChR positively stained cells were by double immunofluorescence identified as primarily macrophages and fibroblasts, with the majority of these cells expressing the receptor. These results indicate the importance of α7nAChR and cholinergic mechanisms in arthritis pathogenesis and implicate specific cholinergic modulation as a potential anti‐inflammatory therapeutic strategy in joint inflammation.

FOXP3 expression in blood, synovial fluid and synovial tissue during inflammatory arthritis and intra-articular corticosteroid treatment

Objective: To analyse the distribution of FOXP3+CD25+CD4+ regulatory T cells (Treg) in peripheral blood, synovial fluid and tissue of patients with rheumatic disease during relapse and after local treatment. Methods: FOXP3 expression was assessed by flow cytometry, immunohistochemistry, immunofluorescence and real-time polymerase chain reaction (RT-PCR). The functional suppressive capacity of Treg was analysed after co-culture with effector CD4+CD25− T cells through assessment of proliferation and cytokine secretion. Results: It was shown that FOXP3 protein and mRNA expression in synovial fluid T cells was not confined solely to CD25bright T cells as seen in blood, but included CD25intermediate and even CD25neg T cells. Indeed, synovial fluid CD25high T cells showed similar suppressive capacity as CD25bright T cells, indicating the presence of functional Treg in T cells with lower intensity of CD25. In synovial tissue, FOXP3+ cells were present in low numbers within T-cell infiltrates and decreased further after intra-articular glucocorticosteroid administration, in parallel with the general reduction in inflammation. Conclusions: Identification of synovial fluid FOXP3+ Treg with varying intensities of CD25 opens up possibilities for thorough characterisation of this important T-cell subset in the inflammatory compartment. However, only scarce synovial membrane expression of FOXP3 was found even in the absence of overt inflammation, suggesting that the synovial membrane is a site that would benefit therapeutically from Treg expansion.

High synovial expression of the inhibitory FcγRIIb in rheumatoid arthritis

Activating Fc gamma receptors (FcγRs) have been identified as having important roles in the inflammatory joint reaction in rheumatoid arthritis (RA) and murine models of arthritis. However, the role of the inhibitory FcγRIIb in the regulation of the synovial inflammation in RA is less known. Here we have investigated synovial tissue from RA patients using a novel monoclonal antibody (GB3) specific for the FcγRIIb isoform. FcγRIIb was abundantly expressed in synovia of RA patients, in sharp contrast to the absence or weak staining of FcγRIIb in synovial biopsies from healthy volunteers. In addition, the expression of FcγRI, FcγRII and FcγRIII was analyzed in synovia obtained from early and late stages of RA. Compared with healthy synovia, which expressed FcγRII, FcγRIII but not FcγRI, all activating FcγRs were expressed and significantly up-regulated in RA, regardless of disease duration. Macrophages were one of the major cell types in the RA synovium expressing FcγRIIb and the activating FcγRs. Anti-inflammatory treatment with glucocorticoids reduced FcγR expression in arthritic joints, particularly that of FcγRI. This study demonstrates for the first time that RA patients do not fail to up-regulate FcγRIIb upon synovial inflammation, but suggests that the balance between expression of the inhibitory FcγRIIb and activating FcγRs may be in favour of the latter throughout the disease course. Anti-inflammatory drugs that target activating FcγRs may represent valuable therapeutics in this disease.

Local administration of glucocorticoids decreases synovial citrullination in rheumatoid arthritis.

IntroductionProtein citrullination is present in the rheumatoid synovium, presumably contributing to the perpetuation of chronic inflammation, in the presence of specific autoimmunity. As a result, the present study examined the possibility that effective antirheumatic treatment will decrease the level of synovial citrullination.MethodsSynovial biopsies were obtained from 11 rheumatoid arthritis (RA) patients before and after 8 weeks of treatment with 20 mg methotrexate weekly, 15 RA patients before and 2 weeks after an intraarticular glucocorticoid injection, and eight healthy volunteers. Synovial inflammation was assessed with double-blind semiquantitative analysis of lining thickness, cell infiltration, and vascularity by using a 4-point scale. Expression of citrullinated proteins (CPs) with the monoclonal antibody F95 and peptidylarginine deiminase (PAD) 2 and 4 was assessed immunohistochemically with double-blind semiquantitative analysis. In vitro synovial fluid (SF), peripheral blood (PB), mononuclear cells (MCs), and synovial explants obtained from RA patients were incubated with dexamethasone and analyzed with immunohistochemistry for expression of CP as well as PAD2 and PAD4 enzymes.ResultsThe presence of synovial CP was almost exclusive in RA compared with healthy synovium and correlated with the degree of local inflammation. Treatment with glucocorticoids but not methotrexate alters expression of synovial CP and PAD enzymes, in parallel with a decrease of synovial inflammation. Ex vivo and in vitro studies suggest also a direct effect of glucocorticoids on citrullination, as demonstrated by the decrease in the level of citrullination and PAD expression after incubation of SFMC and synovial explants with dexamethasone.ConclusionSynovial citrullination and PAD expression are dependent on local inflammation and targeted by glucocorticoids.

Signs of immune activation and local inflammation are present in the bronchial tissue of patients with untreated early rheumatoid arthritis

Objectives Events in the lungs might contribute to generation of anticitrullinated protein antibodies (ACPA) in rheumatoid arthritis (RA). We investigated if signs of immune activation are present in bronchial biopsies and bronchoalveolar lavage (BAL) of patients with early-untreated RA without clinical signs of lung involvement. Methods Twenty-four patients with RA with symptom duration <1 year and naïve to disease-modifying antirheumatic drugs were subjected to bronchoscopy where BAL and mucosal bronchial biopsies were retrieved. For comparison, 15 bronchial biopsies and 79 BAL samples from healthy volunteers were available. Histological examination was performed to evaluate lymphocyte infiltration, presence of immune cells (T and B cells, plasma cells, dendritic cells and macrophages) and immune activation markers. Cell composition of BAL samples was analysed by differential counting and T cell subsets by flow cytometry. Results Lymphocyte infiltration was more frequently found in ACPA-positive patients (50%) as compared with ACPA-negative patients (17%) and controls (13%). Germinal centres, B cells and plasma cells were only found in ACPA-positive patients. The frequency of T cells in bronchial biopsies of patients with ACPA-positive RA was positively associated with expression of immune activation markers. BAL samples of patients with ACPA-positive, but not ACPA-negative, RA had significantly higher relative numbers of lymphocytes and expressed higher levels of activation markers compared with controls. Conclusions Signs of immune cell accumulation and activation are present both in the bronchial tissue and in BAL of untreated patients with early RA without concomitant lung disease, strengthening the role of the lung compartment as an important player in ACPA-positive RA.