<p>Induction of collagen-induced arthritis (CIA), an animal model for human rheumatoid arthritis, is dependent on anti-collagen type II (CII) antibodies. The effector mechanism by which autoantibodies contribute to inflammatory reactions in autoimmune diseases is not well understood. In this thesis I have studied the effector pathways used by IgG anti-CII antibodies to initiate arthritis, namely the IgG Fc receptors (FcγRs) and the complement system. We have found that FcγRIII is crucial for development of CIA, as CII-immunized mice lacking this receptor do not develop arthritis and IgG1 and IgG2b anti-CII antibodies require FcγRIII to trigger arthritis when transferred to naïve mice. The antibody-mediated arthritis was further enhanced in mice deficient in the inhibitory FcγRIIB, indicating that FcγRIIB regulates the activation of FcγRIII. Furthermore, we demonstrate that FcγRIII exist as three distinct haplotypes in mice, FcγRIII:H, FcγRIII:V and FcγRIII:T. Mice expressing the FcγRIII:H haplotype are more susceptible to CIA than mice expressing the FcγRIII:V haplotype, indicating that certain FcγRIII haplotype predisposes for CIA. We also show that the most likely FcγRIII-expressing effector cell in CIA is the macrophage, since FcγRIII-expressing macrophages exclusively can induce arthritis in FcγRIII-deficient mice challenged for CIA.</p><p>The complement system was also investigated in development of CIA. We found that this effector pathway is also necessary for onset of arthritis, as CIA was inhibited by treatment with anti-complement factor 5 (C5) antibodies. C5-deficient mice could neither develop CIA unless provided with C5-containing sera. </p><p>Taken together, the work presented in this thesis indicates that FcγRs and the complement system are crucial for the induction of experimental arthritis. These findings are important for understanding the mechanisms behind rheumatoid arthritis and blocking of these effector pathways may in the future be used as treatment of rheumatoid arthritis. </p>

The T cell function of a proliferation inducing ligand (APRIL or TNFSF13) remains unclear. By comparing APRIL-/- mice with wild type (WT) mice, we have investigated the roles of APRIL in Th1, Th2 and Th17 responses. With regard to APRIL in Th1 responses, cultured APRIL-/- CD4+ T cells showed increased IFN-gamma production under non-polarizing, but not under Th1 polarizing, conditions. No difference in antigen-specific IgG2a levels existed between APRIL-/- and WT mice immunized with ovalbumin (OVA) and complete Freund's adjuvant (CFA) which induces Th1 polarization. Our data indicate that APRIL represses Th1 responses only under non-polarizing conditions. As for APRIL in Th2 responses, cultured APRIL-/- CD4+ T cells exhibited enhanced Th2 cytokine production under non-polarizing conditions, and augmented IL-13 production under Th2 polarizing conditions. Upon immunization with OVA and aluminum potassium sulfate (alum) which induces Th2 polarization, APRIL-/- mice responded with an increased antigen-specific IgG1 response. In the OVA-induced allergic lung inflammation model which is mediated by Th2 responses, APRIL-/- mice had significantly aggravated allergic lung inflammation. Accordingly, a decoy receptor-Ig fusion protein, TACI-Ig, treatment to block APRIL in WT mice enhanced allergic lung inflammation. In agreement with the role of APRIL in CD4+ T cells, the transfer of APRIL sufficient, OVA-specific, TCR transgenic CD4+ T (OT-II) cells to APRIL-/- mice restored the suppressive effect of APRIL on allergic lung inflammation. Mechanistically, the expression of c-maf, the IL-4 gene transcription factor, was markedly enhanced in APRIL-/- CD4+ T cells under non-polarizing and Th2 polarizing conditions. Our data suggest that APRIL inhibits Th2 responses and allergic lung inflammation by suppressing IL-4 production in CD4+ T cells via diminished c-maf expression, and by suppressing IL-13 production in CD4+ T cells via an IL-4 independent, IL-13 specific pathway. Regarding APRIL in Th17 responses, the incidence of Th17-mediated collagen-induced arthritis (CIA) in APRIL-/- mice was reduced, in parallel with diminished levels of antigen-specific IgG2a autoantibody and IL-17 production. Our data indicate that APRIL promotes IL-17 production, and that APRIL-triggered signals contribute to arthritis. Our data clearly show that APRIL is important in T cell immunity, inhibitory in Th2 responses and costimulatory in Th17 responses.

The human being and other animals contain immensely complex biochemical processes that govern their function on a cellular level. It is estimated that several thousand small molecules (metabolites) are produced by various biochemical pathways in humans. Pathological processes can introduce perturbations in these biochemical pathways which can lead to changes in the amounts of some metabolites.Developments in analytical chemistry have made it possible measure a large number metabolites in a single blood sample, which gives a metabolic profile. In this thesis I have worked on establishing and understanding metabolic profiles from patients with rheumatoid arthritis (RA) and from animal models of the autoimmune diseases diabetes mellitus type 1 (T1D) and RA.Using multivariate statistical methods it is possible to identify differences between metabolic profiles of different groups. As an example we identified differences between patients with RA and healthy volunteers. This can be used to elucidate the biochemical processes that are active in a given pathological condition.Metabolite concentrations are affected by a many other things than the presence or absence of a disease. Both genomic and environmental factors are known to influence metabolic profiles. A main focus of my work has therefore been on finding strategies for ensuring that the results obtained when comparing metabolic profiles were valid and relevant. This strategy has included repetition of experiments and repeated measurement of individuals’ metabolic profiles in order to understand the sources of variation.Finding the most stable and reproducible metabolic effects has allowed us to better understand the biochemical processes seen in the metabolic profiles. This makes it possible to relate the metabolic profile differences to pathological processes and to genes and proteins involved in these.The hope is that metabolic profiling in the future can be an important tool for finding biomarkers useful for disease diagnosis, for identifying new targets for drug design and for mapping functional changes of genomic mutations. This has the potential to revolutionize our understanding of disease pathology and thus improving health care.

Induction of collagen-induced arthritis (CIA), an animal model for human rheumatoid arthritis, is dependent on anti-collagen type II (CII) antibodies. The effector mechanism by which autoantibodies contribute to inflammatory reactions in autoimmune diseases is not well understood. In this thesis I have studied the effector pathways used by IgG anti-CII antibodies to initiate arthritis, namely the IgG Fc receptors (FcγRs) and the complement system. We have found that FcγRIII is crucial for development of CIA, as CII-immunized mice lacking this receptor do not develop arthritis and IgG1 and IgG2b anti-CII antibodies require FcγRIII to trigger arthritis when transferred to naïve mice. The antibody-mediated arthritis was further enhanced in mice deficient in the inhibitory FcγRIIB, indicating that FcγRIIB regulates the activation of FcγRIII. Furthermore, we demonstrate that FcγRIII exist as three distinct haplotypes in mice, FcγRIII:H, FcγRIII:V and FcγRIII:T. Mice expressing the FcγRIII:H haplotype are more susceptible to CIA than mice expressing the FcγRIII:V haplotype, indicating that certain FcγRIII haplotype predisposes for CIA. We also show that the most likely FcγRIII-expressing effector cell in CIA is the macrophage, since FcγRIII-expressing macrophages exclusively can induce arthritis in FcγRIII-deficient mice challenged for CIA. The complement system was also investigated in development of CIA. We found that this effector pathway is also necessary for onset of arthritis, as CIA was inhibited by treatment with anti-complement factor 5 (C5) antibodies. C5-deficient mice could neither develop CIA unless provided with C5-containing sera. Taken together, the work presented in this thesis indicates that FcγRs and the complement system are crucial for the induction of experimental arthritis. These findings are important for understanding the mechanisms behind rheumatoid arthritis and blocking of these effector pathways may in the future be used as treatment of rheumatoid arthritis.

B cells are normally regulated to prevent activation against self-proteins through tolerance mechanisms.  However, occasionally there is a break in tolerance and B cells can become self-reactive, which might lead to the development of autoimmune disease. The activation of self-reactive B cells is regulated by receptors on the B cell surface, such as Fc gamma receptor IIb (FcγRIIb), complement receptor type 1 (CR1), and CR type 2 (CR2). In this thesis I have studied the role of FcγRIIb, CR1 and CR2 on B cells in autoimmune arthritis. By using a model for rheumatoid arthritis, I discovered that the initial self-reactive B cell response in arthritis was associated with the splenic marginal zone B cell population. Marginal zone B cells express high levels of CR1/CR2 and FcγRIIb, suggesting that they normally require high regulation. Further, female mice deficient in CR1/CR2 displayed increased susceptibility to arthritis compared to CR1/CR2-sufficient female mice. When investigating whether sex hormones affected arthritis susceptibility, we found that ovariectomy, of the otherwise fairly resistant CR1/CR2-sufficient mice, reduced the expression of CR1 on B cells and rendered the mice more susceptible to arthritis. In humans, a significantly reduced CR1 and FcγRIIb expression was found on B cells in aging women, but not in men. This may contribute to the increased risk for women to develop autoimmune disease as reduced receptor expression may lead to the activation of self-reactive B cells. In agreement, lower CR1, CR2 and FcγRIIb expression was seen in patients with rheumatoid arthritis.   Finally, a soluble form of FcγRIIb was used to investigate FcγRIIb’s ability to bind self-reactive IgG in an attempt to treat autoimmune arthritis. Treatment of mice with established arthritis was associated with less self-reactive IgG antibodies and consequently less disease, suggesting that soluble FcγRIIb may be used as a novel treatment in arthritis.

Plasmin, the central molecule of the plasminogen activator system, is a broad-spectrum serine protease. Plasmin is important for the degradation of fibrin and other components of the extracellular matrix (ECM) during a number of physiological and pathological processes. The aim of this thesis was to elucidate the functional roles of plasmin during pathological inflammation and infection in autoimmune and non-autoimmune diseases. For this purpose, mouse models of rheumatoid arthritis (RA), bacterial arthritis, infection, and sepsis have been used. Previous studies from our laboratory have shown that plasminogen-deficient mice are resistant to the development of collagen type II-induced arthritis (CIA). In contrast, others have shown that plasmin plays a protective role in antigen-induced arthritis (AIA). To investigate the contrasting roles of plasminogen deficiency in models of CIA and AIA, a new animal model of arthritis called local injection-induced arthritis (LIA) was developed. In this model, we replaced methylated bovine serum albumin, which is normally used as an immunogen in the AIA model, with collagen type II (CII) to induce arthritis. When wild-type and plasminogen-deficient mice were injected intra-articularly with CII or 0.9% NaCl following CIA induction, plasminogen-deficient mice developed typical CIA, but the disease was less severe than in wild-type mice and was restricted to the injected joints. When the AIA model was used, plasminogen-deficient mice developed a much more severe arthritis than the wild-type mice. These results indicate that both the antigen and joint trauma caused by the local injection are critical to explaining the contrasting roles of plasminogen deficiency in CIA and AIA. This indicates that CIA and AIA have distinct pathogenic mechanisms and plasmin plays contrasting roles in different types of arthritis models. To study the functional roles of plasmin in the host inflammatory response during infectious arthritis, a Staphylococcus aureus-induced bacterial arthritis model was established. When wild-type mice were injected intra-articularly with 1 × 106 colony-forming units (CFU) of S. aureus per joint, all the bacteria were completely eliminated from the injected joints in 28 days. However, in the plasminogen-deficient mice, the S. aureus counts were 27-fold higher at day 28 than at day 0. When human plasminogen was given to the plasminogen-deficient mice daily for 7 days, the bacterial clearance was greatly improved and the necrotic tissue in the joint cavity was also completely eliminated. Supplementation of plasminogen-deficient mice with plasminogen also restored the expression level of interleukin-6 (IL-6) in the arthritic joints. In summary, plasmin has protective roles during S. aureus-induced arthritis by enhancing cytokine expression, removing necrotic tissue, and mediating bacterial killing and inflammatory cell activation. The functional roles of plasmin during infection and sepsis were also studied in mice. Infection was induced by injecting 1 × 107 CFU of S. aureus intravenously and the sepsis model was induced by injecting 1.6 × 108 CFU of S. aureus. In the infection model, the wild-type mice had a 25-day survival rate of 86.7%, as compared to 50% in the plasminogen-deficient group. However, when sepsis was induced, the average survival for plasminogen-deficient mice was 3 days longer than for wild-type mice. Twenty-four hours after the induction of sepsis, the serum levels of IL-6 and IL-10 as well as the bacterial counts in all organs investigated were significantly higher in wild-type mice than in plasminogen-deficient mice. In wild-type mice, blockade of IL-6 by intravenous injection of anti-IL-6 antibodies significantly prolonged the onset of mortality and improved the survival rate during sepsis. These data indicate that plasmin plays different roles during infection and sepsis. Furthermore, plasmin appears to be involved in the regulation of inflammatory cytokine expression during sepsis. Taken together, our data indicate that plasmin plays multifunctional pro-inflammatory roles in different autoimmune and non-autoimmune diseases. The pro-inflammatory roles of plasmin include activation of inflammatory cells, regulation of cytokine expression, and enhancement of the bacterial killing ability of the host.

In autoimmune diseases such as rheumatoid arthritis (RA), self-reactive antibodies are present at high levels, which contributes to disease pathogenesis. The antibodies mediate their effect predominantly by binding to Fc gamma receptors (FcγR) on various leukocytes, such as monocytes, macrophages and mast cells, where FcγR ligation leads to cell activation. In this thesis the role of FcγR in RA was investigated. We could, for the first time, demonstrate an increased expression of the inhibitory FcγRIIb in RA synovial tissue, while this receptor as well as FcγRI were almost absent in healthy synovial tissue. The enhanced FcγRI expression in RA synovia was reduced by one intraarticular injection of glucocorticoids, indicating that FcγRI participates in the joint inflammation. Interestingly, RA patients with an ongoing joint inflammation exhibited blood monocytes with immune compromised features, such as decreased FcγR binding of IgG1-IC and reduced TNF production. These effects were associated with high levels of auto-antibodies in the patients, implying that the monocyte FcγR are saturated with IgG. In order to investigate whether soluble FcγR could be used as a therapy in arthritis, we injected human soluble FcγR into mice with collagen-induced arthritis (CIA). The soluble FcγR reduced the levels of pathogenic IgG anti-collagen type II (CII) antibodies, arthritis severity and pro-inflammatory cytokines. Thus, suggesting that soluble FcγR may represent a novel therapeutic agent in RA. We also studied the disease-aggravating role of mast cells in arthritis by investigating mouse mast cell protease-4 (mMCP-4) in CIA. We found that mMCP-4 deficient mice displayed a reduced IgG anti-CII response and reduced arthritis severity. This indicates a role for mMCP-4 in adaptive immunity. In conclusion, these data demonstrate that IgG occupancy of FcγR and mast cell secretion of mMCP-4 play vital roles in the development of autoimmune arthritis.

Objective to systematically review randomized trials that compared methotrexate (MTX) monotherapy to MTX in combination with other non-biologic disease-modifying anti-rheumatic Drugs (DMARD) and to compare the performances of PubMed versus MEDLINE (Ovid®) and EMBASE. Methods We performed a systematic review of randomized trials comparing MTX alone and MTX in combination with other non-biologic DMARDs. Heterogeneity was investigated and explored. The performances of Pubmed and MEDLINE were evaluated. The EMBASE unique trials were identified and investigated. Results A total of 19 trials were included and grouped by the type of patients randomized. Trials in DMARD naive patients showed no significant advantage of the MTX combination versus monotherapy. The recall was 85% vs. 90% for Ovid and PubMed, respectively, while the precision and number-needed-to read of Ovid and Pubmed were comparable. Only 23% of trials were EMBASE unique trials Conclusions In DMARD naive patients, the balance of efficacy/toxicity favours MTX monotherapy.

Objective to systematically review randomized trials that compared methotrexate (MTX) monotherapy to MTX in combination with other non-biologic disease-modifying anti-rheumatic Drugs (DMARD) and to compare the performances of PubMed versus MEDLINE (Ovid®) and EMBASE. Methods We performed a systematic review of randomized trials comparing MTX alone and MTX in combination with other non-biologic DMARDs. Heterogeneity was investigated and explored. The performances of Pubmed and MEDLINE were evaluated. The EMBASE unique trials were identified and investigated. Results A total of 19 trials were included and grouped by the type of patients randomized. Trials in DMARD naive patients showed no significant advantage of the MTX combination versus monotherapy. The recall was 85% vs. 90% for Ovid and PubMed, respectively, while the precision and number-needed-to read of Ovid and Pubmed were comparable. Only 23% of trials were EMBASE unique trials Conclusions In DMARD naive patients, the balance of efficacy/toxicity favours MTX monotherapy.

SAMMANFATTNING Hydroterapi har under en längre tid använts som behandlingsform, men det är brist på forskning inom området. Syftet med denna studie var att undersöka upplevelsen av hydroterapi hos personer med reumatisk sjukdom. En kvalitativ ansats anlades och enskilda semistrukturerade intervjuer användes. Deltagarna rekryterades genom bekvämlighetsurval via en kontakt på en reumatologklinik. Sex personer inkluderades: fem hade reumatoid artrit och en hade pelvospondylit. Resultatet tolkades med hjälp av en kvalitativ innehållsanalys, som visade på att deltagarna i denna studie var positivt inställda till hydroterapi. Deltagarna upplevde att hydroterapin ledde till minskad smärta, ökad rörlighet samt ett ökat välmående. Utöver de upplevda positiva effekterna återfanns även upplevda negativa effekter i form av träningsvärk och en ökad stelhet under sommaruppehållen. Det sociala samspel som uppstod i samband med hydroterapin upplevdes av de flesta deltagare ha haft positiv betydelse. Deltagarnas upplevelser kunde i diskussionen kopplas till operant och respondent inlärningsteori, där tänkbara konsekvenser och stimulin identifierades. Slutsatsen som drogs var att deltagarna upplevde hydroterapin som ett sätt att få tillbaka sin friska kropp genom att symtomen minskade. Då deltagarna upplevde att uppehåll orsakade försämring behövs hydroterapi som kontinuerlig behandlingsform för dem. Vidare forskning inom området bör fokuseras på enskilda patientgrupper samt inkludera ett större antal deltagare per patientgrupp. Nyckelord: hydroterapi, intervju, kvalitativ, pelvospondylit, reumatoid artrit.