Chemokines and chemokine receptors in arthritis.

Chemokines are involved in leukocyte recruitment to inflammatory sites, such as the synovial tissue in rheumatoid arthritis (RA). There is a structural and a functional classification of chemokines. The former includes four groups: CXC, CC, C and CX3C chemokines. Chemokines may also be either inflammatory or homeostatic, however, these functions often overlap. Anti-chemokine and anti-chemokine receptor targeting may be therapeutically used in the future biological therapy of arthritis. Most data in this field have been obtained from animal models of arthritis as only very few human RA trials have been completed. However, it is very likely that various specific chemokine and chemokine receptor antagonists will be developed and administered to RA patients.

Effects of biologics on vascular function and atherosclerosis associated with rheumatoid arthritis.

Endothelial dysfunction and accelerated atherosclerosis lead to increased cardiovascular morbidity and mortality in rheumatoid arthritis (RA). Sustained inflammation is a major risk factor. Apart from traditional vasculoprotective agents, biologics may also exert favorable effects on the vasculature. Indeed, tumor necrosis factor-alpha (TNF-alpha) inhibitors agents may transiently improve endothelial function. There are conflicting data regarding the effects of biologics on atherosclerosis and arterial stiffness. Infliximab stimulates the number and differentiation of endothelial progenitor cells that lead to vascular repair. There may be differences in the effects of TNF blockers on dyslipidemia, as long-term infliximab therapy may be proatherogenic, while some studies suggest that etanercept and adalimumab may exert beneficial effects on lipids. TNF blockers may decrease the incidence of cardiovascular events in RA. Preliminary data suggest that rituximab may also improve endothelial function and dyslipidemia. Further studies are needed to determine the net effects of biologics on the vasculature.

The genetic background of ankylosing spondylitis.

It has long been known that the major histocompatibility complex (MHC) is essentially involved in genetic susceptibility to ankylosing spondylitis (AS). The HLA-B27 antigen has been accounted for 20 to 50% of the total genetic risk for this disease. However, susceptibility to AS cannot be fully explained by associations with the MHC. Recent studies including linkage analyses as well as candidate gene and, most recently, genome-wide association studies indicate significant associations of the interleukin-1 gene cluster, interleukin-23 receptor and ARTS1 genes as well as other possible loci with AS. In the murine model of proteoglycan-induced spondylitis, two susceptibility loci termed Pgis1 and Pgis2 were identified. Thus, AS is not a single-gene disease and the involvement of multiple non-MHC genes may account for the individual as well as geographical differences seen in AS.

Effects of rituximab treatment on endothelial dysfunction, carotid atherosclerosis, and lipid profile in rheumatoid arthritis.

Increased cardiovascular mortality has been associated with rheumatoid arthritis (RA). There have been reports indicating that tumor necrosis factor blockers may exert favorable but transient effects on lipid profile, flow-mediated vasodilation (FMD) of the brachial artery, and common carotid intima-media thickness (ccIMT) in RA. In this study, we assessed the effects of rituximab on FMD, ccIMT, and lipid profile. Five female RA patients received two infusions of 1000 mg rituximab i.v. High-resolution B-mode ultrasound was used to assess brachial FMD and ccIMT. We also determined plasma total cholesterol (TC), HDL-C, LDL-C, and triglyceride (Tg) levels. Assessments were performed at baseline, as well as at weeks 2, 6, and 16 after the first infusion. Rituximab (RTX) treatment resulted in a rapid and sustained improvement in FMD. The mean improvement was 30%, 22%, and 81% at weeks 2, 6, and 16, respectively. RTX had little effect on atherosclerosis within this short period of time; however, we observed 10%, 9%, and 2% decreases in ccIMT at weeks 2, 6, and 16, respectively. RTX therapy resulted in 3-11% decrease in TC, as well as 14-35% increase in HDL-C levels. Two infusions of RTX exerted early and sustained favorable effects on endothelial dysfunction, as well as plasma TC and HDL-C levels. RTX may also decrease ccIMT; however, longer follow-up is needed to assess the prolonged effects of RTX on vascular function and lipid profile in RA patients.

Combined plasmapheresis and high-dose intravenous immunoglobulin treatment in systemic sclerosis for 12 months: follow-up of immunopathological and clinical effects.

Systemic sclerosis (SSc) is an autoimmune disease which involves the skin, as well as several internal organs. Most therapies available in this disease are symptomatic. Authors present a case of diffuse SSc with progressive disease not responding to currently available treatments. Therefore a 12-month protocol of repeated plasmapheresis and high-dose intravenous immunoglobulin treatment was administered with good clinical efficacy. Apart from monitoring the clinical symptoms throughout the treatment, authors also assessed a number of humoral and cellular immunolaboratory markers in order to obtain information on the immunomodulatory effects of this combined treatment in SSc.

Intracytoplasmic cytokine expression and T cell subset distribution in the peripheral blood of patients with ankylosing spondylitis.

OBJECTIVE: To determine the role of inflammatory mediators in the pathogenesis of ankylosing spondylitis (AS), we investigated peripheral blood lymphocyte subsets and their intracellular cytokine production. METHODS: The percentages of T and B lymphocytes, natural killer (NK) cells, activated T lymphocytes, CD4+ T helper (Th), and CD8+ T cytotoxic (Tc) cells were determined by flow cytometry in 42 patients with AS compared to 52 healthy controls. In order to assess circulating Th1/Th2 and Tc1/Tc2 subsets, we used a whole-blood cytometric assay based on the intracellular interferon-gamma, interleukin 4 (IL-4), and IL-10 expression of the cells. RESULTS: In the peripheral blood, the frequencies of CD4+ T helper and CD56+ NK cells were higher in AS (54.8% and 16.2%, respectively) compared to controls (45.3% and 10.8%) (p < 0.05). The frequencies of Th0 (1.9% vs 0.8%) and Tc0 (2.1% vs 0.8%) cells were higher, while that of Tc1 cells was lower (26.6% vs 40.1%) in patients with AS versus controls (p < 0.05). The percentage of IL-10-producing Tc cells was significantly higher in AS (18.4%) versus controls (8.5%) (p < 0.05). Finally, the active phase of AS was associated with significantly lower percentage of IL-10-producing Tc cells in the peripheral blood (6.6%) compared to patients with inactive AS (23.1%). CONCLUSION: Our results provide further evidence for an altered T cell subset distribution and intracytoplasmic cytokine balance in AS.

Two loci on chromosome 15 control experimentally induced arthritis through the differential regulation of IL-6 and lymphocyte proliferation.

Using genetic linkage analysis of proteoglycan-induced arthritis (PGIA), a murine model for rheumatoid arthritis, we identified two loci, Pgia8 and Pgia9, on chromosome 15 (chr15) that appear to be implicated in disease susceptibility. Immunization of congenic strains carrying the entire chr15 and separately each of the two loci of DBA/2 arthritis-resistant origin in susceptible BALB/c background confirmed locations of two loci on chr15: the major Pgia9 and lesser Pgia8 locus. Distal part of chr15 (Pgia9) showed a major suppressive effect on PGIA susceptibility in females (40%, p < 0.001), whereas the effect of this locus in congenic males was still significant but weaker. Proximal part of chr15 (Pgia8) demonstrated mild and transient effect upon arthritis; this effect was PGIA-promoting in males and suppressive in females. Pgia8 and Pgia9 loci demonstrated an additive mode of inheritance, since when they were both incorporated in consomic chr15 strain, the total effect was a sum of the two loci. Using F(2) population of the intercross of wild-type and chr15 consomic strain, we confirmed and refined quantitative trait locus positions and identified a strong correlation between disease susceptibility and lymphocyte-producing cytokines of TNF-alpha and IL-6. Both Pgia8 and Pgia9 loci on chr15 appear to control IL-6 production in spleen cultures of arthritic mice, providing an important link to the mechanism of autoimmune inflammation.

Anti-citrullinated protein antibodies in rheumatoid arthritis: as good as it gets?

Anti-citrullinated protein antibodies (ACPAs) have recently emerged as sensitive and specific serological markers of rheumatoid arthritis (RA), providing superior alternative of the rheumatoid factor (RF) test in the laboratory diagnostics of RA. The first members of this autoantibody family were anti-perinuclear factor (APF) and anti-keratin antibodies (AKA). It became evident that both APF and AKA recognize citrullinated epitopes of filaggrin. Citrullination is a post-translational modification of arginine by deimination, physiologically occurring during apoptosis, inflammation or keratinization. The presence of several citrullinated proteins has been demonstrated in the RA synovium. The identification of citrullinated epitopes as targets for anti-filaggrin antibodies led to the development of the first and later second generation anti-cyclic citrullinated peptide (anti-CCP) antibody assays. The widely used anti-CCP2 assays have high diagnostic sensitivity and specificity, and they also show important predictive and prognostic value in RA. The anti-Sa antibody has been identified a decade ago; however, recent studies confirmed that anti-Sa is directed against citrullinated vimentin, hence it is a new member of the family of ACPAs. The newly developed anti-mutated citrullinated vimentin (anti-MCV) assay has similar diagnostic performance than the anti-CCP2 ELISA; however, the diagnostic spectrum of the anti-MCV test is somewhat different from that of anti-CCP2. It's especially useful in the diagnosis of RA in RF and anti-CCP2 seronegative patients. The combined application of anti-CCP2 and anti-MCV assays can improve the laboratory diagnostics of RA. The family of ACPAs is expected to expand; there is an increasing need for developing new diagnostic strategies after careful evaluation of the characteristics of the available assays.

Associations between serum anti-CCP antibody, rheumatoid factor levels and HLA-DR4 expression in Hungarian patients with rheumatoid arthritis.

BACKGROUND: The presence of anti-cyclic citrullinated peptide autoantibody is highly specific for rheumatoid arthritis. Certain HLA-DR4 (HLA-DRB1*04) alleles, also known as the "shared epitope," are associated with increased susceptibility to RA. In addition, these alleles may also have relevance for disease outcome. Anti-CCP antibody positivity has been associated with the presence of HLA-DR4 alleles in patients with RA. However, there is little information regarding a relationship between quantitative anti-CCP production (serum anti-CCP concentrations) and the shared epitope. OBJECTIVES: To determine the association between anti-CCP antibody production and various HLA-DRB1 alleles. METHODS: Serum anti-CCP, rheumatoid factor and C-reactive protein levels were assessed in 53 RA patients. All these patients underwent HLA-DRB1 genotyping. RESULTS: Of the 53 patients 33 (62%) were positive for anti-CCP antibody. We found significant correlations between anti-CCP and RF positivity (chi-square = 6.717, P < 0.01), as well as between anti-CCP and HLA-DRB1*04 positivity (chi-square = 5.828, P < 0.01). There was no correlation between RF positivity and serum levels, CRP serum levels and HLA-DRB1*04 positivity. When quantitatively comparing serum anti-CCP levels with shared epitope positivity, patients carrying one or two copies of HLA-DRB1*04 alleles had significantly higher anti-CCP concentrations (530.0 +/- 182.6 U/ml) compared to DRB1*04-negative patients (56.8 +/- 27.4 U/ml) (P < 0.01). There was no difference in serum anti-CCP antibody concentrations between patients carrying only one HLA-DRB1*01 allele but no HLA-DRB1*04 allele (12.0 +/- 8.6 U/ml) compared to SE-negative patients (76.8 +/- 56.2 U/ml). Regarding non-SE HLA-DRB1 genotypes, all 6 patients (100%) carrying DRB1*15 alleles and 6 of 7 (85%) patients carrying DRB1*13 were anti-CCP positive. In addition, patients with HLA-DRB1*13 (282.5 +/- 23.8 U/ml) and DRB1*15 (398.7 +/- 76.2 U/ml) produced significantly more anti-CCP than did any other non-SE HLA-DRB1 subtypes (P < 0.01). CONCLUSIONS: There is significant association between anti-CCP and RF, as well as between anti-CCP and SE positivity in RA. In addition, the presence of one or two copies of HLA-DRB1*04 alleles has been associated with higher serum anti-CCP antibody levels. Thus, patients carrying HLA-DRB1*04 alleles exhibited an overall tenfold increase in serum anti-CCP antibody levels in comparison to HLA-DRB1*04-negative subjects. Increased anti-CCP production may also be associated with other non-SE HLA-DRB1 genotypes, such as DRB1*13 or DRB1*15. In reports by other investigators, both anti-CCP concentrations and SE positivity were related to more rapid disease progression and unfavorable outcome.

Accelerated atherosclerosis in rheumatoid arthritis.

Cardiovascular disease is a leading cause of mortality in rheumatoid arthritis (RA). Endothelial dysfunction often precedes manifest atherosclerosis. Both traditional, Framingham risk factors and inflammation-associated factors are involved in RA-associated atherosclerosis. Among imaging techniques, the early determination of common carotid intima-media thickness (ccIMT), flow-mediated vasodilation (FMD), and nitroglycerine-mediated vasodilation (NMD) may be useful to determine atherosclerosis and endothelial dysfunction. We and others found increased ccIMT and impaired FMD in RA patients. Among immunological and metabolic laboratory markers, anticyclic citrullinated peptide (anti-CCP) antibodies, IgM rheumatoid factor, circulating immune complexes, pro-inflammatory cytokines including tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), Th0/Th1 T cells, homocysteine, dyslipidemia, decreased folate and vitamin B12 production, and impaired paraoxonase activity may all be involved in the development of vascular disease in RA. The early diagnosis of endothelial dysfunction and atherosclerosis, active immunosuppressive treatment, the use of drugs that control atherosclerosis, changes in sedentary lifestyle, and the close follow-up of RA patients may help to minimize cardiovascular risk in these individuals.

Clinical evaluation of anti-mutated citrullinated vimentin by ELISA in rheumatoid arthritis.

OBJECTIVE: Anti-cyclic citrullinated peptide (CCP) antibodies have emerged as sensitive and specific serological markers of rheumatoid arthritis (RA). However, antibodies to several other citrulline-containing proteins, including citrullinated fibrin and vimentin, have been detected in patients with RA, suggesting that citrulline is an essential constituent of autoantigens for RA-specific autoantibodies. We examined the diagnostic performance of the newly developed anti-mutated citrullinated vimentin (MCV) antibody assay. METHODS: Concentrations of anti-MCV, anti-CCP2, and rheumatoid factors (RF) were determined in the sera of 237 individuals: 119 patients with RA and 118 controls, including patients with other rheumatic diseases and healthy subjects. Diagnostic properties were compared by receiver-operating characteristic curve analysis. RESULTS: Using manufacturer's recommended cutoff values, sensitivity and specificity of anti-MCV antibodies were 75.6% and 91.5% in RA, compared to 66.4% and 98.3% for anti-CCP2. Introducing cutoff values to obtain the same 95% specificity resulted in decreased sensitivity of the anti-MCV test (69.7%) and increased sensitivity of the anti-CCP2 test (74.8%). At optimal cutoff levels, 29.4% of IgM RF-negative cases as well as 13.3% of anti-CCP2-negative cases in the RA group were anti-MCV-positive. Double-positivity for anti-MCV and anti-CCP2 provided 98.3% specificity with 97.5% positive predictive value in RA. CONCLUSION: Overall, the performance of the novel anti-MCV ELISA for the diagnosis of RA is similar to that of the anti-CCP2 test [area under the curve 0.853 (95% CI 0.801-0.905) vs 0.910 (95% CI 0.873-0.946); p not significant]. As the diagnostic spectrum of the anti-MCV assay is somewhat different from that of anti-CCP2, the combined application of the 2 assays can improve the laboratory diagnostics of RA.

T-cell recognition of differentially tolerated epitopes of cartilage proteoglycan aggrecan in arthritis.

Proteoglycan (PG) aggrecan, a major macromolecular component of cartilage, is highly immunogenic; it induces arthritis in genetically susceptible BALB/c mice. The present study maps the T-cell epitope repertoire of cartilage PG by identifying a total of 27 distinct T-cell epitopes. An epitope hierarchy, accounting for the different effector functions of PG-specific T cells, and determinant spreading, has been found. T-cell responses to four epitopes were associated with arthritis induction. Some of the T-cell epitopes were full T-cell activators, whereas a number of subdominant and cryptic epitopes proved to be partial activators in vitro, inducing either cytokine secretion or T-cell proliferation, but not both. A few T-cell epitopes of the core protein of cartilage PG were clearly recognized by T cells in PG-immunized arthritic animals, but the corresponding peptides did not induce T-cell responses when injected into naive BALB/c mice; thus these T-cell epitopes were designated as "conditionally immunogenic."

Genetic control of experimental spondylarthropathy.

OBJECTIVE: To characterize experimentally induced spondylarthropathy (SpA) in arthritis-susceptible inbred mice and in their F(1) and F(2) hybrid generations of susceptible and resistant mouse strains. METHODS: SpA was induced in susceptible BALB/c and C3H/HeJCr (C3H) strains of mice, and in their F(1) and F(2) generations derived from intercrosses with arthritis- and/or spondylitis-resistant DBA/2 and DBA/1 parent strains, by systemic immunization with cartilage proteoglycan (PG) aggrecan. The incidence and severity of PG-induced spondylitis (PGIS) were scored histologically, and these scores for spine involvement were correlated with serum antibody and cytokine levels and with in vitro T cell responses to cartilage PG. RESULTS: PGIS was induced by systemic immunization with cartilage PG in adjuvant, and approximately 60-70% of susceptible mouse strains and their F(2) hybrids developed spondylitis either with or without arthritis. Adjuvants, particularly those activating the innate immune system and enforcing the Th1 dominance, had significant effects on the outcome and progression of SpA. The DBA/1 strain appeared to carry genes protecting this strain and its F(1) and F(2) hybrids from spondylitis, whereas the DBA/2 strain, although resistant to PGIS, harbored genes permitting PGIS in its hybrid generations. Arthritis- and/or spondylitis-susceptible BALB/c and C3H parent strains and their F(2) hybrids exhibited the highest incidence and severity of spondylitis. CONCLUSION: PGIS, a murine model of autoimmune spondylitis, shows similarities to ankylosing spondylitis. Segregation of susceptibility to PG-induced arthritis (PGIA) from that to PGIS in different genetic crosses suggests that PGIA and PGIS are separate diseases. Therefore, this model allows for the elucidation of genetic components involved in the etiology of SpA, independent of those controlling the susceptibility to PGIA.

Two major interacting chromosome loci control disease susceptibility in murine model of spondyloarthropathy.

Autoimmune spondylitis was induced in BALB/c mice and their MHC-matched (BALB/c x DBA/2)F1 and F2 hybrids by systemic immunization with cartilage/intervertebral disk proteoglycan (PG). As in human ankylosing spondylitis, the MHC was the major permissive genetic locus in murine PG-induced spondylitis (PGIS). Two major non-MHC chromosome loci with highly significant linkage were found on chromosomes 2 (Pgis2) and 18 (Pgis1) accounting for 40% of the entire F2 trait variance. The dominant spondylitis-susceptibility allele for Pgis2 locus is derived from the BALB/c strain, whereas the Pgis1 recessive allele was present in the disease-resistant DBA/2 strain. The Pgis1 locus significantly affected the disease-controlling Pgis2 locus, inducing as high incidence of spondylitis in F2 hybrids as was found in the spondylitis-susceptible parent BALB/c strain. Additional disease-controlling loci with suggestive linkage were mapped to the chromosomes 12, 15, and 19. Severity of spondylitis in F2 mice positively correlated with serum levels of amyloid A, IL-6, and Pg-specific Abs, and showed negative correlation with Ag-induced T cell proliferation, IFN-gamma, IL-4, and TNF-alpha production. A major locus controlling serum IL-6 was found on chromosome 14 near osteoclast differentiation factor Tnfsf11. Locus on chromosome 11 near the Stat3 and Stat5 genes controlled serum level of the Ig IgG2a isotype. The two major genetic loci Pgis1 and Pgis2 of murine spondylitis were homologous to chromosome regions in human genome, which control ankylosing spondylitis in human patients. Thus, this animal model of experimentally induced spondylitis might facilitate the identification of spondylitis-susceptibility genes in humans.

Association of rheumatoid arthritis with HLA-DR1 and HLA-DR4 in Hungary.

Susceptibility to and outcome for rheumatoid arthritis (RA) have been associated with particular HLA-DR alleles, but these alleles vary among ethnic groups and geographic areas. The frequency of HLA-DR1 (HLA-DRB1*0101, DRB1*0102) and HLA-DR4 (DRB1*0401, DRB1*0404) alleles is elevated among Caucasian patients with RA. We studied a northeastern Hungarian population of RA patients to determine the frequency of HLA-DR1 and HLA-DR4 phenotypes in this population and to compare it with healthy control subjects, as well as to investigate whether the presence of these alleles could be a marker for RA. We performed HLA-DRB1 genotyping (DRB1*01-DRB1*16) in 83 RA patients and 55 healthy controls using polymerase chain reaction with sequence-specific primers (PCR-SSP). In the case of HLA-DR1- or HLA-DR4-positive patients, the DR1 and DR4 subtypes were also determined. The frequency of HLA-DR4 alleles was significantly higher in RA patients than in controls (31.3 vs. 10.9%; P <.05). HLA-DR1, in particular, tended to be more frequent in patients than in controls (32.5 vs. 18.1%). Among the HLA-DR4 subtypes, DRB1*0401 and DRB1*0404 were the most common alleles found in both groups. However, no significant differences were seen in the frequency of HLA-DRB1*0401 and HLA-DRB1*0404 between RA patients and controls. In contrast, HLA-DRB1*0405 and HLA-DRB1*0408 were significantly more common in RA patients than in control subjects. Among HLA-DR1 subtypes, the DRB1*0101 allele was most commonly detected, but HLA-DRB1*0101 as well as DRB1*0102 and DRB1*0105 were similarly frequent in RA patients and controls. HLA-DR12 was more common among controls than in RA patients (18.1 vs. 0%; P <.05). Our results generally agree with the findings in other Caucasian populations. Nonetheless, we found differences in the frequency of HLA-DR1 and HLA-DR4 subtypes among Hungarian patients compared with reports from other geographic regions (e.g., Finland and Asia). Our data suggest that in northeastern Hungary, HLA-DR4 as well as its subtypes DRB1*0405 and DRB1*0408 may be involved in susceptibility to RA, but HLA-DR1 may not. In addition, the presence of HLA-DR12, at least in Hungary, may protect from this disease.