A shift in the balance of inhibitory and activating Fcgamma receptors on monocytes toward the inhibitory Fcgamma receptor IIb is associated with prevention of monocyte activation in rheumatoid arthritis

OBJECTIVE: To assess surface expression of the inhibitory receptor for IgG (Fcgamma receptor IIb [FcgammaRIIb]) in relation to activating FcgammaR on monocyte/macrophages from patients with rheumatoid arthritis (RA) and healthy controls and to study the influence of proinflammatory and antiinflammatory cytokines on the balance of inhibitory and activating FcgammaR. METHODS: Using a combination of genotyping and phenotyping, surface expression of FcgammaRIIb on monocytes from healthy control subjects and RA patients was demonstrated. Expression of FcgammaR on CD14+ monocytes was assessed by flow cytometry. Regulation of inhibitory and activating FcgammaR on monocytes by proinflammatory (interferon-gamma [IFNgamma], tumor necrosis factor alpha [TNFalpha]) and antiinflammatory (interleukin-4 [IL-4], IL-10) cytokines was studied. A functional change in cytokine-modulated monocytes was assessed in secondary cultures by their ability to produce TNFalpha upon FcgammaR crosslinking by IgG. RESULTS: Monocytes from healthy controls and RA patients expressed FcgammaRIIb at similar levels, in contrast to the higher levels of activating FcgammaRI and FcgammaRIIa in RA patients. The regulation of FcgammaR expression was comparable for patients and controls. IFNgamma selectively up-regulated FcgammaRI. TNFalpha down-regulated expression of FcgammaRIIb and the activating FcgammaR, whereas IL-10 up-regulated expression of monocytic FcgammaRIIb and all activating FcgammaR. Increased or sustained levels of activating over inhibitory FcgammaR induced by IFNgamma, TNFalpha, and IL-10 alone were associated with enhanced IgG-triggered TNFalpha production. In contrast, IL-4 and, more specifically, IL-4 plus IL-10 altered the FcgammaR balance in favor of FcgammaRIIb and completely prevented IgG-triggered TNFalpha production. CONCLUSION: The altered balance of FcgammaR in favor of activating receptors in RA may contribute to increased activation of monocyte/macrophages. A change in the FcgammaR balance toward the inhibitory FcgammaRIIb may offer a novel treatment strategy for preventing the pleiotropic activity of FcgammaR-triggered macrophages.     

Treatment of rheumatoid arthritis patients with anti-TNF-alpha monoclonal antibody is accompanied by down-regulation of the activating Fcgamma receptor I on monocytes

OBJECTIVES: To study the effect of anti-TNF-alpha therapy on activating IgG Fc receptor (FcgammaR) expression on monocytes of RA patients in relation to changes in disease activity. METHODS: RA patients were treated with anti-TNF-alpha mAb (infliximab). At baseline, 2 and 14 weeks after the start of anti-TNF-alpha treatment, FcgammaR expression levels on circulating monocytes were evaluated. Changes in expression were correlated to changes in disease parameters. To study the direct effects of TNF-alpha blockade on monocytic FcgammaR expression levels, monocytes were isolated and cultured with anti-TNF-alpha mAb. The effects were compared with those induced by TNF-alpha. RESULTS: Two weeks after the start of anti-TNF-alpha mAb therapy, monocytic FcgammaRI expression levels were decreased, whereas FcgammaRIIa and IIIa expression levels were unchanged. At 14 weeks, 8 weeks after the last gift of anti-TNF-alpha mAb, FcgammaRI expression levels returned to baseline levels. FcgammaRIIa and IIIa expression levels remained unchanged. The change in FcgammaRI correlated with changes in CRP and ESR levels. In vitro, anti-TNF-alpha mAb treatment did not alter expression of FcgammaRI on monocytes, but increased FcgammaRIIa and IIIa. TNF-alpha down-regulated all activating FcgammaRs, mainly FcgammaRIIa and IIIa, but also the inhibitory FcgammaRIIb. CONCLUSION: Anti-TNF-alpha mAb treatment of RA patients is accompanied by down-regulation of FcgammaRI expression levels on monocytes. This is likely an indirect effect of TNF-alpha blockade on disease activity, since in vitro anti-TNF-alpha mAb does not directly change FcgammaRI expression on monocytes. In contrast, TNF-alpha down-regulated all activating FcgammaRs. Thus, blocking TNF-alpha may relieve the negative feedback mechanism of TNF-alpha as down-regulator of FcgammaRs. Strategies to reduce activating FcgammaRs may have additional value in the treatment of RA patients with TNF-alpha blockade by diminishing immune complex-mediated activation of monocytes/macrophages.     

Increased FcgammaRII expression and aberrant tumour necrosis factor alpha production by mature dendritic cells from patients with active rheumatoid arthritis

OBJECTIVES: To investigate potential differences in phenotype and behaviour of immature (iDC) and mature dendritic cells (mDC) from patients with RA and healthy subjects. METHODS: iDC and mDC were derived from blood monocytes of patients with RA and healthy controls following standardised protocols. FACS was used to analyse expression of FcgammaRI, II, and III and molecules to characterise DC. Discrimination between FcgammaRIIa and FcgammaRIIb was achieved by RT-PCR. Immunohistochemistry was performed on synovial biopsy specimens of three patients with RA and three healthy controls. TNFalpha production by iDC and mDC upon FcgammaR dependent stimulation was compared between patients with RA and controls by ELISA. RESULTS: iDC from patients with active RA but not from patients with inactive RA or healthy controls markedly up regulated FcgammaRII. mDC from patients with active RA also lacked the physiological down regulation of FcgammaRII that occurs upon maturation in both control groups. RT-PCR analysis confirmed the increased expression of FcgammaRII in RA-especially marked for FcgammaRIIb. FcgammaR dependent stimulation of DC using antigen-IgG immune complexes (IC) significantly increased TNFalpha production by DC from healthy subjects, but significantly decreased TNFalpha by DC from patients with RA. Overlapping expression patterns between FcgammaRII and DC-LAMP in the synovial tissue of patients with RA imply that in vivo, also, mature DC express increased levels of FcgammaRIIb. CONCLUSION: The presence and altered characteristics of DC during active RA suggest that DC help to modulate autoimmunity in RA. Further studies should elucidate the role of local factors in altering the function of DC in RA and in increasing expression of FcgammaRII.     

Fcgamma receptor expression levels on monocytes are elevated in rheumatoid arthritis patients with high erythrocyte sedimentation rate who do not use anti-rheumatic drugs

OBJECTIVES: Levels of immunoglobulin G (IgG) Fc receptors (FcgammaRs) affect the activity and function of monocytes/macrophages when binding IgG-containing immune complexes. Hence, the expression level of FcgammaRs on monocytic cells may influence inflammation in patients with rheumatoid arthritis (RA). In this study the expression levels of FcgammaRI, IIa and IIIa on peripheral blood monocytes of RA patients were compared with those of healthy controls and related to patient and disease characteristics and the use of disease-modifying anti-rheumatic drugs (DMARDs). In addition, FcgammaR expression levels were determined on RA synovial fluid macrophages and compared with those in RA peripheral blood. METHODS: Mononuclear cells from peripheral blood and synovial fluid were isolated and FcgammaR expression levels on CD14-positive cells were analysed by flow cytometry. The effects of patient and disease characteristics and the use of DMARDs were assessed. RESULTS: A high expression level of FcgammaRIIa and high percentages of FcgammaRIIIa-expressing monocytes were found in RA patients with a high erythrocyte sedimentation rate. DMARD-naive early RA patients had higher FcgammaRIIa expression levels but a similar amount of FcgammaRIIIa-positive monocytes compared with RA patients using DMARDs. In synovial fluid, FcgammaRIIa expression levels were lower than in RA peripheral blood, whereas the percentage of FcgammaRIIIa-positive monocytic cells was higher in synovial fluid than in peripheral blood. CONCLUSIONS: These data point to the involvement of FcgammaRs, specifically FcgammaRIIa and IIIa, in the immune response of RA and suggest that FcgammaR expression levels are susceptible to modulation by DMARD therapy.     

Increased expression of Fcgamma receptors II and III on macrophages of rheumatoid arthritis patients results in higher production of tumor necrosis factor alpha and matrix metalloproteinase

OBJECTIVE: To evaluate Fcgamma receptor (FcgammaR) expression on synovial macrophages from rheumatoid arthritis (RA) patients and to determine whether this expression correlates with the production of the proinflammatory cytokines tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), IL-12, and matrix metalloproteinase 1 (MMP-1). We also sought to determine whether mature macrophages from RA patients express aberrant levels of FcgammaRI, FcgammaRII, and FcgammaRIII, and to determine the production of inflammatory mediators after immune complex (IC) stimulation. METHODS: Immunohistochemistry was performed on cryostat sections of synovial biopsy specimens obtained from 27 RA patients and 5 controls. FcgammaR I, II, and III were detected, as well as the proinflammatory mediators IL-1, TNFalpha, IL-12, and MMP-1. Monocytes were isolated from the blood of 10 RA patients and 10 healthy controls and cultured for 7 days with macrophage colony-stimulating factor to obtain macrophages. Using fluorescence-activated cell sorting, the expression of FcgammaRI, FcgammaRII, and FcgammaRIII was determined. On day 7, macrophages were stimulated with heat-aggregated gamma globulins (HAGGs) for 24 hours. Production of cytokines was measured using enzyme-linked immunosorbent assay, and production of gelatinases/collagenases was measured by degradation of fluorescent gelatin. RESULTS: Immunohistochemistry showed higher FcgammaRII and FcgammaRIII expression in RA synovium than in controls. FcgammaRII and FcgammaRIII, but not FcgammaRI, were highly correlated with the number of synovial macrophages. Consistent with this, TNFalpha expression correlated positively with FcgammaRIII expression. Moreover, MMP-1 expression strongly correlated with FcgammaR I, II, and III expression. Mature macrophages from RA patients showed significantly enhanced expression of FcgammaRII and FcgammaRIII compared with controls. Twenty-four hours after stimulation of RA macrophages with HAGGs, significantly higher production of TNFalpha and gelatinase/collagenase was measured. CONCLUSION: RA synovium and mature RA macrophages express significantly elevated levels of FcgammaRII and FcgammaRIII, resulting in much higher production of TNFalpha and gelatinase/collagenase after IC stimulation. These data suggest that disturbed expression of FcgammaR on mature synovial macrophages is involved in the pathology of RA.     

Intravenous (IV) anti-D and IV immunoglobulin achieve acute platelet increases by different mechanisms: modulation of cytokine and platelet responses to IV anti-D by FcgammaRIIa and FcgammaRIIIa polymorphisms

Intravenous (IV) anti-D and IV immunoglobulin (IVIG) slow the Fcgamma receptor (FcgammaR)-mediated destruction of antibody-coated platelets in patients with immune thrombocytopenic purpura (ITP). This pilot study explored the mechanism of these immunoglobulin preparations by measuring interleukin-10 (IL-10), monocyte chemoattractant protein-1 (MCP-1), IL-6 and tumour necrosis factor alpha (TNFalpha), before and after infusion and by assessing the effect of FcgammaRIIa and FcgammaRIIIa polymorphisms on both cytokine and haematologic responses to anti-D. Following IVIG, only IL-10 was increased at 2 h and MCP-1 on day 7 (P < 0.05). In contrast, 2 h after anti-D infusion, plasma levels of all four cytokines were increased (P < 0.01); five of six patients with the highest MCP-1, IL-6 and TNFalpha levels had chills. Higher IL-10 levels correlated with platelet increases at 24 h and haemoglobin decreases at day 7 (P < 0.025). Patients with the FcgammaRIIa-131HH genotype had significantly higher MCP-1, IL-6 and TNFalpha levels. Patients with the FcgammaRIIIa-158VF genotype had higher platelet increments at day 7 (P < 0.05). Soluble CD16 (sCD16) was increased 2 h after IV anti-D; day 7 levels correlated with day 7 haemoglobin decreases (P < 0.01). In conclusion, the relationship of FcgammaRIIa and FcgammaRIIIa polymorphisms with both cytokine levels and platelet increments implicated these receptors in responses to anti-D and supported different mechanisms of FcgammaR interaction to those seen with IVIG.     

The membrane proteinase 3 expression on neutrophils was downregulated after treatment with infliximab in patients with rheumatoid arthritis.

Proteinase 3 (PR3) expression on neutrophils was examined in rheumatoid arthritis (RA) patients before and after antitumor necrosis factor (TNF)-alpha therapy. Membrane PR3 expression from patients with either an infection or RA significantly increased. Membrane PR3 expression on neutrophils from RA patients treated with infliximab (anti-TNF-alpha antibody) therapy was less than in those without such treatment in a resting state, but the expression later increased after stimulation in vitro. Membrane PR3 expression increased because of the stimulation of TNFalpha, whereas it was significantly suppressed by plasma or alpha(1)-proteinase inhibitor. The condition of patients with RA improved after treatment with infliximab. Membrane PR3 expression on neutrophils in RA patients was downregulated by infliximab. As a result, PR3 might play an important role in the neutrophil-mediated inflammatory reaction in patients with either RA or an infection.     

Induction of macrophage secretion of tumor necrosis factor alpha through Fcgamma receptor IIa engagement by rheumatoid arthritis-specific autoantibodies to citrullinated proteins complexed with fibrinogen

OBJECTIVE: Macrophage-derived tumor necrosis factor alpha (TNFalpha) is a dominant mediator of synovitis in rheumatoid arthritis (RA). This study was undertaken to assess whether and how immune complexes (ICs) formed by the interaction of disease-specific autoantibodies to citrullinated proteins (ACPAs) with their main synovial target antigen, citrullinated fibrin, contribute to TNFalpha production by macrophages. METHODS: An in vitro human model was developed in which monocyte-derived macrophages were stimulated with ACPA-containing ICs that were generated by capturing ACPAs from RA sera on immobilized citrullinated fibrinogen. Cellular activation was evaluated by TNFalpha assay in culture supernatants. Selective blockade of IC interactions with the 3 classes of Fcgamma receptors (FcgammaR) was used to assess the contribution of each receptor to macrophage activation. In addition, 2 citrullinated fibrin-derived peptides bearing major ACPA epitopes were tested for their capacity to inhibit formation of macrophage-activating ACPA-containing ICs. RESULTS: ACPA-containing ICs induced a dose-dependent TNFalpha secretion by macrophages from 14 of 20 healthy donors. The macrophage response was systematically higher than that of the paired monocyte precursors. TNFalpha secretion was not reduced by blockade of FcgammaRI or FcgammaRIII, but was strongly repressed when interaction of ICs with FcgammaRII was prevented. The 2 citrullinated peptides significantly inhibited ACPA reactivity to citrullinated fibrinogen and, when tested together, almost completely abolished formation of macrophage-activating ICs, thereby diminishing the secreted TNFalpha levels. CONCLUSION: Our model demonstrates the inflammatory potential of ACPA-containing ICs via engagement of FcgammaRIIa at the surface of macrophages, strongly supporting their pathophysiologic involvement. Continuing dissection of these molecular pathways could open the way to new therapeutic approaches in patients with RA.     

Effects of the protein tyrosine phosphatase CD45 on FcgammaRIIa signaling and neutrophil function

OBJECTIVE: Neutrophil receptors for the Fc portion of IgG (FcgammaR) trigger immune responses following cross-linking by IgG-coated foreign particles or immune complexes. Membrane-associated CD45, a protein tyrosine phosphatase termed leukocyte common antigen, has been shown to be essential for antigen receptor kinase mediated signaling in lymphocytes, and we hypothesized that CD45 may play a similar role in FcgammaR-mediated signaling and immune function in human neutrophils. METHODS: The experimental approach was that of cell surface molecule ligation via cross-linking with specific antibodies. Antibody dependent cellular cytotoxicity (ADCC) was assessed using a single-cell plaque assay and IL-6 production measured using ELISA. Tyrosine phosphorylation levels were assessed with anti-phospho-tyrosine blots and F-actin polymerization by flow cytometry and confocal microscopy. RESULTS: Neutrophils pretreated with anti-CD45 had a reduced ability to perform ADCC compared to untreated neutrophils. FcgammaRIIa cross-linking resulted in significantly increased concentrations of secreted IL-6 compared to untreated neutrophils, and IL-6 production was further enhanced by cocross-linking CD45 with FcgammaRIIa. Cross-linking CD45 alone also induced IL-6 production. FcgammaRIIa cross-linking resulted in increased protein tyrosine phosphorylation and F-actin polymerization in neutrophils. Cocross-linking CD45 with FcgammaRIIa resulted in abrogation of FcgammaRIIa mediated tyrosine phosphorylation and F-actin polymerization. CONCLUSIONS: These data provide evidence that CD45 can regulate or enhance the stimulation and function of human neutrophils mediated through FcgammaR(s). In addition, CD45 ligation may play an essential role in cytokine induction pathways that lead to inflammatory reactions in vivo.     

High production of proinflammatory and Th1 cytokines by dendritic cells from patients with rheumatoid arthritis, and down regulation upon FcgammaR triggering

OBJECTIVE: To assess whether DC from RA produce altered cytokine levels and whether this is regulated by triggering of Fc gamma receptors (FcgammaR). METHODS: The production of proinflammatory (TNFalpha, IL1, IL6), Th1 (IL12, IFNgamma), and Th2 (IL10) cytokine profiles of immature DC (iDC) from patients with RA and healthy subjects upon triggering of FcgammaR dependent and independent pathways was investigated. iDC, derived from blood monocytes by standardised protocols, were stimulated with immune complexes (IC) at day 6 for 48 hours and, subsequently, for 2 days with LPS in the presence or absence of IC or IFNgamma, resulting in fully matured DC (mDC). IL1, IL6, TNFalpha, IFNgamma, IL12, and IL10 levels in supernatants were measured by ELISA and RIA. RESULTS: mDC from patients with RA showed a markedly increased production of IL1, IL6, TNFalpha, and IL10 compared with DC from healthy donors. Triggering of FcgammaR decreased the production of proinflammatory cytokines IL1, IL12, and IFNgamma by iDC and mDC in RA and controls. The production of IL6 and TNFalpha decreased in patients with RA, whereas it was increased in controls. Triggering of FcgammaR independent mechanisms using IFNgamma increased the production of proinflammatory and Th1 cytokines, which was more pronounced in RA. CONCLUSION: FcgammaR dependent pathways influence cytokine production by DC. A skewed balance towards proinflammatory and Th1 cytokines in RA can, at least partly, be restored by triggering FcgammaR on DC in RA. Insight into the mechanism which determines the FcgammaR balance might lead to new strategies to abrogate Th1 driven inflammatory processes in RA.     

Down-regulation of activating Fcgamma receptors on monocytes of patients with rheumatoid arthritis upon methotrexate treatment

OBJECTIVE: To determine the effect of methotrexate (MTX) on expression levels of activating receptors for IgG (FcgammaRs) on monocytes of rheumatoid arthritis (RA) patients in relation to changes in disease activity. METHODS: The effect of MTX on FcgammaRs on monocytes of RA patients was evaluated ex vivo as well as in vitro. Recently diagnosed, disease-modifying antirheumatic drug (DMARD)-naive RA patients were treated with low-dose MTX. At baseline and 16 weeks after the start of MTX treatment, changes in FcgammaR expression levels on peripheral blood monocytes were evaluated by fluorescence-activated cell sorting analysis and were correlated to changes in disease parameters. To study the direct effects of MTX on monocytes, these cells were isolated from peripheral blood monocytes of healthy controls and cultured with MTX. Other monocyte surface molecules (CD40, CD80, CD86, MHC class II) were also determined to test the specificity of the effect on FcgammaR expression levels. RESULTS: Eleven out of 15 patients improved clinically (mean disease activity score before 6.2 +/- 0.8 vs 4.3 +/- 1.7 after). Sixteen weeks after the start of MTX therapy, the expression levels of FcgammaRI and IIa on monocytes were significantly decreased, whereas the decreases in FcgammaRIIIa expression levels on monocytes were less marked. The percentage decrease in FcgammaRI expression correlated with the percentage decrease in CRP and well-being. In vitro MTX selectively decreased FcgammaRI and FcgammaRIIa expression levels of isolated monocytes, in contrast to other surface molecules. CONCLUSION: The disease-modifying effect of MTX in the treatment of RA is accompanied by down-regulation of activating FcgammaRI and IIa on monocytes, which could be a direct effect of MTX on monocytes. This down-regulation represents a new mode of action of MTX which should be considered in RA patients, especially during conditions that could give rise to monocyte activation by IgG-containing immune complexes, e.g. during antibody-based therapy of RA.     

Selective blockade of inhibitory Fcgamma receptor enables human dendritic cell maturation with IL-12p70 production and immunity to antibody-coated tumor cells

The final differentiation or maturation of dendritic cells (DCs) in response to environmental stimuli influences their ability to both initiate immunity and determine the quality of the response to antigens. Circulating immune complexes and cell-bound immunoglobulins present in normal human sera represent a potential stimulus for inadvertent DC activation in the steady state and during autoimmunity. Here, we show that selective blockade of the inhibitory Fcgamma receptor (FcgammaR) FcgammaRIIb with recently developed monoclonal antibodies leads to maturation of human monocyte-derived DCs, which depends on the presence of IgG in normal human plasma. Plasma, in the presence of an FcgammaRIIb blockade, caused the DCs to up-regulate the expression of costimulatory molecules and to produce the inflammatory mediator IL-12p70. FcgammaRIIb blockade of DCs loaded with tumor cells led to increased tumor-specific T cell immunity without the need for exogenous stimuli other than human plasma. Therefore, the activation status of DCs in the presence of normal human serum depends on the balance between activating and inhibitory FcgammaRs and can be enhanced by new antibodies that react selectively with FcgammaRIIb. These data suggest an approach for modifying this balance to enhance immunity to immune complexes and antibody-coated tumor cells and to silence DC activation by immune complexes in autoimmune states.     

Tumor necrosis factor alpha blockade treatment down-modulates the increased systemic and local expression of Toll-like receptor 2 and Toll-like receptor 4 in spondylarthropathy

OBJECTIVE: Abnormal host defense against pathogens has been implicated in the pathogenesis of spondylarthropathy (SpA), a disease characterized by abundant synovial infiltration with innate immune cells. Given the role of Toll-like receptors (TLRs) in activation of innate inflammation and the occurrence of TLR-dependent infections after tumor necrosis factor alpha (TNFalpha) blockade treatment, the present study was undertaken to analyze TLRs and their modulation by TNFalpha blockade in SpA. METHODS: Peripheral blood mononuclear cells (PBMCs) were obtained from SpA and rheumatoid arthritis (RA) patients during infliximab therapy, and from healthy controls. TLR-2 and TLR-4 expression and TNFalpha production upon lipopolysaccharide (LPS) stimulation were analyzed by flow cytometry on different monocyte subsets. Synovial biopsy specimens from 23 SpA patients before and after infliximab or etanercept treatment, from 15 RA patients, and from 18 osteoarthritis (OA) patients were analyzed by immunohistochemistry. RESULTS: Expression of TLR-4, but not TLR-2, was increased on PBMCs from patients with SpA, whereas both TLRs were increased in RA patients. TLR expression was particularly increased on the CD163+ macrophage subset. Infliximab reduced TLR-2 and TLR-4 expression on monocytes of SpA and RA patients, leading to lower levels than in controls and to impaired TNFalpha production upon LPS stimulation. In inflamed synovium, the expression of both TLRs and of CD163 was significantly higher in patients with SpA than in those with RA or OA. Paralleling the systemic effect, TLRs in synovium were down-regulated following treatment with infliximab as well as etanercept, indicating a class effect of TNFalpha blockers. CONCLUSION: Inflammation in SpA is characterized by increased TLR-2 and TLR-4 expression, which is sharply reduced by TNFalpha blockade. These findings suggest a potential role of innate immunity-mediated inflammation in SpA and provide an additional clue regarding the mechanism of action as well as the potential side effects of TNFalpha blockade.     

Variant genotypes of the low-affinity Fcgamma receptors in two control populations and a review of low-affinity Fcgamma receptor polymorphisms in control and disease populations

Fcgamma-receptors (FcgammaR) provide a critical link between humoral and cellular immunity. The genes of the low-affinity receptors for IgG and their isoforms, namely, FcgammaRIIa, FcgammaRIIb, FcgammaRIIIa, FcgammaRIIIb, and SH-FcgammaRIIIb, are located in close proximity on chromosome 1q22. Variant alleles may differ in biologic activity and a number of studies have reported the frequencies of variant FcgammaR alleles in both disease and control populations. No large study has evaluated the possibility of a nonrandom distribution of variant genotypes. We analyzed 395 normal individuals (172 African Americans [AA] and 223 Caucasians [CA]) at the following loci: FcgammaRIIa, FcgammaRIIIa, and FcgammaRIIIb, including the SH-FcgammaRIIIb. The genotypic distributions of FcgammaRIIa, FcgammaRIIIa, and FcgammaRIIIb conform to the Hardy-Weinberg law in each group. There was no strong evidence that combinations of 2-locus genotypes of the 3 loci deviated from random distributions in these healthy control populations. The distribution of SH-FcgammaRIIIb is underrepresented in CA compared with AA (P < .0001) controls. A previously reported variant FcgammaRIIb was not detected in 70 normal individuals, indicating that this allele, if it exists, is very rare (<1%). In conclusion, we present data that should serve as the foundation for the interpretation of association studies involving multiple variant alleles of the low-affinity FcgammaR.     

Effect of the first infliximab infusion on sleep and alertness in patients with active rheumatoid arthritis

OBJECTIVE: To assess the benefit of the first infliximab infusion on sleep disturbances in patients with RA Material and methods: Evaluation of RA activity, sleepiness (Epworth scale and multiple sleep latency test), alertness (steer clear test), and sleep structure (polysomnography) were conducted before and after the first infusion of infliximab in six female patients with RA. RESULTS: The day after the first infliximab infusion, the mean (SD) number of tender (20 (2.4)) or swollen (15.3 (2)) joints and the morning stiffness (140 (61.9) min) had not changed. There were significant improvements in the median number of total sleep stage transitions per hour (median (IR) before v after infusion: 20.5 (43) v 7.5 (6); Wilcoxon paired test, p = 0.014), median percentage of phase I+II (83.5 (8) v 54.5 (24); p = 0.023), percentage of REM stages (2 (10) v 11.5 (8); p = 0.014), median percentage sleep efficiency (44 (22) v 75 (18); p = 0.014), median sleep latency (77.5 (150) v 25.5 (23) min; p = 0.023), and median number of hits in the steer clear test (48.5 (86) v 6 (45); p = 0.023). Neither objective nor subjective daytime sleepiness was noted. One obese patient had obstructive sleep apnoea syndrome. CONCLUSIONS: Sleep and the alertness disturbances in RA improve with infliximab treatment. Improvement appears unrelated to joint discomfort amelioration but suggests a central effect through inhibition of circulating TNFalpha levels.     

Fcgamma receptor polymorphisms in systemic lupus erythematosus: association with disease and in vivo clearance of immune complexes

OBJECTIVE: Fc receptors for IgG (FcgammaR) play a prominent role in the clearance of immune complexes in systemic lupus erythematosus (SLE). Polymorphisms of FcgammaR have been proposed as genetic factors that influence susceptibility to SLE. We analyzed 3 functional FcgammaR polymorphisms in a strictly Caucasian population of SLE patients, and determined the influence of these polymorphisms on the clearance of immune complexes in vivo. METHODS: Genomic DNA was isolated from 230 Caucasian patients with SLE and 154 controls. Amplification of FcgammaR-genomic regions in allotype-specific polymerase chain reactions was used to distinguish the genotypes. In addition, we analyzed the FcgammaR genotypes of 13 patients with SLE who participated in a study determining the half-life of IgG-coated erythrocytes in the blood. RESULTS: We found a strong trend toward skewing of FcgammaRIIa, with an enrichment of the homozygous FcgammaRIIa-R/R131 genotype in patients compared with controls. We did not find a correlation between this genotype and the development of lupus nephritis. However, we established that the half-life of IgG-coated erythrocytes in the blood was prolonged in patients expressing the FcgammaRIIa-R/R131 genotype. The homozygous FcgammaRIIIa-F/F158 genotype was found more frequently in patients with arthritis and/or serositis. CONCLUSION: In Caucasian populations, the R/H polymorphism of FcgammaRIIa is a minor determinant in susceptibility to SLE, whereas the V/F polymorphism of FcgammaRIIIa is associated with a set of disease manifestations. Notably, the R/H polymorphism of FcgammaRIIa affects the clearance of immune complexes in vivo, which may influence the course of a disease such as SLE.     

Standardised assessment of membrane proteinase 3 expression. Analysis in ANCA-associated vasculitis and controls

OBJECTIVES: Increased numbers of neutrophils expressing proteinase 3 on their membrane (mPR3) have been reported in anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV) and are suggested to be involved in AAV immunopathogenesis. In most studies, neutrophils were analysed for mPR3 expression without priming with TNFalpha, suggesting that mPR3 expression on neutrophils is dependent on other priming events, such as isolation procedures . These priming events can be variable. Therefore, we analysed mPR3 expression on neutrophils before and after priming with TNFalpha to assess whether standardised assessment of mPR3 expression requires priming. Using neutrophils before and after priming with TNFalpha, we assessed percentages of mPR3(+) neutrophils in patients with AAV and in disease and healthy controls. METHODS: Neutrophils from patients with PR3-AAV and MPO-AAV, systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), and from healthy controls were analysed before and after priming with TNFalpha for mPR3 expression. RESULTS: 42% of all individuals analysed showed minimal expression for mPR3 on all neutrophils before priming with TNFalpha, whereas after priming a clear mPR3(+) subset was observed next to mPR3(-) neutrophils, corresponding to bimodal mPR3 expression. In patients with PR3-AAV or MPO-AAV, the percentage of mPR3(+) neutrophils after priming with TNFalpha was significantly increased (p<0.01 and p<0.05, respectively) compared with healthy controls. Percentages of mPR3(+) PMN were also increased in patients with SLE (p<0.01) but not in RA. CONCLUSION: Standardised assessment of proteinase 3 on the membrane of neutrophils requires priming with TNFalpha. Percentages of mPR3(+) PMN are increased in AAV and SLE, but not in RA.     

FcgammaRIIa polymorphism in Japanese patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an immune complex-mediated disease and organ damage is caused by the deposition of immune complex. Receptors which recognize the Fc portion of immunoglobulin G (FcgammaR) play a key role in the phagocytosis of immune complexes. As the gene encoding for FcgammaR of class IIa (FcgammaRIIa) has two allelic forms, H131 and R131, which differ in their affinity to IgG2, this polymorphism might have implications in handling immune complex. We studied the distribution of the FcgammaRIIa polymorphism in 90 Japanese patients with SLE. We also examined the association between FcgammaRIIa polymorphism and the disease activity of SLE and the histopathological findings of lupus nephritis. FcgammaRIIa polymorphism was determined by PCR and dot blot analysis. The allelic frequency of H131 in patients with SLE was significantly lower (H131/R131 = 0.44/0.56) than that of normal controls (H131/R131 = 0.62/0.38; P < 0.05). No significant association was observed between FcgammaRIIa polymorphism and the clinical parameters for the activity of SLE. There was no association between FcgammaRIIa polymorphism and the histological findings in lupus nephritis. The difference in the distribution of FcgammaRIIa alleles between patients with SLE and normal subjects indicates that this polymorphism is a candidate of susceptibility gene for SLE in Japanese.