Analysis of the HLA-DR gene frequencies in Japanese cases of juveniles rheumatoid arthritis and rheumatoid arthritis by oligonucleotide DNA typing

Summary HLA-DR gene frequencies in 59 Japanese children with juvenile rheumatoid arthritis (JRA) and 62 Japanese adults with rheumatoid arthritis (RA) were analyzed by oligonucleotide DNA typing. As in other studies, the frequency of DRB1*0405 in RA patients was significantly higher than in the Japanese controls. In a comparison of non-calssified JRA patients with Japanese controls, no significant differences were observed in the frequency of DR types. However, when the JRA patients were classified into four clinical types, i.e., a rheumatoid factor-positive [RF(+)] polyarticular type, a rheumatoid factor-negative [RF(-)] polyarticular type, a pauciarticular type, and a systemic onset type, DRB1*0405 was found to be significantly higher in the RF(+) polyarticular JRA patients than in the controls (P>0.05). Thus, the RF(+) polyarticular type of JRA had the same HLA association as RA. This result is consistent with the fact that both RF(+) polyarticular JRA and RA cases have a similar clinical course.     

Clinical features of patients with systemic sclerosis accompanied by rheumatoid arthritis

OBJECTIVE: To determine the incidence of patients with both systemic sclerosis and rheumatoid arthritis (SSc-RA) and the clinical features of those with SSc-RA. METHODS: All 173 patients with systemic sclerosis in our clinic were investigated. RESULTS: Of the 173 patients with systemic sclerosis, 9 (5.2%) developed rheumatoid arthritis (RA). At the first visit, arthritis prior to Raynaud's phenomenon, increased C-reactive protein (CRP), and elevated rheumatoid factor (RF) were seen in patients with SSc-RA at a significantly higher incidence than in those without (44.4% versus 4.8%, p < 0.01; 55.6% versus 13.6%, p < 0.001; 247.2 +/- 312.1 versus 47.9 +/- 54.3 IU/ml, p < 0.001, respectively). Furthermore, in 8 of the 9 patients with SSc-RA, CRP was increased before the diagnosis of RA. CONCLUSION: These results suggest that systemic sclerosis patients with elevated RF and a history of arthralgia prior to Raynaud's phenomenon should be followed up with serial measurements of CRP due to their risk of developing RA.     

Clinical features, autoantibodies and HLA-DR antigens in rheumatoid arthritis

HLA-DR4 was associated with seropositive but not seronegative disease in 105 Caucasians with rheumatoid arthritis (RA). There were no clinical or radiological differences between DR4 positive and negative RA groups, although 7 of 8 patients with early disease onset (less than 30 yr) were DR4 positive. High rheumatoid factor (RF) titers were more frequent in DR4 negative RA. A plot of the frequency distribution of RF titers in DR4 negative disease showed a bimodal distribution with seronegative and high titer groups. HLA-DR3 was not associated with high RF titers but was associated with high titers of antinuclear antibodies.     

Relationship between urinary sialylated saccharides, serum amyloid A protein, and C-reactive protein in rheumatoid arthritis and systemic lupus erythematosus.

The urinary excretion of sialic-acid-containing oligosaccharides, total sialic acid, serum amyloid A protein (SAA), and C-reactive protein (CRP) has been studied in 48 patients with rheumatoid arthritis (RA) and in 17 patients with systemic lupus erythematosus (SLE). Linear regression analysis revealed a close positive correlation between serum SAA and CRP levels in both RA (r = 0.71, p less than 0.001) and SLE (r = 0.86, p less than 0.001). The urinary excretion of sialyl lactose showed a positive correlation with the serum levels of SAA and CRP in RA (r = 0.45 and r = 0.45, respectively, p less than 0.01) but not in SLE (r = 0.05 and r = 0.10 respectively). Changes in serum total sialic acid levels paralleled those in CRP and SAA in RA as well as in SLE. Patients with very active RA had higher urinary sialyl oligosaccharide excretion (p less than 0.001), higher CRP levels (p less than 0.01), and higher SAA levels ( p less than 0.05) than those with moderately active disease.     

The association between rheumatoid arthritis and the HLA antigen DR4.

One hundred and forty-two patients with 'definite' or 'classical' rheumatoid arthritis (RA) were studied for the frequency and possible prognostic significance of HLA DR4. Of these, 122 were seropositive, while 20 were negative for rheumatoid factor (RF) in serum. The HLA antigen DR4 was significantly increased in the seropositive group (65%) as well as in the seronegative group (55%) in comparison with a frequency of 27% in 116 healthy controls. Among seropositive patients a higher frequency of DR4 was found in females (73%) than in males (50%), the difference being statistically significant (p less than 0.01). DR4 was more frequent among patients with a family history of RA (74%) than among patients without affected relatives (57%). DR4 appeared to be associated with an early onset of RA. No significant differences in general disease activity or functional capacity between DR4-positive and DR4-negative RA patients were found. Patients without the HLA antigen DR4 had a significantly (p less than 0.05) higher mean titre of RF than those with the antigen.     

IgM rheumatoid factor (RF), IgA RF, IgE RF, and IgG RF detected by ELISA in rheumatoid arthritis.

One hundred patients with rheumatoid arthritis (RA), of whom 73 were seropositive by latex or Waaler-Rose (WR) assays, or both, 100 healthy subjects, and 102 diseased controls (22 patients with systemic lupus erythematosus (SLE) and 80 with bronchial asthma) were evaluated for the presence of IgM rheumatoid factor (RF), IgA RF, IgE RF, and IgG RF by an enzyme linked immunosorbent assay (ELISA). Ninety two per cent, 65%, 68%, and 66% of the patients with RA were found to be positive for IgM, IgA, IgE, and IgG respectively. A positive correlation existed between the levels of IgM RF and IgA RF on the one hand and disease activity on the other, and the levels of IgM RF and IgA RF correlated with the levels of circulating immune complexes as measured by a C1q binding assay. The presence of extra-articular features also correlated positively with the levels of IgA RF and IgE RF. Five out of six patients with Sjögren''s syndrome had very high levels of IgA RF. Of 47 patients typed for HLA-DR, DR1 and DR2 were significantly more frequent in those with the highest levels of IgM RF. Conversely, DR3 was associated with low levels or absence of IgA RF and IgE RF. These results suggest that immune response genes may regulate the level of different RF isotypes. The frequencies of IgM, IgA, IgE, and IgG RF were 59%, 36%, 9%, and 27% respectively in SLE and 25%, 2.5%, 70%, and 59% in bronchial asthma.     

DR4 prevalence related to the age at disease onset in female patients with rheumatoid arthritis.

The prevalence of HLA-DR4 in relation to age at disease onset was calculated in 226 consecutive female patients with definite or classical rheumatoid arthritis (RA). A slight increase in the prevalence of DR4 with age at RA onset was found. This appeared to be due to the low percentage of rheumatoid factor (RF) positivity in the youngest age groups; the DR4 prevalence in the RF positive patients was constant for all decades--that is, approximately 60%. A previously reported declining trend of DR4 prevalence in women with RA in relation to age of RA onset may be due to the disease heterogeneity of the patients included in that study.     

Potential relationship between herpes viruses and rheumatoid arthritis: analysis with quantitative real time polymerase chain reaction

OBJECTIVE: To determine whether the human herpes viruses, cytomegalovirus (CMV), Epstein-Barr virus (EBV), and human herpesvirus 6 (HHV-6), are detectable in serum and peripheral blood mononuclear cells (PBMCs) of patients with rheumatoid arthritis (RA). METHODS: 133 PBMC samples (61 RA, 72 healthy donors) and 136 serum samples (59 RA, 77 healthy donors) were analysed by quantitative real time polymerase chain reaction for DNA prevalence and viral load of HHV-6, EBV, and CMV. RESULTS: For PBMC samples significant differences were found for EBV in DNA prevalence (56% in RA v 33% in controls, p = 0.009) and viral load (copies/microg DNA 0-592.3 for RA v 0-40.4 for controls, p = 0.001). For serum samples a significant difference was found for HHV-6 DNA prevalence (10% in RA v 0% in controls, p = 0.006) and viral load (copies/microg DNA 0-529.1 for RA v 0 for controls, p = 0.007). CONCLUSIONS: Herpes viruses may have a role in RA, although alternative explanations are possible: (a) defects in cellular immunity in patients with RA may result in a relatively high viral load; (b) patients with RA may be more prone to infection/reactivation. The usefulness of monitoring the DNA viral load in patients with RA is questioned by these data.     

Immunogenetic markers and seropositivity predict radiological progression in early rheumatoid arthritis independent of disease activity

OBJECTIVE: A prospective clinical study of patients with recent onset rheumatoid arthritis (RA) to examine the relationship between inflammatory disease activity and joint destruction in a 4 year followup, and to evaluate prognostic markers for severe joint erosions early in the disease. METHODS: Eighty-seven patients with RA according to the American College of Rheumatology criteria and a disease duration < 2 years were followed for an observation time of 2 to 4 years (mean 3.1 yrs). Variables of clinical and laboratory disease activity were monitored, and HLA-DRB1 alleles were determined. Hand and foot radiographs were taken every 6 months. RESULTS: Multivariate analysis of independent contributions of covariates to progression of joint destruction resulted in a mixed effect regression model with significant influences for the presence of a shared epitope (SE) positive DR4 allele (SE+ DR4+; p = 0.007), rheumatoid factor (RF) IgA (p = 0.01), and sex (p = 0.059), but not for clinical variables or acute phase reactants. The odds ratio to reach a Larsen score above 32 during the observation period of 4 years was increased in patients positive for RF IgM (OR 2.7, p = 0.019), for the shared epitope on a DR4 allele (OR 8.6, p < 0.005), and in patients with erosions already at study entry (OR 11.9, p = 0.001). The highest sensitivity and specificity for the prediction of severe bone destruction (84% and 79%) were found when the presence of either a SE+ DR4 allele or of early erosions was used as a prognostic marker (OR 20.4, p < 0.0001). CONCLUSION: Our results show the pace of joint destruction in RA to be influenced by the presence of SE+ DR4 alleles, RF production, and sex and by the presence of erosive disease at presentation. Those prognostic markers exert their influence independently from the inflammatory disease activity.     

ELISA determined IgM, IgG and IgA rheumatoid factors in rheumatoid arthritis and in other connective tissue diseases

We used an adaptation of an enzyme-linked immunoadsorbent assay (ELISA) to determine serum levels of IgM, IgG and IgA rheumatoid factors (RF) in 50 patients with classic or definite rheumatoid arthritis (RA) according to the ARA criteria, balanced for positive or negative-routine Latex-RF reaction. A control group of 50 young normal subjects and a reference group of 44 patients with other connective tissue diseases (OCTD) were also studied. We confirmed the high sensibility of the method, together with its good specificity and reproducibility. For the IgM RF a very significant correlation was found between ELISA results and Latex-RF titration (p less than 0.001). Many Latex-RF negative RA patients had high ELISA levels of IgM RF, suggesting that this assay reveals, at least in part, hidden or non-agglutinating IgM RF. Among the OCTD group only some SLE cases, mainly Latex-RF positive, had enhanced IgM RF on ELISA. Considered quantitatively, IgG RF did not play a significant diagnostic role for RA (p greater than 0.05), because they were also found, with widely dispersed values, in normal subjects, and because the mean increase in RA patients was relatively small. Interestingly, IgA RF were above the normal range in many RA patients, both Latex-RF positive or negative. The mean values differed significantly from those of controls (p less than 0.005), and a correlation was observed between IgA RF levels and IgA containing immune-complexes. Normal IgA RF values were observed in SLE patients, even if Latex-RF positive, suggesting that their increase in RA patients is not the mere expression of a polyclonal B cell activation.     

HLA-DR alleles with naturally occurring amino acid substitutions and risk for development of rheumatoid arthritis

To determine the HLA-DR4 subtypes associated with rheumatoid arthritis (RA), we performed amplification of DR4 DRB1 genes by the polymerase chain reaction and dot-blots with oligonucleotide probes. In 52 HLA-DR4+ RA patients, Dw4 was the predominant subtype. This subtype was found in 45 of 52 patients (86.5%) compared with 33 of 59 DR4+ controls (55.9%; P less than 0.001). In the whole population, Dw4 also gave the highest relative risk for RA (RR = 5.31). Relative risk was also associated with DR1.1, the common white DR1 (Dw1) type, which has a third hypervariable region amino acid sequence similar to some forms of DR4 and has glycine at position 86. Variants of DR1 (DR1.2) or DR4 (Dw13.1, Dw14.1) with valine at position 86 appeared less able to confer risk for RA. Substitution of residues in the third hypervariable region of the first domain of DRB1 appeared to correlate with relative risk for RA. Among subjects having 0-1 amino acid substitutions, RA developed in 53%, whereas in subjects with 2-4 amino acid changes, RA was present in only 17.4% (P less than 0.00001). DQw7 (formerly DQw3.1) was slightly increased in DR4+ RA patients compared with controls, but a striking excess of Dw4,DQw7 homozygous patients was observed. The results suggest that DQw7 may have an additional effect, possibly with a recessive mechanism, since it was observed only in DR4 homozygous patients.     

D-Penicillamine induced toxicity in rheumatoid arthritis: the role of sulphoxidation status and HLA-DR3

Sulphoxidation of carbocysteine, a drug structurally similar to D-penicillamine, displays a skewed distribution within a population. In 66 patients with rheumatoid arthritis (RA) a significant association between impaired sulphoxidation and toxicity (p less than 0.001) was found; HLA-DR3, although associated with toxicity (p less than 0.05), appeared to be an independent risk factor of most importance in the group with extensive sulphoxidation. The relative risk of toxicity in a patient possessing either DR3 or impaired sulphoxidation was 25. The prevalence of poor sulphoxidizers within this group of RA patients was increased compared to that in a previous population study and requires further investigation. Our findings explain a number of the toxic phenomena associated with D-penicillamine administration in RA.     

Association of HLA-DR with susceptibility to and clinical expression of rheumatoid arthritis: re-evaluation by means of genomic tissue typing

The clinical expression of rheumatoid arthritis (RA) varies considerably among individual patients. Genetic variations in human leucocyte antigen (HLA) may influence clinical expression. We re- examined the association of HLA-DR with susceptibility to and clinical expression of RA using genomic tissue typing, since most studies were based on (less reliable) serological techniques. Seventy-eight patients with recent-onset RA, all participating in a clinical trial on therapeutic strategies, were HLA-DR typed by means of low-resolution genomic typing. Cumulative disease activity within the first 3 yr of disease was measured. Of the RA patients, 54% expressed DR4 (DR4+) vs 26% of healthy controls. Rheumatoid factor (RF)-positive patients had a higher cumulative disease activity than RF-negative patients. Patients who were either DR1+ or DR4+ had a higher cumulative disease activity than those who expressed neither DR1 nor DR4. This association was less obvious after correction for RF status. The association of DR52+ (DR3, 5, 6) and a lower cumulative disease activity could also not be demonstrated after correction for RF status. Among RF-negative patients, DR51+ (or DR2+) was associated with a higher cumulative disease activity. Other HLA-DR types (including DR1 and DR4 separately) were not associated with the severity of RA. DR4 was associated with susceptibility to RA in our patients; HLA-DR low-resolution genomic tissue typing did not yield additional information to RF status for the clinical identification of individual patients with a poor prognosis.      

Smoking is a strong risk factor for rheumatoid nodules in early rheumatoid arthritis

OBJECTIVE: To examine whether smoking is a risk factor for rheumatoid nodules in early rheumatoid arthritis, and if so to determine the quantitative effect of smoking. METHODS: From a cohort (n = 1589) in a structured programme for follow up of newly diagnosed cases of rheumatoid arthritis (symptoms of swollen joints < or =12 months), 112 individuals with rheumatoid nodules at inclusion were identified. Nodular patients were each compared with two age and sex matched controls without nodules from the same cohort. A detailed self administered tobacco use questionnaire was answered by 210 patients (63%). RESULTS: Seventy patients were current smokers, 71 former smokers, and 69 had never smoked. Current smoking and former smoking were more common in patients with rheumatoid nodules compared with controls (86% v 59%) in both sexes. Positive rheumatoid factor (RF) was found more often among cases with nodules than controls (78% v 64%). Using detailed information from the questionnaires with conditional logistic regression analyses, ever having smoked was associated with an increased risk of the presence of rheumatoid nodules (odds ratio (OR) = 7.3 (95% confidence interval, 2.3 to 23.6); p = 0.001). The risk of having nodules was not obviously dose dependent when smoking duration as well as smoking amount were examined. A stratified analysis showed that only RF positive smokers had an increased risk of rheumatoid nodules. Smoking was associated with rheumatoid nodules among both men (p = 0.006) and women (p = 0.001). Tobacco use other than smoking (n = 31) was not associated with an increased risk of nodules (OR = 0.8 (0.2 to 3.4); p = 0.813). CONCLUSIONS: There is a strong association between smoking and rheumatoid nodules in early seropositive rheumatoid arthritis.     

HLA associations with rheumatoid arthritis in African blacks

The HLA-A, B, C and DR antigens were determined in a group of 100 blacks with classical or definite rheumatoid arthritis (RA) in Durban, South Africa. Fifty-six of these patients were also tested for the DQ antigens. There was a significant association of HLA-DR4 with RA (chi 2 = 77.2; p less than 0.0001). The frequency of DR4 in RA was 44% in comparison with 10% in controls (relative risk 7.4). An unusual finding was a significant increase in the frequency of HLA-B8 in 35% of patients with RA compared to 12.5% of controls (p less than 0.001; relative risk 3.8). There was no linkage disequilibrium between DR4 and B8 to explain the latter association.     

Increased serum levels of interleukin-15 in rheumatoid arthritis with long- term disease

OBJECTIVE: To study the serum levels of IL-15 in patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), seronegative spondyloarthropathies (SSd) and healthy donors. METHODS: The IL-15 serum levels were measured by ELISA in sera from 50 RA patients, 30 patients with SLE, 30 patients with SSd and 30 healthy donors. In RA patients, several clinical and demographic parameters were also obtained at the time of sample collection. IL-15 levels were compared in different RA subpopulations (positive or negative rheumatoid factor [RF], long term or recent onset disease, high or low disease activity). In addition, the possible association with other demographic and clinical parameters (gender, age, disease duration, etc) was also analysed. RESULTS: RA patients had significantly higher serum levels of IL-15 (102.4 +/- 150 pg/ml; p = 0.0001) than SLE patients (9.8 +/- 15.3 pg/ml), SSd patients (7.9 +/- 14.6 pg/ml) and healthy donors' (5.2 +/- 11.6 pg/ml). RA patients with a disease evolution less than 2 years showed lower IL-15 levels (33.7 +/- 62.2 pg/ml) than those with long-term disease (152.4 +/- 64.6 pg/ml; p = 0.004). In addition, a significant correlation between IL15 in serum and the number of disease-modifying antirheumatic drugs (DMARDs) prescribed was detected in RA patients (r = 0.42; p = 0.002). No association between IL-15 levels and age, gender, RF or disease activity was observed in this group. CONCLUSION: IL-15 is elevated in RA patients, specially in those with long term disease, compared to other rheumatic disorders. This finding supports that IL-15 is involved in the perpetuation of RA synovitis.     

The immunoregulation of rheumatoid factor by the CD8 T lymphocyte subset in rheumatoid arthritis.

Flow cytometric analysis on 81 peripheral blood samples from patients with rheumatoid arthritis (RA) showed that levels of serum IgM rheumatoid factor (RF) were associated with the CD8+ cell level. A significant elevation of natural killer (CD56) cell levels was also observed in RA peripheral blood. Using in vitro antibody production techniques, CD8+ cells from patients with RA appeared to act as suppressors of RF production. Paired blood and synovial fluid samples from 9 patients with RA indicated a significant increase in SF CD8+ cells and DR+ T cells over the corresponding peripheral blood levels. The data suggest that CD8+ cells in RA may respond to immunological abnormalities occurring during the course of the disease.     

Seronegative rheumatoid arthritis.

Measurement of serum rheumatoid factor (RF) by conventional methods in patients with rheumatoid arthritis (RA) has repeatedly identified a subpopulation of patients without detectable RF. Previous investigators have consistently confirmed the association of HLA–DR4 with seropositive RA, but studies of seronegative RA have been limited and contradictory. We studied 140 randomly selected patients from Alabama, all of whom had either classic or definite RA, and we were able to obtain complete HLA typing for 110 of these individuals. Eighty were consistently seropositive (on at least 2 separate occasions) and 30 were consistently seronegative (on at least 3 separate determinations). There was no statistically significant difference between the seronegative RA patients and 123 control subjects in the distribution of DR antigens. In seropositive RA, there was a significant increase in DR4 (P<0.001; relative risk = 8.02; attributable risk = 49.2%) and a significant decrease in DR3 (P<0.001; relative risk = 0.14) and DR7 (0.01>P>0.001; relative risk = 0.33). The clinical data also distinguished between seropositive RA and seronegative RA; subcutaneous nodules (37.5%) and vasculitis (6.3%) were present only in seropositive RA. DR4 positivity did not correlate with any of the clinical variables measured in the seropositive RA group. In contrast, DR4 in the seronegative RA group was associated with more destructive disease. The data suggest that seronegative RA represents a disease entity clinically and immunogenetically distinct from seropositive RA. Moreover, our results indicate that DR4 may be a previously undisclosed marker for disease severity in seronegative RA.     

DR antigen distribution in Blacks with rheumatoid arthritis

DR antigen distribution was studied in 85 Blacks with either classical or definite rheumatoid arthritis (RA). Sixty-three were seropositive and 22 were seronegative by latex fixation titer. The frequency of DR4 was 7.41% in our control Black population (n = 162), 22.22% in the seropositive (0.01 greater than p greater than 0.001; RR 3.57) and 22.72% in the seronegative RA (0.01 greater than p greater than 0.001; RR 3.67). Our data confirm the association of DR4 with seropositive RA in Blacks, and suggest a similar association of DR4 with seronegative RA. It also suggests that DR4 may be a risk factor for more aggressive disease in Blacks with seropositive RA.     

Associations of HLA-DR 4, DR 2, DRw 9 with clinical findings in rheumatoid arthritis

HLA-DR antigens were determined in 128 patients with classical or definite rheumatoid arthritis (RA) according to American Rheumatism Association criteria (1957). HLA-DR 4 was significantly (p less than 0.01) increased in patients with RA (60%) compared with Japanese control (40%). In radiological changes, the frequency of stage II to IV were significantly greater in DR 4 positive patients (87.1% (67/77)) than in negative patients (70.6%) (36/51)). An early onset of disease was significantly (p less than 0.05) associated with DR 4 positive patients with duration of 4 years or more. Erythrocyte sedimentation rate was significantly (p less than 0.05) less in DR 2 positive patients (8.0% +/- 8.6 (39)) than DR 2 negative patients (12.2 +/- 11.9 (84)). Frequency of Sjogren's syndrome was more in DR 2 positive patients (41.3% (12/29)) than in DR 2 negative (29.2% (19/65)), and less in DR 4 positive (25.4% (15/59)) than in DR 4 negative (45.7% (16/35)), so the complication of Sjogren's syndrome showed a trend against the severity of RA. There were no associations between rheumatoid factor and HLA-DR phenotypes, but the frequency of anti-nuclear anti-nuclear antibody was significantly (p less than 0.01) lower in DRw 9 positive patients (38.4% (15/39)) than in DRw 9 negative (62.7% (54/86)). In both DR 4 and DRw 9 positive patients (16 cases), onset of disease (38.9 years-old +/- 15.9 (16)) was significantly earlier and frequency of Sjogren's syndrome (10.0% (1/10)) was significantly lower than those in DR 4 negative patients (48.5 years-old +/- 12.5 (51): 45.7% (15/35) respectively). The frequency of HLA-DRw 9 was greater in Japanese than people in the other countries and there was the close association between pathogenesis of RA and HLA-DRw 9 as well as DR 4 and DR 2 in Japan.