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Rheumatoid arthritis (RA) is associated with HLA-DR4, which is encoded by the DRB1 gene. This genetic predisposition has been shown to lie within the sequence motif present in the third hypervariable region of the DRB1 gene.1 This sequence of amino acids has been called the “disease epitope” and can be encoded by DR4 subtypes as well as non-DR4 alleles; DR1 subtypes, DR10 and DR14 subtypes .1 Hence, it is also termed the shared epitope. In addition to imparting susceptibility to RA, HLA-DR4 has also been shown to be associated with the severity of the rheumatoid disease, including destructive erosive joint disease, rheumatoid factor positivity,2 and extra-articular manifestations, such as rheumatoid nodules, vasculitis, and Felty's syndrome.3 4HLA-DQ genes are in linkage disequilibrium with HLA-DR and subjects who are DR4 positive may either be DQB1*0301 or *0302 positive; certain extra-articular features of RA have been shown to be associated with HLA-DQB1*0301.4 5
Synovial fluid cytology in RA is heterogeneous both with respect to total white cell count and to the proportions of polymorphs, lymphocytes, and ragocytes.6 Ragocytes are leucocytes containing intracytoplasmic granules and have been shown to be present in higher percentages in the joints of patients with RA.6Davis et al showed that when a patient with RA has a relapse affecting the same joint, the proportions of cell types remain similar, though the total white cell count may differ.7 The same study also found a significantly poorer outcome in patients with persistently high ragocyte counts as assessed by the necessity for subsequent joint replacement or synovectomy.
As both synovial cytology and genetic factors have been shown independently to predict joint damage, this study aimed at assessing whether there is a relation between genetic factors and the pattern of synovial fluid cytology which might also be of prognostic significance.
Fifty nine unrelated patients with RA attending the Department of Rheumatology, Hope Hospital, Salford, were studied. All the patients were white and had classic or definite RA according to the 1987 American Rheumatism Association Criteria.8 All had synovial fluid analysed on at least one occasion during articular flares of arthritis. Blood was taken for HLA typing from each patient. Clinical details recorded were age, sex, disease duration, and drug treatment.
All synovial fluid samples collected were placed in 2 ml paediatric lithium heparin bottles and processed the same day. Total white cell count was carried out with a modified Fusch Rosenthal counting chamber and expressed as cells/cm3. Polymorphs, lymphocytes, monocytes, and ragocytes6 were differentiated in a cytocentrifuged and wet preparation using simple morphology and expressed as percentages of all nucleated cells. Genomic DNA was extracted as described previously.9 DRB, DQA and B were typed by polymerase chain reaction, followed by hybridisation to specific oligonucleotide probes.3 Rheumatoid factor was measured by the sheep cell agglutination test and titres ⩾1/32 were considered positive. Data for white blood cells, polymorphs, lymphocytes, ragocytes, and monocytes were not normally distributed and were expressed as medians (interquartile range). The Mann-Whitney U test was used to compare these variables between the different patient groups. When more than two groups were compared the Kruskal-Wallis one way analysis of variance was used. Corrections for multiple comparisons were also made.
The 59 patients (45 female, 14 male) had a median age of 63 years (range 27–85) and median duration of disease of 16.5 years (range 2–53). Of these 59 patients, 55 were seropositive for rheumatoid factor, 52 had erosive disease, 27 had extra-articular features (nodules, Felty's syndrome, Sjögren's syndrome, vasculitis, peripheral neuropathy, pulmonary, renal disease, and/or eye disease), and 43 were receiving a disease modifying antirheumatic drug (DMARD) with or without oral steroids at the time of the study.
HLA-DR typing showed that 44 (75%) patients were DR4 positive. Eight (18%) of the DR4 positive patients were compound heterozygotes (DRB1*0401/0404). Forty five (76%) patients were “RA epitope” positive (DR4/DRB1*0401, DR4/DRB1*0404, DR4/DRB1*0405, or DR1) and 24 (41%) were heterozygous for the “RA epitope”. HLA-DQ typing showed that 25 (42%) patients were positive for DQB1*0301.
When the patients were subdivided into those who were positive or negative for HLA-DR4, there were no differences in the total white cell count or proportion of the cell types between the two groups, and these were also similar between patients who were or were not compound heterozygotes.
However, when the patients were subdivided into those who were epitope positive or negative, there was an increased median percentage lymphocyte count in patients who were positive (median 11%) compared with those who were negative (median 3%, p=0.036, pcorr>0.05). There were no differences in the other cell type counts between the two groups.
When the patients were subdivided into those who were positive or negative for HLA-DQB1*0301, patients who were positive for the allele (n=25) had an increased median percentage ragocyte count (50%) compared with those who were negative (n=20, 10%, p=0.033, pcorr>0.05). The percentages of the other cell types were similar in the two groups. When the HLA-DR4 status was also taken into account, there was a higher median percentage of white cells typed as ragocytes (57.5%) in DR4+/DQB1*0301+ patients (n=20) than in those who were DR4+/DQB1*0301− (3.8%, n=14, p=0.017, pcorr>0.05). The DR4+/DQB1*0301+ group also had a reduced median lymphocyte percentage (4%) and monocyte count (1.5%) in comparison with DR4+/DQB1*0301− patients (22.5%, p=0.02, 8.5%, p=0.027, pcorr>0.05). Table 1 summarises these results.
There were no statistical differences in synovial fluid cytology depending on whether the patients were positive or negative for rheumatoid factor, those with erosive or non-erosive disease, presence or absence of extra-articular features, and treatment with or without DMARDs and/or steroids.
Even though the corrected p values were not significant, this study suggests that there is a genetic influence on the expression of synovial fluid cytology in patients with RA. The presence of HLA-DR4 together with DQB1*0301 leads to an increase in ragocyte cells in the synovial fluid and a reduced lymphocyte and monocyte count. We speculate that the increase in activity of joint disease, and hence severity of the disease, might be due to the presence of excess ragocytes in the synovial fluid or to the loss of dampening effect owing to the reduced number of lymphocytes/monocytes. Ragocytes are leucocytes with intracytoplasmic granules and their function is unknown.7 In contrast, there was an increase in the percentage of lymphocytes in the synovial fluid of patients who were positive for the rheumatoid epitope. Once again the type of lymphocytes was unknown, but we can speculate that the presence of HLA-DR1 must have been the influencing allele because there were no significant differences in the cell counts when patients who were positive or negative for HLA-DR4 were compared.
The patients in this study were a selected group as they were hospital attenders with joint effusions requiring aspirations and thus had more severe disease than patients without joint effusions who could be managed as outpatients. In this selected group we found no association between synovial fluid cytology and the presence or absence of rheumatoid factor or extra-articular features, erosive or non-erosive disease, and the treatment with DMARDs and/or steroids. This is perhaps expected as patients with negative rheumatoid factor can develop severe joint disease. Although the presence of extra-articular features contributes to the overall morbidity of RA, it is not always associated with severe articular disease. Finally, our treatment of patients with DMARDs with and without steroids does not depend on the severity of articular disease alone and is based on the overall activity of the rheumatoid disease. On the other hand, treatment with DMARDs and steroids may modify synovial fluid findings, but we cannot draw any conclusions from this study as it is a cross sectional study and previous treatment was not taken into account.
Thus we have shown that synovial fluid cytology is influenced by genetic factors, but larger studies are needed to confirm these results. Further studies at the molecular level will help to determine the exact mechanism of action by which the presence or absence of certain cell types has an effect on the joint pathology of patients with RA. These studies may throw light on the pathogenesis of the joint disease in these patients.
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