Immunoglobulin E (IgE) Levels in Serum and Synovial Fluid in Rheumatoid Arthritis

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1 Immunoglobulin E (IgE) Levels in Serum and Synovial Fluid in Rheumatoid Arthritis Gene G. Hunder and Gerald J. Gleich Serum and synovial fluid levels of immunoglobulin E (IgE) were significantly increased in patients with rheumatoid arthritis (RA) but were normal in degenerative arthritis (DJD) and in samples taken at autopsy in cases without joint disease. No significant correlation was found between serum IgE concentration and severity of RA nor was evidence found of the local production of IgE in synovial fluid in RA. The increased IgE in RA may be the result of a general immune response seen in this disease. Immunologic processes involving immunoglobulins may be important in the pathogenesis of rheumatoid arthritis (RA) (1). Demonstrated alterations have been mainly in the IgG and IgM classes. These two immunoglobulins are synthesized by the rheumatoid synovium (24), and the immune complexes found in synovial fluid and serum from patients with RA contain IgG and IgkI molecules (5-7). However, little consideration has been given to the role of IgE antibodies in this disease. IgE antibod- From the Mayo Clinic and Mayo Foundation, Rochester, Minnesota. This investigation was supported in part by a grant from the Minnesota Chapter of The Arthritis Foundation. GENE G HUNDER, MD, MS in Medicine, FACP: Consultant, Division of Rheumatology and Internal Medicine, Mayo Clinic and Mayo Foundation; Associate Professor of Medicine, Mayo Medical School, Rochester, Minnesota; GERALD J GLEICH, MD, FACP: Consultant, Department of Microbiology and the Division of Allergic Diseases and Internal Medicine, Allergic Diseases Research Laboratory, Mayo Clinic and Mayo Foundation; Associate Professor of Medicine and Microbiology, Mayo Medical School, Rochester, Minnesota. Address reprint requests to Dr Hunder. Submitted for publication December 18, 1973; accepted May 13, ies fix to mast cells and, after reaction with antigen, trigger the release of histamine and thereby increase vascular permeability (8,9). IgE antibodies are involved in the pathogenesis of atopic diseases (8-ll), and serum concentrations of IgE tend to be increased in patients with an atopic illness (1,12,13). To determine whether abnormalities of IgE might be associated with RA, we measured this immunoglobulin in serum and synovial fluid specimens from patients with RA and compared the values to those obtained on a group of patients with degenerative joint disease (DJD) and on specimens taken at autopsy. Concentrations of IgE in serum and synovial fluid were significantly higher in patients with RA, but no evidence of IgE production by synovial tissue was found. MATERIALS AND METHODS Serum samples were obtained from 51 adults with definite or classic rheumatoid arthritis (14); synovial fluid samples were obtained from 3 of these patients. Paired synovial fluid and serum samples were obtained on the same day from 12 patients. In addition, serum samples were obtained from 15 patients and synovial fluid samples from 32 patients Arthritis and Rheumatism, Vol. 17, No. 6 (November-December 1974) 955

2 HUNDER AND GLEICH Table 1. Serum IgE Concentrations Degenerative Rheumatoid Joint Parameter Normal' Arthritis Disease No. of patients Mean (ng/ml) Range (ng/ml) 1-2, , No. and percent >54 ng/ml 5 (5%) 14 (27%) (%) Pt <.1 NS *From a previous study (13) $For difference from normal, by t test: NS = not significant. The t test was performed on logla transformed IgE levels. with DJD; paired serum and synovial fluid samples were obtained on the same day from 14 of these patients. To determine the validity of using the DJD specimens as a control for RA specimens, serum (heart blood) and synovial fluid (knee joint) samples were obtained at autopsy in 7 cases in which patients died of various diseases but did not have clinically symptomatic arthritis. Samples were rejected if there was evidence of contamination or hemolysis or if the patients were edematous or had been given blood transfusions within 24 hours before death. All living patients had reported no personal or family history of allergy. A complete history was taken on each patient and all had general physical and joint examinations; laboratory tests were performed as dictated by the patient's problems. In 36 patients with RA, leukocyte total and differential counts were obtained on the same day as, or within 1 or 2 days after blood was drawn for the IgE determination. The eosinophil count was calculated by multiplying the total leukocyte count by the percentage of eosinophils. IgE in serum and synovial fluids was measuwd by radioimmunoassay using rabbit antibody to IgE (ND) (13). Age and sex have not been found to influence IgE blood levels and were ignored in this study (13). Addition of two different purified IgM rheumatoid factors [kindly supplied by Drs HV I.uthra and FC McDuffie (15)] did not influence the xra Normal %AM I&, dtt Fig 1. Cumulative frequency distribution of IgE in normal subjects and patients with rheumatoid arthritis. results of IgE measurement in normal serum. Albumin, a,-macroglobulin, IgA, I@, and IgM were measured by radial immunodiffusion using specific antibodies to each protein (16). Total protein was measured by the biuret method (17). Serum and synovial fluids from 2 patients were analyzed by sucrose density gradient ultracentrifugation, as described previously (18). Fractions were analyzed for IgG, IgA, IgM, and albumin by radial immunodiffusion and for IgE by radioimmunoassay. For albumin, I@, IgA, and IgE, no difference was found between sedimentation rates in serum and synovial fluids. The concentration of IgM was too low to measure by radial immunodiffusion in density gradient fractions; however, results of Kushner and Somerville (4) indicate that in patients with RA, serum and synovial fluid IgM has the same diffusion coefficient and therefore can be quantitated by radial immunodiffusion. RESULTS Serum IgE concentrations in RA were significantly (P <.1) higher than normal (Table 1). In more than one-fourth of the patients with RA, concentrations of IgE were higher than the normal ninety-fifth percentile (Figure 1). No correlation was found between serum IgE concentration and eosinophil count (number per cubic millimeter) in the 36 patients with RA in whom data were available for comparison (Y =.16). There was no significant difference between patients with RA who had normal IgE levels and those with increased levels with regard to rheumatoid nodules, 956 Arthritis and Rheumatism, Vol. 17, No. 6 (November-December 1974)

3 ~ SERUM AND SYNOVIAL FLUID IgE IN RA Table 2. Synovial Fluid IgE Concentrations Degenerative Rheumatoid Joint Parameter Arthritis Disease No. of patients 3 32 Mean (ng/ml) 167' 42 Range (ng/ml) 1-1, 'For difference from DJD, P <.2 by t test. The t test was performed on loglo transformed IgE levels. ARA functional class (l), ARA anatomic stage (1). extraarticular manifestations of RA, or the presence of rheumatoid factor in serum. Of 14 patients with increased serum JgE, only 2 had received gold therapy. One of these, the patient with the highest serum concentration (1 1,546 ng/ml), had stopped receiving gold injections 5 months previously because he had developed leukopenia. No other possible reaction to gold was noted, and after our examination we thought the leukopenia was related to Felty's syndrome. This patient also had other marked immunologic abnormalities and was included as case 3 in another report (19). Serum IgE concentrations in patients with DJD were somewhat lower than those in normal subjects but not significantly so. Synovial fluid IgE concentrations were significantly (P <.2) higher in RA than in DJD (Table 2). In neither group was synovial fluid IgE greater than serum IgE. Measurements on paired specimens of serum and synovial fluid are shown in Table 3. Differences among results were tested by analysis of variance and by Student's t test. In serum, aside from IgE, the only differences in concentrations of various proteins, among the groups, were albumin, which was significantly lower in RA than in DJD (P <.5) or in autopsy cases (P <.1), and IgM, which was lower in autopsy cases than in RA (P <.5). In synovial fluid, Table 3. Results of Analysis of Paired Synovial Fluid (SF) and Serum (S) Specimens Degenerative Joint Rheumatoid Arthritis Disease Autopsy Cases Normal (N = 12) (N = 14) (N = 7) Variable S SF S Total protein (mg/ml) (6-77) (29-61) (38-81) Albumin (mg/ml) (3-4) (8-23) (13-31) IgG (mg/ml) ( ) ( ) ( ) IgA (mg/ml) (1.-3.5) (.3-3.1) (15-52) lg E (ng/ml) (6-78) (4-1,573) (5-2,244) wnacroglobuiin (mg/ml) (153.6) ( ) ( ) IgM (mg/ml) (.8-2.1) (.4-2.7) (.8-2.8) Data shown as mean and range 'For difference from rheumatoid arthritis, P <.5 by t test SF S 32' 71 (26-51) (63-78) 16 31' (12-23) (22-37) 4.9* 1.4 ( ) ( ) (.3-2.9) (.8-4.8) 2, 91' (1-54) (6-299).5' 1.8 ( ) ( ).43' 1.43 (.6-1.1) (.4-5.5) SF 26' (9.3-51) 17 (5.-4) 2.9* (.5-4.1).43* ( ) 14* (3.7-46).34' ( ).4* (.1-1.O) S 8 (51-1 9) 37' (21-45) 14.6 (9.2-22) 2.8 ( ) 88 ( ) 2.4 (.9-4.).74' (.2-1.5) Arthritis and Rheumatism, Vol. 17, No. 6 (November-December 1974) 957

4 m L 4 (D I f p Table 4. Synovial Fluid/Serum Ratios for Various Proteins Rheumatoid Arthritis Degenerative Joint Disease Autopsy Cases Albumin.69 (.42-1.O) IgG.76 ( ) IgA.58 (.31-1.l) IgE.52 ( ) adnacroglobulin.44 ( ) IgM.65 ( ) Total protein.68 ( ) 1.11 ( ).87 ( ).74 ( ).67 (.42-.9).93 (.24-2.).53. ( ).46 ( ).44 (.1-1.5).19' (.8-.4).29* ( ).48' (.8-.7).54* ( ).88 ( ).87 (.3-2.4).36 ( ).55 ( ).77 ( ).47' ( ).2* (.5-.36).16' (.4-.5).22' (.3-.4).18' (.8-.4).7' (.2-.14).32* ( ).45 ( ).31 ( ).48 (.6-1.O).4' (.9-.64).19 (.8-.48) Data shown as mean and range 'For difference from rheumatoid arthritis, P <.5 by t test I c z B rn

5 SERUM AND SYNOVIAL FLUID IgE IN RA concentrations of proteins were significantly higher in the RA than in the DJD or postmortem group, with the exceptions of albumin in both DJD and autopsy cases, and of LgA in DJD. For most synovial fluid proteins, means were lower in autopsy cases than in DJD, except for albumin in which they were essentially the same. Because of the small number of autopsy cases, these differences were not statistically significant except for IgA (P <.5). In RA serum and synovial fluid protein concentrations were positively correlated as follows: IgE, IgM, IgA, and a,-macroglobulin, P <.1; IgG, P <.2; and albumin, P <.5. Among patients with DJD, the positive correlation between synovial fluid and serum levels was not as striking and was statistically significant for IgG and IgE (P <.1) only. In postmortem specimens, a positive correlation was found only for total protein (P <.5). To determine whether synovial fluid protein concentrations were greater than might be explained by diffusion alone, the ratio of concentration in synovial fluid to concentration in serum was examined for each protein. These synovial fluid/serum concentration ratios (SFJS,) and the ratio of these ratios to the ratio for albumin (SF,/S,: SFalb/Salb) are shown in Table 4. The synovial membrane was able to discriminate between small and large proteins regarded as inert in this system, albumin and a,- macroglobulin. By the rank sum test, the SF/S ratio for a,-macroglobulin was significantly (P <.1) less than the SF/S ratio for albumin in all three groups. The SF/S ratios for these proteins were greater (P <.1) in RA than in DJD. SF/S ratios for total protein, IgG, and IgE (P <.1) and IgM (P =.1) also were greater in RA than in DJD. Although the SF/S ratios were lower in postmortem specimens than in DJD (ex- cept for IgE), the differences were not significant except for total protein and IgG (P <.1). Thus, the synovial membrane is more permeable to these proteins in RA than it is in DJD and more permeable in DJD than in the autopsy cases. In Figures 2 and 3 the SFp/S,:SFa,b/Sal, ratios for the different proteins in individual patients with RA and DJD are plotted against their molecular weights (albumin, 66,; IgG, 15,; IgA 17,; IgE, 19,; a,-macroglobulin, 9,; and IgM, 1,,). The ratios for all proteins, except IgG in RA and LgE in DJD, are scattered relatively evenly on both sides of the line from albumin to a,-macroglobulin, which is the expected distribution when a protein enters the synovial cavity by passive diffusion alone. The distribution of IgG values in RA shows concentrations greater than expected by diffusion and suggests that the excess IgG results from local production by synovial tissues. In degenerative joint disease, IgE is present in lower amounts than would be expected by diffusion alone. In Table 4, the SFp/Sp:SFalb/Salb ratio for IgE was the only such ratio lower in DJD than in autopsy cases. The amount of any protein produced locally also can be estimated from these data (2). If one assumes that IgM and a,- macroglobulin (a-m) are filtered similarly, the amount of synovial fluid IgM arising from filtration is given by x (SF,.,/ Sa.m). The IgM locally produced may be calculated by subtracting this value from the observed concentration of IgM in synovial fluid. If the filtration of other immunoglobulins is regarded as halfway between that of albumin and that of a,-macroglobulin, the quantity of immunoglobulin derived from serum may be calculated by the following formula (2): Sp x 1/2 (sf,m/saib + SFa-m/Sa-m) Arthritis and Rheumatism, Vol. 17, No. 6 (November-December 1974) 959

6 HUNDER AND GLEICH?Fb Sp Salb 3* t o :: i I I 1 1 I I I I Fig 2. Rheumatoid arthritis. Ratio of synovial fluidlserum ratios (SFp/S,,:SFalb/&lb) for the various proteins plotted against molecular weight of the proteins. IgE concentration ratios are not greater than expected from passive diffusion alone. * ein x 15 M W pro ein x 15 Fig 3. Degenerative joint disease. Ratio of synovial fluid/serum ratios (SFp/Sp:SF.lb/S.lb) for the various proteins plotted against molecular weight of the proteins. IgE concentration ratios are less than expected from passive diffusion. These values subtracted from SF, provide estimates for locally produced immunoglobulin. Values for all immunoglobulins were calculated in this manner for RA and DJD. By the rank sum test, synovial fluid IgE concentrations were not greater than would be expected on the basis of diffusion from serum alone. In RA synovial fluid IgG concentration was significantly increased (P <.1). Although in most instances the Ighl values were greater than expected by diffusion alone, as shown in Figure 2, the differences were not statistically significant with the number of patients in this study. IgE values in DJD were all lower than what might be expected by diffusion into the synovial cavity (P <.2). Therefore it appears that in DJD, IgE may be relatively excluded from synovial fluid. 96 Arthritis and Rheumatism, Vol. 17, No. 6 (November-December 1974)

7 SERUM AND SYNOVIAL FLUID IgE IN RA DISCUSSION It is not rare for patients with early RA to have transient or episodic attacks of joint swelling and pain (21,ZZ). At times these attacks may resemble the angioneurotic edema seen in allergic or atopic diseases. In atopic disease, IgE antibodies appear to participate in pathogenesis by triggering the release of histamine and other mediators (8-13). The pathophysiologic mechanisms mediating the transient attacks of joint swelling in RA are unknown but could involve IgE antibodies. In addition, studies in experimental vasculitis have shown that the histamine released in response to antibody can influence deposition of immune complexes (23). Through such pathways, IgE antibodies could play a pathogenetic role in RA. especially in patients with complications such as vasculitis or pleurisy in which deposition of immune complexes may be important (7,19,24,25). Histamine has been identified in synovial tissues (26), and the serum concentration of histidine, a histamine precursor, has been found to be decreased in patients with RA (27). The present studies do not define the causes for the increased serum IgE concentrations found in patients with RA, but the increases are consistent with other reports suggesting the occurrence of chronic immunization in RA (19). Because serum IgE concentrations have been found to be increased in some diseases in which IgE is thought to be involved pathogenetically (1,12,13), it is possible that IgE is important in RA. As in atopic diseases, RA may be associated with increases in both IgE concentration and blood eosinophil count (28,29). In the present study, however, no simple relationship between the blood eosinophil count and the IgE concentration was found. IgE concentrations in synovial fluid also were increased in patients with RA when compared to patients with DJD, a condition in which immunologic mechanisms are not thought to be important pathogenetically, and to postmortem specimens, which were studied as an approximation of the normal joint. Because synovial fluid proteins are derived primarily from plasma by diffusion across the synovial membrane, the concentration of a specific protein in syno vial fluid depends on its concentration in serum, on its molecular weight, and on the presence or absence of synovial inflammation (4,ZO). Increasing inflammation of the synovial membrane is accompanied by increasing passage of each protein across the synovial membrane with a relatively greater increase in permeability to large molecules. This was illustrated in the present experiments which showed that synovial fluid protein concentrations were related to the serum protein concentrations in most instances and the SF,/S, ratios were highest in RA, intermediate in DJD, and lowest in the autopsy cases. Thus, in DJD the synovial fluid had the characteristics of a mild inflammatory process, as noted previously by Kushner and Somerville (4) and also reflected by the occasional finding of mild synovitis on histologic examination in such patients (1). These results also serve as a reminder that synovial fluid from patients with DJD cannot be considered normal. Our observation that, in RA, IgG (and possibly IgM) was present in synovial fluids in significantly higher concentrations than could be expected solely from diffusion from plasma is consistent with previous reports (24) of synthesis of IgG and IgM in synovial tissues of patients with classic RA. Synovial fluid IgE concentrations were not increased in RA by this method of analysis. In DJD, the low SF/S ratios for Arthritis and Rheumatism, Vol. 17, No. 6 (November-December 1974) 961

8 HUNDER AND GLEICH IgE indicated that this immunoglobulin was present in lower concentrations than expected from diffusion from plasma and was even lower than in the postmortem specimens. The present experiments did not elucidate the reason for this, but it is possible that IgE either is partly excluded from synovial fluids or is destroyed in the joint space in DJD. The increases in SFI,,/SI,,:SF,,,/Saib ratios for IgE in RA compared with those in DJD were greater than the increases for any other protein. ACKNOWLEDGMENTS We thank Ms. A. K. Averheck for capable technical assistance, Dr. Lila R. Elvehack for help with the statistical analysis, and Dr. Keith E. Holley for obtaining the postmortem specimens. REFERENCES 1. Hollander JL, McCarty DJ, Jr: Arthritis and Allied Conditions: A Textbook of Rheumatology. Eighth edition. Philadelphia, Lea and Febiger, Smiley JD, Sachs C, Ziff M: In vitro synthesis of immunoglobulin by rheumatoid synovial membrane. J Clin Invest 47: , Sliwinski AJ, Zvaifler NJ: In vivo synthesis of IgG by rheumatoid synovium. J Lab Clin Med 76:34-31, Kushner.. I, Somerville J: Permeability of human synovial membrane to plasma proteins: relationship to molecular size and inflammation. Arthritis Rheum 14:56-57, Franklin EC, Holman HR, Muller-Eberhard HJ, et al: An unusual protein component of high molecular weight in the serum of certain patients with rheumatoid arthritis. J Exp Med 15: , Chodirker WB, Tomasi TB, Jr: Low-molecular-weight rheumatoid factor. J Clin Invest 42: , Winchester RJ, Agnello V, Kunkel HG: Gamma globulin complexes in synovial fluids of patients with rheumatoid arthritis: par- tial characterization and relationship to lowered complement levels. Clin Exp Immunol 6:68+76, Ishizaka K, Ishizaka T, Hornbrook MM: Allergen-binding activity of re, rg, and ra antibodies in sera from atopic patients: in vitro measurements of reaginic antibody. J Immunol 98:49-51, Ishizaka T, Ishizaka K, Orange RP, et al: The capacity of human immunoglobulin E to mediate the release of histamine and slow reacting substance of anaphylaxis (SRS-A) from monkey lung. J Immunol 14: , Berg T, Johansson SGO: IgE concentrations in children with atopic diseases: a clinical study. Int Arch Allergy Appl Immunol 36: 21F232, 1969 I. Stanworth DR, Humphrey JH, Bennich H, et al: Specific inhibition of the Prausnitz- Kustner reaction by an atypical human myeloma protein. Lancet 2: Heiner DC, Rose B: Elevated levels of YE (IgE) in conditions other than classical allergy. J Allergy Clin Immunol 45:3-42, Gleich GJ, Averbeck AK, Swedlund HA: Measurement of IgE in normal and allergic serum by radioimmunoassay. J Lab Clin Med 77:69-698, Committee of the American Rheumatism Association: 1958 Revision of diagnostic criteria for rheumatoid arthritis. Arthritis Rheum 2:16-2, Randilla KK, McDuffie FC: Reactivity of rheumatoid factor with autologous IgG antibodies. Arthritis Rheum 12:74-81, Mancini G, Carbonara AO, Heremans JF: Immunochemical quantitation of antigens by single radial immunodiffusion. Int J Immunochem 2: , Kabat EA, Mayer MM: Experimental Immunochemistry. Second edition. Springfield, Illinois, Charles C Thomas, Publisher, Bush ST, Swedlund HA, Gleich GJ: Low molecular weight IgM in human sera. J Lab Clin Med 73:194-21, Hunder GG, McDuffie FC: Hypocomple- 962 Arthritis and Rheumatism, Vol. 17, No. 6 (November-December 1974)

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