RHEUMATOID FACTORS FROM PATIENTS WITH RHEUMATOID ARTHRITIS REACT WITH f2-MICROGLOBULIN* RALPH C. WILLIAMS, JR.**t and (by invitation) CHRISTINE C. MALONE and NAOYUKI TSUCHIYA GAINESVILLE, FLORIDA
INTRODUCTION Rheumatoid factors (RF), found in the serum of most patients with rheumatoid arthritis (RA), probably represent the first human autoantibody ever described (1, 2). More than 50 years ago Professor Waaler working in his Bergen laboratory in Western Norway discovered that serum samples from patients with RA contained an agglutinating principle or antibody which caused marked clumping of particles already sensitized with either human or some other species' IgG antibody (1). Some eight years later this same phenomenon was rediscovered or described again by Drs. Rose, Ragan and others in the rheumatology group at Columbia University College of Physicians and Surgeons (2). The next four decades of clinical observations and immunochemical or serologic studies in this field of rheumatology were filled with literally thousands of scientific papers and astute clinical observations bearing on the possible role, mechanisms of production and pathophysiologic significance of RF during on-going severe rheumatoid disease (3-9). It was found, for instance, that RF occurred in virtually all Ig classes (IgG, IgA and IgM). Moreover, these antibodies appeared to behave as if they were actually autoantibodies capable of reacting with the patient's own (autologous) IgG. Thus during the initial investigations of these curious factors, Drs. Franklin, Kunkel and their colleagues demonstrated that IgM RF were capable of associating and binding to 7S IgG in patients' plasma or serum to form 22S complexes easily visualized in the ultracentrifuge (Figure 1A). Moreover, some patients with RA showed markedly elevated levels of IgG RF in their sera and these RF were also capable of * This work was supported in part by Grants R01AR40438-01 and R55AR13824-21A2 from the National Institutes of Health and in part by the Marcia Whitney Schott endowment to the University of Florida for rheumatic disease research. ** Department of Medicine, Division of Rheumatology and Clinical Immunology, University of Florida, Gainesville. t Address to which requests for reprints should be sent: Ralph C. Williams, Jr., M.D., Department of Medicine, Division of Rheumatology, University of Florida, PO Box 100221, Gainesville, FL 32610. 198
RHEUMATOID FACTORS REACT WITH f2M
199
FIG. 1(A). Ultracentrifuge pattern of a serum from patient with active rheumatoid arthritis showing the 19S peak of IgM rheumatoid factor followed by the 22S complex of 19S rheumatoid factor (arrow) complexed with autologous 7S IgG. Direction of sedimentation is from left to right.
reacting with each other or with other non-RF IgG molecules to form socalled intermediate complexes sedimenting as 11S peaks within the analytic ultracentrifuge (Figure 1B). Besides these striking physical demonstrations that RF from patients with RA behaved like autoantibodies to self IgG, a vast number of studies supported the idea that these strange apparent autoantibodies to autologous IgG were reacting with specific regions on IgG localized mainly within the Fc or non-antibody combining site portion of the molecule. Moreover, RF from patients with RA often showed a curious sort of heteroclitic specificity in which they appeared to react best with IgG molecules containing specific allotypic markers of IgG not present within the RA patient himself (9). Thus many such RF from patients who were Gm(a-), (g-) IgG phenotype showed marked preferential reactivity for IgG molecules which were Gm(a+), (g+). If this were the case, how then could RF be considered an autoantibody? Moreover, a number of studies indicated that RF in sera of patients with RA showed relatively low association constants-in the
200
RALPH C. WILLIAMS, JR
FIG. 1(B). Ultracentrifuge pattern showing two dilutions of serum from a patient with rheumatoid arthritis having large amounts of serum IgG rheumatoid factor. This pattern shows a huge peak sedimenting as 11S representing dimers of 7S IgG RF interacting with itself or other IgG molecules.
order of 10- liters/mol instead of 10-7 or 10-8 liters/mol which would be expected of other IgM antibodies against different antigens such as pneumococcal polysaccharide or other foreign proteins. Could it be that for all these years we have been barking up the wrong tree and that although one can repeatedly see 22S complexes in the ultracentrifuge or even demonstrate activation of the complement cascade by RF (10, 11), that actually these antibodies are directed at something else within the body (not IgG) which has stimulated RF production? The present report provides some preliminary evidence that presently the latter possibility cannot be ignored and now needs to be re-explored further since such a strategy could possibly eventually lead to a major breakthrough in understanding the pathogenesis of the underlying disease process. Work reported here indicates that RF from patients with rheumatoid arthritis react with human f2-microglobulin (f2m), the light chain component of Class I HLA molecules. Since both O2m and IgG (the putative
RHEUMATOID FACTORS REACT WITH 32M
201
major antigen reacting with RF) are members of what is now regarded as the immunoglobulin supergene family (12, 13) and since in this report we describe reactions between RF and 22m, it could also be possible that the original antigenic stimulus for RF production is neither IgG nor f2m but something else bearing strong primary sequence or shape homology to IgG and fl2m but which in turn is directly related to the etiology of RA.
MATERIALS AND METHODS RF were isolated from 18 sera of patients with definite RA (by American Rheumatism Association Criteria) using passage over monomeric IgG-Sepharose 4B affinity columns as previously described (9, 14). All these polyclonal IgM RF from RA patients were also examined for their anti-Gm specificity using both polyclonal and monoclonal human antiRh antibodies as previously described (9). A summary of the anti-Gm specificities as well as the relative degrees of reactivity of the RFs with isolated myeloma proteins of H-chain groups 1, 2, 3 and 4 to assess possible anti-Ga (anti-IgG-1, 2, and 4) specificity (14) is shown in Table 1. No results with monoclonal RF are reported here since our previous studies had indicated that unlike polyclonal RF from the sera of RA patients, monoclonal RF showed little or no positive reactivity with f2m
(15). f2m was obtained from Sigma Chemical Co., St. Louis, Missouri. In addition recombinant f2m was also generously provided by Dr. Kenneth C. Parker, Biological Resources Branch, NIH. Both the Sigma and recombinant f2m were re-purified by elution at pH 3.5 from affinity columns of goat anti-human f32m antibody as previously described (15). Anti-fl2m ELISA. Antibody to 32m was assayed by ELISA using j32m coated on microtiter plates (Dynatech Labs, Inc.) at 5 ,g/ml in carbonate buffer pH 9.6. After washing in phosphate buffered saline (PBS) containing 1% BSA and 0.05% Tween-20, non-specific sites were blocked with PBS containing 2% BSA for 1 hour. RF isolated by adsorption to and elution from IgG or f2m affinity columns were tested at 5 ,ug/ml. After application of RF to f2m-coated plates for 1.5 hours, plates were washed and reactions completed with peroxidase-conjugated F(ab')2 fragments of goat IgG anti-human Fc of IgM. Normal human IgM as well as monoclonal human IgM paraproteins without detectable RF activity were included as controls (15). Overlapping 7-mer pin ELISA. The entire 99 amino acid sequence of f2m was synthesized using the Cambridge Epitope Mapping Kit (Cambridge Research, Cambridge, MA) in overlapping 7-mers as previously described (15, 16, 17). The pin ELISA was employed to test reactivity of
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INTRODUCTION Rheumatoid factors (RF), found in the serum of most patients with rheumatoid arthritis (RA), probably represent the first human autoantibody ever described (1, 2). More than 50 years ago Professor Waaler working in his Bergen laboratory in Western Norway discovered that serum samples from patients with RA contained an agglutinating principle or antibody which caused marked clumping of particles already sensitized with either human or some other species' IgG antibody (1). Some eight years later this same phenomenon was rediscovered or described again by Drs. Rose, Ragan and others in the rheumatology group at Columbia University College of Physicians and Surgeons (2). The next four decades of clinical observations and immunochemical or serologic studies in this field of rheumatology were filled with literally thousands of scientific papers and astute clinical observations bearing on the possible role, mechanisms of production and pathophysiologic significance of RF during on-going severe rheumatoid disease (3-9). It was found, for instance, that RF occurred in virtually all Ig classes (IgG, IgA and IgM). Moreover, these antibodies appeared to behave as if they were actually autoantibodies capable of reacting with the patient's own (autologous) IgG. Thus during the initial investigations of these curious factors, Drs. Franklin, Kunkel and their colleagues demonstrated that IgM RF were capable of associating and binding to 7S IgG in patients' plasma or serum to form 22S complexes easily visualized in the ultracentrifuge (Figure 1A). Moreover, some patients with RA showed markedly elevated levels of IgG RF in their sera and these RF were also capable of * This work was supported in part by Grants R01AR40438-01 and R55AR13824-21A2 from the National Institutes of Health and in part by the Marcia Whitney Schott endowment to the University of Florida for rheumatic disease research. ** Department of Medicine, Division of Rheumatology and Clinical Immunology, University of Florida, Gainesville. t Address to which requests for reprints should be sent: Ralph C. Williams, Jr., M.D., Department of Medicine, Division of Rheumatology, University of Florida, PO Box 100221, Gainesville, FL 32610. 198
RHEUMATOID FACTORS REACT WITH f2M
199
FIG. 1(A). Ultracentrifuge pattern of a serum from patient with active rheumatoid arthritis showing the 19S peak of IgM rheumatoid factor followed by the 22S complex of 19S rheumatoid factor (arrow) complexed with autologous 7S IgG. Direction of sedimentation is from left to right.
reacting with each other or with other non-RF IgG molecules to form socalled intermediate complexes sedimenting as 11S peaks within the analytic ultracentrifuge (Figure 1B). Besides these striking physical demonstrations that RF from patients with RA behaved like autoantibodies to self IgG, a vast number of studies supported the idea that these strange apparent autoantibodies to autologous IgG were reacting with specific regions on IgG localized mainly within the Fc or non-antibody combining site portion of the molecule. Moreover, RF from patients with RA often showed a curious sort of heteroclitic specificity in which they appeared to react best with IgG molecules containing specific allotypic markers of IgG not present within the RA patient himself (9). Thus many such RF from patients who were Gm(a-), (g-) IgG phenotype showed marked preferential reactivity for IgG molecules which were Gm(a+), (g+). If this were the case, how then could RF be considered an autoantibody? Moreover, a number of studies indicated that RF in sera of patients with RA showed relatively low association constants-in the
200
RALPH C. WILLIAMS, JR
FIG. 1(B). Ultracentrifuge pattern showing two dilutions of serum from a patient with rheumatoid arthritis having large amounts of serum IgG rheumatoid factor. This pattern shows a huge peak sedimenting as 11S representing dimers of 7S IgG RF interacting with itself or other IgG molecules.
order of 10- liters/mol instead of 10-7 or 10-8 liters/mol which would be expected of other IgM antibodies against different antigens such as pneumococcal polysaccharide or other foreign proteins. Could it be that for all these years we have been barking up the wrong tree and that although one can repeatedly see 22S complexes in the ultracentrifuge or even demonstrate activation of the complement cascade by RF (10, 11), that actually these antibodies are directed at something else within the body (not IgG) which has stimulated RF production? The present report provides some preliminary evidence that presently the latter possibility cannot be ignored and now needs to be re-explored further since such a strategy could possibly eventually lead to a major breakthrough in understanding the pathogenesis of the underlying disease process. Work reported here indicates that RF from patients with rheumatoid arthritis react with human f2-microglobulin (f2m), the light chain component of Class I HLA molecules. Since both O2m and IgG (the putative
RHEUMATOID FACTORS REACT WITH 32M
201
major antigen reacting with RF) are members of what is now regarded as the immunoglobulin supergene family (12, 13) and since in this report we describe reactions between RF and 22m, it could also be possible that the original antigenic stimulus for RF production is neither IgG nor f2m but something else bearing strong primary sequence or shape homology to IgG and fl2m but which in turn is directly related to the etiology of RA.
MATERIALS AND METHODS RF were isolated from 18 sera of patients with definite RA (by American Rheumatism Association Criteria) using passage over monomeric IgG-Sepharose 4B affinity columns as previously described (9, 14). All these polyclonal IgM RF from RA patients were also examined for their anti-Gm specificity using both polyclonal and monoclonal human antiRh antibodies as previously described (9). A summary of the anti-Gm specificities as well as the relative degrees of reactivity of the RFs with isolated myeloma proteins of H-chain groups 1, 2, 3 and 4 to assess possible anti-Ga (anti-IgG-1, 2, and 4) specificity (14) is shown in Table 1. No results with monoclonal RF are reported here since our previous studies had indicated that unlike polyclonal RF from the sera of RA patients, monoclonal RF showed little or no positive reactivity with f2m
(15). f2m was obtained from Sigma Chemical Co., St. Louis, Missouri. In addition recombinant f2m was also generously provided by Dr. Kenneth C. Parker, Biological Resources Branch, NIH. Both the Sigma and recombinant f2m were re-purified by elution at pH 3.5 from affinity columns of goat anti-human f32m antibody as previously described (15). Anti-fl2m ELISA. Antibody to 32m was assayed by ELISA using j32m coated on microtiter plates (Dynatech Labs, Inc.) at 5 ,g/ml in carbonate buffer pH 9.6. After washing in phosphate buffered saline (PBS) containing 1% BSA and 0.05% Tween-20, non-specific sites were blocked with PBS containing 2% BSA for 1 hour. RF isolated by adsorption to and elution from IgG or f2m affinity columns were tested at 5 ,ug/ml. After application of RF to f2m-coated plates for 1.5 hours, plates were washed and reactions completed with peroxidase-conjugated F(ab')2 fragments of goat IgG anti-human Fc of IgM. Normal human IgM as well as monoclonal human IgM paraproteins without detectable RF activity were included as controls (15). Overlapping 7-mer pin ELISA. The entire 99 amino acid sequence of f2m was synthesized using the Cambridge Epitope Mapping Kit (Cambridge Research, Cambridge, MA) in overlapping 7-mers as previously described (15, 16, 17). The pin ELISA was employed to test reactivity of
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