Ann Rheum Dis: first published as 10.1136/ard.22.1.1 on 1 January 1963. Downloaded from Ann. rheum. Dis. (1963), 22, 1. AN IMMUNOFLUORESCENCE STUDY OF RHEUMATOID FACTOR* BY J. N. McCORMICK Oxford Regional Rheumatic Diseases Research Centre, Stoke Mandeville Hospital, Aylesbury, Bucks. The rheumatoid factor group of macroglobulins globulin apparently located rheumatoid factor at is a characteristic concomitant of rheumatoid sites in perivascular collagenous connective tissue in arthritis and can be detected readily in the serum rheumatoid synovium (Kaplan and Vaughan, 1959). of most rheumatoid patients by a variety of sero- More recently, Hess and Ziff (1961) have used logical techniques employing as reactant either fluorescent aggregated human gamma globulin to human gamma globulin (Heller, Jacobson, Kolodny, detect rheumatoid factor on leucocytes and platelets and Kammerer, 1954; Singer and Plotz, 1956; from patients with juvenile and adult rheumatoid Epstein, Johnson, and Ragan, 1956) or erythrocytes arthritis. sensitized with rabbit antibody (Waaler, 1940; The present investigation has been concerned Rose, Ragan, Pearce, and Lipman, 1948). Various mainly with the distribution of rheumatoid factor copyright. workers applying the immunofluorescence technique in lymph nodes and synovium and, although similar of Coons and Kaplan (1950) have studied the techniques were employed, the results obtained are distribution of rheumatoid factor in the tissues of not entirely in agreement with those of Mellors and rheumatoid patients (Kaplan and Vaughan, 1959; his colleagues. In addition, further observations Mellors, Heimer, Corcos, and Korngold, 1959; have been made which may allow the elaboration Kaplan, Suchy, and Meyeserian, 1960; Mellors, of an hypothesis concerning the nature and bio- Nowoslawski, Korngold, and Sengson, 1961b; logical function of rheumatoid factor. http://ard.bmj.com/ Mellors, Nowoslawski, and Korngold, 1961a). Employing aggregated human gamma globulin and Methods and Materials rabbit immune complexes labelled with fluorescent Preparation of Conjugates tracers and known to react with rheumatoid factor In earlier experiments, the protein reagents were in serological studies (Edelman, Kunkel, and conjugated with fluorescein isocyanate by the method of Franklin, 1958; Christian, 1958), Mellors and his Coons and Kaplan (1950), but at a later stage, con- associates located rheumatoid factor reactive with jugation with fluorescein isothiocyanate, as described by both types of reagent in plasma cells in synovium Riggs, Seiwald, Burckhalter, Downs, and Metcalf (1958), on September 27, 2021 by guest. Protected and lymph nodes as well as in the intrinsic cells of was used routinely. Conjugation with lissamine rhod- germinal centres in lymphoid follicles. From the amine B 200 was carried out by modification of the results of their mixed staining experiments with technique of Chadwick, McEntegart, and Nairn (1958). con- In each case, a protein concentration of 10 mg./ml. was contrastingly labelled reagents (1961b), they employed. Free dye was removed by absorption on a cluded that there were at least two types of cellular column of Dowex ion-exchange resin (1 x 10, 200-400 rheumatoid factor which occur separately or simul- mesh) equilibrated in 0O 1 M phosphate buffered isotonic taneously in individual plasma cells, but that only saline (pH-7-2) followed by dialysis against the same one or the other was present in germinal centres. buffer for 24 hours. All conjugates were absorbed twice Kaplan and his colleagues reported that rabbit with acetone-dried guinea-pig liver powder and three gamma globulin as well as aggregated human times with washed human AB cells, and in addition all gamma globulin reacted with plasma cells and except the anti-human globulin conjugates were absorbed germinal centre cells in rheumatoid lymph nodes with lyophilized whole human spleen. (Kaplan and others, 1960) and that rabbit gamma Fluorescent Aggregated Human Gamma Globulin * Based on a paper read at a meeting of the Heberden Society The human gamma globulin (HGG) used routinely on September 28, 1962. was obtained from a pool prepared by the method of Ann Rheum Dis: first published as 10.1136/ard.22.1.1 on 1 January 1963. Downloaded from 2 ANNALS OF THE RHEUMATIC DISEASES Kekwick and Mackay (1954). A small quantity of human Fluorescent Anti-human Globulin Antisera Cohn fraction 11 was available for comparison with (I) A potent anti-human globulin antiserum pool was this preparation and was found to give similar results. obtained from rabbits immunized with alum-precipitated The absorbed conjugates were heated in a water bath at human globulin. 63- C. for 5-10 minutes to aggregate the HGG and were centrifuged in the cold for 30 minutes at 16,000 r.p.m. (2) An anti-human gamma globulin (7S) antiserum before use. Sodium sulphate precipitation as used by was raised in rabbits against a pure preparation of 7S Mellors and others (1959) was not employed routinely as gamma globulin obtained by fractionation on DEAE the redissolved precipitates tended to be unstable. cellulose. Immunoelectrophoresis showed no evidence Similarly, the aggregates were unstable if the protein of other antibodies. concentration exceeded 10 mg./mi. or if the heating was (3) An anti-19S gamma globulin antiserum was prolonged. The reactivity of the heated HGG con- obtained from a rabbit injected with cryoglobulin from jugates with rheumatoid factor was assessed by their a patient with macroglobulinaemia and was absorbed ability in high dilution (e.g. 1/1,000) to inhibit the with pure 7S gamma globulin and the serum of a patient agglutination of sensitized sheep cells by two agglu- with hypogammaglobulinaemia. This antiserum gave a tinating doses of a standard rheumatoid serum. Heat reaction of identity with an antiserum raised against aggregation of the labelled HGG was not essential for normal 19S gamma globulin and was regarded as specific the subsequent staining reactions, however, as even the for this class of globulin. unheated conjugates produced convincing although less The labelled anti-human globulin antiserum (1) was intense staining. Presumably this was due to some used routinely for general assessment of the globulin denaturation of the HGG during conjugation and content of tissues, while the more immunospecific anti-7S dialysis, etc. and anti-19S gamma globulin antisera were reserved for detailed examination of selected tissues. Fluorescent Rabbit Globulin Reagents Tissues Examined copyright. In preliminary experiments where the distribution of (1) Rheumatoid Biopsy Material. Fourteen lymph globulin in rheumatoid tissues was being studied, fluores- nodes (3 epitrochlear, 10 axillary, I cervical) from nine cent rabbit anti-human globulin antiserum exhaustively patients with sero-positive rheumatoid arthritis; three absorbed with normal human serum was used as a control subcutaneous nodules; two specimens of muscle; of specificity. Although this absorbed conjugate did not eighteen specimens of synovium obtained at arthrotomy react with normal human tissues, it was found that some from sixteen rheumatoid patients, one of whom was sero- staining persisted in rheumatoid tissues. When rheuma- negative although the diagnosis of rheumatoid arthritis toid serum was used to absorb the the residual antiserum, was unequivocal. http://ard.bmj.com/ staining was reduced but not abolished. It was thought that traces of soluble antigen-antibody complexes in the (2) Rheumatoid Post-mortenm Material. 41 lynph absorbed antiserum might be responsible for the reaction nodes obtained from two subjects with sero-positive with rheumatoid tissues but, as fluorescent normal rabbit arthritis. globulin produced similar staining, it appeared that (3) Control Material. Eighteen assorted lymph nodes aggregation of the rabbit globulin in an immune complex from five normal subjects, two mesenteric nodes from a was not essential for this reaction. case of rheumatic carditis, and two hilar nodes from In order to compare our findings with those of Mellors two subjects with bronchial carcinoma were obtained at on September 27, 2021 by guest. Protected and others (1961a), however, soluble complexes of rabbit autopsy; ten specimens of normal synovium from ten anti-bovine albumin and labelled bovine albumin (BSA) meniscectomies; two specimens from osteo-arthritic were prepared according to their technique. In all, knees and one from the knee of a patient with psoriatic four types of rabbit globulin reagent were used: arthritis were obtained at exploratory arthrotomy. Small blocks of tissue were quick-frozen at 70- C. (l) Labelled rabbit anti-human globulin antiserum in a mixture of dry ice and acetone. The specimens absorbed with normal human serum; of synovium were suspended in 5 per cent. buffered normal rabbit gelatin during freezing, which facilitated subsequent (2) Labelled pooled globulin; sectioning of the blocks. All blocks were stored in the (3) Rabbit anti-BSA/labelled BSA complexes; deep-freeze at -20- C. until required. In addition, portions of each biopsy specimen and a selection of the (4) Labelled rabbit anti-BSA globulin, absorbed autopsy material were fixed in formalin and embedded in with normal human serum to remove cross- paraffin for routine histological staining with haematoxy- reacting antibodies (presumably traces of soluble lin and eosin and with methyl green-pyronin. Where antigen-antibody complexes may have been indicated, sections from the frozen blocks were also present).