Immune Complexes, Rheumatoid Factors, and Cellular Immunological Parameters in Patients with Giant Cell Arteritis
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Ann Rheum Dis: first published as 10.1136/ard.40.3.276 on 1 June 1981. Downloaded from Annals of the Rheumatic Diseases, 1981, 40, 276-280 Immune complexes, rheumatoid factors, and cellular immunological parameters in patients with giant cell arteritis BO-ERIC MALMVALL1, BENGT-AKE BENGTSSON2, LARS-AKE NILSSON3, AND LARS-MAGNUS BJURSTEN3 From the 'Department of Infectious diseases, East Hospital, the 2Departments of Medicine I and II, Sahlgren's Hospital, and the 3Department ofMedical Microbiology, University ofGdteborg, Sweden SUMMARY Circulating immune complexes were found in 2 of 15 patients with giant cell arteritis (GCA) by using a solid phase Clq enzyme linked immunoabsorbent assay (ELISA). The prevalence in controls was 5 %Y. Rheumatoid factor could be demonstrated in 2 out of 27 patients and in 11 % of the controls by using a similar ELISA technique. The prevalence of T cells in blood was similar in 25 patients with GCA and in controls. The blood lymphocyte blastogenic response to the mitogens, phytohaemagglutinin, concanavalin-A, and pokeweed mitogen did not differ in 25 untreated patients compared with controls. Stimulation of lymphocytes by arterial homogenates was copyright. tested in 8 patients. In no case could a significant stimulation be obtained. We conclude that immune complexes and rheumatoid factors are present in the same low frequency in GCA patients as in the normal population, and that the studied parameters ofcellular immunity appear to be normal. Several authors have suggested that immunological of T cells in peripheral blood and the reactivity of http://ard.bmj.com/ mechanisms may be involved in the pathogenesis of blood lymphocytes to different mitogens and to giant cell arteritis (GCA).1-4 Our group has found arterial wall homogenate. that serum levels ofIgG and total serum complement, as well as factors C3 and C4, are slightly elevated Materials and methods in this disease.5 Other workers have demonstrated immunoglobulin and anti-immunoglobulin (rheuma- PATIENTS toid factors) in the arterial wall,6 7 findings which The studies were performed on untreated GCA were interpreted as indicating either deposition of patients during the years 1975-8 at the Department on September 25, 2021 by guest. Protected immune complexes or the occurrence of antigen- of Internal Medicine III, Sahlgren's Hospital, and at antibody reactions within the vessel wall. the Department ofInfectious Diseases, East Hospital, Cell mediated immune mechanisms have also been Goteborg. A temporal artery biopsy was performed proposed as being involved in the pathogenesis of on all patients. If the biopsy showed mononuclear GCA. Hazleman et al.8 found that blood cell infiltration and destruction of the internal elastic lymphocytes from patients with GCA in vitro membrane, the patients were regarded as biopsy- showed a higher transformation response to artery positive. Biopsy-negative patients were included if antigen than lymphocytes from controls, a finding certain clinical criteria, defined earlier, were which has been questioned by others, however.9 10 fulfilled.11 The patients were classified by symptoms In this study the presence of circulating immune at diagnosis into 4 clinical groups: temporal arteriiis, complexes and rheumatoid factors (RF) was combined temporal arteritis and polymyalgia investigated by 2 enzyme linked inmumosorbent rheumatica, polymyalgia rheumatica, and general assays (ELISA). We have also studied the prevalence symptoms only.12 The mean age was 71*6 years (range 58-85 years). Not all the immunological tests Accepted for publication 2 July 1980 were available during the entire period of study, and Correspondence to Dr Bo-Eric Malmvall, Goteborgs Universitet, Infektionskliniken, Ostra sjukhuset, 416 85 the number of patients studied with the different tests Goteborg, Sweden. therefore varies. 276 Ann Rheum Dis: first published as 10.1136/ard.40.3.276 on 1 June 1981. Downloaded from Immune complexes, rheumatoidfactors, and cellular immunologicalparameters in patients 277 CONTROLS factor. Values above 7 % for IgG, 6 % for IgA, and People aged 70 were selected at random from a 7 % for IgM were considered as positive.15 gerontological and geriatric population study in G6teborg"s and used as controls. PREPARATION OF LYMPHOCYTES Lymphocytes were prepared from fresh heparinised IMMUNE COMPLEXES venous blood samples by centrifugation on Determination of immunecomplexes by ELISA was Metrizoate-Ficoll (Lymphoprep, Nyegaard) as performed as previously described.14 Polystyrene described by B6yum.16 After washing, the cells in the tubes were coated by incubation with a solution intermediary phase were suspended in minimal containing 50 ng purified Clq in 0-5 ml of 0-05 M essential medium (MEM) and used for lymphocyte phosphate buffered saline, pH 7*2, for 3 hours at stimulation tests with various mitogens and arterial 37°C. After rinsing away the excess of Clq, 0 5 ml of antigens (see below). the serum samples was added in 10-fold serial dilutions. Incubation was performed by gently MITOGENS shaking the tubes at 370C for 3 hours. After a further Phytohaemagglutinin (PHA) (Wellcome Ltd, rinsing rabbit antihuman Clq (kindly provided by Beckenham, Kent, England) was used in a final Dr Bengt Johansson, Lund, Sweden) was added to concentration of 1:100. Concanavalin-A (con-A) the tubes to cover unoccupied Clq antigenic (Miles-Yeda Ltd, Rehovot, Israel) was used in determinants. The tubes were incubated for another a final concentration of 75 ,1g/ml. Pokeweed mitogen 3 hours at 370C. After a further rinsing rabbit (PWM) (Grand Island Biological Co, Paisley, antisera against human IgG, IgA, and IgM Scotland) was used in a final concentration of 1:10. (Dakopatts AS, Copenhagen, Denmark), conjugated Arterial antigens were prepared from aortic tissues with alkaline phosphatase, were added. After obtained from a 20-year-old man who was killed in incubation on a roller drum for 16 hours at room an accident and from a 50-year-old woman who temperature and subsequent rinsing the remaining had died from a brain tumour. The tissues were copyright. enzyme activity was determined by adding para- frozen within 3 and 12 hours respectively after death nitrophenyl phosphate (Sigma Chemical Company, and stored at -700C. After thawing, the tissue was Saint Louis, Missouri, USA) 1 mg/ml in 1 M first cut into small pieces with scissors, dispersed in diethanolamine buffer, pH 9*8, containing 0-0011 an equal volume of PBS, then finely dispersed in an M MgC12. The enzymatic reaction was interrupted Omnimixer. The mixture was centrifuged at 23 500 g after 100 minutes by addition of 0-1 ml 3 M NaOH, (14 000 rpm) for 30 minutes. The clear supernatant and the light absorption at 405 nm was registered. was stored frozen in suitable portions at -200C http://ard.bmj.com/ Optical densities (OD) were recorded for 1:10 until used. Another piece of aortic tissue was treated dilutions of the tested serum samples and compared as described above, but after grinding in the with the OD obtained for a pool of positive sera.15 Omnimixer the tissue homogenate was subjected to Sera giving an OD higher than 25 % for IgG, 35 % for freeze-pressing in an X-press.'7 The freeze-pressed IgA, and 75% for IgM of the OD for the reference material was centrifuged at 23 050 g for 30 minutes pool of sera were regarded as positive. All dilutions and the clear supernatant frozen and stored at -200C. and rinses were done with 0 05 M phosphate buffered The sediment was suspended in an equal volume saline, pH 7-2, containing 0 5% Tween 20, except of MEM, and after division into suitable portions on September 25, 2021 by guest. Protected for the coating procedure, where Tween 20 was it was frozen and stored at -200C. All 3 antigen excluded. preparations were used undiluted and diluted 1:10, 1:100, and 1:1000 in lymphocyte stimulation tests. RHEUMATOID FACTORS Polystyrene tubes were coated by incubation with LYMPHOCYTE STIMULATION TESTS 10 ,ug bovine serum albumin (BSA) in 0 5 ml 0 15 M Lymphocyte stimulation was performed according phosphate buffered saline for 2 hours at room to the routine procedures of the immunological temperature. After rinsing, rabbit anti-BSA serum laboratory. Briefly, washed lymphocytes were diluted in 1/5000 in PBS-Tween was added. After suspended in Mishell-Dutton medium18 supple- further rinsing, the serum sample to be tested, mented with 10% pooled human AB serum, 2 mM diluted 1:100 or 1:1000, was added and the presence 1-glutamine, 100 U/ml penicillin, 100 ,tg/ml of bound human immunoglobulins was determined streptomycin, and 20 mM Hepes buffer, and adjusted as described above for the Clq immunosorbent test. to a concentration of 2 x 106 cells. Cultivations were The results were expressed as the OD at 405 nm performed in Linbro Tissue Culture grade microtitre for 1/100 dilution of the tested samples in per cent of plates at 370C in air containing 5 % CO2. 10 1ul of the the OD of a pool of sera containing rheumatoid mitogens or arterial antigen preparations were Ann Rheum Dis: first published as 10.1136/ard.40.3.276 on 1 June 1981. Downloaded from 278 Malmvall, Bengtsson, Nilsson, Bjursten added to 0-2 ml of the lymphocyte suspensions. radioactivity was determined by liquid scintillation Each mitogen and the different antiigen dilutions counting. The stimulation index is given as the ratio were tested in triplicate. 1 ,uCi 3H-thymidine between means for triplicates obtained in cultures (Radiochemical Centre, Amersham, Bucks) was with and without mitogens or antigens. added to each well on the third day (of culture for PHA and con-A and on the sixth day ffnrior 'PWMArwIvi1 enALnu T LYMPHOCYTES arterial antigens. After further incubat;ion overnight Owing to a change of routine laboratory procedures the cultures were harvested, and the incorporated during the course of this investigation T cells were quantitated by 2 different methods during different T cells phases of the study.