Circulating Immune Complexes and Monocyte Fc Function in Autoimmune Diseases

Ann Rheum Dis: first published as 10.1136/ard.38.1.79 on 1 February 1979. Downloaded from

Annals of the Rheumatic Diseases, 1979, 38, 79-83

Circulating immune complexes and monocyte Fc function in autoimmune diseases

MARIA KAVAI, KATALIN LUKACS, ILDIKO SONKOLY, KATALIN PALOCZI, AND GY. SZEGEDI From the Department ofPulmonary Diseases, Division of Medicine, University Medical School of Debrecen, Hungary

SUMMARY The phagocytosis of separated and adherent monocytes of patients with systemic lupus erythematosus is subnormal as compared to controls on the basis of latex and yeast uptake. The monocytes from the same patients react with antibody-coated sheep red blood cells in a significantly higher degree than normal monocytes. There is a correlation between the percentage of reactive monocytes and the serum immune complex content. After brief treatment ofpatients with levamisole the phagocytic function of monocytes is restored and at the same time the circulating immune complex content is decreased.

Patients with autoimmune connective tissue dis- CIRCULATING IMMUNE COMPLEXES orders display immunological hyperreactivity char- Immune complex assays were performed by a acterised by the formation of numerous autoanti- modification of the Onyewotu et al. (1974) technique. copyright. bodies and immune complexes (Johnson et al., The ingestion of labelled aggregated IgG by guinea- 1975; Mulli and Cruchaud, 1977; Winfield et al., pig peritoneal macrophages was inhibited in the 1977). In addition to the alteration of humoral presence of serum containing IC. 106 macrophages immunity there is an impairment of cell mediated in 0 5 ml medium 199 (Flow) modified with Hanks's immunity (CMI) (Utsinger, 1976). Recently Landry salts and HEPES buffer were incubated for 75 min (1977) has shown that the impaired function of at 37°C with 4 .*g 1251-aggregated human IgG and phagocytes rather than of lymphocytes may be with 10 ,ul of a 1:5 dilution of SLE or control serum responsible for alteration of CMI in systemic lupus (Kaivai et al., 1977). All tests were performed in http://ard.bmj.com/ erythematosus (SLE). triplicate in siliconised glass tubes. After incubation In this study we report impaired function of blood the cells were washed 3 times, and the radioactivity monocytes and the presence of circulating immune of pellets was counted in a Packard automatic complexes (CIC) in the same patients with SLE. counter. The IC content of the sera was expressed Alteration of monocyte function may be directly in inhibition %. related to serum immune complexes (Rabinovitch et al., 1975). SEPARATION OF MONOCYTES on September 29, 2021 by guest. Protected Human monocytes were obtained by the method of Patients and methods Weston et al. (1975). 15 ml of heparinised venous blood from patients and controls was sedimented in PATIENTS Ficoll-Uromiro gradients. After washing, the cells Twenty-nine patients with SLE were selected, 2 men were suspended in 1 ml of medium 199 and counted and 27 women. Twenty of the patients were not with a stardard haemocytometer. Trypan blue receiving treatment at the time of the test. The viability and neutral red phagocytosis were per- patients fulfilled the preliminaryARA criteria for SLE formed on the cells to determine the percentage of (Cohen et al., 1971). Nine patients had received leva- phagocytic cells and their viability. misole 150 mg daily, every other day for a week. Six- teen controls were selected from among volunteers. MONOCYTE PHAGOCYTOSIS A monolayer technique was used with the cells Accepted for publication 15 March 1978. adherent to a slide in chamber. After Correspondence to Dr Maria Kavai, Department of Pul- glass Boyden monary Diseases, University Medical School, 4004 Deb- washing, the monocytes (0 5-1.0 x 105 cells) were recen 4, Hungary. incubated with 25 u1l of a 02 Y% suspension of latex 79 Ann Rheum Dis: first published as 10.1136/ard.38.1.79 on 1 February 1979. Downloaded from

80 Kdvai, Lukacs, Sonkoly, Pd16czi, Szegedi particles (Dow, 0-481 ,t q) or with 25 V1 of a sus- analysed. In Fig. 1 it is shown that the phagocytosis pension of baker's yeast containing 5 x 106 parti- of monocytes is subnormal in patients with SLE as cles, together with 0 5 ml medium 199. Incubation compared with controls. was at 370C for 60 min in 5 % CO2 and 100% humidity with constant rocking. After washing, REACTIVE MONOCYTES the monolayers were stained with Turk's or Wright's In this experiment the separated and adherent stain, and cells containing ingested particles were monocytes from the same patients and controls were counted. The percentage of cells ingesting latex incubated with sensitised SRBC or with opsonised particles, and of cells ingesting or attaching yeast, yeast. Monocytes from the patients reacted with and also the number of yeast particles ingested per antibody-coated SRBC to a significantly greater cell (phagocytic index) was determined. extent than normal monocytes (Fig. 2).

MONOCYTE OPSONISATION Monolayers of adherent monocytes were incubated as above at 37°C for 60 min with 25 V.1 of 2 x 108 0 70- particles/ml yeast which had been treated with 0 1 P<0.01 P

-02copyright. antisera (Hyland) and examined by fluorescent microscopy. Latex Yeast Yeast MONOCYTE EA ROSETTE Fig. I In vitro phagocytic activity of monocytes from Monocyte Fc receptor activity was assayed by patients v and cotitrols. 3 adherence of sheep red blood cells (SRBC) coated with a subagglutinating dilution (1:128) of an IgG

fraction isolated on Sephadex G-200 from a rabbit http://ard.bmj.com/ anti-SRBC serum. The adherent monocytes were 2 2 incubated with 0-2 ml of 2% sensitised SRBC in 0* 3 ml medium 199 at 370C for 30 min. After wash- 100 - 2 0 ing, the percentage of cells attaching or ingesting 90 18 -v 3 or more SRBC was determined. a 80 16 'O 0 lb 70- 0 1 4- on September 29, 2021 by guest. Protected Results l2< L 14 60 = CIRCULATING IMMUNE COMPLEXES 3E4. The patients' sera inhibited the ingestion of labelled .10 x aggregated IgG by macrophages to a significantly L,_ 08 greater extent (48. 1 ± 22 - 8 % inhibition) than normal sera (7 8 ± 5 9% inhibition). The individual ,_, 30' 06 results are shown in Fig. 3. 20 0*4 PHAGOCYTOSIS OF MONOCYTES 10' 02 Separated mononuclear leucocyte suspensions de- rived from peripheral blood by Ficoll-Uromiro Sens. SRBC Opsonised yeast density gradient centrifugation contained 25-30% monocytes. Monolayers of these mononuclear cells Fig. 2 Percentages ofmlonocytes from patientsm were prepared. The non-adherent cells were re- and controls reactive with sensitised SRBC or moved, then adherent cells, 85-99 % monocytes, were opsonised yeast particles. Ann Rheum Dis: first published as 10.1136/ard.38.1.79 on 1 February 1979. Downloaded from

Circulating immune complexes and monocyte Fc function in autoimmune diseases 81 No significant difference between the 2 groups was found in their ability to attach or phagocytose 262 6 opsonised yeast. By immunofluorescence C3 could 2 4 be demonstrated on the surface of each opsonised yeast particle but IgM only on 28% of the opsonised P

u tD~~~~~~~~~ 170 -50 0 ~~~~600 ~~~~~~~~~~~~L 40-0 z5 *30- 0-6 80- 0 0 t 70- P<0 01 (Fig.patients'monocyteswit20h4). The reactivity of -' * 10- 0.2 Fig.30-3L60reatobewe1hecnag fmn-Fg hostecreain2eweh ecn selm IC conen o1tn sm ate SRBC ~~~~~~~8020T~~ ~ ~~ ooye Senswith inrese OpsonisedFcatiiyyeast 60 Fig. 5 Effects of levanyisole on patients' monocyte

-+ iptake of sensitised SRBC and opsonised yeast particles, copyright. 60- -i----- befj-8e' lZLEAIOLtreatment,dRAMETO.PTETafterttreatmentt. 10 20 30 40 50 60 70 80 90 100 0/ IC Fig. 3 Correlation between the percentage of mono- Fig. 3 shows the correlation between the percen- cytes attaching or ingesting sensitised SRBC and the tage of monocytes ingesting or attaching sensitised serum IC content of the same patient. SRBC and the IC content of the same patients' sera.

Patients having high circulating IC in most cases had http://ard.bmj.com/ 100 monocytes with increased Fc activity. 90 ~~~~~~~~LEVAMISOLE TREATMENT OF PATIENTS Fig.4 EDectsofFollowing levamisole treatment for 1 week phago- U) 80 cytosis of latex and yeast increased to normal levels LI P<0.01 The of with 70 (Fig. 4). reactivity patients' monocytes for y opsonised yeast particles increased as well, but on the S. basis of reactivity with sensitised SRBG monocyte on September 29, 2021 by guest. Protected 60 P<001 ~~~~p<0*01 Fc activity was unchanged (Fig. 5). L5!YJ ~~~T-u1-0 In parallel with increased phagocytosis we obser- ON4 8 ved decreased serum IC content except in 1 case Ln 0~~~~~~~~~(Table 1). The Table shows that the monocyte FC G'30 4 -6.0 = receptor activity was altered together with GIG of C). patients in 7 cases out of 9 after levamisole treat- 10- Ftd 0x Discussion LatexYeast Yeast ~~~Adequate phagocytic capacity is essential for develop- Fig. 4 Effects of levamnisole on phagocytic activity ment of delayed hypersensitivity reactions. The for latex and yeast of moniocytes fromn patients.~Freduced cutaneous response of patients with SLE before treatment, = after treatment. may result from the reduced phagocytic ability of the Ann Rheum Dis: first published as 10.1136/ard.38.1.79 on 1 February 1979. Downloaded from

82 Kdvai, Lukacs, Sonkoly, Pl16czi, Szegedi Table 1 Effects of levamisole on monocyte IgG particles. The IgG; was only occasionally on a few receptor activity and on circulating immune complexes particles. This was evidently too low a level of coat- M-EA %, CIC Y. ing to reveal the increased reactivity demonstrated with sensitised SRBC. Patients Before After Before After treatment treatment treatment treatment Schmidt and Douglas (1976) have reported that levamisole increases in vitro phagocytosis of bacteria 1 58-9 71.0 0 0 2 35-2 65-2 0 72 by mononuclear phagocytes. After brief treatment 3 43-3 42-0 23 16 of the patients in the present study with levamisole 4 50.6 51-0 63 40 5 75-0 32-3 67 22 the phagocytic function of their monocytes was 6 56*3 24-1 65 51 restored, as reported by Symoeus (1976), and in 7 55-9 32-7 40 25 parallel the CIC content decreased. After levamisole 8 82-5 13.4 33 12 9 72-3 41.3 38 24 treatment the increased reactivity of the monocytes was demonstrable even with opsonised yeast particles. M-EA %==Y monocytes with erythrocyte-antibody. CIC %=circulating immune complex Y. The authors are grateful to Dr E. J. Holborow for his helpful discussion and kind revision of the manuscript. polymorphonuclear leucocytes and monocyte- macrophages. Impaired phagocytic ability of polymorphonuclear leucocytes in SLE has been reported References (Besana et al., 1975; Zurier, 1976), and the phagocytic function of monocytes in SLE has also been Besana, C., Lazzarin, A., Capsoni, F., Caradda, F., and Maroni, M. (1975). Phagocyte function in systemic lupus questioned (Wenger and Bole, 1973; Landry, 1977). erythematosus. Lancet, 2, 918-918. The present studies demonstrate impaired phago- Douglas, S. D., and Daughaday, C. C. (1976). Kinetics of cytosis of latex particles and baker's yeast by mono- monocyte receptor activity for immunoproteins in patients cytes in 29 patients with SLE. The CIC content of the with sarcoidosis. Annals of the New York Academy of

Sciences, 278, 190-200. copyright. same patients was significantly higher than that of Haakenstad, A. O., and Mannik, M. (1974). Saturation ofthe controls. The data of Haakenstad and Mannik reticuloendothelial system with soluble immune complexes. (1974) support our findings. In vivo administration Journal of Immunology, 112, 1939-1948. of IC to the subject results in a subsequent decrease Huber, H., and Fudenberg, H. H. (1968). Receptor sites of human monocytes for IgG. International Archives of in the clearance rate of particulate matter from Allergy and Applied Immunology, 34, 18-31. circulation. Cohen, A. S., Reynolds, W. E., Franklin, E. C., Kulka, J. P., We have also shown, however, that monocytes Ropes, M. W., Shulman, L. E., and Wallace, S. L. (1971). from with SLE react with Preliminary criteria for the classification of systemic lupus patients antibody-coated http://ard.bmj.com/ erythematosus. Bullefin on Rheumatic Diseases, 21, 643- SRBC to a significantly greater extent than normal 648. monocytes, indicating increased monocyte IgG Johnson, P. M., Watkins, J., and Holborow, E. J. (1975). receptor activity. These data of normal monocytes Antiglobulin production to altered IgG in rheumatoid are well supported by the same experiments of arthritis. Lancet, 1, 611-614. reason this Fc Kavai, M., Dank6, K., Kalmar, E., Francia, I., and Szegedi, Reikvam (1977). The for receptor Gy. (1977). Radioassay of soluble immune complexes activation in SLE is unknown. using their uptake by macrophage Fc receptors. Immuno- Increased activity of monocytes to bind antibody- logy, 32, 617-621. coated SRBC was reported in patients with malig- Landry, M. (1977). Phagocyte function and cell-mediated on September 29, 2021 by guest. Protected immunity in systemic lupus erythematosus. Archives of nant lymphoma (LoBuglio, 1970; Saragome et al., Dermatology, 113, 147-154. 1977) and in sarcoidosis (Douglas and Daughaday, LoBuglio, A. (1970). Effect of neoplasia on human macro- 1976). phage membrane activity. Journal ofLaboratory and Clini- As the Fc receptor has a specially important cal Medicine, 76, 888. Mannik, M., Haakenstad, A. O., and Arend, W. P. (1974). role in the ingestion of IgG antibody-antigen The fate and detection of circulating immune complexes. complexes (Mannik et al., 1974), the permanent Progress in Immunology, II, 5, p. 91. North-Holland presence of IC may elicit an increased net synthesis Publishing Co.: Amsterdam. of membrane which contributes to activation of Mulli, J. C., and Cruchaud, A. (1977). Immunoreactivity to for differences nuclear antigens in systemic lupus erythematosus, with or Fc receptor sites. This may account without nephritis, and in other connective tissue diseases, in the immune and non-immune interaction of the with particular reference to the RNA-protein antigen. monocyte-macrophage system with particles (Weiner International Archives of Allergy and Applied Immunology, and Bandieri, 1977; Walker, 1974). 53, 279-289. Onyewotu, I. I., Holborow, E. J., and Johnson, G. D. (1974). In SLE Fc receptor activity towards opsonised Detection and radioassay of soluble circulating immune yeast is not increased, as shown in Fig. 2. We could complexes using guinea pig peritoneal exudate cells. detect mainly C3 and IgM on the opsonised yeast Nature, 248, 156-159. Ann Rheum Dis: first published as 10.1136/ard.38.1.79 on 1 February 1979. Downloaded from

Circulating immune complexes and monocyte Fc function in autoimmune diseases 83 Rabinovitch, M., Manejias, R. E., and Nussenzweig, V. Wenger, M. E., and Bole, G. G. (1973). Nitroblue tetra- (1975). Selective phagocytic paralysis induced by immobil- zonium dye reduction by peritoneal leukocytes from rheu- ized immune complexes. Journal ofExperimental Medicine, matoid arthritis and systemic lupus erythematosus patients 142, 827-838. measured by a histochemical and spectrophotometric Reikvam, A. (1977). The phagocytic capacity of macro- method. Journal of Laboratory and Clinical Medicine, phages in S phase of the cell cycle. Cellular Immunology, 82, 513-521. 31, 199-204. Weston, W. L., Duskin, R. D., and Hecht, S. K. (1975). Saragone, A. L., Jr., Kamps, S., Campbell, R., and King, Quantitative assays of human monocyte-macrophage G. W. (1977). Lack of correlation of activated monocytes function. Journal ofImmunological Methods, 8, 213-222. with lymphocyte function in patients with lymphoma. Journal of the Reticuloendothelial Society, 21, 377-383. Wiener, E., and Bandieri, A. (1977). Differences in the immune Schmidt, M. E., and Douglas, S. D. (1976). Effect of leva- and nonimmune interaction of peritoneal macrophages misole on human monocyte function and immuno- fromBiozzi high and low responder lines of mice with sheep protein receptors. Clinical Immunology and Immuno- red blood cells. Journal of the Reticuloendothelial Society, pathology, 6, 299-305. 21, 331-342. Symoeus, J. (1976). Levamisol technical report. Janssen Winfield, J. B., Faiferman, I., and Koffler, D. (1977). Avidity Pharmaceutica Publications: Beerse, Belgium. of anti-DNA antibodies in serum and IgG glomerular Utsinger, P. D. (1976). Lymphocyte responsiveness in systemic eluates from patients with systemic lupus erythematosus. lupus erythematosus. Arthritis and Rheumatism, 19, 88-92. Journal of Clinical Investigation, 59, 90-96. Walker, W. S. (1974). Functional heterogeneity of macro- Zurier, R. B. (1976). Reduction of phagocytosis and lyso- phages: subclasses of peritoneal macrophages with somal enzyme release from human leukocytes by serum different antigen-binding activities and immune complex from patients with systemic lupus erythematosus. Arth- receptors. Immunology, 26, 1025-1037. ritis and Rheumatism, 19, 73-78. copyright. http://ard.bmj.com/ on September 29, 2021 by guest. Protected