Clin. exp. Immmnnzol. (1979) 35, 62-66.

Immune complexes in peripheral blood polymorphonuclear leucocytes of patients with Raynaud's phenomenon J. VAN DER MEULEN, A. A. WOUDA, E. MANDEMA & T. H. THE Clinical Immunology Unit, Department ofInternal Medicine and Department ofCardiology, University ofGroningen, The Netherlands

(Received 20 June 1978)

SUMMARY

Phagocytosed immune complexes in polymorphonuclear (PMN) leucocytes of sixty-nine patients with Raynaud's phenomenon were studied by scoring the cells for IgG and complement inclusions. The results were compared with the degree of vasospasm, as measured by photoelectric plethysmography of the fingers after cooling. Median IgG and complement scores of PMN cells were significantly elevated in patients with Raynaud's phenomenon when compared with those of the control group (P< 0-01). Patients with a positive (n = 35) or a strongly positive (n = 11) plethysmographic test showed significantly higher median IgG and complement scores than those with a negative plethysmographic test (n = 23). They also showed elevated IgG and complement scores in a greater proportion (89 and 100%, respectively, vs 48%). Thus, a positive relationship was shown to exist between the degree of vasospasm and the levels of phagocytosed immune complexes in circulating PMN leucocytes. In secondary Raynaud's phenomenon high levels of phagocytosed immune complexes were found. In primary Raynaud's, patients with some clinical or laboratory signs of an auto-immune disease (n = 12), who were classified as 'suspected secondary', had higher IgG and complement scores than those without such signs (n = 37). This suggests that the test for immune complex detection may distinguish patients with connective tissue disease-associated Raynaud's phenomenon from those with truly primary Raynaud's, in an early stage of the disease. The clinical and patho-physiological implications of these findings with regard to the Raynaud phenomenon are discussed.

INTRODUCTION Raynaud's phenomenon, an intermittent spasm of the digital arteries with subsequent acral pallor and cyanosis on exposure to cold, followed by a reactive hyperaemia when warming up, has an obscure aetiology. There is considerable variability in the prognosis of the phenomenon: it may present as 'a fleeting harmless colour change' in anaemic hypometabolic girls, or alternatively, it may be a forerunner of an ominous connective tissue disease (De Takatas & Fowler, 1962), such as scleroderma or systemic lupus erythematosus (SLE). Subjects with Raynaud's phenomenon, however, often show evidence of autoimmunity and other immunological abnormalities upon investigation. Experimental studies in animal models suggest that immune complexes play an important role in the pathogenesis of the vascular lesions leading to the disease symptoms (Cochrane, 1968). In addition, a correlation has been found between Raynaud's phenomenon and immune complexes in vinyl chloride disease, an occupational hazard for workers in vinyl chloride plants, with multiple system involvement (Ward et al., 1976). Correspondence: Dr T. H. The, Clinical Immunology Unit, Department of Internal Medicine, University Hospital, Oostersingel 59, Groningen, The Netherlands. 0099-9104/79/0010-0062S02.00 (© 1979 Blackwell Scientific Publications

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63 Immune complexes and Raynaud's phenomenon Immune complexes can be detected in patients' sera, biopsies of kidney or skin and also in circulating polymorphonuclear (PMN) leucocytes (Steffelaar, De Graaff-Reitsma & Feltkamp-Vroom, 1976; Steffelaar et al., 1977). In an attempt to elucidate the possible role of immune complexes in patients with Raynaud's phenomenon, we have studied the degree of vasospasm of the fingers due to cold provocation by photo-electric plethysmography, and the levels of phagocytosed immune complexes in sixty-nine patients and sixteen controls. We have also studied the level of phagocytosed immune complexes in different disease groups: patients with primary Raynaud's have been compared with those classified as secondary Raynaud's with scleroderma or SLE. Within the primary Raynaud's, patients showing minor symptoms of a systemic autoimmune disease, called 'suspected secondary', were considered separately. MATERIALS AND METHODS Subjects. The study included sixty-nine patients with a typical history of Raynaud's phenomenon. Cases with a history of vascular compression, trauma due to vibrating tools and drugs known to provoke the phenomenon were excluded. Twenty patients, classified as secondary Raynaud's, consisted of fourteen with scleroderma (diagnosed by the presence of acrosclerosis, telangiectasia and/or diagnostic skin biopsy) and six with SLE, according to the preliminary ARA criteria (Cohen et al., 1971). Forty-nine patients, classified as having primary Raynaud's, included twelve with 'suspected secondary Raynaud's' (two had pulmonary fibrosis, three had telangiectasia, two had periods of arthralgia without symptoms of rheumatoid arthritis, one had discoid lupus, one had pleuritis of unknown origin, one had cryoglobulinaemia, one had hypomotility of the oesophagus on X-ray and one had the sicca syndrome). Sixteen apparently healthy individuals acted as controls. Detection of immune complexes. A modification (The et al., 1978) of the granulocyte phagocytosis test described by Steffelaar et al. (1976) was used to detect IgG and complement in PMN leucocytes. Briefly, 10 ml of fresh venous blood was defibrinated by gentle rotation in sterile flasks with glass beads. Buffy-coat PMN leucocytes were obtained after subsequent centrifugation in a haematocrit tube. The contaminating red blood cells were lysed in a buffered NH4C1 solution at 0WC. After washing the cells in HEPES buffered RPMI medium, cytocentrifuge slides were made and fixed in acetone. Intracellular IgG and complement were detected using specific rabbit antisera and a FITC-conjugated sheep anti-rabbit immunoglobulin serum in a double layer system. Cells containing at least six fluorescent granules were regarded as positive. They were graded with respect to the number of fluorescent inclusions. A total of 1000 PMN cells in fields chosen at random were observed at 10 x 40 magnification. Two observers without prior knowledge of the diagnosis read and scored the results independently. Plethysmographic test. Severity of vascular spasm of the digital arteries was measured by photo-electric plethysmography of the fingers during cold provocation, described in detail elsewhere (Wouda, 1977). A negative plethysmographic test indicated that during cooling of the hands the plethysmogram was unchanged. A positive test indicated that vascular spasm in two or more fingers occurred during cooling, and a strongly positive test indicated that this occurred above 18°C. Statistical analysis. Statistical analysis of the results was performed with the Wilcoxon rank-sum test; P values less than 0 01 were considered significant.

RESULTS Fifty-three of the sixty-nine patients (77%o), but none of the controls showed elevated levels of phagocytosed immune complexes in PMN leucocytes (IgG and complement scores >20). The relationship between IgG inclusions and the degree of vasospasm is illustrated in Fig. 1. A greater proportion of patients with a positive (n = 35) or a strongly positive (n = 11) cold-provoked plethysmographic test showed elevated IgG scores (89 and 100% respectively) compared with those with a negative plethysmographic test (48%). Moreover, their median IgG scores were significantly higher: 98 and 120 vs 20. The relationship between complement inclusions and the degree of vasospasm is shown in Table 1. Patients with positive tests had significantly higher median complement scores than those with negative plethysmographic tests. IgG scores in the different groups of patients classified according to underlying disease are shown in Fig. 2. In secondary Raynaud's both the scleroderma group (median score 94) and the SLE group (median score 118) were significantly higher than the controls. In primary Raynaud's, subdivided into true primary Raynaud's and 'suspected secondary Raynaud's', both subgroups were also significantly higher than the controls. However, patients with true primary Raynaud's, who had a negative plethysmographic test (n = 17), showed significantly lower IgG scores (median 20) than patients with a positive

64

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FIG. 1. Relationship between immune complexes in PMN leucocytes and degree of vasospasm measured by plethysmography cold provocation in patients with Raynaud's phenomenon and in controls. The number of individuals and the median score in each group is given. The statistical significance of the differences between the median scores (indicated by cross bars) of the patients' groups and the controls was calculated with the Wilcoxon rank-sum test. TABLE 1. Relationship between complement scores in PMN leucocytes and degree of vasospasm measured by the plethysmographic test

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plethysmographic test (n = 20, median 72). Furthermore, in the primary Raynaud's group all patients with a positive plethysmographic test had elevated levels of immune complexes (20/20 = 100%); in contrast, only eight of the seventeen patients (450%) with a negative plethysmographic test had elevated levels. The patients with 'suspected secondary Raynaud's' (n = 12, median score 100) showed significantly higher immune complex levels than primary Raynaud's patients having a negative plethysmographic test (n = 17, median score 20). The patients with secondary Raynaud's also had significantly higher scores than patients with a negative plethysmographic test and primary Raynaud's phenomenon.

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FIG. 2. immune complexes in patients with primary and secondary Raynaud's and in controls. Within the group of primary Raynaud's, patients showing minor symptoms of a systemic autoimmune disease, called 'suspected secondary', were considered separately. For further details see legend to Fig. 1.

DISCUSSION The finding in PMN leucocytes of both IgG and complement inclusions suggests the presence of phagocytosed immune complexes, although the possibility of the presence of phagocytosed immunoglobulin aggregates must also be considered. In this and previous studies (Jansen et al, 1977; The et al., 1978), however, no relation between IgG scores of PMN cells and serum IgG levels could be demonstrated. Furthermore, a clear disease association was found. The specificity of the antigen(s) involved has still to be shown, however. The finding of increased levels of phagocytosed immune complexes in circulating PMN leucocytes in a high proportion of patients with Raynaud's phenomenon may contribute to a better understanding of some of the clinical and patho-physiological aspects of the disease. A classification of patients in primary and secondary Raynaud's seems useful, for secondary Raynaud's associated with connective tissue diseases appears to be resistant to therapy and carries the worse prognosis (Lancet, 1977). As with earlier findings in SLE (Steffelaar et al, 1976), the present study has shown that in patients with secondary Raynaud's associated with SLE, high levels of phagocytosed immune complexes in PMN leucocytes are detectable. This is in agreement with the finding of immune complexes deposited in the glomeruli of kidneys or other affected organs of SLE patients. In scleroderma, phagocytosed immune complexes in PMN leucocytes have not been demonstrated before, although circulating immune complexes in this disease have been reported (Hallgren & Wide, 1976). E

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J. van der Meulen et al.

Primary Raynaud's is a heterogenous group with respect to the future course and prognosis of the disease, and this diversity seems to be reflected in the level of phagocytosed immune complexes in PMN leucocytes of individual patients. The results obtained suggest that this immunological test might distinguish cases with associated connective tissue disease from those with primary Raynaud's in an early phase of the disease. This would follow from the demonstration of high levels of phagocytosed immune complexes in a high frequency of primary Raynaud's cases, which are included in a 'suspected secondary' group because these patients showed some clinical or laboratory signs of a systemic connective tissue disease. A follow-up study is necessary to prove the validity of our supposition that primary Raynaud's patients with normal immune complex levels have a better prognosis. Previous studies have demonstrated the importance of immune complexes in the development of vascular lesions. In this process polymorphonuclear leucocytes with phagocytosed immune complexes probably represent important mediators of the inflammatory lesions, because of the liberation of proteolytic enzymes (Cochrane, 1968). A striking improvement in patients with Raynaud's phenomenon as a result of plasmapheresis has been reported (Talpos et al., 1978). This is not unexpected in view of our findings and the beneficial effect of this kind of treatment as in acute SLE (Lancet, 1976; Verrier Jones et al., 1976), is likely to be the removal of immune complexes. In the present study a positive relationship between the degree of vasospasm and the incidence and levels of phagocytosed immune complexes in PMN leucocytes of patients with Raynaud's phenomenon has been demonstrated. This finding suggests a possible patho-physiological relationship with the vascular abnormality of Raynaud's phenomenon (Birnstingl, 1971). We are grateful to Dr P. C. Das and Dr M. van der Giessen for correcting the manuscript.

REFERENCES BIRNSTINGL, M. (1971) The Raynaud syndrome. Postgrad. STEFFELAAR, J.W., TEN KATE, F.J.W., NAP, M., SWAAK, Med.I. 47, 297. A.J.G., DE GRAAFF-REITSMA, C.B., VAN ELVEN, E.H. & CoCHRANE, C.G. (1968) Immunologic tissue injury mediated FELTKAMP-VROOM, TH. M. (1977) Immune complex detection by immunofluorescence on polymorphonuclear by neutrophilic leucocytes. Adv. Immunol. 9, 97. leucocytes. Clin. exp. Immunol. 27, 391. CoHEN, A.S., REYNoLDS, W.E., FRANKLIN, E.C., KuLKA, J.P., RoPES, M.W., SHuLmAN, L.E. & WALLACE, S.L. TALPOS, G., HonRocKS, M., WHITE, J.M., & COTTON, L.T. (1971) Preliminary criteria for the classification of syste(1978) Plasmapheresis in Raynaud's disease. Lancet i, 416. THE, T.H., VAN DER GIESSEN, M., HuIGEs, H.A., SCHRAFmic lupus erythematosus. Bull. Rheum. Dis. 21, 643. FORDT Koops, H. & VAN WINGERDEN, I. (1978) Immune DE TAKATS, G. & FowuLR, E.F. (1962) Raynaud's phenomenon. . Amer. Med. Ass. 179,99. complex in peripheral blood polymorphonuclear leucoHALLGREN, R. & WIDE, L. (1976) Detection of circulating cytes of malignant melanoma patients. Clin. exp. Immunol. 32,387. IgG aggregates and immune complexes using 1251 protein A from staphylococcus aureus. Ann. Rheum. Dis. 35, 306. VERRIER JONES, J., BUCKNALL, R.C., CUMMING, R.H., JANSEN, H.M., TH, T.H., DE GAST, G.C., HUIGES, H.A., ASPLIN, C.M., FRASER, I.D. BOTHAMLEY, Joy, DAVIS, P. & HAMBLIN, T.J. (1976) Plasmapheresis in the manageESSELINK, M.T., VAN DER WAL, A.M. & ORIE, N.G.M. ment of acute systemic lupus erythematosus? Lancet, (1977) Immunoglobulin and complement inclusions in i,709. peripheral blood polymorphonuclear leucocytes of patients WARD, A.M., UDNOON, S., WATKINS, J., WALKER, A.E. & with bronchial carcinoma. Thorax, 32, 706. LANCET (1976) Plasmapheresis and immunosuppression. DARKE, C.S. (1976) Immunological mechanisms in the pathogenesis of vinyl chloride disease. Brit. Med. 3. Editorial. Lancet, i, 1113. i,936. LANCET (1977) Episodic digital vasospasm-the legacy of WOUDA, A.A. (1977) Raynaud's phenomenon. Acta Med. Maurice Raynaud. Editorial. Lancet, i, 1039. Scand. 201,519. STEFFELAAR, J.W., DE GRAAFF-REITSMA, C.B. & FELTKAMPVROOM, TH. M. (1976) Immune complex detection by immunofluorescence on peripheral blood polymorphonuclear leucocytes. Clin. exp. Immunol. 23, 272.