Journal of Autoimmunity (1990) 3, 789-792
Circulating Immune Complexes in Systemic Necrotizing Vasculitis of the Polyarteritis Nodosa Group. Comparison of HBV-related Polyarteritis Nodosa and Churg Strauss Angiitis
L. Guillevin,* P. R o n c o t and P. V e r r o u s t t *H6pital Avicenne, Department of Internal Medicine, 125 rue de Stalingrad, 93009, Bobigny, France; tH6pital Tenon, 4 rue de la Chine, 75020, Paris, France (Received5 February 1990 and accepted27June 1990) Levels of i m m u n e complexes (IC) were m e a s u r e d before t r e a t m e n t in 16 patients affected with classic p o l y a r t e r i t i s nodosa (PN) or C h u r g Strauss Angiitis (CSA). The six patients with P N were positive for HBV m a r k e r s . The others p r e s e n t e d severe a s t h m a . IgG containing i m m u n e complexes were m e a s u r e d using Raji cell assay. N o r m a l level was 4,642+509 (X_+ SEM). IC levels were significantly different in patients with and without HBV m a r k e r s . When HBV was present, m e a n IC level was 7,185 _+2,472. In the absence o f HBV m a r k e r s , m e a n IC level was 26,462_+ 10,796. These results c o n f i r m that systemic vasculitls is an heterogeneous group o f diseases a n d f u r t h e r suggest that pathogenesis o f vasculitis is different in patients with a s t h m a and those with HBV m a r k e r s .
Polyarteritis nodosa (PN) is an heterogeneous group of connective tissue diseases which is the consequence o f irnmunoregulation disorders. Vascular damage mediated by i m m u n e complexes (I C) has been extensively discussed and studied [ 1]. Such damage would be initiated by antigens which in most cases remain unknown. T h e pathogenesis of P N related to H B V and o f Churg Strauss Angiitis (CSA) is p r e s u m e d to be different: humoral i m m u n i t y might be a p r o m i n e n t factor in P N related to HBV; in contrast, cellular i m m u n i t y might be predominantly involved in CSA and would explain the presence of granulomas around vessels in such patients. Hepatitis B virus (HBV) is considered a major aetiological factor in P N and, in a few cases [2], viral components have been demonstrated in i m m u n e complexes. T h e pathogenetic role of the virus was also suggested by the demonstration o f viral particles in the vessel wall of one patient [3]. T h i r t y - s i x per cent [4] o f patients affected with Phi present an infection with H B V , but in other cases it is difficult to 789 0896--8411/90/060789+ 04 $03.00/0
9 1990Academic Press Limited
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L. Guillevin et al.
determine its aetiology. In patients with CSA, aetiological factors remain unknown, but there are arguments to suggest the responsibility of vaccinations or desensitization [5] which may introduce the antigen initiating the vascular damage. Although belonging to the same group of systemic necrotizing vasculitis (SNV), P N related to H B V and C S A also present differences in evolution. In most cases, P N related to H B V occurs a few months after infection [6], whereas CSA occurs several years after onset of severe asthma and sometimes needs a triggering factor as mentioned above. T h e aim of this study was to compare the level of I g G containing C I C according to the presence of H B V markers or the existence of asthma in order to approach the pathogenesis of vasculitis. Patients and methods Patients
Sixteen patients, nine men and seven women, observed in our D e p a r t m e n t of Internal Medicine, were studied before treatment. Six were affected with H B V related P N and 10 with CSA. In each case, diagnosis was proven by histology which showed fibrinoid necrosis of medium-sized arteries. Diagnosis of CSA was assessed by histology and clinical and biological symptoms: presence of asthma and eosinophilia over 1,500/mm 3 associated with other symptoms of systemic vasculitis. T h e presence of peri- or extravascular granulomas was not observed in every case and was not selected for eligibility for the study as its sensitivity has been demonstrated to be poor [7, 8]. Patients in whom asthma or H B V were not found were excluded from this study because aetiology in these patients is heterogeneous and cannot be determined. Immune complexes
Samples were obtained before the beginning of treatment. Detection of i m m u n e complexes was performed with the Raji cell radioassay described by Theofilopoulos, Wilson and Dixon [9] with some modifications [10]. Briefly, 2 x 1 0 6 Raji cells were incubated for 30 min at 37~ with 25 ~tl of 1/4 dilution of the plasma to be tested. After three washings, the cells were incubated with an excess of 125I radiolabeled protein A from Staphylococcus aureus (Pharmacia Fine Chemical, France). After three further washings, the cell pellets were counted in a gamma counter. All plasma samples tested in duplicate were assessed in the same session as well as 10 samples from healthy individuals. Results were expressed as cell-bound 12sI counts per min (cpm). Statistical results were expressed as mean_+ standard error of the mean. Statist.ical analysis used Student's t-test. Results
Results obtained in a control group of normal subjects were 4,642_+509 (range 2,315-6,549). Results obtained in patients with CSA were 26,462_+ 10,796 (range 5,887-128,640) and in patients with H B V markers 7,185_+2,472 (range 2,546-
Immune complexes in SNV
791
18,550). Circulating immune complex (CIC) levels in patients with CSA were significantly higher ( P < 0 . 0 1 ) than in patients with HBV markers and normal subjects. In contrast, there was no significant difference between patients with HBV markers and normal subjects. Rheumatoid factor was not found in these patients.
Comments Pathogenesis of SNV is likely to implicate several mechanisms, one of which could be predominant in one type of SNV and ancillary in another. T h e group of PN is heterogeneous and associates diseases which have common clinical and biological symptoms, the same basic vascular signature but which might be the consequence of different pathogenetic mechanisms. This study shows that levels of I gG containing C I C detected before treatment by protein A binding are markedly different in patients with CSA and HBV-related PN. In CSA, CIC are more than five times the normal range whereas in HBV-related PN, CIC are close to normal range. This suggests that in CSA, CIC may appear paradoxical in view of histological lesions in which granulomas are usually considered a hallmark of the disease. As a matter of fact, our data and those of Lanham [8] show that granuloma is not a constant feature in SNV with asthma and is not a requirement for CSA diagnosis. In addition, as proposed by Fauci [I 1], phagocytosis of certain types of immune complexes by macrophages may initiate the chain of events leading to granuloma formation. In contrast, IgG containing CIC do not seem to be implicated in the pathogenesis of HBV-related PN. However, the detection system used in this study does not permit to exclude a role for IgA containing CIC.
Acknowledgements This study was presented at the 1st International Congress of Immunointervention and Autoimmune Diseases, Paris, 16-17 June 1988, supported by a grant from the Institut National pour la Sant6 et la Recherche M6dicale ( I N S E R M ) and the Caisse Nationale des Travailleurs Salari6s ( C N A M T S ) . Research was carried out with the help of the Cooperative Study Group for Polyarteritis Nodosa, Soci&~ Nationale Franqaise de M6decine Interne.
References 1. Nydegger, U. E. and P. H. Lambert. 1980. The role of immune complexes in the pathogenesis ofnecrotizing vasculitides. Clin. Rheum. Dis. 6:255-278 2. Trepo, C. G., A. S. Zuckerrnan, R. C. Bird, and A. M. Prince. 1974. The role of circulating hepatitis B antigen/antibody immune complexes in the pathogenesis of vascular and hepatic manifestations in polyarteritis nodosa. J. Clin. Pathol. 27:863-868 3. Gower, R. G., W. F. Sausker, P. F. Kohler, G. E. Thome, and R. M. McIntosh. 1978. Small vessel vasculitis caused by hepatitis B virus immune complexes. J. Allergy Clin. Immunol. 62:222-228 4. Guillevin, L., D. U. Le Thi Huong, P. Godeau, Ph Jais, and B. Wechsler. 1988. Clinical findings and prognosis ofpolyarteritis nodosa and Churg-Strauss angiitis: a study in 165 patients. Br. ft. Rheumatol. 27:258-264
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5. Guillevin, L,, T. Guittard, O. Bletry, P. Godeau, and P. Rosenthal. 1987. Systemic necrotizing angiitis with asthma: causes and precipitating factors in 43 cases. Lung 165: 165--172 6. Guillevin, L., Y. Merrouche, M. Gayraud, B. Jarrousse, I. Royer, A. Leon, and J. Baudelot. 1988. P~riart~rite noueuse li~e au virus de l'h~patite B. D~termination d'une strat~gie th~rapeutique nouvelle: 13 observations. Presse M~d. 17:1522-1526 7. Finan, M. C. and R. K. Winkelmann. 1983. The cutaneous extravascular necrotizing granuloma (Churg-Strauss granuloma) and systemic disease: a review of 27 cases. Medicine 62:142-158 8. Lanham, J. G., K. B. Elvon, L. D. Pusey, and G. R. Hughes. 1984. Systemic vasculitis with asthma and eosinophilia: a clinical approach to the Churg-Strauss syndrome. Medicine 63:65-81 9. Theofilopoulos, A. N., C. B. Wilson, and F. J. Dixon. 1976. The Raji cell radioimmune assay for detecting immune complexes in human sera. ft. Clin. Invest. 57:169-182 10. Verroust, P. J., C. Adam, M. D. Smith, D. Richard-Lenoble, O. Kourilsky, and L. Morel-Maroger. 1979. Circulating immune complexes and C3d in human parasitosis. Kidney Int. 16: 9-14. 11. Fauci, A. S., B. F. Haynes, and P. Katz. 1978. The spectrum of vasculitis. Clinical, pathologic, immunologic and therapeutic considerations. Ann. Int. Med. 89:660-676
Circulating Immune Complexes in Systemic Necrotizing Vasculitis of the Polyarteritis Nodosa Group. Comparison of HBV-related Polyarteritis Nodosa and Churg Strauss Angiitis
L. Guillevin,* P. R o n c o t and P. V e r r o u s t t *H6pital Avicenne, Department of Internal Medicine, 125 rue de Stalingrad, 93009, Bobigny, France; tH6pital Tenon, 4 rue de la Chine, 75020, Paris, France (Received5 February 1990 and accepted27June 1990) Levels of i m m u n e complexes (IC) were m e a s u r e d before t r e a t m e n t in 16 patients affected with classic p o l y a r t e r i t i s nodosa (PN) or C h u r g Strauss Angiitis (CSA). The six patients with P N were positive for HBV m a r k e r s . The others p r e s e n t e d severe a s t h m a . IgG containing i m m u n e complexes were m e a s u r e d using Raji cell assay. N o r m a l level was 4,642+509 (X_+ SEM). IC levels were significantly different in patients with and without HBV m a r k e r s . When HBV was present, m e a n IC level was 7,185 _+2,472. In the absence o f HBV m a r k e r s , m e a n IC level was 26,462_+ 10,796. These results c o n f i r m that systemic vasculitls is an heterogeneous group o f diseases a n d f u r t h e r suggest that pathogenesis o f vasculitis is different in patients with a s t h m a and those with HBV m a r k e r s .
Polyarteritis nodosa (PN) is an heterogeneous group of connective tissue diseases which is the consequence o f irnmunoregulation disorders. Vascular damage mediated by i m m u n e complexes (I C) has been extensively discussed and studied [ 1]. Such damage would be initiated by antigens which in most cases remain unknown. T h e pathogenesis of P N related to H B V and o f Churg Strauss Angiitis (CSA) is p r e s u m e d to be different: humoral i m m u n i t y might be a p r o m i n e n t factor in P N related to HBV; in contrast, cellular i m m u n i t y might be predominantly involved in CSA and would explain the presence of granulomas around vessels in such patients. Hepatitis B virus (HBV) is considered a major aetiological factor in P N and, in a few cases [2], viral components have been demonstrated in i m m u n e complexes. T h e pathogenetic role of the virus was also suggested by the demonstration o f viral particles in the vessel wall of one patient [3]. T h i r t y - s i x per cent [4] o f patients affected with Phi present an infection with H B V , but in other cases it is difficult to 789 0896--8411/90/060789+ 04 $03.00/0
9 1990Academic Press Limited
790
L. Guillevin et al.
determine its aetiology. In patients with CSA, aetiological factors remain unknown, but there are arguments to suggest the responsibility of vaccinations or desensitization [5] which may introduce the antigen initiating the vascular damage. Although belonging to the same group of systemic necrotizing vasculitis (SNV), P N related to H B V and C S A also present differences in evolution. In most cases, P N related to H B V occurs a few months after infection [6], whereas CSA occurs several years after onset of severe asthma and sometimes needs a triggering factor as mentioned above. T h e aim of this study was to compare the level of I g G containing C I C according to the presence of H B V markers or the existence of asthma in order to approach the pathogenesis of vasculitis. Patients and methods Patients
Sixteen patients, nine men and seven women, observed in our D e p a r t m e n t of Internal Medicine, were studied before treatment. Six were affected with H B V related P N and 10 with CSA. In each case, diagnosis was proven by histology which showed fibrinoid necrosis of medium-sized arteries. Diagnosis of CSA was assessed by histology and clinical and biological symptoms: presence of asthma and eosinophilia over 1,500/mm 3 associated with other symptoms of systemic vasculitis. T h e presence of peri- or extravascular granulomas was not observed in every case and was not selected for eligibility for the study as its sensitivity has been demonstrated to be poor [7, 8]. Patients in whom asthma or H B V were not found were excluded from this study because aetiology in these patients is heterogeneous and cannot be determined. Immune complexes
Samples were obtained before the beginning of treatment. Detection of i m m u n e complexes was performed with the Raji cell radioassay described by Theofilopoulos, Wilson and Dixon [9] with some modifications [10]. Briefly, 2 x 1 0 6 Raji cells were incubated for 30 min at 37~ with 25 ~tl of 1/4 dilution of the plasma to be tested. After three washings, the cells were incubated with an excess of 125I radiolabeled protein A from Staphylococcus aureus (Pharmacia Fine Chemical, France). After three further washings, the cell pellets were counted in a gamma counter. All plasma samples tested in duplicate were assessed in the same session as well as 10 samples from healthy individuals. Results were expressed as cell-bound 12sI counts per min (cpm). Statistical results were expressed as mean_+ standard error of the mean. Statist.ical analysis used Student's t-test. Results
Results obtained in a control group of normal subjects were 4,642_+509 (range 2,315-6,549). Results obtained in patients with CSA were 26,462_+ 10,796 (range 5,887-128,640) and in patients with H B V markers 7,185_+2,472 (range 2,546-
Immune complexes in SNV
791
18,550). Circulating immune complex (CIC) levels in patients with CSA were significantly higher ( P < 0 . 0 1 ) than in patients with HBV markers and normal subjects. In contrast, there was no significant difference between patients with HBV markers and normal subjects. Rheumatoid factor was not found in these patients.
Comments Pathogenesis of SNV is likely to implicate several mechanisms, one of which could be predominant in one type of SNV and ancillary in another. T h e group of PN is heterogeneous and associates diseases which have common clinical and biological symptoms, the same basic vascular signature but which might be the consequence of different pathogenetic mechanisms. This study shows that levels of I gG containing C I C detected before treatment by protein A binding are markedly different in patients with CSA and HBV-related PN. In CSA, CIC are more than five times the normal range whereas in HBV-related PN, CIC are close to normal range. This suggests that in CSA, CIC may appear paradoxical in view of histological lesions in which granulomas are usually considered a hallmark of the disease. As a matter of fact, our data and those of Lanham [8] show that granuloma is not a constant feature in SNV with asthma and is not a requirement for CSA diagnosis. In addition, as proposed by Fauci [I 1], phagocytosis of certain types of immune complexes by macrophages may initiate the chain of events leading to granuloma formation. In contrast, IgG containing CIC do not seem to be implicated in the pathogenesis of HBV-related PN. However, the detection system used in this study does not permit to exclude a role for IgA containing CIC.
Acknowledgements This study was presented at the 1st International Congress of Immunointervention and Autoimmune Diseases, Paris, 16-17 June 1988, supported by a grant from the Institut National pour la Sant6 et la Recherche M6dicale ( I N S E R M ) and the Caisse Nationale des Travailleurs Salari6s ( C N A M T S ) . Research was carried out with the help of the Cooperative Study Group for Polyarteritis Nodosa, Soci&~ Nationale Franqaise de M6decine Interne.
References 1. Nydegger, U. E. and P. H. Lambert. 1980. The role of immune complexes in the pathogenesis ofnecrotizing vasculitides. Clin. Rheum. Dis. 6:255-278 2. Trepo, C. G., A. S. Zuckerrnan, R. C. Bird, and A. M. Prince. 1974. The role of circulating hepatitis B antigen/antibody immune complexes in the pathogenesis of vascular and hepatic manifestations in polyarteritis nodosa. J. Clin. Pathol. 27:863-868 3. Gower, R. G., W. F. Sausker, P. F. Kohler, G. E. Thome, and R. M. McIntosh. 1978. Small vessel vasculitis caused by hepatitis B virus immune complexes. J. Allergy Clin. Immunol. 62:222-228 4. Guillevin, L., D. U. Le Thi Huong, P. Godeau, Ph Jais, and B. Wechsler. 1988. Clinical findings and prognosis ofpolyarteritis nodosa and Churg-Strauss angiitis: a study in 165 patients. Br. ft. Rheumatol. 27:258-264
792
L. G u i l l e v i n et aL
5. Guillevin, L,, T. Guittard, O. Bletry, P. Godeau, and P. Rosenthal. 1987. Systemic necrotizing angiitis with asthma: causes and precipitating factors in 43 cases. Lung 165: 165--172 6. Guillevin, L., Y. Merrouche, M. Gayraud, B. Jarrousse, I. Royer, A. Leon, and J. Baudelot. 1988. P~riart~rite noueuse li~e au virus de l'h~patite B. D~termination d'une strat~gie th~rapeutique nouvelle: 13 observations. Presse M~d. 17:1522-1526 7. Finan, M. C. and R. K. Winkelmann. 1983. The cutaneous extravascular necrotizing granuloma (Churg-Strauss granuloma) and systemic disease: a review of 27 cases. Medicine 62:142-158 8. Lanham, J. G., K. B. Elvon, L. D. Pusey, and G. R. Hughes. 1984. Systemic vasculitis with asthma and eosinophilia: a clinical approach to the Churg-Strauss syndrome. Medicine 63:65-81 9. Theofilopoulos, A. N., C. B. Wilson, and F. J. Dixon. 1976. The Raji cell radioimmune assay for detecting immune complexes in human sera. ft. Clin. Invest. 57:169-182 10. Verroust, P. J., C. Adam, M. D. Smith, D. Richard-Lenoble, O. Kourilsky, and L. Morel-Maroger. 1979. Circulating immune complexes and C3d in human parasitosis. Kidney Int. 16: 9-14. 11. Fauci, A. S., B. F. Haynes, and P. Katz. 1978. The spectrum of vasculitis. Clinical, pathologic, immunologic and therapeutic considerations. Ann. Int. Med. 89:660-676
