Deficiency of c7 with systemic lupus erythematosus. solubilization of immune complexes in complement-deficient seraкод для вставкиСкачать
87 DEFICIENCY OF C7 WITH SYSTEMIC LUPUS ERYTHEMATOSUS Solubilization of Immune Complexes in Complement-Deficient Sera HOWARD J. ZEITZ, GARY W. MILLER, THOMAS F. LINT, MIR A. ALI, and HENRY GEWURZ A woman with systemic lupus erythematosus (SLE) was found to have undetectable serum total complement (C) activity with marked reductions in levels of several C components. Recognition of rising C4, C3, and C5 levels coupled with sustained absence of hemolytic activity led to studies of individual C components that identified a persisting selective and complete absence of C7 hemolytic activity. Review of the literature indicated that the frequency of SLE is increased in patients with complete deficiency of a late-acting C component (7% as compared to 0.05% in the general population); however, the disease frequency is less than that observed in patients with complete deficiency of an early-acting C component (41% incidence in patients lacking C1, C4, or C2). In order to explore one possible basis for this association, the capacity of serum from the patient described herein and from patients with other C component deficiencies to mediate in vitro solubilization of immune complexes was studied by using a recently developed asFrom the Institute of Allergy and Clinical Immunology, Grant Hospital of Chicago; La Rabida-University of Chicago Institute; and the Department of Immunology, Rush Medical Center, Chicago, Illinois. This work was supported in part by a grant (#I R 0 1 CA26143) from the NCI, DHEW. Howard J. Zeitz, MD: Institute of Allergy and Clinical Immunology; Gary W. Miller, PhD: La Rabida (present address, Loyola University School of Dentistry, Maywood, Illinois); Thomas F. Lint, PhD: recipient of NCI Research Career Development Award 0 1 R 0 4 CA00558, Rush Medical Center; Mir A. Ali, MD: Institute of Allergy and Clinical Immunology (present address, Ravenswood Hospital Medical Center, Chicago, Illinois); Henry Gewurz, MD: holder of Thomas J. Coogan, Sr., Chair in Immunology established by Marjorie Lindheimer Everett, Rush Medical Center. Address reprint requests to Howard J. Zeitz, MD, Institute of Allergy and Clinical Immunology, Grant Hospital of Chicago, 550 W. Webster, Chicago, IL 60614. Submitted for publication January 22, 1980; accepted in revised form August 1 1, 1980. Arthritis and Rheumatism, Vol. 24, No. 1 (January 1981) say. The patient’s serum had a markedly decreased capacity to induce solubilization of immune complexes, as did sera of other patients with active SLE and sera of individuals with inborn deficiencies of C1, C2, or C3 without SLE. By contrast, the sera of patients with a selective terminal C component deficiency (C5, C6, C7, or C8) but without SLE readily supported immune complex solubilization. These results suggest that patients lacking a terminal C component are predisposed to the development of SLE by a process which is independent of C-mediated immune complex solubilization. Whether the compromised capacity for C-mediated immune complex solubilization contributes to the even greater frequency of SLE in patients lacking an early-acting C component remains to be established. In recent years, hereditary abnormalities of the complement (C) system have been recognized with increasing frequency (1,2). Selective C component deficiencies have been noted in individuals who are apparently healthy at the time of study (3-9, but most reports have suggested that patients with inborn complement errors are predisposed both to repeated and/or unusual infection (6,7) and to a variety of inflammatory diseases, especially systemic lupus erythematosus (SLE) (1,8). Although the association of SLE or SLE-like syndromes with hereditary deficiency of an early-acting (Cl, C4, or C2) component of the classical complement pathway (9-14) (Table 1) is well known, the relationship between SLE and complete deficiency of a late-acting (C5, C6, C7, C8, or C9) component has been less well characterized. Patients with selective deficiency of a late-acting C component have a very high frequency of Neisseria infection (15); in addition, two previously reported patients have developed SLE (16,17). ZEITZ ET AL 88 The nature of the association between SLE and complete deficiency of a C component is not established. One possibility is that C-deficient patients might also be deficient in the ability to dispose of circulating immune complexes, with subsequent development of the lupus process. Previous studies have demonstrated that C-mediated solubilization of immune complexes proceeds via the alternative activation pathway alone, but this is substantially enhanced by concomitant participation of the classical activation pathway (18,19). The late-acting C components are thought to be nonessential for immune complex solubilization (18,20); however, studies of solubilization in sera of patients with late-acting C component deficiencies have not yet been reported. We present here a patient with SLE who has selective absence of C7, and we report the results of studies of the capacity of serum from this patient and individuals with other C component deficiencies to mediate solubilization of immune complexes in vitro. CASE REPORT GS, a 27-year-old Mexican woman, was hospitalized in April 1977 after 10 days of fever and cough which had failed to respond to acetylsalicylic acid and ampicillin. She had been well until December 1974 when she developed premature labor in the 18th week of her second pregnancy and gave birth to a female infant who died shortly after delivery; the placenta exhibited hydatid mole formation without evidence of choriocarcinoma. During the next 4 months, she noted symmetrical joint pain and swelling in her knees, feet, and hands, with subsequent loss of weight (10 kg) and hair. There was no history of Raynaud's phenomenon, dysphagia, or diarrhea; a urinalysis 4 months prior to admission had revealed mild (1+) proteinuria. She had received treatment with prednisone (5 mg twice daily) for 5 months until abruptly electing to halt this therapy 1 month prior to admission. On admission, the patient was febrile (38.9"C) and tachypneic. She had diminished breath sounds at the right posterior lung base, mild bifrontal alopecia, and severe oral ulceration, but the joints appeared normal. No other abnormalities were noted. The initial hematocrit was 31%, and the white blood cell count was 3,800/mm3 (70% neutrophils and 8% band forms). There were 8-10 fine granular casts/ high power field, with quantitative urinary protein excretion ranging between 1.2 and 4.0 gm/24 hours. The blood urea nitrogen, serum creatinine, and creatinine clearance levels were within normal limits. Definitive renal biopsy study was precluded by the patient's religion (Jehovah's Witness). No chorionic gonadotropins were detected in the urine. The Westergren erythrocyte sedimentation rate was 6 mm/hour, and serum C-reactive protein (CRP) was undetectable by immunoprecipitation assay. LE cell preparations were positive on several occasions. Indirect immunofluorescence demonstrated antinuclear antibodies in a relatively high titer (1 :500) with a homogeneous and peripheral staining pattern. Hemagglutinating antibodies to double-stranded DNA (dsDNA) were also detected in a high titer (1 :640), while rheumatoid factor and antibodies to smooth muscle, mitochondria, and parietal cells were undetectable. Histocompatibility typing revealed only single specificities at the A and B loci-Aw24 and B12. Most strikingly, serum hemolytic C activity was absent; detailed studies of the complement system are described below. Admission chest x-ray revealed bilateral pulmonary infiltrates. Pure cultures of Escherichia coli were isolated from the blood, and treatment with intravenous antibiotics (first cephalothin and gentamycin, and later tobramycin) was initiated. Icteric hepatitis and thrombocytopenia were noted shortly after admission, but hepatitis-associated antigen was not detected. Corticosteroid therapy resulted in slow but steady clinical improvement accompanied by a marked reduction of the anti-dsDNA antibody level. She became totally afebrile on the thirty-seventh hospital day and was discharged 10 days later. MATERIALS AND METHODS Total serum C hemolytic activity via both the primary and alternative pathways was assayed semiquantitatively in gels containing sensitized sheep or unsensitized rabbit erythrocytes, respectively, as previously described (2 1). Determination of the hemolytic levels of components C1-C9 and protein levels of Clq, C4, C3, C5, properdin, and factor B were performed by previously described methods (7). Complement-mediated solubilization of immune precipitates or complex release activity (CRA) was quantitated by a new, standardized procedure (22). Briefly, IZ5I-labeled bovine serum albumin (BSA) and guinea pig anti-BSA antiserum were precipitated at equivalence to form appropriate immune complexes. The complexes were washed four times in isotonic phosphate-buffered saline (PBS) and adjusted to 25 pg BSA/ml. For the assay of solubilization, precipitates containing 0.15 pg '251-BSAwere added to 75 pl of test serum in a total volume of 0.6 ml PBS containing 0.15 mM CaCl, and I .O mM MgC12. Tubes were incubated at 37"C, and 50 p1 aliquots were removed at various times up to 30 minutes, diluted in 2.0 ml ice-cold saline, and centrifuged 20 minutes at 1200g. Radioactivity was counted in both the supernatants and the pel- C7 DEFICIENCY WITH SLE w a 53 89 4c 2%- 39 -a* x w o an 38 Temperature 55 37 1 PREDNISONE I PREDNISOLONE I 100 > 80 t L c0 ; 60 ? € 2: wz 6 8 CL- 40 n V 20 Hemolytic Activity C d 10 20 30 40 50 60 DAY OF OBSERVATION Figure 1. Persisting inability of patient’s serum to lyse sensitized sheep erythrocytes (classical complement pathway = 0-0)and unsensitized rabbit erythrocytes (alternative pathway = UU),even while C3 and CS protein, anti-dsDNA antibody, and maximum daily temperature were returning to normal in response to steroid therapy. lets to calculate the amount of immune precipitate solubilized after each interval. For each serum, a plot of “% soluble versus time” was constructed. The areas under the curves were measured and were divided by the area for the standard control serum (area = 100%by definition) run at the same time. Solubilization by patient GS serum was compared to solubilization by a series of C-deficient sera and sera from patients with active SLE. Sera with selective deficiency of C2, C3, C6, or C7 were obtained from patients previously studied in our laboratory. In addition, sera from patients with deficiency of Clr (9,23), C5 (16), or C8 (24) were generously supplied by Drs. R.P. Young, F. Schmid, J.P. Leddy, and B. Petersen, respectively. RESULTS Analysis of complement activity in patient GS serum. The patient’s serum was unable to lyse either sensitized sheep or unsensitized rabbit erythrocytes suspended in agarose gels, reflecting deficiencies in function of the primary and alternative complement pathways, respectively. While prolonged incubation of gels containing patient and normal serum in adjacent wells resulted in no lysis around the well containing the pa- tient’s serum, distinct lines of lysis developed between the wells containing patient and normal serum. These lines are indicative of the presence of an activated complex of C5 and C6 (CS), known to be formed when serum which contains C5 and C6 in excess of C7 is added to gels such as those used in the present study (25,26). Analysis of the C component hemolytic activities and protein levels on day 84 (Table 2) revealed selective total deficiency of C7. Hemolytic activities of several other individual components (C 1, C4, C3, and C6) were present but reduced to 23-54% of normal. C4, C3, C5, properdin, and factor B protein levels were markedly reduced during the first 3 weeks of hospitalization (levels on day 14 are shown in Table 2), with the depletion pattern indicating substantial activation via the alternative complement pathway. With administration of corticosteroid therapy and in association with the reduction of anti-dsDNA antibodies and the disappearance of fever, complement components (with the exception of C7) returned to normal or near-normal levels, but without restoration of hemolytic activity via either the classical or alternative pathway (Figure 1). Solubilization of immune complexes by sera of patient G S and other C-deficient individuals. C-mediated solubilization was measured in sera from several patients with a selective C component deficiency. As seen in Figure 2, sera from patients deficient in Clr, C2, or C3 had <5%, <15%, and <5%, respectively, of the immune complex release activity (CRA) of a standard normal serum. CRA activities of 96 f 15% were obtained for a group of 10 normal sera under identical conditions. These results are consistent with the hypothesis that in this assay C3 activation by the classical path- -2 E 100 0 0 -8a IX V 50 ClrD C2D C3D C5D C6D C7D C8D (C A )(G.S.) SLE SERA Figure 2. Solubilization of immune precipitate by sera from patients with defined C component deficiencies or active SLE. Solubilization (CRA) activity is expressed as a percentage of a standard normal control serum. The shaded area defines the mean f standard deviation for a group of 10 normal sera run under identical conditions. Each circle represents an individual patient. ZEITZ ET AL 90 Table 1. Lupus erythematosus and Neisseriu infection in individuals with isolated complement component deficiencies* Deficient component Reported individuals c1 'i ) - Lupus erythematosust 55 8 (62) 3(1m) 18 (33) ) c3 5 0 (0) c5 c7 C6 9 C8 c9 8 2 ] c4 c2 71 44 1 (11) 01 (0) (9) 1 (12) 0 (0) Neisseriu infection? 29 (41) 1(20) 3) 5 (56) 83 (27)] (57) 19(43) 3 (38) 0 (0) * Individuals reported in the literature through January 1, 1980. t % indicated in parentheses. f Including C7-deficient patient reported herein. way is required for efficient immune complex solubilization (20). By contrast, sera from patients deficient in C5, C6, or C8 possessed CRA activities within the normal range. Serum taken from patient GS on four separate occasions over an 8-week period (day 7 to day 60), even when C3 levels had returned to 70% of normal, showed no CRA activity, while serum from another C7deficient individual (CA) without evidence of SLE showed normal releasing activity. Two sera from patients with active SLE but without an inborn C defect, shown for comparison, had 4% CRA activity. DISCUSSION The patient reported here was found to have Gram negative septicemia on hospital admission, along with subsequently diagnosed SLE. The diagnosis of SLE was supported by a group of clinical features including arthralgias, fever, thrombocytopenia, leukopenia, abnormal urinary sediment, and proteinuria; when occurring together, these features clearly meet the proposed criteria for the diagnosis of SLE (27). Circu- lating antibodies to dsDNA were detected in high titer and provided the single laboratory finding most indicative of SLE (28). Complement component analysis of our patient strongly suggested that the C cascade was being activated via both the primary and the alternative pathways in association with the active SLE process. Levels of properdin, factor B, and C4, as well as of C3 and C5, were markedly depressed during the initial investigations, but the values returned to normal or near-normal after several weeks of therapy. Recognition of rising C component protein levels coupled with persistent absence of hemolytic activity led directly to tests to determine whether a selective C component deficiency was present, and isolated deficiency of C7 was subsequently observed in 8 separate samples taken over a 15-week interval. C7 deficiency was also suggested by the reaction of lysis in gels. Previous studies have demonstrated that C7-deficient serum has a marked capacity to generate persisting C% in agarose and thus form a distinctive pattern of reactive lysis in the absence of primary lysis, which seems to be diagnostic of C7 deficiency (26). Table 2. Complement component profile of patient GS Hemolytic C % normal Day 84, C protein Day 14, Pdml Day 84, Pg/ml c1 c4 c2 c3 c5 C6 c7 C8 c9 54 24 72 40 90 23 C h c4 c3 c5 Factor B Properdin 181 48 330 37 58 <2 173 134 450 91 140 5 <o. I 67 346 Normal values, Pg/ml 110-210 200-800 800-1,800 70- 170 175-275 10-20 C7 DEFICIENCY WITH SLE Identical studies in the patient reported here confirmed the previous findings and emphasized the ease of demonstration of this deficiency. The inability to obtain serum samples from family members in Mexico precluded definitive identification of the hereditary nature of this deficiency; however, an autosomal codominant pattern of inheritance has been demonstrated in 9 other individuals with C7 deficiency (26,29-32). Studies of HLA antigens in those patients and their families demonstrated the Aw24 and B12 alleles in only 1 other patient (26); linkage between the HLA-A and B loci and the C7 deficiency locus was not observed in 4 unrelated families (26, 30-32). None of the previously reported individuals with C7 deficiency (26,29-34) were known to have developed SLE. However, 2 patients having SLE in association with the isolated absence of another late-acting complement component have been reported previously (Table 1): a 20-year-old woman with C5 deficiency (16) and a 56-year-old woman with C8 deficiency (17). The occurrence of SLE in 3 (including the patient reported herein) of the 44 reported individuals (7%) with a terminal C component deficiency suggests an increased prevalence of this disease in these patients, since the prevalence of SLE in the general population is only 0.05% (35). SLE has been noted even more frequently (41%) (Table 1) in patients wi&%ereditary deficiency of C1, C4, or C2 (9-14, 21, 23). While patients with rheumatic complaints undergo coniplement analysis more frequently than the general population, it is unlikely that selection bias alone can explain the markedly increased frequency of SLE in C-deficient patients. Since in the general population the approximate frequency of C deficiency is 0.03% (36) and that of SLE is 0.05% (35), the probability that both SLE and C deficiency will be found in the same individual is of the order of one in six million. Thus, there appears to be a unique susceptibility to the development of SLE in all patients with an inborn C component deficiency, which is particularly marked in individuals lacking an early-acting classical pathway component. This is reflected in the difference in frequency of SLE in patients with early-acting (41%) versus late-acting (7%) component deficiency. The mechanism whereby an inborn deficiency of a C component might contribute to the development of SLE is uncertain. As suggested elsewhere (1,8,12), the development of immune complex disease in C-deficient patients might be due to an unusual host propensity to infection, attributable to the lack of complement per se or to genetic linkage with another factor predisposing to 91 SLE. Alternatively, C-deficient patients might lack the ability to dispose of circulating immune complexes in a normal manner, with consequent tissue damage. To examine this latter possibility, we measured C-mediated solubilization in the sera of 13 patients with various selective C component deficiencies, including 2 with C7 deficiency, and 2 patients with SLE in the absence of inborn C deficiency. In this assay, which requires participation of the classical complement pathway for optimal activity, sera from patients with deficiency of C 1r, C2, or C3 lacked normal CRA activity, as predicted by the finding (20) that C3b fixation is required for immune complex solubilization. (It is of interest that in preliminary experiments performed by GWM, the sera deficient in C l r and C2 demonstrated normal or near-normal CRA activity in an assay involving activation via the alternative pathway alone.) Sera from our patient GS and 2 other patients with SLE but with no inborn C defect also lacked solubilization activity. By contrast, sera from patients deficient in C5, C6, C7, or C8 but without SLE demonstrated normal CRA activity. Thus, our results support the concept that immune complex solubilization does not require participation of the late-acting C components, and suggest that the defective solubilization in the C7-deficient serum described herein is due to an as yet undefined defect associated with SLE rather than to deficiency of C7 per se. The findings of a more detailed study of immune complex solubilization in patients with SLE, which will be the subject of a separate report (37), indicate that CRA depression correlates directly with the severity of clinical disease in patients with active SLE, while SLE patients in complete remission display completely normal CRA. In conclusion, studies prompted by the identification of our C7-deficient patient with SLE revealed that patients with a selective homozygous terminal C component deficiency have an increased frequency (7%) of SLE despite a normal ability to solubilize immune complexes. Patients with selective homozygous deficiency of an early-acting C component have an even higher frequency (41%) of SLE, as well as a markedly reduced capacity to solubilize immune complexes. The data pesented here suggest that patients with inborn terminal C component deficiencies are predisposed to the development of SLE by a process which is independent of the C-mediated ability to solubilize immune complexes. Whether the compromised capacity for C-mediated immune complex solubilization in patients lacking ZEITZ ET AL an early-acting C component contributes to the greater incidence of SLE in these patients remains to be established. ACKNOWLEDGMENTS The authors wish to thank Lisbeth Suyehira for thoughtful assistance and Dr. Zakira Khan for referring this patient for study. REFERENCES 1. Agnello V: Complement deficiency states. Medicine 57: 123, 1978 2. Jersild C, Rubinstein P, Day NK: The HLA system and inherited deficiencies of the complement system. 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