Clin. exp. Immunol. (1979) 37, 221-227.
Circulating immune complexes in acute schistosomiasis T. J. LAWLEY, E. A. OTTESEN, R. A. HIATT & L. A. GAZZE The Dermatology Branch, National Cancer Institute and the Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA, and San Juan Laboratories, Center for Disease Control, San Juan, Puerto Rico
(Acceptedfor publication 22 December 1978)
SUMMARY
The sera of patients with acute and chronic schistosomiasis were tested for the presence of circulating immune complexes with the 125I-Clq binding assay. Fourteen out of fifteen (93%°) patients
with acute schistosomiasis had elevated 125I-Clq binding activity, while only two out of eleven
(18%) patients with chronic disease had Clq binding complexes. This difference was significant (P< 0.001) and paralleled the degree of clinical disease activity between the two groups of patients. IgG and IgM were readily detected in all of these circulating complexes but the specific parasite antigens initiating their formation could not be defined. The level of circulating immune complexes was inversely correlated with the absolute eosinophil counts for individuals in the acutely infected group, an observation compatible with the hypothesis that a functional role for the eosinophil is the destruction and elimination of immune complexes.
INTRODUCTION In recent years evidence has accumulated which indicates that circulating immune complexes may be involved in the pathogenesis of human schistosomiasis. A number of workers have demonstrated the presence of granular basement membrane deposits in the renal glomeruli of patients with chronic infection, and immunoglobulins, complement and even parasite antigens have on occasion been identified at these sites (Andrade & Quieroz, 1968; DeBrito et al., 1970; DaSilva et al., 1970; Andrade, Andrade & Sodigursky, 1971; Quieroz et al., 1973; Falcao & Gould, 1975; Hoshino-Shimizu et al., 1976). Furthermore, following the recent development of sensitive assays for the detection of circulating immune complexes, two groups also studying individuals with chronic infection have reported the presence of low levels of such complexes in the sera of a variable percentage of their patients (Smith, Verroust & MorelMaroger, 1977; Bout et al., 1977). In contrast to chronic schistosomiasis, the clinical manifestations of the acute phase of the infection in man (the Katayama fever syndrome) include certain features similar to those found in serum sickness and other diseases associated with immune complexes (Warren, 1976). It has, therefore, been suspected that such complexes might play a role in the pathogenesis of this syndrome, but the presence of immune complexes during this early period of infection has not been previously documented, both for technical reasons and because of the difficulty in identifying and then studying patients when they first acquire this infection in endemic regions. In the present report we describe our findings from studies of fifteen patients with well documented acute Schistosomiasis mansoni and contrast the very high incidence of '25I-Clq binding immune complexes found in the sera of these patients with the relatively low frequency of such complexes found in individuals with chronic schistosome infections. The nature of the complexes and their relationship to clinical disease activity in individual patients are also explored. Correspondence: Dr T. J. Lawley, Dermatology Branch, National Institutes, of Health, National Cancer Institute, Building 10, Room 12N238, Bethesda, Maryland 20014, USA. 0099-9104/79/0800-0221$02.00 A) 1979 Blackwell Scientific Publications
221
222
T. 9. Lawley et al. MATERIALS AND METHODS
Patient population. The fifteen patients with acute schistosomiasis were part of a larger study group reported in detail elsewhere (Hiatt et al., 1979). They were primarily city dwellers from Puerto Rico who had well-defined, isolated exposures to fresh-water streams documented to contain schistosome-infected snails. All manifested the signs and symptoms of acute schistosomiasis including fever, arthralgias, myalgias, diarrhoea and peripheral blood eosinophilia and all had viable Schistosoma mansoni eggs in their stools. The intensity of infection (determined by stool egg counts using the Kato and modified Ritchie techniques) varied from light to heavy (Hiatt et al., 1979). Their ages ranged from 11 to 29 years. Geometric mean egg count of the acute infection group was 124 eggs/g of stool with a range of 1-4192 eggs/g. The eleven patients with chronic infection included nine Puerto Ricans from a well-characterized endemic area (Cline et al., 1977) and two North Americans. All had had infection documented for 3 or more years but were asymptomatic at the time of study and treatment at the National Institutes of Health. Their ages ranged from 11 to 33 years. Geometric mean egg count in this chronic infection group was 907 eggs/g of stool with a range of 100-2250 eggs/g. Serum samples. Sera were collected from acutely infected individuals 6-15 weeks after their exposure to infection and from chronically infected patients just prior to treatment. They were stored at - 70'C and thawed immediately before testing. The sera of thirty-one blood bank donors, which served as controls, were handled in the same way. Purification of human Clq. Clq was purified from freshly drawn human blood according to the method of Yonemasu & Stroud (1971). Purity was checked by double diffusion in gel against rabbit antisera to human Clq, IgG, IgA, IgM and C3; immunoelectrophoresis (IEP) against these same rabbit antisera as well as against anti-whole human serum; and radial immunodiffusion on LC-partigen plates (Behring Diagnostics). No contaminating IgG, IgA, IgM or C3 was found and the Clq preparation gave a single precipitin line against anti-whole human serum on IEP. Purified Clq was aliquoted and stored at -70'C in 0-75 M NaCl, 0 01 M EDTA, 0-02 M sodium acetate buffer pH 7-5. Radioiodination of Clq. Radiolabelling of purified human Clq with 1251 was performed with the Bolton-Hunter reagent (iodinated p-hydroxyphenylpropionic acid, N-hydroxysuccinimide ester) (New England Nuclear, Boston, Massachusetts) using a modification of the technique of Bolton & Hunter (1973). Specific activity of the 125I-Clq used in these assays was 0 1-02 puCi/pg Clq. 125I-Clq binding assay (polyethyleneglycolprecipitation). The Clq binding assay was modified from that described by Zubler & Lambert (1976). In brief, 0-2 ml of test serum was incubated with 0 4 ml of Na2EDTA (0-2 M, pH 7-5) for 30 min at 37°C. After the addition of 200 ng of 125I-Clq, 1 ml of 4% (w/v) polyethylene glycol (PEG) (carbowax, mol.wt 6000, Fischer Scientific Co., Fairlawn, New Jersey) was added to the mixture to make a final concentration of 2.5% PEG. The mixture was incubated on ice for 1 hr and then centrifuged at 1000 g for 20 min. The supernatant was decanted and the residual radioactivity was counted without washing the pellet. Results are expressed as a percentage of the protein-bound'251 precipitated (as determined by 10% trichloroacetic acid precipitation). All samples were tested in duplicate. A standard normal human serum (NHS) was used as a negative control in each assay. Positive controls consisted of serial dilutions of heataggregated human gammaglobulin (HGG) (Miles Laboratories, Inc., Kanakee, Illinois) added to a standard normal human serum. The upper 95% confidence limit of Clq binding activity (BA) in the control population was 10%. Immunoglobulin, complement and antibody derminations. Serum levels of IgG, IgA, IgM and C3 were measured by radial immunodiffusion (Behring Diagnostics, Somerville, New Jersey). Serum IgE levels were measured by radioimmunoassay (Pharmacia Fine Chemicals, Piscataway, New Jersey). The presence of IgG, IgA, IgM and C3 in the Clq precipitates was assayed for by radial immunodiffusion of the re-solubilized precipitate on LC-Partigen plates (Behring Diagnostics). Anti-schistosome antibody titres were measured by a complement fixation (CF) test employing adult worm antigen as described previously (Chaffee, Bauman & Shapilo, 1954). Other laboratory tests. Absolute peripheral blood eosinophil counts were determined on all patients using pre-diluted counting vials (Unopette, Becton-Dickinson Co., Rutherford, New Jersey). Liver function tests (serum alkaline phosphatase, serum glutamic oxaloacetic transaminase and serum lactic dehydrogenase) and renal function assessments (urinalysis, blood urea nitrogen and serum creatinine) were carried out for all individuals. Statistical analyses. Laboratory data was analyzed by Spearman rank correlation, the Fisher exact test and Student's t-test.
RESULTS Immune complexes Elevated '25I-Clq BA (> 10%) was found in the sera of fourteen out of fifteen (930%) patients with acute schistosomiasis (Fig. 1). The geometric mean Clq BA was 340% with a range of 2-65%. In chronic schistosomiasis only two out of eleven patients (18%) had increased Clq BA and the geometric mean was 900 with a range of 2-49%. The incidence of immune complexes in these two groups was significantly different at the P< 0-001 level (Fisher exact test). Clq precipitates were analysed for the presence of immunoglobulin and complement (C3) in all patients with Clq BA > 10%. Moderate amounts of both IgM (> 17.6 mg/dl) and IgG (> 13.3 mg/dl)
Imuuiune complexes in schist(osonuasmis
223
70 60
_~50 50 *, 40_
230 20 10
__ ____.__-._-
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___
__
Chronic Acute Schistosomiosis Schistosomissis
1* 1C. 1. 1251-Clq binding activity in the sera of patients with acute and chronic schistosomiasis and controls.
could be detected in these complexes, but only trace amounts of IgA, IgE and C3. Efforts Were made using double diffusion in gel and enzyme-linked immunosorbant techniques to detect schistosome antigens in these complexes, but the results were equivocal.
Serunm immunoglobulins, complemzent and anti-schistosome antibodies Serum IgM levels were elevated (>280 mg/dl) in most individuals from both patient groups, but the geometric mean level of IgM in patients with acute disease (634 mg/dl) was significantly higher than that in patients with chronic infection (282 mg/dl P< 0 0005) (Fig. 2). Increased serum IgG (> 1800 mg/dl) was also found in the majority of individuals and geometric mean levels for both patient groups were not significantly different (Fig. 2). Serum IgE levels were elevated (>200 u/ml) in most patients of both groups, but the geometric mean IgE level in patients with acute schistosomiasis (755 u/ml) was significantly lower than that in patients with chronic infection (2033 u/ml) (P< 005) (Fig. 2). Serum IgA levels were normal in all patients. Serum C3 levels were elevated (> 120 mg/dl) in eight out of fourteen patients with acute disease, but were normal in all but one patient with chronic infection. Anti-schistosome antibodies could be demonstrated by the complement fixation technique in the sera of all patients. Those with acute schistosomiasis had significantly higher titres (geometric mean titre 338; range, 64-1024) than did chronically infected individuals (geometric mean titre, 87; range, 8-2048)
(P500/mm3) peripheral blood eosinophil counts. Eosinophilia ranged from 266/mm3 to 10,567/mm3 with a mean of 4503/mm3. Ten out of eleven patients with chronic schistosomiasis had increased absolute eosinophil counts. Their range was from 383/mm3 to 1894/mm3, with a mean of 862/mm3. Although elevated in both groups, the absolute eosinophil count was significantly higher (P< 0-005) in patients with acute schistosomiasis than in those with chronic infection. Correlation Of l2 5JIC I q BA mit/i other disease paraimeters The results of the 125II-Clq binding assay in patients with acute schistosomiasis were evaluated ill relation to the individuals' clinical signs and symptoms, serum immunoglobulin and C3 levels, antischistosome antibody titres, liver function tests, renal function parameters and peripheral blood eosinophil counts. As can be seen in Fig. 3, a significant inverse correlation weas noted between the Clq BA
224
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counts in
patients with acute schistosomiasis
and peripheral blood eosinophilia (rho =- 50554, P< 0025). No other meaningful correlation could be found between the levels of these complexes and the various clinical and laboratory parameters of infection studied.
DISCUSSION Acute schistosomiasis occurs 1-4 months after cercarial penetration in previously uninfected individuals. Its clinical manifestations are varied but may include fever, cough, arthralgia, diarrhoea, lymphadenopathy, urticaria and eosinophilia. Warren (1976) has suggested that the pathogenesis of the syndrome may
Immune complexes in schistosomiasis
225
be associated with the formation of circulating immune complexes. Antibody formation commences shortly after cercarial penetration and is further stimulated at the time of initial oviposition (5-8 weeks) when thousands of eggs may be produced daily. In such a setting of antibody production in the presence of presumptive antigen excess one could anticipate the formation of circulating immune complexes capable of giving rise to a serum sickness-like clinical picture. Clq, a subcomponent of the first component of complement, is known to bind to immune complexes whose antibody moiety is either IgM or IgG (subclasses 1-3) and can precipitate them in the test conditions utilized. Although various polyanions, particularly DNA and endotoxin, can also bind Clq, they will not precipitate in this test and thus do not cause false positive results (Zubler & Lambert, 1976; T. J. Lawley, unpublished observation). The finding of increased '25I-Clq binding activity (Clq BA) in the sera of fourteen out of fifteen patients with acute schistosomiasis is the first demonstration of circulating immune complexes in man during the acute phase of this infection. The high incidence (930%) of elevated Clq BA in these individuals contrasts with the significantly lower incidence (18%) in patients with chronic schistosomiasis and indeed parallels the differences in clinical activity found between the acute and chronic stages of the disease. None of our chronically infected patients was symptomatic from his schistosome infection, save for the occasional complaint of increased fatigue. There was neither hepatosplenic nor renal disease in this group of patients. It is unlikely, however, that the absence of such patients with overt pathology introduced bias into our estimate of the low incidence of elevated Clq BA in chronically infected patients for two reasons. Firstly, Bout et al. (1977) have indicated that patients with hepatosplenic disease do not actually have higher levels of circulating immune complexes than other individuals with chronic schistosomiasis without hepatosplenic disease. Secondly, immune complex nephropathy, itself a relatively rare manifestation of chronic schistosomiasis, has been shown to be associated with even lower levels of circulating immune complexes than are found in patients with chronic infection without renal injury (Brito et al., 1979). It is interesting that the two chronically infected patients in our study with elevated Clq BA did not differ clinically from the rest of the chronic infection group. In contrast to these chronically infected patients, all of our patients with acute schistosomiasis were symptomatic with the findings already described above and elsewhere (Hiatt et al., 1979). Unfortunately, however, despite our analyses of various clinical parameters including the presence of fever, diarrhoea, nausea, vomiting, arthralgias and myalgias, percentage weight loss and intensity of infection, we were unable to correlate the presence of specific clinical findings in acutely infected individuals with their levels of circulating immune complexes. Such correlations might well imply important aetiological relationships, and if they do exist our inability to detect them could have resulted from the relatively restricted period of time each person's clinical activity and immune complex levels were evaluated. Serial observations on individual patients over a longer period of time might help to resolve this important issue. One of the most intriguing observations from the present study was the inverse correlation seen between the absolute eosinophil level and the amount of circulating immune complexes in acutely infected patients. It has long been postulated from the results of experiments both in animal models and in vitro that a potential functional role for the eosinophil is the ingestion and destruction of immune complexes (Ishikawa, Wicher & Arbesman, 1974). While much remains unknown about even the basic mechanisms which underlie the generation and regulation of the eosinophilia developing during acute schistosomiasis, the data shown in Fig. 3 certainly suggest a functional relationship between eosinophils and immune complexes during this natural infection of man. Furthermore, although the preponderance of normal levels of Clq BA in the patients with chronic disease does not allow a meaningful statistical comparison with the eosinophil counts, it should be noted that the two chronic patients with evidence of circulating immune complexes also had the two lowest eosinophil counts in that group of patients. The differences in immunoglogulin and antibody levels for patients with acute and chronic schistosomiasis (Fig. 2) are consistent with previous observations which have contrasted features of the host's immune response during the two stages of this parasitic helminth infection (Ottesen et al., 1978; Hiatt
226
7'. 5. Laiwlcy et ai.
et al., 1979). It was interesting that no patients in either group had depressed serum C3 levels; indeed over half of the acute cases studied had elevations of C3. Although depressed C3 levels in immune complex disease might be anticipated, it is known from C3 turnover studies that serum C3 levels are relatively insensitive parameters of complement activation because of their role as acute phase reactants. In addition in several connective tissue and infectious diseases normal or elevated serum C3 levels have been found, despite the presence of high levels of circulating immune complexes (Nydegger et al., 1977; Walker et al., 1978). At present the composition of these complexes is largely undefined. While all complexes contained appreciable amounts of IgM and IgG, the nature of the antigen initiating formation of the complexes is unknown. Whether there are specific types of parasite antigens which tend to form immune complexes, or whether any of the circulating antigens described in schistosome infections (Berggren & Weller, 1967; Nash, Prescott & Neva, 1974) are related to the complexes detected in this study, or even whether host antigens and autoantibodies were being detected by our Clq binding assay, all are unknown. It is clear, then, that subsequent studies of these circulating immune complexes in acute and chronic schistosomiasis must focus onl two important issues-firstly, the problem of the immunochemical composition of the complexes, and secondly, a more extensive evaluation of their relationship to the pathophysiology involved in the clinical manifestations of this important parasitic disease. We would like to thank Dr Z. Roberto Sotomavor, Chief Bilharzia Clinic, University of Puerto Rico MNledical Center for his generous assistance in the study of the patients with acute schistosomiasis.
REFERENCES ANDRADE, Z.A. & QUEIROZ, A.C. (1968) Lesoes renais an equistosomose hepatosplenica. Rev. Inst. Med. Trop.
Sao Paulo, 10, 36. ANDRADE, Z.A., ANDRADE, S.G. & SODIGURSKY, MN1. (1971) Renal changes in patients with hepatosplenic schistosomiasis. Amer. 3. Trop. Med. Hyg. 20, 77. BERGGREN, W.L. & WELLER, T.H. (1967) Immunoelectrophoretic demonstration of specific circulating antigen in animals infected with Schisloswma miiauisow. Amier. 3. Ty-op. Med. Hyg. 16, 606. BOLTON, A.E. & HUNTER, W.M. (1973) The labelling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agent. Application to the radioimmunoassay. Bilochem. 7. 133, 529. BOUT, D., SANTORO, F., CARLIER, Y., BINA, J.C. & CAPRON, A. (1977) Circulating immune complexes in schistosomiasis. Immunology,, 33, 17. BRITo, E., SANTORO, F., ROCHA, H., DUTRA, M. & CAPRON, A. (1978) Circulating immune complexes in schistosomiasis patients with and without nephropathy. Bilomedicine. (In press.) CHAFFEE, E.F., BAUNIAN, P.M. & SHAPILO, J.J. (1954) Diagnosis of schistosomiasis by complement fixation. Amer. 3. Trop. Med. Hyg. 3, 905. CI.INE, B.L., RYZMO, W.T., HIATT, R.A., KNIGHT, W.B. & BERRIOS-DURAN, L.A. (1977) Morbidity from Schistosoma manisoni in a Puerto Rican community: a population based study. Amer. 3. Trop. Med. Hyg. 26, 109. DASILVA, L.C., DEBRITO, T., CAMARGO, M.E., BONI, D., 1)ELOPEZ, J.D. & GUNJI, J. (1970) Kidney biopsy in the hepatosplenic form of infection with SclhistosoKna mansoin: in man. Bull. WIt'd HIh Org. 42, 907. DEBRITO, T., GUNJI, J., CAMARGO, MNI.D., PENNA, D.O. & DASILVA, L.C. (1970) Advanced kidney disease in patients with hepatosplenic Manson's schistosomiasis. Rev. Inist. Med. Trop. Sao Paulo, 12, 225.
FALCAO, H.R. & GOULD, D.B. (1975) Immune complex nephropathy in schistosomiasis. Ann. Int. Med. 83, 148. HIATT, R.A., SOTOMAYOR, Z.R., SANCHEZ, G., ZAMBRANA, MI. & KNIGHT, W.B. (1979) Factors in the pathogenesis of acute Schist(osomiasis mansoni.3. infect. Dis. (In press.) HOSHINO-SHINIZU, S., DEBRITO, T., KANAMURA, H.Y., CANTO, RO.., SILVA, A.O., CAMPOS, A.R., PENNA, D.O. & DASILVA, L.C. (1976) Human schistosomiasis: Schistosoma mansoni antigen detection in renal glomeruli. Trans. roy. Soc. Trop. Med. Hyg. 70, 492. ISHIKAWA, T., WICHER, K. & ARBESMAN, C.E. (1974) In vitro and iln vivo studies on uptake of antigen-antibody complexes by eosinophils. Int. Arch. Allerigy appl. Immunol. 46, 230. NASH, T.E., PRESCOTT, B. & NEVA, F.A. (1974) The characteristics of a circulating antigen in schistosomiasis. 7. Immunol. 112, 1500. NYDEGGER, U.E., ZUBLER, R.H., GABAY, R., JOLIAT, G., KARAGEVREKIS, LAMBERT, P.H. & MIESCHER, P.O. (1977) Circulating complement breakdown products in patients with rheumatoid arthritis. 7. c/in. Invest. 59, 862. OTTESEN, E.A., HIATT, R.A., CHEEVER, A.W., SOTOMAYOR, Z.R. & NEVA, F.A. (1978) The acquisition and loss of antigen-specific cellular responsiveness in acute and chronic schistosomiasis in man. C/in. exp. Immitnol. 33, 38. QUIEROZ, F.P., BRITO, E., MARTINELLI, R. & ROCHA, H. (1973) Nephrotic syndrome in patients with Schistosoma mansoni infection. Amer.]. 7rop. Med. Hyg. 22, 622. SMITH, M.D., VERROUST, P.J. & MOREL-NMAROGER, L. (1977) A study of the presence of circulating immune complexes in schistosomiasis. 7Trans. roy. Soc. 7rop. Med. Hyg. 70, 492. WALKER, L.C., EHLIN, T.D., TUNG, K.S.K. & WIl.LIAMS, R.C. (1978) Circulating immune complexes in disseminated gonorrheal infection. Ann. Int. Med. 89, 28.
Immune complexes in schistosomiasis WARREN, K.S. (1976) Schistosomiasis: A multiplicity of immunopathology. J. Invest. Dermatol. 67, 464. YoNEMAsu, K. & STROUD, R.M. (1971) Clq: rapid purification method for preparation of monospecific antisera and for biochemical studies. J. Immunol. 106, 304.
227 2 5I-Clq
ZUBLER, R.H. & LAMBERT, P.H. (1976) The binding test for the detection of soluble immune complexes. In Vitro Methods in Cell-Mediated and Tumor Immunity (ed. by B. R. Bloom and J. R. David), p. 565. Academic Press, New York.
Circulating immune complexes in acute schistosomiasis T. J. LAWLEY, E. A. OTTESEN, R. A. HIATT & L. A. GAZZE The Dermatology Branch, National Cancer Institute and the Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA, and San Juan Laboratories, Center for Disease Control, San Juan, Puerto Rico
(Acceptedfor publication 22 December 1978)
SUMMARY
The sera of patients with acute and chronic schistosomiasis were tested for the presence of circulating immune complexes with the 125I-Clq binding assay. Fourteen out of fifteen (93%°) patients
with acute schistosomiasis had elevated 125I-Clq binding activity, while only two out of eleven
(18%) patients with chronic disease had Clq binding complexes. This difference was significant (P< 0.001) and paralleled the degree of clinical disease activity between the two groups of patients. IgG and IgM were readily detected in all of these circulating complexes but the specific parasite antigens initiating their formation could not be defined. The level of circulating immune complexes was inversely correlated with the absolute eosinophil counts for individuals in the acutely infected group, an observation compatible with the hypothesis that a functional role for the eosinophil is the destruction and elimination of immune complexes.
INTRODUCTION In recent years evidence has accumulated which indicates that circulating immune complexes may be involved in the pathogenesis of human schistosomiasis. A number of workers have demonstrated the presence of granular basement membrane deposits in the renal glomeruli of patients with chronic infection, and immunoglobulins, complement and even parasite antigens have on occasion been identified at these sites (Andrade & Quieroz, 1968; DeBrito et al., 1970; DaSilva et al., 1970; Andrade, Andrade & Sodigursky, 1971; Quieroz et al., 1973; Falcao & Gould, 1975; Hoshino-Shimizu et al., 1976). Furthermore, following the recent development of sensitive assays for the detection of circulating immune complexes, two groups also studying individuals with chronic infection have reported the presence of low levels of such complexes in the sera of a variable percentage of their patients (Smith, Verroust & MorelMaroger, 1977; Bout et al., 1977). In contrast to chronic schistosomiasis, the clinical manifestations of the acute phase of the infection in man (the Katayama fever syndrome) include certain features similar to those found in serum sickness and other diseases associated with immune complexes (Warren, 1976). It has, therefore, been suspected that such complexes might play a role in the pathogenesis of this syndrome, but the presence of immune complexes during this early period of infection has not been previously documented, both for technical reasons and because of the difficulty in identifying and then studying patients when they first acquire this infection in endemic regions. In the present report we describe our findings from studies of fifteen patients with well documented acute Schistosomiasis mansoni and contrast the very high incidence of '25I-Clq binding immune complexes found in the sera of these patients with the relatively low frequency of such complexes found in individuals with chronic schistosome infections. The nature of the complexes and their relationship to clinical disease activity in individual patients are also explored. Correspondence: Dr T. J. Lawley, Dermatology Branch, National Institutes, of Health, National Cancer Institute, Building 10, Room 12N238, Bethesda, Maryland 20014, USA. 0099-9104/79/0800-0221$02.00 A) 1979 Blackwell Scientific Publications
221
222
T. 9. Lawley et al. MATERIALS AND METHODS
Patient population. The fifteen patients with acute schistosomiasis were part of a larger study group reported in detail elsewhere (Hiatt et al., 1979). They were primarily city dwellers from Puerto Rico who had well-defined, isolated exposures to fresh-water streams documented to contain schistosome-infected snails. All manifested the signs and symptoms of acute schistosomiasis including fever, arthralgias, myalgias, diarrhoea and peripheral blood eosinophilia and all had viable Schistosoma mansoni eggs in their stools. The intensity of infection (determined by stool egg counts using the Kato and modified Ritchie techniques) varied from light to heavy (Hiatt et al., 1979). Their ages ranged from 11 to 29 years. Geometric mean egg count of the acute infection group was 124 eggs/g of stool with a range of 1-4192 eggs/g. The eleven patients with chronic infection included nine Puerto Ricans from a well-characterized endemic area (Cline et al., 1977) and two North Americans. All had had infection documented for 3 or more years but were asymptomatic at the time of study and treatment at the National Institutes of Health. Their ages ranged from 11 to 33 years. Geometric mean egg count in this chronic infection group was 907 eggs/g of stool with a range of 100-2250 eggs/g. Serum samples. Sera were collected from acutely infected individuals 6-15 weeks after their exposure to infection and from chronically infected patients just prior to treatment. They were stored at - 70'C and thawed immediately before testing. The sera of thirty-one blood bank donors, which served as controls, were handled in the same way. Purification of human Clq. Clq was purified from freshly drawn human blood according to the method of Yonemasu & Stroud (1971). Purity was checked by double diffusion in gel against rabbit antisera to human Clq, IgG, IgA, IgM and C3; immunoelectrophoresis (IEP) against these same rabbit antisera as well as against anti-whole human serum; and radial immunodiffusion on LC-partigen plates (Behring Diagnostics). No contaminating IgG, IgA, IgM or C3 was found and the Clq preparation gave a single precipitin line against anti-whole human serum on IEP. Purified Clq was aliquoted and stored at -70'C in 0-75 M NaCl, 0 01 M EDTA, 0-02 M sodium acetate buffer pH 7-5. Radioiodination of Clq. Radiolabelling of purified human Clq with 1251 was performed with the Bolton-Hunter reagent (iodinated p-hydroxyphenylpropionic acid, N-hydroxysuccinimide ester) (New England Nuclear, Boston, Massachusetts) using a modification of the technique of Bolton & Hunter (1973). Specific activity of the 125I-Clq used in these assays was 0 1-02 puCi/pg Clq. 125I-Clq binding assay (polyethyleneglycolprecipitation). The Clq binding assay was modified from that described by Zubler & Lambert (1976). In brief, 0-2 ml of test serum was incubated with 0 4 ml of Na2EDTA (0-2 M, pH 7-5) for 30 min at 37°C. After the addition of 200 ng of 125I-Clq, 1 ml of 4% (w/v) polyethylene glycol (PEG) (carbowax, mol.wt 6000, Fischer Scientific Co., Fairlawn, New Jersey) was added to the mixture to make a final concentration of 2.5% PEG. The mixture was incubated on ice for 1 hr and then centrifuged at 1000 g for 20 min. The supernatant was decanted and the residual radioactivity was counted without washing the pellet. Results are expressed as a percentage of the protein-bound'251 precipitated (as determined by 10% trichloroacetic acid precipitation). All samples were tested in duplicate. A standard normal human serum (NHS) was used as a negative control in each assay. Positive controls consisted of serial dilutions of heataggregated human gammaglobulin (HGG) (Miles Laboratories, Inc., Kanakee, Illinois) added to a standard normal human serum. The upper 95% confidence limit of Clq binding activity (BA) in the control population was 10%. Immunoglobulin, complement and antibody derminations. Serum levels of IgG, IgA, IgM and C3 were measured by radial immunodiffusion (Behring Diagnostics, Somerville, New Jersey). Serum IgE levels were measured by radioimmunoassay (Pharmacia Fine Chemicals, Piscataway, New Jersey). The presence of IgG, IgA, IgM and C3 in the Clq precipitates was assayed for by radial immunodiffusion of the re-solubilized precipitate on LC-Partigen plates (Behring Diagnostics). Anti-schistosome antibody titres were measured by a complement fixation (CF) test employing adult worm antigen as described previously (Chaffee, Bauman & Shapilo, 1954). Other laboratory tests. Absolute peripheral blood eosinophil counts were determined on all patients using pre-diluted counting vials (Unopette, Becton-Dickinson Co., Rutherford, New Jersey). Liver function tests (serum alkaline phosphatase, serum glutamic oxaloacetic transaminase and serum lactic dehydrogenase) and renal function assessments (urinalysis, blood urea nitrogen and serum creatinine) were carried out for all individuals. Statistical analyses. Laboratory data was analyzed by Spearman rank correlation, the Fisher exact test and Student's t-test.
RESULTS Immune complexes Elevated '25I-Clq BA (> 10%) was found in the sera of fourteen out of fifteen (930%) patients with acute schistosomiasis (Fig. 1). The geometric mean Clq BA was 340% with a range of 2-65%. In chronic schistosomiasis only two out of eleven patients (18%) had increased Clq BA and the geometric mean was 900 with a range of 2-49%. The incidence of immune complexes in these two groups was significantly different at the P< 0-001 level (Fisher exact test). Clq precipitates were analysed for the presence of immunoglobulin and complement (C3) in all patients with Clq BA > 10%. Moderate amounts of both IgM (> 17.6 mg/dl) and IgG (> 13.3 mg/dl)
Imuuiune complexes in schist(osonuasmis
223
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230 20 10
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Chronic Acute Schistosomiosis Schistosomissis
1* 1C. 1. 1251-Clq binding activity in the sera of patients with acute and chronic schistosomiasis and controls.
could be detected in these complexes, but only trace amounts of IgA, IgE and C3. Efforts Were made using double diffusion in gel and enzyme-linked immunosorbant techniques to detect schistosome antigens in these complexes, but the results were equivocal.
Serunm immunoglobulins, complemzent and anti-schistosome antibodies Serum IgM levels were elevated (>280 mg/dl) in most individuals from both patient groups, but the geometric mean level of IgM in patients with acute disease (634 mg/dl) was significantly higher than that in patients with chronic infection (282 mg/dl P< 0 0005) (Fig. 2). Increased serum IgG (> 1800 mg/dl) was also found in the majority of individuals and geometric mean levels for both patient groups were not significantly different (Fig. 2). Serum IgE levels were elevated (>200 u/ml) in most patients of both groups, but the geometric mean IgE level in patients with acute schistosomiasis (755 u/ml) was significantly lower than that in patients with chronic infection (2033 u/ml) (P< 005) (Fig. 2). Serum IgA levels were normal in all patients. Serum C3 levels were elevated (> 120 mg/dl) in eight out of fourteen patients with acute disease, but were normal in all but one patient with chronic infection. Anti-schistosome antibodies could be demonstrated by the complement fixation technique in the sera of all patients. Those with acute schistosomiasis had significantly higher titres (geometric mean titre 338; range, 64-1024) than did chronically infected individuals (geometric mean titre, 87; range, 8-2048)
(P500/mm3) peripheral blood eosinophil counts. Eosinophilia ranged from 266/mm3 to 10,567/mm3 with a mean of 4503/mm3. Ten out of eleven patients with chronic schistosomiasis had increased absolute eosinophil counts. Their range was from 383/mm3 to 1894/mm3, with a mean of 862/mm3. Although elevated in both groups, the absolute eosinophil count was significantly higher (P< 0-005) in patients with acute schistosomiasis than in those with chronic infection. Correlation Of l2 5JIC I q BA mit/i other disease paraimeters The results of the 125II-Clq binding assay in patients with acute schistosomiasis were evaluated ill relation to the individuals' clinical signs and symptoms, serum immunoglobulin and C3 levels, antischistosome antibody titres, liver function tests, renal function parameters and peripheral blood eosinophil counts. As can be seen in Fig. 3, a significant inverse correlation weas noted between the Clq BA
224
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and peripheral blood eosinophilia (rho =- 50554, P< 0025). No other meaningful correlation could be found between the levels of these complexes and the various clinical and laboratory parameters of infection studied.
DISCUSSION Acute schistosomiasis occurs 1-4 months after cercarial penetration in previously uninfected individuals. Its clinical manifestations are varied but may include fever, cough, arthralgia, diarrhoea, lymphadenopathy, urticaria and eosinophilia. Warren (1976) has suggested that the pathogenesis of the syndrome may
Immune complexes in schistosomiasis
225
be associated with the formation of circulating immune complexes. Antibody formation commences shortly after cercarial penetration and is further stimulated at the time of initial oviposition (5-8 weeks) when thousands of eggs may be produced daily. In such a setting of antibody production in the presence of presumptive antigen excess one could anticipate the formation of circulating immune complexes capable of giving rise to a serum sickness-like clinical picture. Clq, a subcomponent of the first component of complement, is known to bind to immune complexes whose antibody moiety is either IgM or IgG (subclasses 1-3) and can precipitate them in the test conditions utilized. Although various polyanions, particularly DNA and endotoxin, can also bind Clq, they will not precipitate in this test and thus do not cause false positive results (Zubler & Lambert, 1976; T. J. Lawley, unpublished observation). The finding of increased '25I-Clq binding activity (Clq BA) in the sera of fourteen out of fifteen patients with acute schistosomiasis is the first demonstration of circulating immune complexes in man during the acute phase of this infection. The high incidence (930%) of elevated Clq BA in these individuals contrasts with the significantly lower incidence (18%) in patients with chronic schistosomiasis and indeed parallels the differences in clinical activity found between the acute and chronic stages of the disease. None of our chronically infected patients was symptomatic from his schistosome infection, save for the occasional complaint of increased fatigue. There was neither hepatosplenic nor renal disease in this group of patients. It is unlikely, however, that the absence of such patients with overt pathology introduced bias into our estimate of the low incidence of elevated Clq BA in chronically infected patients for two reasons. Firstly, Bout et al. (1977) have indicated that patients with hepatosplenic disease do not actually have higher levels of circulating immune complexes than other individuals with chronic schistosomiasis without hepatosplenic disease. Secondly, immune complex nephropathy, itself a relatively rare manifestation of chronic schistosomiasis, has been shown to be associated with even lower levels of circulating immune complexes than are found in patients with chronic infection without renal injury (Brito et al., 1979). It is interesting that the two chronically infected patients in our study with elevated Clq BA did not differ clinically from the rest of the chronic infection group. In contrast to these chronically infected patients, all of our patients with acute schistosomiasis were symptomatic with the findings already described above and elsewhere (Hiatt et al., 1979). Unfortunately, however, despite our analyses of various clinical parameters including the presence of fever, diarrhoea, nausea, vomiting, arthralgias and myalgias, percentage weight loss and intensity of infection, we were unable to correlate the presence of specific clinical findings in acutely infected individuals with their levels of circulating immune complexes. Such correlations might well imply important aetiological relationships, and if they do exist our inability to detect them could have resulted from the relatively restricted period of time each person's clinical activity and immune complex levels were evaluated. Serial observations on individual patients over a longer period of time might help to resolve this important issue. One of the most intriguing observations from the present study was the inverse correlation seen between the absolute eosinophil level and the amount of circulating immune complexes in acutely infected patients. It has long been postulated from the results of experiments both in animal models and in vitro that a potential functional role for the eosinophil is the ingestion and destruction of immune complexes (Ishikawa, Wicher & Arbesman, 1974). While much remains unknown about even the basic mechanisms which underlie the generation and regulation of the eosinophilia developing during acute schistosomiasis, the data shown in Fig. 3 certainly suggest a functional relationship between eosinophils and immune complexes during this natural infection of man. Furthermore, although the preponderance of normal levels of Clq BA in the patients with chronic disease does not allow a meaningful statistical comparison with the eosinophil counts, it should be noted that the two chronic patients with evidence of circulating immune complexes also had the two lowest eosinophil counts in that group of patients. The differences in immunoglogulin and antibody levels for patients with acute and chronic schistosomiasis (Fig. 2) are consistent with previous observations which have contrasted features of the host's immune response during the two stages of this parasitic helminth infection (Ottesen et al., 1978; Hiatt
226
7'. 5. Laiwlcy et ai.
et al., 1979). It was interesting that no patients in either group had depressed serum C3 levels; indeed over half of the acute cases studied had elevations of C3. Although depressed C3 levels in immune complex disease might be anticipated, it is known from C3 turnover studies that serum C3 levels are relatively insensitive parameters of complement activation because of their role as acute phase reactants. In addition in several connective tissue and infectious diseases normal or elevated serum C3 levels have been found, despite the presence of high levels of circulating immune complexes (Nydegger et al., 1977; Walker et al., 1978). At present the composition of these complexes is largely undefined. While all complexes contained appreciable amounts of IgM and IgG, the nature of the antigen initiating formation of the complexes is unknown. Whether there are specific types of parasite antigens which tend to form immune complexes, or whether any of the circulating antigens described in schistosome infections (Berggren & Weller, 1967; Nash, Prescott & Neva, 1974) are related to the complexes detected in this study, or even whether host antigens and autoantibodies were being detected by our Clq binding assay, all are unknown. It is clear, then, that subsequent studies of these circulating immune complexes in acute and chronic schistosomiasis must focus onl two important issues-firstly, the problem of the immunochemical composition of the complexes, and secondly, a more extensive evaluation of their relationship to the pathophysiology involved in the clinical manifestations of this important parasitic disease. We would like to thank Dr Z. Roberto Sotomavor, Chief Bilharzia Clinic, University of Puerto Rico MNledical Center for his generous assistance in the study of the patients with acute schistosomiasis.
REFERENCES ANDRADE, Z.A. & QUEIROZ, A.C. (1968) Lesoes renais an equistosomose hepatosplenica. Rev. Inst. Med. Trop.
Sao Paulo, 10, 36. ANDRADE, Z.A., ANDRADE, S.G. & SODIGURSKY, MN1. (1971) Renal changes in patients with hepatosplenic schistosomiasis. Amer. 3. Trop. Med. Hyg. 20, 77. BERGGREN, W.L. & WELLER, T.H. (1967) Immunoelectrophoretic demonstration of specific circulating antigen in animals infected with Schisloswma miiauisow. Amier. 3. Ty-op. Med. Hyg. 16, 606. BOLTON, A.E. & HUNTER, W.M. (1973) The labelling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agent. Application to the radioimmunoassay. Bilochem. 7. 133, 529. BOUT, D., SANTORO, F., CARLIER, Y., BINA, J.C. & CAPRON, A. (1977) Circulating immune complexes in schistosomiasis. Immunology,, 33, 17. BRITo, E., SANTORO, F., ROCHA, H., DUTRA, M. & CAPRON, A. (1978) Circulating immune complexes in schistosomiasis patients with and without nephropathy. Bilomedicine. (In press.) CHAFFEE, E.F., BAUNIAN, P.M. & SHAPILO, J.J. (1954) Diagnosis of schistosomiasis by complement fixation. Amer. 3. Trop. Med. Hyg. 3, 905. CI.INE, B.L., RYZMO, W.T., HIATT, R.A., KNIGHT, W.B. & BERRIOS-DURAN, L.A. (1977) Morbidity from Schistosoma manisoni in a Puerto Rican community: a population based study. Amer. 3. Trop. Med. Hyg. 26, 109. DASILVA, L.C., DEBRITO, T., CAMARGO, M.E., BONI, D., 1)ELOPEZ, J.D. & GUNJI, J. (1970) Kidney biopsy in the hepatosplenic form of infection with SclhistosoKna mansoin: in man. Bull. WIt'd HIh Org. 42, 907. DEBRITO, T., GUNJI, J., CAMARGO, MNI.D., PENNA, D.O. & DASILVA, L.C. (1970) Advanced kidney disease in patients with hepatosplenic Manson's schistosomiasis. Rev. Inist. Med. Trop. Sao Paulo, 12, 225.
FALCAO, H.R. & GOULD, D.B. (1975) Immune complex nephropathy in schistosomiasis. Ann. Int. Med. 83, 148. HIATT, R.A., SOTOMAYOR, Z.R., SANCHEZ, G., ZAMBRANA, MI. & KNIGHT, W.B. (1979) Factors in the pathogenesis of acute Schist(osomiasis mansoni.3. infect. Dis. (In press.) HOSHINO-SHINIZU, S., DEBRITO, T., KANAMURA, H.Y., CANTO, RO.., SILVA, A.O., CAMPOS, A.R., PENNA, D.O. & DASILVA, L.C. (1976) Human schistosomiasis: Schistosoma mansoni antigen detection in renal glomeruli. Trans. roy. Soc. Trop. Med. Hyg. 70, 492. ISHIKAWA, T., WICHER, K. & ARBESMAN, C.E. (1974) In vitro and iln vivo studies on uptake of antigen-antibody complexes by eosinophils. Int. Arch. Allerigy appl. Immunol. 46, 230. NASH, T.E., PRESCOTT, B. & NEVA, F.A. (1974) The characteristics of a circulating antigen in schistosomiasis. 7. Immunol. 112, 1500. NYDEGGER, U.E., ZUBLER, R.H., GABAY, R., JOLIAT, G., KARAGEVREKIS, LAMBERT, P.H. & MIESCHER, P.O. (1977) Circulating complement breakdown products in patients with rheumatoid arthritis. 7. c/in. Invest. 59, 862. OTTESEN, E.A., HIATT, R.A., CHEEVER, A.W., SOTOMAYOR, Z.R. & NEVA, F.A. (1978) The acquisition and loss of antigen-specific cellular responsiveness in acute and chronic schistosomiasis in man. C/in. exp. Immitnol. 33, 38. QUIEROZ, F.P., BRITO, E., MARTINELLI, R. & ROCHA, H. (1973) Nephrotic syndrome in patients with Schistosoma mansoni infection. Amer.]. 7rop. Med. Hyg. 22, 622. SMITH, M.D., VERROUST, P.J. & MOREL-NMAROGER, L. (1977) A study of the presence of circulating immune complexes in schistosomiasis. 7Trans. roy. Soc. 7rop. Med. Hyg. 70, 492. WALKER, L.C., EHLIN, T.D., TUNG, K.S.K. & WIl.LIAMS, R.C. (1978) Circulating immune complexes in disseminated gonorrheal infection. Ann. Int. Med. 89, 28.
Immune complexes in schistosomiasis WARREN, K.S. (1976) Schistosomiasis: A multiplicity of immunopathology. J. Invest. Dermatol. 67, 464. YoNEMAsu, K. & STROUD, R.M. (1971) Clq: rapid purification method for preparation of monospecific antisera and for biochemical studies. J. Immunol. 106, 304.
227 2 5I-Clq
ZUBLER, R.H. & LAMBERT, P.H. (1976) The binding test for the detection of soluble immune complexes. In Vitro Methods in Cell-Mediated and Tumor Immunity (ed. by B. R. Bloom and J. R. David), p. 565. Academic Press, New York.
