492 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE, VOL. 70. Nos. 516.1976.

Human

schistosomiasis:

SUMIE HOSHINO-SHIMIZU,

Schistosoma mansoni in renal glomeruli”

antigen

detection

THALES DE BRITO, HERMMIA Y. KANAMURA, ABAETE L. CANTO, ADAVIO 0. SILVA, AFON~~ R. CAMPOS, DECIO 0. PENNA AND Lutz C. DA SILVA

Laboratory

of Immunology, “Institute de Medicina Tropical de S&o Paulo” and Department of Pathology (Medical School of Sao Paulo) S. Paul0 University, SLio Paulo, Brazil; Instituto de Medicina Tropical de Sao Pa&o, Av. Dr. Endas C. Aguiar, 470, Caixa Postal, 2921, Srio Paulo, Brazil SUIIUIXWY Twelve kidney, five biopsy and seven necropsy specimens, all from schistosomiasis mansoni patients were studied by light and immunofluorescent microscopy in an attempt to detect antigen in the glomerular walls. Deposits of IgM, IgG,I gA, IgE, complement C, and fibrinogen were observed in most cases. Antigen was successfully detected in two cases (one biopsy and one necropsy specimen), both exhibiting proliferative glomerulonephritis. The only clinical manifestation was a slight proteinuria. IgG antibodies eluted from the autopsy kidney homogenates showed specific binding mostly to Schistosoma mansoni gut, thus suggesting that the fixed antibodies (eluates) are, at least partially, constituted by antibodies similar to the anti-circulating antigen. These data reinforce the hypothesis that renal injury in schistosomiasis is mediated through an immune complex disease. Introduction

Previous workers have consistently shown deposits of IgG, IgM, IgA, IgE and complement C, in glomeruli of animals experimentally infected with S. mansoni (BRITO et al., 1971; ANDRADE & SUSIN, 1974; CAVALLO et al., 1974) and in kidney biopsies of patients with the hepatosplenic form of the disease (SILVA et al., 1970; BRITO et al., 1970). Moreover, circulating specific immune complexes were demonstrated in patients and animals infected with S. mansoni (BERGGREN & WELLER, 1967; CARLIER et al., 1975; SMITH et al., 1975; VOLLER et al., 1976). Rena1 disease in schistosomiasis is probably an immune complex nephritis, but antigen demonstration is necessary in order to confirm this theory. In experimental infections S. japonicum (TADA et al., 1975) and S. mansoni (NATALI & CIOLI, 1974) antigens have been demonstrated by immunofluorescent techniques. However, it is only recently that such findings have been reported in man. Antigen was shown in the glomeruli of a transplanted kidney in a patient with hepatosplenic schistosomiasis mansoni (FALC~O & GOULD, 1975).

The purpose of this paper is to complete a preliminary account of the demonstration of schistosomal antigens (S. mansoni) in human kidneys obtained at autopsy, and in percutaneous biopsies of patients with heavy schistosomal infections (HOSHINO-SHIMIZU et al., 1975). In *This work was partially supported by a Grant from CNPq.

addition, an attempt has been made to characterize the specificity of the antigens and antibodies of the immune complexes localized in the kidney. Materials

and methods

Kidney Specimens These were from two sources: (a) Five patients, four with the hepatosplenic and one with the intestinal form of schistosomiasis mansoni were submitted to percutaneous kidney biopsies. Except for a slight proteinuria, none had clinical evidence of renal disease; (b) Seven kidneys were obtained at autopsy from patients with the hepatosplenic form of the disease. Autopsy was performed six to 24 hours after death. Three kidney autopsies from non-schistosomiasis patients were used as controls. Light Microscopy One part of the tissue fragments was fixed in 10% formalin or Zenker’s fluid, embedded in paraffin, sections cut at 3 to 4 pm and routinely stained-by HE, PAS (periodic-acid. Schiff reagent) and PAMSM (periodic-acid-methenamine silve; counterstained by Masson’s trichrome stain). Immunofluorescence Test (a) Another kidney fragment obtained through biopsy was quickly frozen in liquid nitrogen and 4 pm sections were cut in a cryostat, dried on microscope slides at room temperature and stored at -70°C until used. Direct immunofluorescence test was performed after absorption of the conjugates with normal human kidney powder from autopsy. (b) Cryostat sections of adult S. mansoni embedded in Ames OCT Compound (WILSON et al., 1974) and sections of infected hamster’s liver with eggs and granulomata were tested as antigen against globulins eluted from the kidney. Antiserum Sheep antiserum to S. mansoni antigens was prepared by repeated injections of homogenate of recently harvested adult worms from infected mice. The first dose of the homogenate was mixed with complete, and subsequent doses with incomplete, Freund’s adjuvant. After three doses of this homogenate, the sheep were twice given adult worm incubation products (BIGUET et al., 1962), followed by three doses of the polysaccharide-like fraction of adult worm, corresponding to trichloroacetic acid-soluble chloroform extracted material from adult worms (NASH et al., 1974).

et

SUMIE HOSHINO-SHIMIZU

El&ions In order to test the specificity of the fixed globulins in the kidney, acid elutions were made according to LAMBERT & DIXON (1968). The eluates were lyophilized and diluted to 4 mg protein per ml. Kidney sections underwent the same acid elution to remove the globulins from the glomeruli and expose more antigen from within the complexes. In addition, 2M NaCI, 2M MgCb, 3M urea were used to dissociate the specific antibodies from the antigen. Gel Diffusion Test Sheep antiserum was collected after the 10th immunization and tested in immunodiffusion plates (SILVA et al., 1965) against the following S. mansoni antigens: adult worm extracts; incubation products of adult worms; polysaccharide-like material extracted from adult worms (NASH et al., 1974), cercariae, and sera from mice infected with S. mansoni and containing circulating antigenic material. Before the test, mouse sera and sheep antiserum were absorbed respectively with sheep and mouse blood cells to remove nonspecific precipitin lines (BERGGREN

& WELLER,

1967).

Conjugates Fluorescein isothiocyanate labelled sheep antiserum to S. mansoni antigens was used. Commercially* available conjugates to human IgG, IgM, IgA, IgE, complement Cn and fibrinogen were used after testing their specificity by immunoelectrophoresis. *Hyland Division Travenol Laboratories, Inc. Costa Mesa, California 92626, U.S.A. Serological Studies Sera collected from the biopsied patients were submitted to the indirect haemagglutination and immunoTable I - Results of the immunofluorescence of patients infected with S. mansoni

493

al.

fluorescence tests according to HOSHINO et al. (1970) and COUDERT et al. (1968). No serum was available from the autopsied cases. Rheumatoid Factor A search for rheumatoid factor was carried out in both the kidney eluates from the autopsies and the sera from the biopsied patients, according to SINGER & PLOTZ (1956).

Egg Counting It was performed according to

et al. (1972).

KATZ

Renal Function This was studied by creatinine and phenolsulphonphthalein clearances. At least four urinary sediments were performed in each of the biopsied patients. Results The main results both of the immunofluorescence tests and histopathological findings are presented in Table I. The kidney injury as seen by light microscopy in the necropsy material ranged from different degrees of proliferative to mixed proliferative and membranous glomerulonephritis. Proliferative glomerulonephritis was characterized by hypertrophy and hyperplasia of axial cells. When present, the membranous component of the mixed glomerulonephritis was usually seen at the peripheral loops of the glomeruli. Only in case No. 6A was it marked and diffuse, prevailing over the proliferative component. Viable eggs surrounded by a slight granulomatous reaction were detected in the kidney parenchyma from the autopsy specimen No. 3A, from a patient with proliferative glomerulonephritis. One egg, without granulomatous reaction, was seen impacted at the afferent glomerular artery (Fig. 1, C).

test and histopathological

findings in kidney of seven autopsies and five biopsie

Eluates of kidney fixed-globulinsCase No. 1A 2A 3A 4A 5A

6A

Id+

4+

2+ 4+

I@

2Ik 2+ 2+

2+

-

3+ 4f

2+ 2+

3+

2+ _ 3+ 3+ 3+ 2+ -

7A

2+

-

1B 2B 3B

3f 3+ 4+ 3+ 4+

2+ 2+ 4+ -

2+

2+

3+

1_

1_

4B

%A c3AC 2,4

4+ 2+ 3+ _ 3+

2+ 4+

2+

-

2+

_x

C3

Fibr.

Ag.

4+ -

2t 4+ 4

2+

2+

3+ 3+ 2+ 4+ -

2+

2+

3+ -

-

-

2+

2+

2+

A 3+

-1

-3f

-

Glomerular changes IgG ab to IgG ab to worm miracidium

2+

PG 4+

2+

PGl+

-

-

PG 3+ MG

3+ -

2+

PGl+

-

-

nd nd nd nd nd

nd nd nd nd nd

-

-

N

PG4+ nd PG l+ N

2+

PG l+ PG l+ N

_1

:

-

-

-

PG - proliferative glomerulonephritis; MG - mixed (proliferative and membranous) glomerulonephritis; N - normal; C - control cases; A - Autopsy; Ab - antibody; Fibr. - fibrinogen; Ag - antigen; nd -not done. The intensity of the proliferative changes seen in the glomeruli were graded from 1 to 4.

494

Schistosoma mansoni ANTIGEN DETECTION IN RENAL GLOMERULI

The biopsy material, however, showed less well marked lesions. Three cases were of slight proliferative glomerulonephritis. One case showed normal kidney. In the remaining case only kidney medulla was obtained for light microscopy. IgM fluorescent deposits were found in all 12 cases, IgG in nine cases, IgE and complement C3 in eight cases, and IgA and fibrinogen in seven cases. In case No. 4A, a patient with severe injury, only IgM was detected (Table I). Immunoglobulins, complement C3 and fibrinogen showed a confluent granular pattern mainly along the mesangium. Antigenic material was demonstrated in case No. 3A and 5B. A faint reaction (l+) was observed in cases No. 1A and 3B but was not considered positive for discussion (Table I). The antigenic material appeared as fluorescent small granules along the mesangium and also focally on the glomerular walls (Fig. 1, A and B), roughly following the same pattern of the immunoglobulin deposits. Elutions of the fixed globulins from the kidney sections failed to reveal more antigen. Sheep antiserum developed 11 precipitin lines in gel diffusion test against adult worm extract, three against incubation products of adult worms, and one against polysaccharide-like material from adult worms or cercariae. Also, the circulating antigenic material in the mice sera showed one precipitin line against this antiserum. Reactions of identity among these S. mansoni antigens were seen. Conjugate prepared with the sheep antiserum revealed antigen in the kidney, This conjugate did not stain antigen in the glomeruli after absorption with lyophilized adult worms, or incubation products of worms, polysac&ride-like materials from worms or cercariae. This shows that the small fluorescent dots were not artefacts. Specific IgG antibodies were eluted from kidney homogenates in cases Nos. 1A and 3A. Bright fluorescence of the digestive tract and a weak fluorescence of the integment of the worm sections were observed. (Fig. 1, D). Miracidia were also stained in the sections of infected hamster’s liver. As seen in the Table II, slight proteinuria was observed in all biopsied patients. Renal clearances were normal. Egg countings showed heavy infection, varying from 380 to 7,000 eggs per gram of faeces. Hypergammaglobulinaemia was observed in all patients. Antibody levels by the immunofluorescence and indirect haemagglutination tests, were high. No rheumatoid factor was detected in serum or in kidney eluates.

Table II - Clinical

Discussion

One of the mechanisms of glomerular disease depends upon the patient producing antibodies which are capable of reacting with non-glomerular antigens in the circulation to form soluble circulating antigen-antibody complexes which are subsequently trapped in the glomerular capillary walls. This appears to be the mechanism of renal injury in chronic infectious diseases like schistosomiasis, malaria and kala-azar (KIBUKAMUSOKE, 1973: BRITO et al.. 1975) where the circulating immunocomplexes are presented over a long period to the kidney, eventually producing glomerular disease which is characterized by various clinical and laboratory features. These range from slight proteinuria, with overall preservation of renal function (SILVA et a/., 1970), to advanced clinical disease (BRITO et al., 1970; LEHMAN et al., 1975). Previous work has consistently shown deposits of immunoglobulins, complement CJ and fibrinogen in glomeruli of animals experimentally infected with S. mansoni (BRITO et al., 1971; ANDRADE et al., 1974; CAVALLOet aI., 1974) and in kidney biopsies of patients with the hepatosplenic form of the disease (SILVA et a/., 1970; BRITO et a/., 1970). Experimentally, antigen can be demonstrated in the circulation (GOLD et a/., 1969; BAWDENet al., 1974) or in the kidney (TADA et al., 1975) early in the disease after heavy inoculations of cercariae. Initially, the immune complexes deposited in the glomeruli are those formed in a zone of antigen or slight antibody excess. Later, the antigenic material is not easily detected, probably because of the formation of larger immune complexes by direct binding of circulating antibodies to the remaining antigen molecules in the glomerular walls (LAMBERTet al., 1973). These experimental studies have their counterpart in the natural disease of man. It should be easier to demonstrate antigen in recent infections, as in experimental animals, or in heavy infections without advanced renal lesions, as in our two patients. One of the main causes of the technical failure to detect antigen in the glomerular deposits in schistosomiasis is the conjugate used. We succeeded only when adequate sheep antiserum to S. mansoni was obtained. IgG antibodies eluted from the kidney homogenates of two autopsies reacted mainly with adult worm gut. These findings strongly suggest that the fixed antibodies (eluates) are, at least partially, constituted by antibodies similar to the anti-circulating antigens (LICHTENBERG et al., 1974). A significant amount of S. mansoni antigen was detected in unisexual infection but involvement of egg

data of patients with S. mansoni infection submitted to kidney biopsy

Serum antibodies Patient 1. HVS MCN JBS MAS

2. 3. 4. 5.

MDX

Sex age twrs)

Egg/g of stools

M-19 F -12 M- 9 F -15 F -13

330 7,000 6,530 506 2,800

IHAT -indirect haemagglutination HI - hepatointestinal.

Mean daily Gamma proteinuria globulins (g %) 0.12 0.15 0.17 o-15 0.18

test (titres);

g g g g g

2.35 2.23 2.89 2.74 240

IHAT

FAT

Clinical form

2,560 640 640 640 640

2,560 1,280 640 640 320

HS HI HS HS HS

FAT - fluorescent antibody

test (titres);

HS - hepatosplenic;

SUMIE HOSHINU-SHIMIZU

Fig. I, A. Antigen granules are demonstrated by direct immunofluorescence along the mesangium and walls of the glomerular loops. Arrows point to main groups of antigenic material. Only part of glomerulus is seen at the picture. Necropsy specimen. x 500. Fig. I, B. Similar distribution of antigen granules in part of the glomerulus in a biopsy specimen. (Arrows).

et d.

Fig. I, C. Kidney exhibiting proliferative glomerulitis. A viable egg of S. mansoni is seen impacted at the afferent glomerular artery. Necropsy specimen. HE, x ZOO. Fig. 1, D. Fluorescent staining of the digestive tract ofadult S. mansoni worms after treatment with eluates of kidney fixed-antibodies and anti-human IgG conjugare. x 80.

496

Schistosoma

mansoni

ANTIGEN

DETECTION

IN RENAL

GLOMERULI

antigen cannot be entirely ruled out. Antigen was demonstrated only in heavily infected patients with the hepatosplenic form of the disease, when there is massive antigen liberation with probable formation of soluble immune complexes. Massive antigen liberation from worms destroyed by chemotherapy is another possible source of soluble immune complexes. In one kidney control, IgM and fibrinogen were detected. The kidney controls were obtained from nonschistosomiasis patients, but the possibility that they had previously suffered from other infectious diseases cannot be discarded. Catabolism of antigen-antibody complexes may exist in man and might be responsible for the occasional occurrence of such immune complexes in normal kidneys.

Hoshino, S., Camargo, M. E. & Silva, L. C. da (1970). Standardization of a haemagglutination test for schistosomiasis with formalin treated human erythrocytes.

References Andrade, Z. & Susin, M. (1974). Renal changes in mice infected with Schistosoma mansoni. American Journal

Lambert, P. H. & Dixon, F. J. (1968). Pathogenesis of the glomerulonephritis of NZBjW mice. Journal of

of Tropical Medicine

and Hygiene, 23,4@%403. Bawden, M. P. & Weller, T. H. (1974). Schistosoma mansoni circulating antigen: detection by complement

fixation in sera from infected hamsters and mice. American Journal of Tropical Medicine and Hygiene, 23,

1077-1084. Berggren, W. L. & Weller, T. H. (1967). Immunoelectrophoretic demonstration of specific circulating antigen in animals infected with Schistosoma mansoni. American Journal 16,606-612.

of Tropical

Medicine

and Hygiene,

Biguet, J., Capron, A. & Tran Van Ky, P. (1962). Les antigknes de Schistosoma mansoni. I. Etude Blectrophor&ique. Caract&isation des antigknes seifiques. knnalesde

I’lnstitut

Pasteur, 103, 7631777.

-

-

Brito, T. de, Gunji, J., Camargo, M. E., Penna, D. 0. & Silva, L. C. da (1970). Advanced kidney disease in patients with hepatosplenic Manson’s schistosomiasis. Revista do Instituto 12,225-235.

de Medicina

Trovical

de Srio Pa&o.

ization, 45,419-422.

Brito, T: de, Hoshino-Shim& S., Amato Neto, V., Duarte. 1. S. & Penna. D. 0. (1975). , Glomerular involvement in human kala-azar. A light, immunofluorescent and electron microscopic study based on kidney biopsies. American Journal of Tropical Medicine and Hygiene, 24,9-18.

Cavallo, T., Galvanek, E. G., Ward, P. A. & Lichtenberg, F. von (1974). The nephropathy of experimental hepatosplenic schistosomiasis. American Journal of 76,433-445.

Coudert, J., Ambroise-Thomas, P., Pothier, M. A. & Kien Truong, T. (1968). Diagnostic sCrologique par immunofluorescence sur coupes li la cong&lation d’infections B S. mansoni, S. haematobium et S. intercalatum. Acta Tropica (Base& 25, 109-132. Faldo, H. A. & Gould, D. B. (1975). Immune complex nephropathy in schistosomiasis. Anna/s of Internal Medicine, 83, 148-l 54. Gold, R., Rosen, F. S. & Weller, T. H. (1969). A specific circulating antigen in hamsters infected with Schistosoma mansoni. Detection of antigen in serum and urine, and correlation between antigenic concentration and worm burden. American Journal of Tropical Medicine

and Hygiene,

463-470. Hoshino-Sbimizu, S., Brito, T. de, Canto, A. L., Kanamura, H. Y. & Silva, L. C. da (1975). Detection of schistosomal antigen (S. mansoni) in human kidneys obtained at autopsy. Revista de Znstituto de Medicina TrooicaI de SCo Paula. 17, 394-397.

Katz,-N., Chaves, A. & Pellegrino, J. (1972). A simple device for quantitative stool thick smear technique in schistosomiasis mansoni. Revista do Znstituto de Medicina

Tropical de Sdo Paulo, 14, 397-400. J. W. (1973). The nephrotic syndrome of quartan malaria. London: Edward Arnold, p. 1.

Kibukamusoke,

Experimental

Medicine,

127, 507-521.

Lambert, P. H., Bricteux, N., Salmon, J. & Miescher, P. A. (1973). Dynamics of immune complex nephritis during antibody excess. International Archives of Allergy, 45, 185. Lehman, J. S. Jr., Mott, K. E., Souza, C. A. M., Leboreiro, 0. & Muniz, T. M. (1975). The association of schistosomiasis mansoni and proteinuria in an endemic area. American Journal of Tropical Medicine and Hygiene, 24,616-618.

Lichtenberg, F. von, Bawden, M. P. & Shealy, S. H. (1974). Origin of circulating antigen from the schistosome gut. An immunofluorescent study. American Journal of Tropical Medicine and Hygiene, 23, 10881091. Nash, T. E., Prescott, B. & Neva, F. A. (1974). The characteristics of a circulating antigen in schistosomiasis. Journal of Immunology, 112, 1500-1507. Natali, P. G. & Cioli, D. (1974). Immune complex nephritis in mice infected with Schistosoma mansoni. Federation Proceedings, 33, 757.

Brito, T. de, Gunji, J., Camargo, M. E., Ceravolo, A. & Silva. L. C. da (1971). Glomerular lesions in exoerimental infections of ‘Schistosoma mansoni in de&us apella monkeys. Bulletin of the World Health Organ-

Pathology,

American Journal of Tropical Medicine and Hygiene, 19,

18, 545-552.

Silva, L. C. da & Ferri, R. G. (1965). Immunodiffusion studies in human schistosomiasis mansoni. I. Hepatointestinal and hepatosplenic forms. Revista do lnstituto de Medicina Tropical de Srio Paula, 7, l-6. Silva, L. C. da, Brito, T. de, Camargo, M. E., Boni, D. R., Lopes, J. D. & Gunji, J. (1970). Kidney biopsy in the hepatosplenic form of infection with Schistosoma mansoni. Bulletin

of the World Health

Organization,

42,907-910. Singer, J. M. & Plotz, C. M. (1956). The latex fixation test. I. Application to the serologic diagnosis of rheumatoid arthritis. American Journal of Medicine, 21,888-892.

Smith, M., Verroust, P. J., Morel-Maroger, L. M., Pasticier. A. & Coulaud. J. P. (1975). Circulating immune ‘complexes in sch&.tosomia&s. British Medi&Z Journal, ii, 274. Tada, T., Kondo, Y., Okumura, K., Sano, M. & Yokogawa, M. (1975). Schistosoma japonicum: immunopathology of nephritis in Macaca fascicularis. Experimental Parasitology, 38, 291-302. Voller, A., Bartlett, A. & Bidwell, D. E. (1976). Enzyme immunoassays for parasitic diseases. Transactions of the Royal Society of Tropical

Medicine

and Hygiene,

70,98-106. Wilson, M., Sulzer, J. A. & Walls, K. W. (1974). Modified antigens in the indirect immunofluorescent test for schistosomiasis. American Journal of Tropical Medicine and Hygiene, 23, 1072-1076.