A N T I B O D I E S T O L A M I N I N I N C H A G A S ' D I S E A S E * BY ANA S Z A R F M A N , V I C T O R P. T E R R A N O V A , S T E P H E N I. R E N N A R D , J E A N - M I C H E L F O I D A R T , M A R I A DE F A T I M A LIMA, J O N I. SCHEINMAN,
AND G E O R G E R. M A R T I N
From the Laboratory of Developmental Biology and Anomalies, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20205; Transplantation Research Branch, Casualty Care Research Program Center, Naval Medical Research Institute, Bethesda, Maryland 20814; Instituto de Microbiologia, Universidade Federal de Rio de Janeiro, Ilha do Funddo, Rio de Janeiro 21910, Brazi# and
Department of Pediatrics, University of Minnesota, Minneapolis 55455
C h a g a s ' disease occurs in C e n t r a l a n d S o u t h A m e r i c a in i n d i v i d u a l s i n f e c t e d w i t h the p r o t o z o a n Trypanosoma cruzL In t h e a c u t e stage of t h e disease, s u b c u t a n e o u s swelling a r o u n d t h e eye ( c h a g o m a ) , m y o c a r d i t i s , e n c e p h a l i t i s , a n d fever a r e observed. T h e s e c o n d i t i o n s are r e l a t e d to t h e i n v a s i o n o f t h e tissues b y t h e p a r a s i t e (1). Years later, in t h e c h r o n i c stage o f C h a g a s' disease, w h e n few, if a n y , p a r a s i t e s are p r e s e n t in t h e tissues, i n f l a m m a t o r y , d e g e n e r a t i v e , a n d fibrotic c h a n g e s are o b s e r v e d , p a r t i c - u l a r l y in the e n d o c a r d i u m , m y o c a r d i u m , e s o p h a g u s , a n d colon (1, 2). S u c h lesions c a n be life t h r e a t e n i n g (1).
I m m u n o l o g i c a l causes are t h o u g h t to u n d e r l i e t h e c h r o n i c lesions in C h a g a s ' disase (3). S e r a from b o t h a c u t e a n d c h r o n i c cases o f C h a g a s ' disease show r e a c t i v i t y w i t h t h e p a r a s i t e as well as w i t h n o r m a l e n d o c a r d i u m , b l o o d vessels, a n d t h e i n t e r s t i t i u m o f s t r i a t e d m u s c l e (the so-called E V I 1 p a t t e r n ) , a n d S c h w a n n s h e a t h s o f p e r i p h e r a l nerve (4-6). S t u d i e s on skeletal m u s c l e biopsies o f i n d i v i d u a l s i n f e c t e d w i t h T. cruzi i n d i c a t e the existence o f i m m u n o g l o b u l i n s (Ig) w i t h a s i m i l a r d i s t r i b u t i o n (4). T h e e x t e n t o f t h e clinical a l t e r a t i o n s f o u n d in i n d i v i d u a l s w i t h c h r o n i c C h a g a s ' disease was c o r r e l a t e d w i t h these t i s s u e - r e a c t i n g a n t i b o d i e s in one (4) b u t not in o t h e r g e o g r a p h i c a l a r e a s (7). T h e s e a n t i b o d i e s were not f o u n d to o c c u r in v a r i o u s o t h e r h e a r t diseases e x a m i n e d (4). H e r e , we d e m o n s t r a t e t h a t these a n t i b o d i e s react w i t h c o n n e c t i v e tissue structures. W e also h a v e a s s a y e d p u r i f i e d c o m p o n e n t s o f c o n n e c t i v e tissues for t h e i r r e a c t i o n w i t h t h e a n t i b o d i e s p r e s e n t in h u m a n s a n d in R h e s u s m o n k e y s i n f e c t e d w i t h T. cruzi. O u r studies i n d i c a t e t h a t these a n t i b o d i e s are specific for l a m i n i n (8), a b a s e m e n t m e m b r a n e g l y c o p r o t e i n , w h i c h m e d i a t e s the a t t a c h m e n t
* Supported in part by funds provided by the Brazilian Research Council (CNPq), Financiadora de Estudos e Projectos (FINEP), Brazil; the United Nations Development Programme World Bank/WHO special program for research and training in tropical diseases; and research task MF58.524.013.1031 from the Naval Medical Research and Development Command. The opinions and assertions contained herein are the private ones of the writers and are not to be construed as official or reflecting the views of the Navy Department of the naval service at large. The experiments reported herein were conducted according to the principles set forth in the current edition of the "Guide for the Care and Use of Laboratory Animals," Institute of Laboratory Animal Resources, National Research Council.
1 Abbreviations used in this paper: ANA, antinuclear antibodies; CHO, chinese hamster ovary; EHS tumor, Engelbreth-Holm-Swarm tumor; ELISA, enzyme-linked immunoabsorbent assay; EVI, endocar- dium, blood vessels and interstitium; FN, fibroneetin; IF, immunofluorescence; LIT, ox liver-infusion tryptose; PBS, phosphate-buffered saline; pepsin fragments of laminin: PI, Mr ~ 285,000; Pli, Mr -~ 272,000; Pb, Mr = 70,000; Pc, Mr ~ 52,000.
o f e p i t h e l i a l a n d e n d o t h e l i a l cells to t y p e I V c o l l a g e n (9). M a t e r i a l s a n d M e t h o d s
Patients. Sera were examined from nine patients, ages 18-40, with chronic Chagas' disease (five with cardiomegaly a n d three with megacolon) in whom the infection had been present for at least 5 yr (mean 7 yr). In addition, sera were studied from six patients between the ages of 3 mo and 4 yr in whom acute Chagas' disease was present. Each child had unilateral chagoma of the eye a n d acute myocarditis within the last 2 mo, a n d the diagnosis was confirmed by identifying parasites in blood smears. All patients had a positive serology (titers _>1:64) for T. cruzi by the indirect immunofluorescence (IF) test using cultured epimastigote forms of the parasite (Tulahuen strain) (10). Sera of 20 normal individuals a n d 10 patients with inflam- matory diseases other than Chagas' disease (3 with osteomyelitis, 4 with paracoccidioidomycosis, a n d 4 with bacterial meningitis) from the same geographical areas (Argentina a n d Brazil), all showed a negative reaction for T. cruzi (10). Additional negative controls included sera from 10 patients with surgical wounds a n d 10 with invasive neoplasms of the breast a n d digestive system, 10 with toxoplasmosis, 10 with rheumatoid arthritis, a n d 10 with malaria, all from the United States.
Infected Monkeys. T h e insect Dzpetalogaster maximus, infected 3 mo earlier with blood from an acute case of Chagas' disease, was kindly supplied by Dr. P. Marsden from the University of Brasilia, Brasilia, Brazil. Two Rhesus monkeys were infected u n d e r the eyelid with 1,000 metacyclic trypomastigotes obtained from the feces of the insect. After infection of the monkey, T. cruzi trypomastigotes were observed in blood smears, a n d 1 mo after infection the animals were found to have developed antibodies against the parasite (titer ___ 1:64) (10).
Parasites. H u m a n foreskin fibroblasts strain C R L 1475 (kindly provided by Dr. W. Gleiber, National Institutes of Health), which do not synthesize laminin, were grown in Dulbecco's modified Eagle's medium (Gibco Laboratories, G r a n d Island Biological Co., G r a n d Island, NY) with 4% fetal calf serum (Gibco Laboratories). When confluent, cells were infected with blood trypomastigote forms of T. cruzi, T u l a h u e n strain (kindly provided by Dr. T. Mercado, National Institutes of Health). T h e amastigote a n d trypomastigote stages were obtained from the supernatant fluids from cultures 5 d after infection. Epimastigotes were cultured in ox liver- infusion tryptose (LIT) medium p H 7.2 as described (11). Blood trypomastigotes were purified as described (12). T h e parasites were fixed with 0.5% paraformaldehyde, allowed to dry on slides, a n d used as antigens in an indirect IF test (10). T h e purified antibodies were used at a concentration of 50 /tg/ml. T h e u n b o u n d fraction was reconstituted to the original serum volume a n d used diluted 1:10.
Laminin. L a m i n i n was extracted from the Engelbreth-Holm-Swarm (EHS) t u m o r grown in C57 BI mice a n d was isolated as previously described (8). For additional purification, 10 mg of laminin, dissolved in 1 ml of 8 M urea, 0.05 M Tris-HCl, p H 8.6, was applied to a column of DEAE-cellulose (2.5 X 25 era) a n d eluted with a gradient of 0-0.3 M NaCI. Portions of the collected fractions were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis or tested as antigen in an enzyme-linked immunoabsorbent assay (ELISA) (sera were diluted 1:50, a n d purified antibodies were used at 50/~g/ml) (see below). Various fragments of mouse l a m i n i n (PI from soluble l a m i n i n a n d Pli, Pb, and Pc from insoluble laminin), obtained from l a m i n i n by digestion with pepsin (Boehringer, Federal Republic of Germany) (20 m g / g laminin), were a gift from Dr. Rupert Timpl, Max Planck Institut fiir Biochemie, Martinsried, Federal Republic of G e r m a n y (13). L a m i n i n was also tested after reaction with an equal weight of trypsin a n d Streptomyces griseus protease (Sigma Chemical Co., St. Louis, MO). Each of these antigens were tested at a concentration of 500 ng of antigen per well.
Gel electrophoresis of the protein precipitated by serum from either control or infected monkeys, or of the affinity-purified antibodies, was carried out as described (8). Rabbits a n d sheep were i m m u n i z e d with l a m i n i n as previously described (8). The sera obtained from these animals, as well as sera from monkeys infected with T. cruzi, were chromatographed on a column of laminin-Sepharose (8). U n b o u n d material was washed from the column with 0.02 M phosphate-buffered saline (PBS), p H 7.4, a n d the b o u n d material containing the affinity- purified antibodies was eluted with 0.5 M NaCI, 0.5 M acetic acid, p H 3. The fractions
SZARFMAN ET AL. 1163 containing protein were adjusted to pH 7, dialyzed against PBS, and concentrated by uhrafihration.
Immunoassays. Indirect IF (4) was used to test the reaction of antibodies obtained from humans and monkeys with freshly prepared cryostat sections of mouse peripheral nerve, skin, kidney, heart, and the EHS mouse tumor. The protease inhibitors p-hydroxymercuribenzoate (50 #g/ml) and phenylmethylsulfonyl fluoride (50 #g/ml) (Sigma Chemical Co.) were present during all steps of the reaction. In some experiments, the same section was exposed first to antibodies from infected monkeys purified by laminin-Sepharose affinity chromatography (50 #g/ml) and then to antilaminin antibodies purified from rabbit serum (50 #g/ml). The tissue distribution of these antibodies was established with species-specific secondary fluorochromes (rhodamine conjugated goat anti-human IgG and fluorescein conjugated goat antirabbit IgG) (N. L. Cappel Laboratories, Inc., Cochranville, PA).
Attempts were made to identify masked antigens by treating the tissue sections for 3 h with chondroitinase ABC (50 U/ml) (Miles Laboratories, Elkhart, IN) (14), testicular hyaluronidase (40 mg/ml) (Type IV, Sigma Chemical Co.), or purified bacterial collagenase form III (500 U / ml), Advance Biofactures, Lynbrook, NY) (15) before reaction with antibodies.
The blocking reaction with collagen types I, III, IV, or V, fibronectin (FN), BM-1 proteo- glycan, chondronectin, laminin, or bovine serum albumin was performed at antigen concentra- tions of 150 btg/ml for 1 h at 37°C and overnight at 4°C. The antigens used were obtained from the following sources: type I collagen from lathyritic rat skin (16), type III collagen from a pepsin digest of fetal calf serum (17), type IV collagen from the EHS tumor (18), type V (cr- AB) from calf skin (19), FN from mouse serum (20), BM-1 proteoglycan from the EHS tumor
(21), and chondroneein from chicken serum (22). When one of these materials inhibited fluorescence, a titer was obtained by absorbing the sera with different concentrations of antigen. The reactivity of sera from humans or Rhesus monkeys infected with T. cruzi with these components of connective tissue and laminin fragments (see above) were also tested by ELISA, using a concentration of 500 ng of antigen per well (23).
The class of antibodies reacting with parasites tissue sections or laminin was assessed by immunofluorescence or by ELISA using fluorescein or peroxidase-conjugated goat antihuman IgG or IgM (N. L. Cappel Laboratories, Inc.). Radial immunodiffusion of antibodies with laminin was examined by Ouchterlony immunodiffusion at 4°C using 1% agarose by standard procedures.
To demonstrate that laminin does not bind nonspecifically to other IgG molecules, we preincubated two sera containing antinuelear antibodies (ANA) with 150 ~g/ml of laminin for 1 h at 37°C and overnight at 4°C, before performing the immunofluorescent reaction. Nuclei of hepatocytes were used as antigen.
Attachment Assays. The attachment to a type IV collagen substrate of endothelial cells from sheep aorta, or of fibroblastic cells of the Chinese hamster ovary (CHO) line to plastic dishes, was measured in the presence and absence of sera or antibodies from infected primates as previously described (9).
R e s u l t s
Distribution of the Antigen. In p r e l i m i n a r y studies, a careful analysis was m a d e o f the histological structures t h a t were s t a i n e d b y t h e tissue-reacting a n t i b o d i e s in C h a g a s ' p a t i e n t s a n d infected m o n k e y sera. In general, t h e y r e a c t e d in those areas o f tissue w h e r e b a s e m e n t m e m b r a n e s were present. T h e p a t t e r n r e s e m b l e d t h a t o b t a i n e d w h e n a n t i b o d i e s to isolated b a s e m e n t m e m b r a n e c o m p o n e n t s , such as l a m i n i n , were used (8). As discussed below, the tissue-reacting a n t i b o d i e s c o u l d be isolated b y l a m i n i n S e p h a r o s e affinity c h r o m a t o g r a p h y .
T h e r e a c t i o n o f a n t i b o d i e s from infected m o n k e y s p u r i f i e d b y l a m i n i n - S e p h a r o s e affinity c h r o m a t o g r a p h y (Fig. 1, first c o l u m n ) was c o m p a r e d w i t h the r e a c t i o n o f r a b b i t a n t i l a m i n i n a n t i b o d i e s (Fig. 1, second c o l u m n ) . B o t h a n t i b o d i e s s h o w e d a n i d e n t i c a l a n d intense s t a i n i n g o f the e x t r a c e l l u l a r m a t r i x o f the E H S t u m o r (not shown), a n d o f p e r i p h e r a l nerve in the p e r i n e u r i u m , e p i n e u r i u m , a n d capillaries, a n d
ANTIBODIES TO LAMININ IN CHAGAS' DISEASE
Ftc. 1. Comparison by double immunofluorescence histochemistry of the tissue distribution of purified antibodies from monkeys infected with T. cruzi (first column) or from rabbits immunized with laminin (second column). Sections of mouse peripheral nerve (A and B), skin (C and D), kidney (E and F), and heart (G and H) are shown.
a less intense s t a i n i n g o f m y e l i n sheaths (Fig. 1 A a n d B). Differences in I F were o b s e r v e d w i t h o t h e r tissues. T h e a n t i b o d i e s from infected m o n k e y s showed a b r o a d d i s t r i b u t i o n a l o n g the e p i d e r m a l - d e r m a l j u n c t i o n o f skin, i n c l u d i n g the e p i d e r m i s (Fig. 1 C), whereas t h e a n t i l a m i n i n a n t i b o d i e s from r a b b i t s s h o w e d a l i n e a r d i s t r i b u - tion a l o n g the e p i d e r m a l - d e r m a l j u n c t i o n (Fig. 1 D). I n the k i d n e y , the a n t i b o d i e s from infected m o n k e y s strongly s t a i n e d p e r i t u b u l a r capillaries, g l o m e r u l a r e n d o t h e - l i u m , a n d m e s a n g i a t m a t r i x ; b u t r e a c t e d o n l y very w e a k l y w i t h the g l o m e r u l a r a n d
S Z A R F M A N E T AL. 1 1 6 5
tubular basement membranes and did not stain Bowman's capsule (Fig. 1 E), whereas rabbit antilaminin antibodies strongly stained all kidney basement membranes (Fig. 1 F). In the heart, the antibodies from infected monkeys localized in the endocardium, vessels, and interstitium in a diffuse distribution (Fig. 1 G) compared with the fine double line in certain regions with rabbit antilaminin antibodies (Fig. 1 H).
Prior treatment of tissue sections with chondroitinase ABC, hyaluronidase, and bacterial collagenase did not change the distribution or the intensity of the staining with antibodies from infected primates. T h e whole sera from monkeys and humans infected with T. cruzi reacted with the same histological structures as the purified antibodies from infected monkeys. Laminin (25 #g/ml) completely blocked the reaction while other purified connective tissue components (i.e., collagen types I, III, IV, and V, FN, BM-1 proteoglycan, or chondronectin) had no effect.
Immunochernical Characterization. ELISA assays indicated that sera from both acute and chronic patients contained antibodies to laminin and that the highest titers were present in the acute phase (Table I). No significant reaction of sera from patients infected with T. cruzi was observed with type I, III, IV, or V collagen, FN, BM-1 proteoglycan, or chondronectin. Sera from the various other healthy and diseased humans or from uninfected monkeys did not react with tissues as judged by immu- nofluorescence and did not contain antibodies to laminin as judged by ELISA tests (titers ~:1:64).
Monkeys and humans with acute T. crvzi infection had high titers (titers _>1:6000 and ~ 1:1024, respectively) of IgM and IgG antibodies, which reacted with tissues and with laminin. T h e sera at the chronic stage of the infection had no reacting IgM (_<I :2) and lower titers of IgG (I :500). T h e shift from IgM and IgG antibodies present
TAaLE I
Immunofluorescence (IF) and ELISA Titers for Antibodies to Connective Tissue Components in Sera from Patients Infected with T~ypanosoma cruzi*
Clinical stage IF:~
ELISA
Collagen type
Laminin b'N
I II1 IV V
BM-I Pro- Chondro- teoglycan nectin C h a g a s ' pa- Acute tients n = 6 Chronic n - ~ 9 Controls N o r m a l from en- n - - 20 demic area With o t h e r in- f l a m m a t o r y diseases n ~ 10 ~1024 --~4096 --<64 --<32 -<64 -<32 -<32 -<32 _<32 (1024-8192) (4096- ~ 5 1 2 ~51.2 -<64 --<32 -<64 -<32 -<32 -<32 -<32 (s12-204e) (512-2048) -<32 ! ----.32 N -<64 -<32 -<32 --<32 -<32 -<32 -<32 -<64 -<64 -<32 -<64 -<32 -<32 -<32 -<32 -<32
Statistically significant values are italicized. * Goat a n t i - h u m a n IgG was used as a second antibody.
:~ Indirect I F test using heart, skin, a n d E H S t u m o r cryostat sections as antigens. Each serum sample showed the same endpoint titer with all of the tissues studied.
i~The n u m b e r s in parenthesis represent the range of values. T w o sera had a n t i b o d y titers of 128.
at 3 m o o f infection to o n l y I g G a n t i b o d i e s at 12 m o after infection in m o n k e y s was verified w i t h the p u r i f i e d a n t i b o d i e s b y i m m u n o e l e c t r o p h o r e s i s a n d r a d i a l i m m u n o - diffusion.
L a m i n i n was p r e c i p i t a t e d from solution b y sheep a n t i l a m i n i n a n t i b o d i e s or b y s e r u m or a n t i b o d i e s p u r i f i e d from infected m o n k e y serum. T h e presence o f l a m i n i n in the p r e c i p i t a t e was e s t a b l i s h e d b y electrophoresis, b a s e d on the presence o f two c h a i n s w i t h M r = 400,000 a n d 200,000 (Fig. 2). In a d d i t i o n , p u r i f i e d a n t i b o d i e s o b t a i n e d from infected m o n k e y s were f o u n d to p r e c i p i t a t e l a m i n i n in a single line o f i d e n t i t y w i t h sheep a n t i l a m i n i n a n t i b o d i e s ( d a t a not shown).
W h e n l a m i n i n was c h r o m a t o g r a p h e d on D E A E - c e l l u l o s e u n d e r d i s s o c i a t i n g con- ditions (8 M urea), a single p e a k was e l u t e d (Fig. 3). Electrophoresis o f the fractions c o r r e s p o n d i n g to this p e a k r e v e a l e d the c h a r a c t e r i s t i c c h a i n s o f l a m i n i n . T h e s a m e p e a k c o n t a i n e d m a t e r i a l t h a t r e a c t e d in E L I S A tests w i t h the s e r u m from infected m o n k e y s a n d sheep a n t i l a m i n i n a n t i b o d i e s (Fig. 3), the p u r i f i e d a n t i b o d i e s from infected monkeys, or s e r u m from infected h u m a n s (not shown). N o r e a c t i o n was
Fro. 2. Gel electrophoresis of laminin immunoprecipitated with normal sheep serum (lane 1), normal monkey serum (lane 2), sheep antilaminin antiserum (lane 3), serum from a molakey infected with T. cruzi (lane 4), antibodies purified by laminin affinity chromatography of 4 (lane 5), and unbound fraction after laminin affinity chromatography of 4 (lane 6). Bands of Mr = 200,000 and 400,000 correspond to laminin.
Fro. 3. Identification of laminin after purification by DEAE cellulose chromatography. Each fraction was subjected to sodium dodecyl sulfate gel electrophoresis (A) and was assayed for laminin by ELISA (B). Std., laminin standard.
SZARFMAN ET AL. 1167 observed with n o r m a l m o n k e y sera (Fig. 3) or with the fraction o f infected m o n k e y serum not b o u n d to laminin-Sepharose.
T h e specific antigenic site that reacted with antibodies from the infected monkeys a n d sheep a n t i l a m i n i n antibodies was further characterized by testing various pro- tease-derived fragments o f laminin in the E L I S A assay. T h e antigenic activity of b o t h antibodies was retained in the PI a n d Pli fragments o f laminin (Mr -- 280,000) p r o d u c e d by digestion with pepsin; however, when laminin was treated with Strepto- myces griseus protease or trypsin, its reactivity was lost (Table II). L a m i n i n did not bind nonspecifically to other I g G molecules, because laminin did not block antinuclear antibodies from b i n d i n g to the nuclei o f hepatocytes.
Reaction with Parasites. Antibodies (both the I g M a n d I g G types) from infected monkeys, purified by laminin-affinity c h r o m a t o g r a p h y , p r o d u c e d a very strong reaction with trypomastigotes a n d amastigotes, but showed only a weak reaction with epimastigotes when used in the indirect IF technique. L a m i n i n (25 # g / m l ) completely blocked this reaction. In contrast, the antibodies present in the infected serum, which did not bind to the laminin affinity c o l u m n a n d which did not stain tissues, intensely stained the epimastigotes a n d amastigotes, b u t only weakly stained the trypomasti- gotes.
Functional Studies. L a m i n i n is the a t t a c h m e n t protein that binds endothelial cells to type I V collagen (9). Serum from infected monkeys (Fig. 4), as well as serum from C h a g a s ' patients, similarly inhibit the a t t a c h m e n t o f endothelial cells to type I V collagen, In addition, purified antibodies from infected monkeys (50 # g / m l ) inhibit the a t t a c h m e n t of these cells. This inhibition was reversed by the addition o f laminin to the m e d i u m in which the cells were suspended (Fig. 4). As expected, the a t t a c h m e n t o f C H O fibroblasts, which is mediated by FN, was not inhibited by these antibodies (Fig. 5).
D i s c u s s i o n
H u m a n s a n d monkeys infected with T. cruzi produce antibodies that react with extracellular structures in some areas where basement m e m b r a n e s are present. Their
TABLE II
Reaction of Laminin and Laminin Fragments with Antibodies Using the ELISA Technique
Substrate Antibodies purified by affinity chromatography to laminin Laminin- T. cruzi- immunized infected sheep* monkey* Normal Normal monkey sheep serum:]: serum::]: Laminin 1.2 I. 4 0.1 0.1
Laminin plus trypsin 0.1 0. I 0. l 0.1
Laminin plus S. griseus protease 0.1 0.1 0.1 0.1
Laminin pepsin-derived fragments
Pb 0.2 0.2 0.1 0.1
Pc 0.1 0.2 0.1 0.1
Pl 0.8 0.4 0.1 0.1
Pli 0.8 0.8 0.1 0.1
Detected with peroxidase-conjugated goat antihuman IgG. significant values are italicized.
* At a concentration of 50 #g/ml. ~z Diluted 1:10.
1168 100 8O Ncxm, a~ ~ MOnkey Serum 40 / MOnkey Serum
20 Me nlk~ect/~ mlnfected ~ + l ~ , g l m l Laminin
0 --// I I I I I
0 "~0 -s 10 -4 10 -3 10 -2 10 -1
Serum Dilution
FxG. 4. Inhibition by monkey serum of attachment of endothelial cells to type IV collagen. The serum from an infected monkey inhibits the attachment to type IV collagen, and is reversed by added laminin. Normal monkey serum has no effect.
100 8O == E 6O "8 20 Infected Monkey Serum N o r m a l Monkey Se~m _ _ / / p i i i t 0 10 -5 10 -4 10 -s 10 -2 10 -1 Serum Dilution
FIo. 5. Lack of inhibition by monkey serum of attachment of CHO cells. Serum from an infected monkey and normal monkey serum has no effect.
sera were f o u n d to c o n t a i n h i g h titers o f a n t i b o d i e s to l a m i n i n b u t not to various o t h e r c o n n e c t i v e tissue c o m p o n e n t s .
Several different tests were used to c o n f i r m t h e i d e n t i f i c a t i o n o f l a m i n i n as the tissue a n t i g e n . T h e s e studies i n c l u d e d i m m u n o p r e c i p i t a t i o n o f l a m i n i n , a b s o r p t i o n o f t h e a n t i b o d i e s to l a m i n i n c o v a l e n t l y b o u n d to S e p h a r o s e b e a d s , r e a c t i o n o f l a m i n i n w i t h t h e a n t i b o d i e s in t h e O u c h t e r l o n y test, b i n d i n g o f a n t i b o d i e s to l a m i n i n - c o a t e d wells (the E L I S A test), a n d i n h i b i t i o n o f t h e a t t a c h m e n t o f e n d o t h e l i a l a n d e p i t h e l i a l ceils (not shown). F o r p r e l i m i n a r y studies using t h e E L I S A t e c h n i q u e , we h a v e f o u n d t h a t l a m i n i n o b t a i n e d from h u m a n a m n i o n is a b l e to b l o c k the r e a c t i v i t y of l a m i n i n a n t i b o d i e s o b t a i n e d from infected p r i m a t e s to l a m i n i n o b t a i n e d from the E H S t u m o r (A. S z a r f m a n , V. P. T e r r a n o v a , a n d G. R. M a r t i n , u n p u b l i s h e d o b s e r v a t i o n ) . T h e P1 a n d Pli f r a g m e n t s o f l a m i n i n p r o d u c e d b y p r o t e o l y t i c d i g e s t i o n shows a h i g h degree o f r e a c t i o n w i t h these a n t i b o d i e s . F u r t h e r m o r e , it is likely t h a t l a m i n i n represents the m a j o r , or even the only, tissue a n t i g e n in the E V I system, b e c a u s e passage o f sera from C h a g a s ' p a t i e n t s or infected m o n k e y s over l a m i n i n - S e p h a r o s e b e a d s e l i m i n a t e s the tissue reactions.
D i r e c t c o m p a r i s o n o f the r e a c t i o n o f these a n t i b o d i e s w i t h r a b b i t a n t i b o d i e s to l a m i n i n in tissue sections r e v e a l e d similar, b u t not i d e n t i c a l , reactions. T h e a n t i g e n in tissues r e a c t i n g w i t h a n t i b o d i e s from infected m o n k e y s e r u m is m o r e l a b i l e t h a n the a n t i g e n d e t e c t e d b y a n t i l a m i n i n a n t i b o d i e s p r o d u c e d in r a b b i t s ( d a t a not shown). F u r t h e r m o r e , r a b b i t a n t i l a m i n i n a n t i b o d i e s s t a i n e d all b a s e m e n t m e m b r a n e s uni-
SZARFMAN ET AL. 1169 formly, whereas the antibodies from infected primates showed a more limited distri- bution. However, both sera stained the matrix of the EHS tumor, which is thought to be solely basement membrane (18). It is likely that these two antibodies recognize different antigenic sites in laminin. T h e sheep or rabbit antibodies are probably heterogeneous and directed against a variety of antigenic sites in the laminin molecule.
The antibodies arising after T. cruzi infection may recognize fewer and different
antigenic determinants in laminin. T h e interaction of laminin with other components of the basement membrane, such as type IV collagen and the heparan sulfate proteoglycan, might mask some antigenic sites and make the protein unreactive with antibodies which recognize a limited number of antigenic determinants. Therefore, laminin may be a heterogeneous family of proteins or could exist in several stages of processing which could affect the reactivity and specificity of the antigen.
After infection by T. cruzi, antibodies are produced against laminin and not against
any of the other known basement membrane or connective tissue components. Furthermore, production of antibodies against laminin is a distinctive feature of T.
cruzi infection in primates. Such antibodies are not present in sera from normal
individuals, persons who have undergone surgery, or patients with various inflam- matory conditions or neoplasms, malaria, toxoplasmosis, or rheumatoid arthritis. However, we have found high titers of antibodies to laminin in Rhesus monkeys
infected with T~ypanosoma rhodesiense (A. Szarfman, K. Esser, V. P. Terranova, and G.
R. Martin, unpublished observation). It is possible that the induction of antibodies to laminin is associated with trypanosomal infections.
It is not clear why T. cruzi infection induces antibodies to laminin. T h e titer of
antibodies is highest during the early phase of the infection as would be expected if laminin or an immunologically related protein were produced by the parasite. In this regard, we have found that the trypomastigote and amastigote forms of the parasite have a higher reactivity with purified antibodies from infected monkeys than the epimastigote forms. Furthermore, recent studies have shown that sera from Chagas' patients increase the attachment and penetration of blood trypomastigotes into WI 38 cells (24), and that purified antibodies to laminin decrease the penetration of the parasites (Dr. G. A. Schmufiis, personal communication). Because the trypomastigote
form of T. cruzi encounters basement membranes during its penetration into the host,
the production of a laminin-like protein and or incorporation of host laminin may permit the parasite to interact with the host tissues.
T h e significance of the antilaminin antibodies to the pathol6gy of Chagas' disease is unclear. As shown here, the antibodies in infected monkeys that react with laminin are able to block the attachment of endothelial cells to basement membranes, and this could damage blood vessels and endocardium leading to the endocardial fibrosis found in Chagas' disease (1) and in African Trypanosomiasis (25). T h e initial changes would arise during the parasitic invasion of the host, and the changes that occur later on would be part of a slow destructive process secondary to autoimmune reactions (26, 27). The study of the antilaminin antibodies that appear after infection with T.
cruzi holds considerable promise of yielding important information of the interactions
of the parasite and it's host. In addition, when immunization with T. cruzi is taken
into consideration, these antibodies can become useful in producing antigenic prep-
1170
S u m m a r y
W e h a v e f o u n d t h a t sera from h u m a n s w i t h C h a g a s ' disease a n d R h e s u s m o n k e y s infected w i t h Trypanosoma cruzi c o n t a i n I g M a n d I g G antibodies, w h i c h react with structures in a v a r i e t y o f c o n n e c t i v e tissues. T h e s e a n t i b o d i e s react w i t h l a m i n i n b u t not w i t h various o t h e r p u r i f i e d c o n n e c t i v e tissue c o m p o n e n t s like c o l l a g e n t y p e s I, III, IV, a n d V, fibronectin, h e p a r a n sulfate (BM-1) p r o t e o g l y c a n , or c h o n d r o n e c t i n . T h e tissue-reacting a n t i b o d i e s were isolated b y a b s o r p t i o n to a l a m i n i n - S e p h a r o s e c o l u m n . T h e b o u n d fraction c o n t a i n e d all t h e tissue-reacting a n t i b o d i e s . T h e s e a n t i b o d i e s s t r o n g l y s t a i n e d t r y p o m a s t i g o t e s a n d a m a s t i g o t e s , b u t w e a k l y s t a i n e d e p i m a s t i g o t e s . T h e s e studies show t h a t sera from T. cruzi-infected p r i m a t e s c o n t a i n a n t i l a m i n i n a n t i b o d i e s , w h i c h m a y be p r o d u c e d b y those host in response to a l a m i n i n - l i k e m o l e c u l e p r e s e n t in t h e p a r a s i t e .
The authors thank Dr. L. R. R. G. Travassos, Dr. R. Wistar, Jr., Dr. D. M. Strong, Dr. F. Neva, and Dr. R. Russell for their advice and assistance; Mr. J. Dietrich and Ms. F. Cannon for excellent technical assistance; Mr. C. Bowen for editorial support; and Dr. J. C. Murray for assistance in the early phases of this work.
Received for publication 14 December 1981.
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