I N T E R A C T I O N O F R H E U M A T O I D F A C T O R A N D E N T A M O E B A H I S T O L Y T I C A
P . ( , . K R I M S N K R (1) & W . G R A M N G K R (2)
S U M M A R Y
The amoebae's cytotoxicity test and the amoebae's lysis test were used to show possible interactions between rheumatoid factor ( R F ) and Entamoeba histolytica. Amoebae's cytotoxic activity ( A C A ) was inhibited by affinity chromatography purified antiamoebae rabbit I g G ( R I g G ) . Enhanced inhibition could be demonstrated with R I g G plus R F . But the same marked inhibition of A C A could be seen when replacing R F by heat inactivated normal human serum as a control.
About 50% amoebae's lysis occurred when amoebae were brought together with native normal human serum ( N N H S ) as a source of complement. Amoebae's lysis increased to 60% when incubated with N H S plus human antiamoebae antibodies. No further augmentation could be obtained by the addition of R F . Using R I g G instead of human antibodies the lysis rate did not increase. Incubation of amoebae, N N H S , R I g G and R F even reduced amoebae's lysis. R F neither has an effect on A C A nor on complement mediated A L in vitro.
K E Y W O R D S : Entamoeba histolytica; Amoebae's lysis; Rheumatoid factor.
I N T R O D U C T I O N
Rheumatoid factors ( R F ) are mostly IgM antibodies directed to various antigenic determinants of the Fc part of autologous or homologous I g G . The occurrence of IgM with R F like activity has not only been reported in collagen vascular diseases like rheumatoid arthritis, but also in numerous infectious diseases, like endocarditis2 and parasitic
infections like malaria, trypanosomiasis, leishmaniasis, schistosomiasis and oncho-c e r oncho-c i a s i s3-1 1 1 2'1 6-1 7" . The large number of
infectious disease states known to be associated with rheumatoid factor production could be taken as evidence that this may be a normal immune response engendered in the host during
many reversible and chronic infectious diseases9.
C L A R K S O N & M E L L O W4 reported on
R F acting in a protective way by showing that R F protects uninfected rat pups and dams from Trypanosoma lewisii infections. G R E E N & P A C K E R1 0 reported an inhibitory effect of R F
on Plasmodium falciparum schizonts in vitro. To our knowledge no studies exist dealing with the interaction of amoebae and R F .
In this work we investigated the in vitro interaction of E . histolytica with antiamoebae antibodies, human complement and rheumatoid factor.
(1) Institute of Specific P r o p h y l a x i s and T r o p i c a l M e d i c i n e , B e r l i n .
(2) Department of C h e m o t h e r a p y , University o f V i e n n a , A u s t r i a .
Address for correspondence: D r . P . G . K r e m s n e r , State Institute o f T r o p i c a l Medicine B e r l i n , K õ n i g i n - E l i s a b e t h - S t r a B e 32
M E T H O D S Amoebae:
F o r all tests 72 hours cultured E . histolytica strain S F L 3 were taken. Modified TY-S-33 Medium was used6-7-8. Trophozoites
were harvested and washed twice in R P M 1640 medium plus 10% fetal calf serum ( R P M I / F C S ) . Then amoebae were adjusted to the wanted cell number per ml.
Target cells:
K 562, a human erythroleukemia cell line was used as target for amoebae's cytotoxicity. K 562 cells were cultured in R P M I / F C S . Rheumatoid Factor ( R F ) :
The source of R F was serum from a patient with inactive rheumatoid arthritis. The titer of R F was 1:1280 as determined by agglutination of Latex particles coated with human IgG (Behring) and 1:20.000 as measured by haemagglutination of erythrocytes coated with rabbit I g G (Raha-Test, Fujirebio). The serum was used after heat inactivation and absorption of I g G by Protein A (Pansorbin, Behring) and centrifugation. The titer after absorption was 1:5000 as measured by haemagglutination. Human serum:
Freshly drawn normal human serum ( N H S ) was used which was negative with respect to all amoeba-specific serological tests ( I I F T , I H A , E L I S A ) . The complement activity was 36 C H5 üU / m l .
One part of the same serum was heat inactivated at 56°C for 30 minutes ( H N H S ) and treated in the same way as the RF-containing serum.
Antibodies:
F o r p r e p a r a t i o n of a n t i a m o e b a e antibodies, 30 ml of the serum from a patient suffering from an amoebic liver abscess was precipitated with a 33% ( N H4)2S 04 solution,
leaving the IgG-fraction in solution. The I g G fraction was dialysed against isotonic phosphate-buffered saline, pH-7.4. A s shown
by Immunoelectrophoresis, this preparation proved to be pure IgG ( H I g G ) and showed a highly positive reaction in the E L I S A2 8
For preparation of rabbit antiamoebae antibodies, we immunized rabbits intramuscularly with amoebic antigens. The immunization schedule was week 0, 1, 2, 6 and a boosting dose 3 months later. Blood was drawn by intracardial punction.
For antigen preparation we took S F L 3 amoebae and homogenized the cells with the Potter-Elvejlham homogenizer at OüC . The
resulting solution we mixed for the first immunizing dose with complete Freund's adjuvant, for the next doses with incomplete Freund's adjuvant.
The rabbit sera were test by the E L I S A technique for antiamoebic activity2 1. A l l four
sera showed positive reactions and were pooled. For getting pure I g G out of sera the 33% ( N H4)2S 04 precipitation method was used and
proved by immunoelectrophoresis after dialysis against P B S .
A F F I N I T Y C H R O M A T O G R A P H Y 109 S F L 3 trophozoites were washed three
times in P B S containing 2 m m o l / i phenyl-methylsulfonylfluoride ( P M S F ) , 10 m m o l / i ethylendiaminotetraacetate ( E D T A ) and 10 m m o l / i E-aminocaproicacid ( E A C A ) and resuspended in this buffer at 4 ° C . Amoebae were homogenized by use of a Potter-Elvejlham homogenizer at 4 ° C . Subsequently, the homogenate was centrifuged at 1.500g for 20 minutes at 4 ° C . Then the supernatant was subjected to centrifugation at 25.000g for one hour at 4 ° C . The pellet (membrane fraction) was
resuspended in coupling buffer and coupled to C N B r activated Sepharose 4 B (Pharmacia Fine Chemicals, Vienna, Austria) according to the manufacturer's order i.a. 10 ml rabbit I g G (20 m g / m l ) were processed over the affinity column. After that the gel was washed by P B S until the base line at the U V monitoring system was reached. The amoebae specific antibodies were eluted with 0.1 m o l / i C H3C O O H , p H
-2.5. After elution, the antibody preparation ( R I g G ) was immediately neutralised and dialysed against several changes of P B S . In the E L I S A we could show a 30 fold enrichment of antiamoebae antibodies in comparison with I g G before affinity cromatography.
Amoebae's lysis test ( A L )
Amoebae's lysis test was performed as described earlier2 1 2 7. Amoebae were labelled
with ? 1C r . Then 5 x 105 amoebae were mixed
with human serum, R F , antibodies and R P M I / F C S and incubated at 37°C for 30 minutes. Each test was carried out six fold.
Amoebae's cytotoxic activity test ( A C A ) A modified microcytotoxicity assay was performed as described for K and N K lymphocytes2 6. K 562 target cells were labelled
with 5 i C r . The effector to target cell ratio was adjusted to 5:1. 100 JLÍ! amoebae (washed twice in R P M I / F C S in a concentration of 5 x lOVml) and 300 \A of R P M I / F C S or antibody or human serum or heat inactivated human serum or R F were mixed and incubated at 37°C for 30 minutes. Then 200 \x\ K 562 (5 x lOVml in R P M I / F C S ) were added. Tubes were cen-trifuged at 200g for 2 minutes and incubated at 37°C for 10, 30 and 60 minutes. The assay was performed in triplicates. Target cell lysis was calculated from the radioactivity released into the supernatant. As a control K 562 were incubated with R P M I / F C S instead of amoebae (spontaneous lysis).
R E S U L T S
Amoebae's cytotoxic activity ( A C A ) against target cells was inhibited by antiamoebae antibodies. Figure 1 shows the
F i g u r e 1
I n h i b i t i o n o f a m o e b a e ' s cytotoxic activity b y antibodies.
i o o " 0 1 T O * 3 0 ' 6 0 ' T~ I fVI hi 1 = S F L 3 amoebae + K 5 6 2 2 = S F L 3 amoebae + K 5 6 2 + R I g G 3 = S F L 3 amoebae + K 5 6 2 + R I g G + R F 4 = S F L 3 amoebae + K 5 6 2 + R I g G + H N H S 5 = K 5 6 2 (spontaneous lysis) T A B L E 1 A m o e b a e ' s L y s i s ( A L ) w i t h h u m a n s e r u m , r h e u m a t o i d factor a n d r a b b i t I g G after 3 0 ' i n c u b a t i o n at 3 7 ° C (% l y s i s ) E X P E R I M E N T S I I I I V S a m p l e s N o . A m + R I g G + + R F + N N H S A m + R I g G + + R P M I + N N H S A m + R P M I + + R P M I + N N H S A m + R P M I + + R P M I + R P M I 3 5 . 7 3 6 . 8 37.6 37.5 37.3 3 7 . 4 5 4 . 9 51.1 52.5 51.1 53.6 5 1 . 8 5 2 . 9 5 0 . 6 515 5 1 . 9 5 1 . 5 5 1 . 7 6.9 6.7 7.2 6.9 7.0 6.8 Mean Median M i n - M a x . 37.1 37.3 3 5 . 7 - 3 7 . 6 52.5 5 2 . 2 5 1 . 1 - 5 4 . 9 5 1 . 7 5 1 . 6 5 0 . 6 - 5 2 . 9 A m (1 m l ) = 5 x 10 S F L 3 a m o e b a e / m l . R F (1 m l ) = R F c o n t a i n i n g heat i n a c t i v a t e d h u m a n s e r u m . R P M I (1 m l ) = R P M I 1 6 4 0 + 1 0 % F C S R I g G (1 ml) — R a b b i t I g G , a n t i a m o e b a e a n t i b o d i e s a f f i n i t y c h r o m a t o g r a p h i c p u r i f i e d , 1 0 0 m c g / m l . 6.9 6.9 6 . 6 - 7 . 1 N N H S (1 m l ) Native n o r m a l h u m a n s e r u m .
T A B L E 2 A m o e b a e ' s L y s i s ( A L ) w i t h h u m a n s e r u m , r h e u m a t o i d f a c t o r and h u m a n I g G after 3 0 ' i n c u b a t i o n at 3 7 ° C (% lysis) E X P E R I M E N T S I I I I I I I V S a m p l e s A m + H I g G + A m + H i g G + A m + R P M I + A m + R P M I + N o . + R F + N N H S + R P M I + N N H S + R P M I + N N H S + R P M I + R P M I 1 6 1 . 2 6 1 . 2 5 4 . 2 6.6 2 62.8 6 1 . 7 5 4 . 6 6.8 3 6 1 . 4 6 1 . 3 5 5 . 6 7.1 4 6 0 . 8 6 2 . 3 5 5 . 0 7.0 5 61.1 62.1 5 4 . 6 6.7 6 63.5 6 2 . 3 5 4 . 8 6.7 Mean 61.8 6 1 . 8 5 4 . 8 6.8 Median 6 1 . 3 6 1 . 9 5 4 . 7 6.8 M i n - M a x . 6 0 . 8 - 6 3 . 5 6 1 . 2 - 6 2 . 3 5 4 . 2 - 5 5 . 6 6 . 7 - 7 . 2 A m ( l m l ) = 5 x 1 0s S F L 3 a m o e b a e / m l . R F (1 m l ) = R F c o n t a i n i n g heat i n a c t i v a t e d h u m a n s e r u m . R P M I (1 m l ) = R P M I 1 6 4 0 + 10% F C S
H I g G (1 m l ) ~ H u m a n I g G f r a c t i o n f r o m a serum f r o m a patient suffering f r o m an a m o e b i c liver abscess, 1 m g / m l . N N H S (1 m l ) = N a t i v e n o r m a l h u m a n s e r u m .
Table 1 shows complement mediated amoebae's lysis after incubation at 37°C for 30 minutes. The addition of R I g G (25 ^g/ml final concentration) to N N H S did not increase the lysis rate compared to human serum alone. Surprisingly, incubation of amoebae, N N H S , R I g G plus R F gave a marked reduction of amoebae's lysis. Control experiments with R F alone revealed A L similar to the spontaneous amoebae's lysis.
In the next set of experiments human anti bodies directed against amoebic antigens ( H I g G ) were used in the amoebae's lysis test together with N N H S as source of complement. Again N N H S alone lysed 55% of amoebae. N H S plus H I g G (0.25 mg/ml) produced about 62% amoebae's lysis. No further increase in the rate of amoebae's lysis could be obtained by the addition of R F (Table 2).
D I S C U S S I O N
The capability of pathogenic strains of Entamoeba histolytica to lyse various cultured target cells has been described by several a u t h o r s u s i n g d i v e r s e e x p e r i m e n t a l approaches1 1 3 1 8'2 3-2 5. Most probably structures
of the amoebae's surface membrane are capacity of R I g G (130 Mg/ml final con
centration) to inhibit the A C A against K 562 target cells. This inhibition of A C A can be enhanced with R I g G plus R F . As a control we replaced R F containing serum by heat inactivated normal human serum. No difference between the two latter assays could be detected with regard to A C A .
Complement in form of native normal human serum ( N N H S ) was able to inhibit the A.CA as well. After 60 minutes incubation K 562 lysis was only 16%. The addition of R I g G led to an augmented inhibition of A C A (10% K 562 lysis after 60 minutes) compared to N N H S alone. No further potentiating effect could be seen by the addition of R F (11% K 562 lysis), (data not shown in detail).
Lysis tests:
Amoebae's lysis test ( A L ) already occurs when amoebae are brought together with N H S as a source of complement. About 50% of amoebae are lysed by the activation of complement. Amoebae's lysis increases to 60% when incubated with N N H S plus human antibodies to amoebae.
involved in the cytotoxic action, presumably by mediating target and effector cell contact via a lectin like interaction1 9. Serum factors capable
to inhibit the amoebae's cytotoxic activity are obviously interfering with the recognition of the target c e l l1 3 1 4.
Antibodies directed against amoebic plasma membrane antigens inhibit the A C A . However, after binding to the membrane the antigen-antibody complexes are capped and partially shedded or i n t e r n a l i z e d . T h e reappearance of a smaller number of new surface antigens are thought to be responsible for A C A inhibition. A further amplification of these effects by R F was considered, because it has been reported that R F react with I g G which has undergone severe conformational change such as antigen binding'. In our assay no modulation of A C A by R F could be detected. These results can also be presumed for the situation in vivo.
The slightly enhanced inhibition of A C A during incubation with R I g G plus H N H S could possibly be due to cytokines, such as interferon-gamma or tumor necrosis factor, which are known to exert antiparasitic activity and occur in small amounts in N H S .
Complement can lyse about 50% of amoebae after 30 or more minutes incubation at 3 7 ° C1 S'2° . Beside this activation of the
complement cascade via the alternative pathway, human antiamoebae antibodies also activate the classical pathway of complement2 4.
A total amoebae's lysis rate of more than 60% after 30 minutes incubation at 37°C could be demonstrated in our assay. We expected R F to act as a second antibody and a complement magnet in form of a Clq binding site. In our hands R F did not show any influence on amoebae's lysis together with N N H S and H I g G . Forty per cent of amoebae are apparently resistant to complement lysis even under this maximally complement activated condition and prolonged incubation.
In presence of R I g G we could not show an activation via the classical pathway of complement in the amoebae's lysis assay. By adding R F we found a reduction of amoebae's lysis. Those findings might be due by a missing activation of the classical pathway of the human complement system by these R I g G . However R I g G may react with R F . Amoebae antigen-RIgG-RF complexes on the amoebae's surface may lead to a transient immobilisation
of the membrane followed by capping phenomena, partial ingestion and partial shedding of these immune complexes which in turn activate and consume complement far away from the amoebae's surface.
Another possible mechanism for our findings is that these complexes on the amoebae's surface membrane may block C3b binding sites and hence inhibit assembling of C3 convertase of the alternative pathway of complement activation. So the complex C 3 B b P cannot be formed sufficiently on the amoebae's surface.
In our study we could show that R F neither has an effect on amoebae's cytotoxic activity nor on complement mediated amoebae's lysis in vitro. Our results suggest that the interaction between antibodies against amoebic antigen and human I g G ( R F ) do not exert a protective role against extraintestinal amoebiasis.
R E S U M O
Interação do fator reumatóide e Entamoeba histolytica
Testes para citotoxicidade e lise amebiana foram utilizados para demonstrar uma possível interação entre o fator reumatóide e a Entamoeba histolytica. A atividade citotóxica amebiana foi inibida pela I g G antiameba de coelho purificada através de cromatografia. Constatou-se inibição aumentada com I g G antiameba de coelho mais fator reumatóide. A mesma inibição acentuada da atividade citotóxica amebiana pôde ser constatada quando se substituiu o fator reumatóide por soro humano normal, inativado pelo calor, como controle. Cerca de 50% de lise amebiana ocorreu quando as amebas foram misturadas com soro normal humano como fonte de complemento. A lise amebiana aumentou para 60% quando incubadas com soro humano normal, acrescido de anticorpos humanos antiameba. Nenhum aumento adicional pode ser obtido pela adição de fator reumatóide. Usando I g G antiameba de coelho em vez de anticorpos humanos, a proporção de lise não aumentou. A incubação de amebas com soro humano normal, I g G antiameba de coelho e fator reumatóide reduziu acentuadamente a lise amebiana. O fator reumatóide não teve efeito na atividade citotóxica amebiana, nem na lise amebiana mediada pelo complemento in vitro.
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