Immunology 1976 30 485

Depression of the plaque-forming cells to sheep red blood cells by the new-born larvae of Trichinella spiralis G. M. FA UB ER T Institute of Parasitology, McGill University, Macdonald College, Province of Quebec, Canada

Received 13 August 1975; acceptedfor publication 3 September 1975

Summary. The number of plaque-forming cells (PFC) per spleen is reduced in mice experimentally infected with 200 Trichinella larvae at 14 and 28 days after infection, whereas no difference is shown at days 7 and 56. The worms of the three different phases of the life cycle of Trichinella have been isolated and kept alive in vitro at 370 in the inner compartment of a Marbrook chamber. The outer compartment of the chamber contained normal spleen cells and sheep erythrocytes. After 4 days, the spleen cells were removed and assayed for PFC. The results indicate that the new-born larvae are capable of producing substances which can diffuse through a Millipore filter and affect the spleen cells forming antibody to sheep erythrocytes. The suppression of antibody to sheep red blood cells in mice infected with Trichinella is a transitory phenomenon and can be related to the migrating phase of the life cycle of the parasite.

mouse sera obtained from a 30-day Trichinella infection. These results suggest that toxic products having an effect on the lymphoid cells appear in the serum of the animals during the course of the infection (Faubert and Tanner, 1975). Barriga (1975) has observed that the production of sheep haemagglutinin is reduced in mice treated with this parasite extract. Cypess, Lubiniecki and Hammon (1973) have reported that Trichinella causes a suppression of the neutralizing and complement-fixing antibody responses to Japanese B encephalitis virus both at 7 or 28 days after infection. The suppression of the immunological system in mice experimentally infected with Trichinella is of additional interest when viewed in the context of the observed immunogenicity of this parasite discernible at both the cellular and humoral levels. It should be remembered, however, that despite this strong immune reaction, the parasite manages to survive and overcome the host defence mechanism. The present study examines the kinetics of the immunosuppression against SRBC in mice infected with Trichinella. It was hoped, therefore, that it would be possible to correlate the suppressed state with a particular phase of the parasite's life cycle and determine whether the immunosuppression is of a permanent or transitory nature. Finally, some preliminary experiments were performed to study the origin of the toxic factor found in the infected mouse serum and in the saline extract of the muscle larvae.

INTRODUCTION The humoral and cellular antibody response to sheep red blood cells (SRBC) is depressed in mice experimentally infected 30 days previously with Trichinella (Faubert and Tanner, 1971; Faubert and Tanner, 1974). This depression can also be observed by injecting normal mice for 7 consecutive days with Correspondence: Dr G. M. Faubert, Institute of Parasitology, McGill University, Macdonald College, Province of Quebec, Canada HOA ICO. 485

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MATERIALS AND METHODS Animals and the parasite The parasite and the mice used in these experiments have been described in earlier papers (Faubert and Tanner, 1971). The mice were inoculated orally without anaesthesia with a dose of 200 larvae.

Plaque-forming cell assay (PFC) The method used for assaying IgM PFC was essentially as described by Kongshavn and Lapp (1972) with certain modifications made in this laboratory. Spleen cell suspensions were prepared by gently sieving each mouse spleen through an 80-mesh stainless steel screen, the cells being collected in culture medium RPMI-1640 (GIBCO) supplemented with 10 per cent heat-inactivated calf serum. The following components were also added to the medium: penicillin and streptomycin, 100 pg/ml; heparin, 1 u/ml; sodium bicarbonate, 2 g/l; Hepes* (GIBCO) 10 ,umoles/l, and the pH adjusted to 7 2. After washing, the spleen cells were made up to 10 ml with this same medium. The test consisted of adding 0-1 ml of the spleen cell suspension to 0-1 ml of SRBC and 0 4 ml of complement (BBL) both diluted 1:5 with the culture medium. In the first experiment, the total number of PFC per spleen was estimated by multiplying the number of PFC in all chambers (0-1 ml of spleen cells) by 100. In the second experiment, the number of PFC is reported per 5 x 106 spleen cells.

Cultivation of the spleen cells and Trichinella in the Marbrook Chamber The cells were cultured in a Marbrook chamber as described by Parthenais, Elie and Lapp (1974). Approximately 5 x 106 SRBC were cultured with 5 x 106 normal spleen cells in the outer chamber and 2000 adult worms, new-born larvae or muscle larvae placed in the inner chamber. The chambers were incubated at 370 in a 5 per cent CO2 incubator for 4 days. Spleen cells from the outer chamber were then assayed for the direct PFC response to SRBC using the technique described above. Isolation of the three different phases of Trichinella The muscle larvae of Trichinella were obtained from the muscle of infected rats after acid-pepsin digestion as described by Tanner (1968). The technique used to recover the adults and migrating larvae has been described by Dennis, Despommier and Davis (1970).

In order to isolate the adult worms before copulation, the rats were killed 4 days after infection instead of 7 days.

Sterilization of the living parasite In order to eliminate any bacterial contamination, the adult worms, the new-born larvae and the muscle larvae were incubated at 370 for 30 min in a special medium before cultivation. The sterilizing medium consists of basic medium 199, to which was added 0-005 g of merthiolate, 0 1 g of kanamycin, 0 1 g of streptomycin and 0-1 g of penicillin per litre of medium. After the incubation, the 'sterilizing' medium was removed and the worms washed twice in basic medium and finally diluted in the culture medium RPMI.

Experimental protocol In the first experiment, forty mice were each infected with 200 muscle larvae. The mice were then divided into four groups and received one intraperitoneal injection of 1 x 108 SRBC, the first group on day 3, the second on day 10, the third on day 24, and the fourth group on day 52. The spleens of all these mice were removed and assayed for PFC to SRBC. The control animals consisted of four groups of six mice each of which were injected with SRBC 4 days before. In the second experiment, the PFC to SRBC was studied in an in vitro system using the Marbrook chamber. The spleen cells of non-infected mice were cultured in vitro in the presence of SRBC for 4 days. The adult worms, the migrating or the muscle larvae were each placed in the inner chamber which was separated from the outer chamber by a 0 45 pm Millipore filter membrane. This was done in order to determine if the viable Trichinella can secrete products which can pass through a Millipore filter and effect the spleen cells located underneath. As a control, medium RPMI was placed in the inner chamber of the Marbrook chamber.

RESULTS Splenic PFC to SRBC in Trichiella-infected mice The total number of PFC per spleen from mice infected with 200 Trichinella muscle larvae is presented in Fig. 1. The mean number of PFC per spleen in this group of mice killed on day 7 (ten

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Figure 1. Mean number of PFC per spleen removed from mice killed at different days post-infection. The mean counts were determined from ten spleens in the infected group (0) and from six in the control group (0). The standard error of the control is indicated only for the group of mice killed at day 14, since it is constant in the remaining group.

mice) is not statistically different from the mean number of PFC in the control group according to Student's t-test (P>0005). However, after 14 days the mean number of PFC in the infected mice is much lower than the mean number in the control group (P 0 05). Production of plaques by normal spleen cells in the of Trichinella

cultured in the Marbrook Chamber. The histogram represents the mean number of PFC obtained from twelve different samples respectively cultured in the presence of culture medium (C), adult worms (A), new-born larvae (N) and muscle larvae (M).

separated from the 2000 living organisms by a Millipore filter in order to eliminate any cell to cell contact while allowing the transfer of soluble substances across the membrane. Fig. 2 shows the effect of 2000 isolated worms, either adults (A), newborn larvae (N) or muscle larvae (M) on the spleen plaque-forming cells. As a control, normal spleen cells cultured in the presence of culture medium were placed in the inner chamber without any Trichinella. The spleen cells cultured in the presence of adult worms (A) or muscle larvae (M) produced as many plaques as the control. However, the number of plaques produced by the spleen cells cultured in the presence of the new-born larvae (N) was at least three times less than either the control or the other two groups. These results indicate, therefore, that only new-born larvae are secreting products which will diffuse through a Millipore filter and are capable oft suppressing the antibody response to SRBC.

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DISCUSSION This experiment was designed to check whether or not the adult worms, the new-born larvae or the encysted muscle larvae have any specific effect on the spleen plaque-forming cells removed from noninfected mice. The three different developmental stages in the life cycle of Trichinella were isolated and maintained in an in vitro system for 4 days. The spleen cells (5 x 106) isolated from non-infected mice were

The object of this study was to determine whether the immunosuppression observed in trichinellosis towards SRBC, as reported by different workers, Faubert and Tanner (1971, 1974), Barriga (1975) and Lubiniecki and Cypess (1975) is associated with a specific developmental phase of the parasite's life cycle. The present results indicate that the production

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G. M. Faubert

of antibody against SRBC as measured by the PFC assay, is depressed in those mice infected with 200 Trichinella larvae, only after day 14, the suppressing effect disappearing by day 56 (Fig. 1). However, the in vitro experiments seem to indicate that only new-born larvae are causing immunosuppression (Fig. 2), suggesting therefore that the immunosuppression observed at day 28 rather than being associated with the muscle phase of the life cycle, could be due to toxic products released by the newborn larvae and still active after that period of time. The number of PFC produced by the spleens removed from mice infected 7 days before is not significantly lower than the control group and therefore confirms the result of Lubiniecki, Cypess and Lucas (1974). In a study on the kinetics of the development of delayed hypersensitivity (D.H.) in the CBA mouse infected with Trichinella, Vernes and co-workers (Vernes, Floch, Biguet and Tailliez, 1975) observed that the DH is depressed by the 14th day. The cells involved in DH and in plaqueforming are probably different but it is worth noting that the results of the present study parallel the findings of Vernes et al. (1975). The suppression of the immunological system in mice infected with Trichinella is paradoxical in view of the strong immune response observed against the parasite at both the cellular and humoral levels. However, the results presented here show quite clearly that the immunosuppression observed in trichinellosis towards unrelated antigens is not permanent but of a transitory nature. Furthermore, it is possible to correlate this immunodepression to a specific phase of the life cycle of the parasite. The antigenicity of the new-born larvae is a controversial subject. Despommier (1971) has reported that the new-born larvae are not immunogenic because the stichocytes do not have Bi granules. On the other hand, James and Denham (1975) have reported that precipitates develop around new-born larvae when they are incubated in vitro in an immune serum. It is the present author's belief that the immunogenicity of new-born larvae is of importance in controlling the subsequent parasitism since the female adult worms are capable of producing larvae despite a strong immune reaction against the adult phase of the life cycle (Larsh, Weatherly, Goulson and Chaffee, 1972; James and Denham, 1975). Therefore, the only chance for the immunological system to stop muscle parasitism is by eliminating the new-born larvae. As far as is

known, the existence of an antibody or cells capable of eliminating new-born larvae has not been reported yet. The reason for this could well be that this larval stage exerts a toxic effect on lymphoid cells. The abnormal suppression of the immunological system produced by the migrating larvae cannot be explained only by the secretion of toxic substances. In a previous paper (Faubert and Tanner, 1974), it was reported that transitory lesions were observed in all the peripheral lymphoid organs. These lesions, it was suggested, might be due to physical damage resulting from migration of larvae en route to the muscles. Antigenic competition is obviously another possible explanation of the immune depression since infection of mice with Trichinella fulfils many of the criteria leading to such competition. For instance antigenic competition can be demonstrated when the first antigen is thymus-dependent, which is the case with Trichinella (Ruitenberg, Teppema and Steerenberg, 1974). The route of injection of the antigen is important (Brody and Siskind, 1972). Recently, Lubiniecki and Cypess (1975) demonstrated this to be also true regarding Trichinella where it was found that the antibody response to SRBC was normal in Trichinella-infected mice if they used the intravenous route but depressed when the intraperitoneal route was employed. Furthermore, the response will be maximal when the antigens presented to the immunological system are separated by a time interval of 1-7 days (Brody and Siskind, 1972). Also the suppression caused by antigenic competition is not merely a delay in the onset of antibody formation to the antigen but represents a quantifiable decrease in the immune response (Eidinger, Pross, Kerbel, Baines, Ackerman and Khan, 1971). Many mechanisms have been proposed to explain antigenic competition: one of these suggests that the increased number of cells arising in response to the first antigen interferes physically with the response to the second, either by crowding out the cells at the follicular level or by blocking thymus-bone marrow cell interactions (Pross and Eidinger, 1974). The results of experiments performed in this laboratory indicate that it may well be this kind of phenomenon that is occuring in trichinellosis (manuscript in preparation). If this is the case, then the term antigenic competition is a misleading term to describe such phenomena associated with trichinellosis. The term antigen-induced suppression proposed by Kerbel and Eidinger (1971) would, in my opinion, seem to be a more accurate description.

T. spiralis larvae effects on PFC to SRBC

ACKNOWLEDGMENTS The author gratefully acknowledges the skilled technical assistance of Mrs H. C. Smith and of Dr J. McLaughlin for assistance in the preparation of the manuscript. This work was made possible by a grant from National Research Council of Canada A-9374 to G.F.; work at the Institute of Parasitology is supported by the National Research Council of Canada. REFEREN CES BARRIGA 0.0. (1975) Selective immunodepression in mice by T. spiralis extracts and infections. Cell. Immunol. 17, 306. BRODY N.L. & SISKIND G.W. (1972) Studies on antigen competition. 1I. Evidence for effect at level of antigen 'processing'. Immunology, 22, 75. CYPEss R.H., LUBINIECKI A.S. & HAMMON W. (1973) Immunosuppression and increased susceptibility to Japanese B. encephalitis virus in Trichinella spiralis-infected mice Proc. Soc. exp. Biol. (N. Y.) 143, 469. DENNIS D.T., DESPOMMIER D.D. & DAVIS N. (1970) Infectivity of the newborn larva of T. spiralis in the rat. J. Parasit. 56, 974. DESPOMMIER D.D. (1971) Immunogenicity of the newborn larva of T. spiralis. J. Parasit. 57, 531. EIDINGER D., PRoss H.F., KERBEL R.S., BAINES M.G., ACKERMAN A. & KHAN S.A. (1971) Further studies of competition of antigens. I. Variation in immunosuppression induced by alterations of dosage, route of injection, nature of antigen and immunological status of host. Canad. J. Microbiol. 17, 803. FAUBERT G. & TANNER C.E. (1971) T. spiralis: inhibition of sheep hemagglutinin in mice. Exp. Parasit. 30, 120. FAUBERT G.M. & TANNER C.E. (1974) The suppression of sheep rosette-forming cells and the inability of mouse bone-marrow cells to reconstitute competence after infection with the nematode Trichinella spiralis. Immunology, 27, 501. FAUBERT G. & TANNER C.E. (1975) Leucoagglutination and cytotoxicity of the serum of infected mice and of extracts

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of T. spiralis larvae and the capacity of infected mouse serum to prolong skin allografts. Immunology, 28, 1041. JAMES E.R. & DENHAM D.A. (1975) Antigenicity of the newborn larvae of T. spiralis. J. Parasit. 61, 354. KERBEL R.S. & EIDINGER D. (1971) Further studies of antigenic competition. III. A model to account for the phenomenon based on a deficiency of cell to cell interaction in immune lymphoid cell populations. J. exp. Med. 133, 1043. KONGSHAVN P.A.L. & LAPP W.D. (1972) Immunosuppressive effect of male mouse submandibular gland extracts on plaque forming cells in mice: abolition by orchidectomy. Immunology, 22, 227. LARSH J.E., WEATHERLY N.F., GOULSON H.T. & CHAFFEE E.F. (1972) Studies on delayed (cellular) hypersensitivity in mice infected with T. spiralis. VII. The effects of ATS injections on the numbers of adult worms recovered after challenge. J. Parasit. 58, 1052. LUBINIECKI A.S. & CYPEss R.H. (1975) Immunological sequelae of T. spiralis infection in mice. Effect on the antibody responses to SRBC and Japanese B. encephalitis virus. Infect. Immun. 11, 1306. LUBINIECKI A.S., CYPEss R.H. & LUCAS J.P. (1974) Immune response to and distribution of sheep enythrocytes in Trichinella spiralis infected mice. Tropenmed. Parasit. 25, 345. PARTHENAIS E., ELIE R. & LAPP W.S. (1974) Soluble factors and the immune response. In vitro studies of the immunosuppression induced by the graft-versus-host reaction. Cell. Immunol. 13, 164. PRoss H.F. & EIDINGER D. (1974) Antigenic competition: a review of nonspecific antigen-induced suppression. Advanc. Immunol. 18,133. RUITENBERG E.J., TEPPEMA J.S. & STEERENBERG P.A. (1974) The intestinal phase of T. spiralis infection. Parasitic Zoonoses (ed. by E. J. L. Sousby), p. 319. Academic Press, New York. TANNER C.E. (1968) Relationship between infecting dose, muscle parasitism and antibody response in experimental trichinosis in rabbits. J. Parasit. 54, 98. VERNEs A., FLOCH F., BIGUET J. & TAILLIEZ R. (1975) Trichinose experimentale. I. Cinetique des phenomenes d'hypersensibilite retard6e chez la souris CBA et le rat Wistar. Int. J. Parasit. 5, 63.

Depression of the plaque-forming cells to sheep red blood cells by the new-born larvae of Trichinella spiralis.

Immunology 1976 30 485 Depression of the plaque-forming cells to sheep red blood cells by the new-born larvae of Trichinella spiralis G. M. FA UB ER...
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