Milan Hašek

Antigen-mediated macrophage adherence inhibition

Abstract Antigen-mediated macrophage adherence inhibition (MAI) was studied in inbred rats immunized with various transplantation, tumour-specific and protein antigens. A macrophage-rich suspension of peritoneal cells (PC) was obtained from the peritoneal cavity of immunized and control animals by washing. The adherence to glass of PC was specifically inhibited by the addition of the antigens used for sensitization of PC donors or by related (cross-reacting) antigens but not with unrelated antigens. The MAI seems to be due to the direct interaction of the respective antigen with a corresponding PC receptor and not due to the humoral factor released from immune lymphocytes of PC population upon contact with the specific antigen.

Transplantation Immunity and Tolerance

Publisher Summary This chapter discusses transplantation immunity and immunological tolerance, which are, under certain conditions, alternative reactions of the same organism to the same antigenic stimulus––namely, transplantation of tissues or cells from an antigenically different individual. Which of the two alternatives will take place is decided by the circumstances of the first experience by the reacting individual of the given antigens, especially important is the stage of development at which the first experience occurs. The chapter describes the methods of inducting tolerance in the prenatal and postnatal periods. It determines five techniques for induction of tolerance in the prenatal period comprising—namely, natural embryonic parabiosis, experimental embryonic parabiosis, the relation of mother and fetus, intraembryonic injections of cells, and transplantation of solid tissue in embryos. It discusses various tests for immunological tests, including tissue transplants, formation of serum antibodies, skin tests, susceptibility to infective agents, and autoimmunity to tissue antigens. It also examines the occurrence of immunological tolerance and the duration of the adaptive period in different species and presents the basic facts of immunological tolerance, along with some possible mechanisms of immunological tolerance.

Immunological tolerance and tumour allografts in the brain

THE brain has been considered to be an immunologically privileged site with regard to tissue transplantation. In addition, a loss of ‘tumour surveillance’ due to a weakening of the immune system in the brain has been postulated. The data on the brain as a privileged site are not unequivocal, however. The evidence for and against this thesis has been discussed by Woodruff1 and Lance2. Barker and Billingham3 also speculated that “reports that the brain can prevent implanted homografts from inciting sensitivity because it lacks a lymphatic drainage, thus having no afferent pathway of the immunological reflex, are more equivocal”. The need for a critical re-evaluation of the brain as a privileged site has been stressed. We report here a study of the possible action of immunological tolerance in the brain and of the sensitivity of normal rats and tolerant rats to inoculation of allogeneic tumour cells into the brain. We show that transplantation tolerance is involved even in the brain; thus demonstrating the presence of specific immunity following inoculation of tumour cells into the brain. This finding suggests that previously held views of the brain as a privileged site may not be entirely valid and that specific immune processes are accomplished in the central nervous system.

Participation ofH-2 regions in neonatally induced transplantation tolerance

At least three strong histocompatibility (H) loci--the H-2K, H-2D, and H-21 (for review see Klein 1975)--have been demonstrated within the H-2 complex of the mouse, and additional subregions have been defined within the 14-21 region (Shreffler and David 1975). The products of individual loci differ in their physicochemical properties (Hess 1976), immunogenicity (McKenzie and Snell 1973, McKenzie and Henning 1976), and ability to induce enhancing antibody formation (McKenzie and Snell 1973, Staines et al. 1974, Davies and Staines 1976, McKenzie and Henning 1977a, b). The ease with which transplantation tolerance can be induced is related to the genetic disparity between the donor-recipient strain combinations (for review see Silvers and Billingham 1969). Which of the H-2 products might be a major obstacle to overcoming the tissue incompatibility is still unexplained, however. Antigens which stimulate in mixed lymphocyte culture (MLC) have recently been thought to provide the major barrier to tolerance induction in H-2-incompatible combinations (Brent et aL 1976, Brooks 1976). We have tested neonatal tolerance induction in six combinations of H-2-congenic strains in such a way as to provide differences in the entire H-2 complex or in only a small proportion of it. The results presented here indicate that tolerance induction is much more difficult when donor and recipient differ at the K region than when they differ at the D and/or 1regions. All mice were from inbred strains maintained at the Institute of Molecular Genetics, Czechoslovak Academy of Sciences, Prague. The following inbred strains, congenic resistant partner strains, and F 1 hybrid mice were employed: B10.A, C57BL/10ScSn (hereinafter abbreviated B10), B10.D2, B10.AQR, B10.A(2R), B10.A(4R), A.TL, A.TH, (B10.A x B10)F~, (B10.A • B10.D2)F1, (B10.A • B10.AQR)F~, [B10.A x B10.A(2R)]F~, [BI0.A • B10.A(4R)]F1, and (A.TL • A.TH)F r The H-2 genotypes of these strains are listed in Table 1. Tolerance was induced in newborn mice (up to 20 hours of age) by the intravenous injection of 1215 • 10 6 living hybrid spleen cells. Cell suspensions were prepared in Hanks buffered solution from spleens of 2-3-month old donors and injected in a volume of 0.07-0.09 ml into the orbital branch of the anterior facial vein. At 8-9 weeks, the neonatally treated recipients were grafted with the respective allogeneic tail skin, according to standard procedure (Billingham et al. 1954). Grafts were inspected daily the first 4 weeks after transplantation and twice a week thereafter. The success of tolerance induction depended on H-2 antigenic difference between donor and recipient.

Radiosensitivity of suppressor cells in neonatally tolerant rats.

SUMMARY Specific suppressor cells were demonstrated in rats that had carried tolerated skin allografts for long periods of time after being rendered tolerant at birth. These suppressor cells were able to transfer tolerance to sublethally irradiated syngeneic recipients and to inhibit cytotoxic antibody production in normal syngeneic recipients. Suppressive activity of these cells was shown to be radiosensitive. The presence of suppressor cells in tolerant animals was attributable to neonatal tolerance induction and not to skin grafting of neonatally treated animals. In some cases spleen cells from tolerant animals transferred adoptively or induced permanent tolerance to skin grafts, which suggests a long-lasting active mechanism of tolerance.

Vegetative hybridization of animals by joint blood circulation during embryonal development. 1953.

We used our method of parabiosis of eggs during embryogenesis (Hasek 1953) as an expression of vegetative hybridization in animals. In this way, we connected the blood circulation between the Rhode Island and Leghorn breeds of chickens and between ducks and chickens. The advantage of joining animals during embryogenesis is that it results in an intensive exchange of blood at a time when the embryo is not able to react antagonistically by producing antibodies against foreign proteins, that is, at a time when there are suitable conditions for mutual assimilation. We confirmed the lack of antibody formation after immunization of chick embryos by inoculation of duck serum into the yolk sack or intravenously. The connected blood circulation was confirmed by histological analysis and by the passage of dye that was injected into one parabiont and then into the partner. We also investigated the intensity of this exchange by immunobiological assays, using specific antibodies against the partners blood proteins. We proved the presence of duck serum in the blood of chickens, parabiotic with ducks, during parabiosis and also shortly after hatching. We also described examples of a tug of body weight between duck and chicken parabionts, in which the hatched chicken was much heavier than expected from its own nutrition. This proves that the chicken used duck materials for its development. Vegetative hybridization was expressed by a change in the color of feathers, greater body weight, and, the permanent nature (as immunologically demonstrated). The parabionts after reciprocal immunization with the partners erythrocytes did not form any antibodies. This outcome is extraordinary when considered in the light of previous findings of other authors and ourselves and indicates a permanent change caused by the exchange of blood during embryogenesis. We are presently investigating whether there is a permanent change in blood groups, that is, whether the partners agglutinogens permanently persist after vegetative hybridization and the exchange of blood during embryogenesis or whether there is a permanent change in reactivity in the sense that the presence of the partners agglutinogens during parabiosis leads to a lack of antibody responsiveness in adult age. In any case, the described effects indicate that vegetative hybridization has a profound and permanent metabolic influence on the vegetative hybrids. This method has great advantages over previous attempts of inducing parabiosis, particularly in animals because there are different conditions for mutual assimilation of two metabolisms between the embryonal and postembryonal stages. Furthermore, the method is not traumatic for the embryo, whereas the surgical joining of adults limits their mobility and with different nutrition is not physiological and traumatizing for the animals. With our method, only the extraembryonal blood circulation is joined, whereas the embryos retain unrestrained mobility. They come out of parabiosis in a natural way by rejecting the umbilical cords from the extraembryonal circuit at the time of hatching. Despite its profound metabolic influence, the very physiological nature of our method is indicated by the high hatching rate of parabionts, which is 80% with good-quality starting materials (corresponding to the generally observed hatching rate) or even 100% in some experiments.

Induction of transplantation tolerance using serum as antigen source

IT has been shown that massive doses of allogeneic serum produce the prolongation of renal allograft survival in adult pigs1. Furthermore, alloantigenic activity was demonstrated in human serum by inhibiting certain reactions in vitro2–6; and in some strains of mice, the presence of histocompatibility (H) antigens in the serum could be demonstrated by inhibition of the cytotoxicity of specific alloantisera7.

The role of h-2 regions in overcoming tissue incompatibility.

Neonatal transplantation tolerance to the products of theH-2b complex was induced in B10.A (H-2a) mice. On the basis of the survival of skin allografts it was found that antigens determined by theD region of theH-2b complex (of the B10.A(2R) strain) were most easily overcome and that tolerance to the products of theD end of theH-2 complex (of the B10.A(4R) strain) was also easy to induce. The antigens produced by theK end ofH-2 (of the B10.A(5R) and B10.A(3R) strains) represented a stronger incompatibility barrier and a difference in the entireH-2b complex caused strongest resistance to tolerance induction. When tolerance to the products of the entireH-2b complex was induced in newborn B10.A mice, and the neonatally treated animals were grafted simultaneously with five different grafts, those disparate at theK end ofH-2 and in the entireH-2 region were rejected in some animals, while the grafts disparate at theD end of H-2 remained intact in the same mice. No dependence on theI-J subregion was observed in this system. Furthermore, tolerance was more easily inducible in male than in female B10.A mice.

The regulatory role of I-J subregion in neonatal tolerance induction to H-2D alloantigens.

The mechanism of neonatally induced transplantation tolerance was studied in two mouse strain combinations involving differences at the D region of the H‐2 complex only or at the same D region plus I‐J subregion (including I‐E, I‐C, S and G regions). In the strain combination with the H‐2D difference only, cells from tolerant mice proliferated markedly in the MLR assay when incubated with antigens tolerated in vivo, whereas the MLR reactions were negative in the combination with D plus I‐J region disparities. In the latter combination cells from tolerant mice also did not respond to third‐party antigens and their incubation with the tolerated antigens led to the suppression of cell proliferation. This non‐specific suppression was absent in cells from tolerant mice in the strain combination, which differed in I‐C, S, G and D alloantigens. Specific suppressor cells, which inhibited the development of cytotoxic cells, were demonstrated in tolerant mice of both strain combinations. The results show that, in addition to the specific suppressor cells induced by H‐2K or H‐2D alloantigens, non‐specific suppressor cells induced by the I‐J region disparity that may regulate the resultant activity against H‐2D (and probably also H‐2K) alloantigens are involved in transplantation tolerance.

Attempts to compare the effectiveness of blocking factors and enhancing antibodies in vivo and in vitro.

Sera from rats carrying tolerated skin allografts were tested for the presence of blocking activity in vitro. Sera with blocking activity had no effect on transplantation tolerance induction in newborn animals. Immunological enhancement of tumor growth was procured by passive transfer of serum from tolerant animals bearing skin allografts. It made no difference whether or not the serum contained blocking activity in vitro. These results suggest that there is no relationship between blocking factors and enhancing activity in vivo.