Immunodeviation by passive antibody, an expression of selective immunodepression. II. Action of guinea pig IgG1 and IgG2 anticarrier antibodies.

The effect of passively administered IgG1 and IgG2 anticarrier antibodies on the IgG1 and IgG2 antihapten response has been studied. Guinea pigs were immunized with dinitrophenylated bovine gamma globulin mixed with purified IgG1 or IgG2 antihuman gamma globulin antibodies, i.e., antibodies directed against a limited range of the carrier determinants. Humoral IgG1 and IgG2 anti-DNP antibody contents were assayed at weekly intervals and 4 to 10 days after a booster injection of antigen in saline given on the 12th wk. The main finding was the sustained suppressive effect of passive IgG1 anti-carrier antibodies on the active IgG1 antihapten response. This result is compared with the enhancing effect of passive IgG1 antihapten antibodies and is discussed in the light of T cell-B cell and hapten-carrier relationships, leading to the proposal of a regulatory function of the Fc portion of the IgG1 anticarrier antibody, combined with the antigen, on the T cell.

Description and comparison of three in vitro methods for the detection of guinea pig MHC antigens.

Two methods (an immunoenzymatic and a cell dot immunobinding assay) are described and compared with a reference protein A rosetting assay, for the detection of guinea pig class I and class II histocompatibility antigens. The main requirement of these three assays for obtaining good results is the use of whole fresh target cells since all tested experimental conditions for fixing cellular antigens (glutaraldehyde, formaldehyde, paraformaldehyde), as well as for obtaining cell monolayers (adherence, desiccation, poly-L-lysine) presented several drawbacks and were unsuccessful in our hands. The comparison between the three methods shows that the immunoenzymatic assay gives good results but that it is less reproducible than the other two. The cell dot immunobinding is highly sensitive, reproducible and economical. It can be used for genetic typing and alloantigen analysis of guinea-pig lines, instead of the rosetting method, which is highly reliable but time consuming.

A rapid and reliable in vivo method for anti-GPLA (class I and class II antigens) antibody titration and GPLA typing.

A reverse Arthus reaction (RAR) may be successfully used in guinea pigs to detect histocompatibility alloantigens of the GPLA type. Epidermal cells carry class I GPLA antigens, and Langerhans cells also bear class II alloantigens. It is therefore possible to elicit an RAR by intradermal injection of relevant alloimmune sera in the skin of guinea pigs of known GPLA haplotype. RAR is detected by increased vascular permeability due to IgG1 antibody and hemorrhage due to IgG2 antibody. Compared with an in vitro protein A-rosetting method RAR is easier and quicker. It proved more sensitive for class II antigens in which Langerhans cells are the target for anti-class II antibody and rather less sensitive for class I antigens. RAR is a convenient method for following the course of GPLA alloimmunization, allowing titration of antibodies against both classes of antigen. It may also be used to type guinea pigs of unknown GPLA haplotype.

Alloantibody bipolar bridging; a new mechanism of cell surface activation.

Bipolar bridging of cellular membrane receptors and epitopes by alloantibodies (Fab bridging the MHC antigens and Fc the Fc receptors) has been shown on a murine mast cell model to be a way of cell signaling and activation. In order to test a possible general significance of this phenomenon, another model was studied, namely guinea pig neutrophils. It was found l(1) that neutrophils from S2, S13 and BIO-AD strains both express class I (B) and class II (Ia) antigens on their surface, as detected by a Prot.A-SRBC rosetting method, after cell incubation with related alloantibodies; (2) that Fc receptors for IgG (Fc gamma R) were specific for IgG2 subclass, as determined by the same rosetting method after binding of preformed immune complexes (IgG1, IgG2 and F(ab')2 anti-DNP-DNP25 BSA); and (3) that specific alloantibodies of IgG2 subclass were able to specifically activate the neutrophil oxidative metabolism as shown by superoxide anion (O2-) release, detected by the luminol-dependent chemiluminescence method. Neither the IgG1 nor F(ab')2 portion were able to trigger O2- release. This demonstrates a second situation of a cell membrane activation through alloantibody bipolar bridging.

17 beta estradiol affects the expression of guinea pig blood leukocyte MHC antigens.

Sex hormones have an effect on various immune responses but the mechanisms of action are unknown. One of these mechanisms might be a modification of expression of major histocompatibility complex (MHC) antigens in blood leucocytes. Estradiol-induced variations of the expression of guinea pig blood leukocytes MHC antigens (GPL-A) was studied. Class I and class II MHC antigens were detected by a sensitive rosetting method using specific alloimmune sera (AIS) and staphylococcal protein A-coated sheep red blood cells (SPA-SRBC) and evaluated by counting the number of bound SPA-SRBC per 100 cells. MHC antigens decreased after estrogen treatment. Estradiol modifies the expression of GPL-A antigens on the mononuclear cells including the Kurloff cells, which are involved in immunity or in a natural killer effect, but did not affect the expression of polymorphonuclear cells, ones which are not involved in immunity.

Study of the antigenic cross reactivity between carcinoembryonic antigen and "nonspecific cross reacting antigens" (NCA and NCA 2).

The immunochemical relationship between CEA, NCA and NCA 2 was studied in guinea-pigs. Strong cross reactions were found between these antigens, either in delayed or anaphylactic reactions. Some specific determinants for each antigen could still be demonstrated. Delayed hypersensitivity is likely to be due to the protein moiety of the molecules while anaphylactic reactivity could probably be related to their glucidic parts. Consequently, CEA and NCA have common antigenic determinants on their glucidic and peptidic moieties, perhaps more on the latter ones.

GPLA antigens are not expressed on guinea-pig epididymal spermatozoa.

The expression of guinea-pig major histocompatibility antigens (class I and II) has been investigated on guinea-pig epididymal spermatozoa. Specific alloantisera (anti-B1, anti-B3, anti-Ia2,4 and anti-Ia1,3,7) were obtained by cross-immunization of strains 2, 13 and BIO-AD animals with relevant spleen cell membranes. These sera were tested on spermatozoa from the same three strains by a protein A rosetting assay and by an indirect immunofluorescence test. The results obtained showed non-strain specific reactions of all the alloantisera tested on epididymal spermatozoa of the three strains. These non-strain specific reactions were absorbed by spermatozoa of any strain but they were not absorbed by the splenic cells of the same animals. On the other hand, the alloantisera specific reactivity on peripheral blood cells was not diminished after incubation of the sera with spermatozoa. Furthermore anti-B1 and anti-B3 antibodies eluted from guinea-pig platelets did not react with any spermatozoa but reacted with the relevant peripheral blood cells. These results indicate that the studied guinea-pig sera contained two types of antibodies: anti-MHC antibodies able to react with relevant blood cells but not with spermatozoa and sperm specific antibodies (also observed in untreated and DNP-BGG immunized guinea-pig sera) reacting with all spermatozoa but not with blood cells. They are not compatible with the expression of MHC antigens at the surface of guinea-pig epididymal spermatozoa.

Genetics of chemical carcinogenesis. 1. Bidirectional selective breeding of susceptible and resistant lines of mice to two-stage skin carcinogenesis.

Six generations of a bidirectional selective breeding model for producing lines of mice susceptible (Car-S) and resistant (Car-R) to two-stage skin carcinogenesis are described. Initiation was with 9,10-dimethyl-1,2-benzanthracene (DMBA single application), and promotion with 12-O-tetradecanoyl-phorbol-13-acetate (TPA twice weekly). The selective breeding was initiated with a highly genetically polymorph foundation population, produced by the intercrossing of eight inbred mouse strains. The Car-S line was produced by assortative mating of the mice presenting the largest number of tumors induced by low DMBA and TPA doses, the Car-R line by mating tumorless mice or mice showing the smallest number of tumors induced by large DMBA and TPA doses. The character investigated was expressed as per cent tumor incidence and as tumor multiplicity per mouse. The mean heritability of the susceptibility character for the two first generations was 0.84 for tumor incidence and 1.3 for tumor multiplicity; these values decreased to 0.53 and 0.44 respectively for the two consecutive generations. The heritability of the resistance character maintained a constant value of 0.29 +/- 0.04 for tumor incidence, and 0.53 +/- 0.08 for tumor multiplicity. The progressive response to selection indicates that the characters investigated are subject to polygenic regulation, even though some genes may have a major effect on the susceptibility character. The interline separation in F5, challenged with the same initiation and promotion schedule, is very large. In the Car-S line, tumor incidence was 82.5% and tumor multiplicity 4.9/mouse on promotion day 49, whereas the corresponding values for the Car-R line were 4.5% and 0.1/mouse on promotion day 81.

Genetics of chemical carcinogenesis--III. Tissue-specificity of the genes controlling susceptibility and resistance to skin carcinogenesis in the mouse.

Carcinogenesis-resistant (Car-R) and carcinogenesis-susceptible (Car-S) mice have been obtained by the method of bi-directional selective breeding. After 10 generations of selection Car-R and Car-S mice show a remarkable difference in their response to chemical carcinogenesis. Car-R and Car-S mice, initiated and promoted by skin application of 9,10-dimethyl-1,2-benzanthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) reach a tumour multiplicity of 0.05 and 6.2, respectively, after 49 days of promotion. When benzo[a]pyrene (B[a]P) is topically applied for initiation, followed by TPA promotion, Car-R and Car-S mice maintain a large difference in sensitivity to skin tumour induction. Car-S mice are also more susceptible than Car-R mice to complete carcinogenesis produced by single or repeated applications of DMBA only. On the contrary, when DMBA or B[a]P are administered by subcutaneous injection rather than by topical application, no significant difference in tumour incidence is observed between the two lines. All tumours induced by topical administration of carcinogens on the skin are of epithelial origin, whereas the tumours produced by subcutaneous injection are of connectival origin. These observations suggest a tissue-specific effect of the selected genes, probably restricted at the skin level.

Genetics of chemical carcinogenesis: analysis of bidirectional selective breeding inducing maximal resistance or maximal susceptibility to 2-stage skin tumorigenesis in the mouse.

We report on bidirectional selective breeding, initiated from a genetically defined foundation population and carried out to selection limit, for producing lines of mice endowed with maximal resistance (Car-R) or maximal susceptibility (Car-S) to 2-stage skin tumorigenesis. The initial population resulted from a balanced intercrossing of 8 inbred strains of mice. The tumors, induced by a single application of DMBA (initiation) and twice weekly applications of TPA (promotion), were benign papillomas; their number at the end of the promotion period was the phenotype chosen for assortative mating. Afterward, the majority of them regressed while others progressed to malignant carcinomas. The Car-R line was selected through a strong challenge, while the Car-S line selection was based on responses to decreasing concentrations of DMBA and TPA. The selection limit was reached after 14 or 15 generations showing progressive interline divergence, which strongly suggests the interaction of several quantitative trait loci (QTL). The phenotypic difference was extremely large: the tumor response was 73 times higher in Car-S than in Car-R mice, though the applied concentrations of DMBA and TPA were 100 and 40 times lower, respectively. The mean heritability realized during the selective breeding was 0.20 in Car-R and 0.49 in Car-S. Our results are compatible with a minimal QTL estimate of 8 in the Car-R line and of 9 or 10 in the Car-S line. The Car-S line is also much more susceptible to carcinoma induction. An association of coat color with tumorigenesis was observed in interline F2 segregants. The Car-R and Car-S lines, obtained through a long-lasting breeding program, are a unique model for identifying the QTL involved in chemical tumorigenesis and will be provided to interested investigators.

Genetics of chemical carcinogenesis--II. Papilloma induction and malignant conversion in susceptible (Car-s) and resistant (Car-R) lines of mice produced by bidirectional selective breeding and in their (Car-S X Car-R) F1 hybrids.

Susceptible (Car-S) and resistant (Car-R) lines of mice separated by 10 consecutive generations of bidirectional selective breeding present a very large difference in responsiveness to two-stage skin carcinogenesis. Car-S mice initiated with 0.5 micrograms 9,10-dimethyl-1,2-benzanthracene (DMBA) and promoted with 0.25 micrograms 12-O-tetradecanoyl-phorbol-13-acetate (TPA) for 77 days showed a papilloma incidence of 88% and a tumour multiplicity of 3.2 +/- 0.4 (mean +/- SE), with a tumour induction rate of 0.415. Car-R mice initiated with larger DMBA and TPA doses (50 micrograms and 20 micrograms respectively) and promoted for 111 days gave a comparable papilloma response: incidence 65%, tumour multiplicity 3.2 +/- 0.6 and tumour induction rate 0.288. The difference in papilloma response between the two lines is due to the interaction of genetic and environmental factors. In order to overcome the genetic effect with environmental factors and induce in Car-R a papilloma response comparable to that of Car-S, the DMBA dose had to be increased up to 100 times, that of TPA 40 times and the promotion time augmented by 44%. Papilloma to carcinoma conversion 112 days after the end of promotion depends on the DMBA and TPA doses applied. The number of carcinomas induced in Car-S mice and in (Car-S X Car-R) F1 hybrids was larger than that induced in Car-R mice, but the ratio of carcinoma conversion was lower, therefore a larger proportion of the small number of papillomas induced in the Car-R mice progressed to malignancy. The dominance effect measured in (Car-S X Car-R) F1 hybrids demonstrated that the susceptibility to papilloma induction was an incomplete dominant character (d/a = 0.38), whereas for carcinoma conversion the resistance was incompletely dominant (d/a = -0.49).

Effect of genetic modification of acute inflammatory responsiveness on tumorigenesis in the mouse.

Two distinct bidirectional selective breedings for quantitative traits were initiated from identical genetically heterogeneous mouse populations. The resulting lines are characterized by maximal or minimal acute inflammatory responsiveness (AIR): AIRmax and AIRmin lines, respectively, and by resistance or susceptibility to chemical skin tumorigenesis: Car-R and Car-S lines, respectively. The AIR response to s.c. injection of polyacrylamide microbeads, measured by cell content in the local exudate, was 10 times higher in AIRmax than in AIRmin mice. The response to selection was asymmetrical: the realized heritability was 0.26 in AIRmax and 0.008 in AIRmin, and resulted from the additive effect of 7-11 quantitative trait loci (QTL). Low responsiveness was globally dominant in F1 and 48% of F2 segregant variance was found to be due to genetic factors. These findings are the first demonstration of innate regulation of AIR by germ line genes. Susceptibility to skin tumorigenesis induced by a two-stage initiation (DMBA)-promotion (TPA) protocol was lower in AIRmax mice than in AIRmin mice, a 6-fold difference in tumor induction rate. Intense AIR was found to be associated with resistance, and low AIR with susceptibility to tumorigenesis, in F2 segregants chosen for extreme AIR phenotypes. At least some of the AIR QTLs therefore contain genes controlling tumorigenesis. Tumor phenotypes differed more in Car-R and Car-S than in AIRmax and AIRmin lines, indicating that QTLs unrelated to AIR, contribute to the host response to tumorigenesis. The extreme phenotypes/genotypes of the four selected lines and the known genetic constitution of their foundation population, offer new possibilities to discriminate the genes/mechanisms controlling two important traits: AIR and response to chemical tumorigenesis. Collaborative projects will be favorably considered. The description of tumor resistance genes in AIRmax and Car-R mice may be helpful for epidemiology and therapy of human cancer.