Skin tumorigenesis by initiators and promoters of different chemical structures in lines of mice selectively bred for resistance (Car-r) or susceptibility (Car-s) to two-stage skin carcinogenesis.

Carcinogenesis-resistant (Car-R) and carcinogenesis-susceptible (Car-S) mice were obtained applying a bi-directional selective breeding approach to a two-stage skin carcinogenesis protocol, using 9,10-dimethyl-1,2-benzanthracene (DMBA) as initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as promoter. Sixteen generations of selection produced a remarkable interline difference in responsiveness to two-stage skin carcinogenesis between Car-R and Car-S: identical DMBA (25 microgram) and TPA (5 microgram) doses induced papillomas in 100% of Car-S compared with 3.3% of Car-R mice and maximal responses of 14.3 or 0.03 papillomas/mouse, respectively, despite the shorter promotion applied to Car-S (49 vs. 208 days). To define the factors determining this great difference, Car-R and Car-S mice were challenged by initiators/promoters chemically unrelated to those used for selection. Both lines were subjected to either initiation by N-methyl-N-nitrosourea (MNU) followed by TPA promotion, or promotion by benzoyl peroxide, or 1,8-dihydroxy-3-methyl-9-anthrone (chrysarobin) following DMBA initiation. Initiation with MNU induced a 10-fold tumour incidence in Car-S compared with Car-R mice, and a 32-fold difference in tumour induction rate. The 2 lines also differed markedly in susceptibility to benzoyl peroxide promotion: Car-S mice initiated with 25 microgram DMBA and promoted with 7.5 mg benzoyl peroxide showed a 12-fold tumour incidence and a 103-fold tumour induction rate compared with the corresponding Car-R group. Both lines, however, were refractory to chrysarobin promotion. The progression of papillomas to carcinomas was examined in all Car-S groups. The incidence of mice that developed carcinomas was 57% in MNU-initiated mice. Benzoyl peroxide was also able to promote carcinoma development in Car-S mice, though with a lower incidence (30.4%) than TPA.

Low antibody responsiveness is found to be associated with resistance to chemical skin tumorigenesis in several lines of Biozzi mice.

High and low antibody responder lines of mice from Selections I, III and G were assayed for two-step skin tumorigenesis using a protocol consisting in initiation with 7,12-dimethylbenz[a]anthracene (DMBA) and promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). Concordant results were obtained in the three selections: low antibody responder mice were shown to be significantly more resistant to tumor induction than the high responder counterparts. The difference was observed for all parameters: kinetics and percentages of tumor incidence and tumor multiplicity. The three bidirectional selective breeding experiments differed in several respects namely, the origin of the foundation populations, the antigens and immunization protocols used during the selection, as well as the breeding unit environments. Therefore, the consistent results relative to tumorigenesis strongly suggest that some of the alleles relevant to multispecific 'low' antibody production could contribute to the resistance to cutaneous chemical tumorigenesis.

Susceptibility and resistance to Leishmania amazonensis in H-2q syngeneic high and low antibody responder mice (Biozzi mice).

H-2 syngeneic H and L (Biozzi) mice provide a model to study Leishmania infections in which polar resistant and susceptible phenotypes are independent from H-2 differences. High-Ab-responder (H) and low-Ab-responder (L) mice syngeneic at the H-2 locus (H-2q) were, respectively, susceptible and highly resistant to Leishmania amazonensis infection. L-mice resistance was associated with high IFN-gamma and transient IL-4 production by lymph node (LN) cells, in contrast with sustained IL-4 and decreasing IFN-gamma production by susceptible H mice. IL-12 production could be detected only in LN from resistant mice. The cytokine production pattern was consistent with preferential progression to a Th1-type response in resistant L-mice, and to a Th2-type response in susceptible H-mice. We also investigated whether this shift towards Th1- or Th2-type cytokine responses was dependent upon H or L antigen presenting cells' (APC) intrinsic ability to preferentially stimulate either T-cell subset. To this end, LN-derived T-cell lines were grown from 12-day infected mice, when both strains produced IFN-gamma and IL-4. L-derived T-cell lines developed a Th2 cytokine pattern whereas H-derived T-cell lines produced IFN-gamma, IL-4 and IL-10 whatever the APC origin (H or L) used for their derivation. This work constitutes the first characterization of cellular immune responses to the intracellular parasite, L. amazonensis in H-2 syngeneic mice, an infection model in which polar resistant and susceptible phenotypes are determined by non-MHC genes.

Identification of two quantitative trait loci involved in antibody production on mouse chromosome 8.

Several quantitative trait loci (QTLs) contributing to the extreme phenotypes of the selected high (H) and low (L) antibody-responder lines of mice were mapped on distinct chromosomes. Successive backcrosses were bred to reduce the length of the QTL-bearing segment detected on chromosome 8 and to produce congenic lines to test gene effect independently of the other QTLs. An increase in antibody responses was repeatedly found to be associated with inheritance of the H-line allele at two markers separated by 30 cM on that chromosome. In the successive backcrosses, background and unlinked involved genes of H-line origin were progressively eliminated; however, unexpected within-progeny variations persisted in the third and even fourth backcross. Nevertheless, the presence of two QTLs within the considered interval was definitely demonstrated in distinct progenies of the fourth backcross which separately inherited one of the two gene-marker H-line alleles. The previously identified chromosome 8 segment therefore contains at least two QTLs involved in antibody responsiveness.

Interactions between H-2 and background genes modulate collagen induced arthritis in high (HI) and low (LI) antibody producer Biozzi mice.

The experimental arthritis (collagen induced arthritis, CIA) induced in mice by heterologous collagen of type II is mainly restricted to the H-2q or H-2r haplotypes. However, data including ours, strongly suggest that CIA is also under non MHC polygenic control. This point has been studied in new sub-strains of high (HI) and low (LI) Biozzi mice made congenic for H-2q and H-2s: the original Biozzi lines HI/H-2q and LI/H-2s and the new HI/H-2s, LI/H-2q congenic mice. 80% of the HI/H-2q mice develop severe chronic inflammatory symptoms with joint deformation and swelling soon after induction of the disease, while 60% of LI/H-2q counterpart develop at a later stage, deformation of joints with no or mild swelling. In the H-2s haplotype, considered to be non or weakly permissive to CIA, 40% of HI/H-2s have strong CIA symptoms; the LI/H-2s being totally refractory. Thus, if MHC products play a crucial role in selecting the arthritogenic epitope of CII; non H-2 genes strongly modulate the severity of experimental arthritis.

Toward genetic dissection of high and low antibody responsiveness in Biozzi mice.

Several distinct chromosomal segments were recently identified by cosegregation analysis of polymorphic markers with antibody responsiveness in an F2 cross between high (H) and low (L) antibody responder lines of Biozzi mice. The effect associated with the relevant markers has now been investigated in backcross populations (toward the L line) bred from H and L mice made coisogenic at the H-2 locus. The antibody titers, measured on days 5 and 14 of the primary response to sheep red blood cells, were considered to be two distinct quantitative phenotypes. The results of single or multilocus analyses demonstrated the significant involvement, at one or the two titration times, of Im gene(s) on four distinct chromosomes: 4, 8, 12, and 18. The regions on chromosomes 6 and 10 have a lesser but still suggestive effect. The contribution of each locus ranged from 3% to 13%, and together these loci accounted for about 40% of the phenotypic variance at each titration time. The data are compatible with an additive effect of the relevant loci and suggestive of some interaction effects. In a second backcross toward L line, the H line alleles of the putative Im genes on chromosomes 6, 8, and 12 were isolated from each other and their effects were still detected.

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.

Variations in susceptibility to Leishmania amazonensis infection in lines of mice selected for high or low immunoresponsiveness.

The degree of resistance to a local Leishmania amazonensis challenge has been compared in lines of mice obtained by selective breeding for high or low immunoresponsiveness: High and Low antibody responder mice of Selections I and II (HI, HII and LI, LII lines) and high and low responder mice to T mitogen PHA (Hi/PHA and Lo/PHA). The aim of this preliminary study was to focus attention on genetic differences related with well defined immune characteristics. Clear-cut results were obtained, both HI and HII mice developed large and disseminating lesions, the rate of symptom aggravation being faster in HII, while LI and LII proved resistant to parasites, only small and transient lesions being observed for them during a 150 days follow up. The outcome of infection also differs in Hi/PHA and Lo/PHA mice, Hi/PHA having a resistant and Lo/PHA a susceptible phenotype.

Isotypic distribution of antibody responses in lines of mice selected for high or low immunoresponsiveness.

1. The isotype distribution of antibody (Ab) responses to Salmonella antigens (Ag) was investigated in high (H) and low (L) Ab responder lines of mice from Selections III and IV carried out for responsiveness to flagellar (f) and somatic (s) Ag, respectively. 2. Primary immunization resulted in higher Ab titers of all isotypes in response to both Ag in H mice from both selections and was confirmed after booster injections. The interline difference (H-L) in response to the distinct isotypes ranged from 3.0 to 7.0 log2 to Ag f in Selection III and from 2.0 to 5.1 log2 to Ag s in Selection IV. 3. Comparison of isotype production to 3 Ag in Selections I, II, III and IV demonstrated that: 1) the highest responses in all mice are those against the selection Ag, 2) the isotypic pattern depends on both the Ag injected and the host's genetic constitution, and 3) the presence or lack of a multispecific effect is not due to isotype-restricted regulation.

Isotypic distribution of antibody responses in lines of mice selected for high or low immunoresponsiveness

The isotype distribution of antibody (Ab) responses to Salmonella antigens (Ag) was investigated in high (H) and low (L) Ab responder lines of mice from Selections III and carried out for responsiveness to flagellar (f) and somatic (s) Ag, respectively. Primary immuniztion resulted in higher Ab titers of all isotypes in response to both Ag in H mice fro m both selections and was confirmed after booster injections. The interline difference (H-L) in response to the distinct isotypes ranged from 3.0 to 7.0 log2 to Ag f in Selection III and from 2.0 to 5.1 log2 to Ag s in Selection IV. Comparison of isotype production to 3 Ag in Selections I,II,III and IV demonstrated that: 1) the highest responses in all mice are those against the selection Ag, 2) the isotypic pattern depends on both the Ag injected and the host's genetic constitution, and 3) the presence or lack of a multispecific effect is not due to isotype-restricted regulation

Spontaneous rheumatoid-like arthritis in a line of mice sensitive to collagen-induced arthritis.

Twenty-eight percent of 13-month-old male mice of the high antibody responder line of Biozzi's selection I (HI) spontaneously developed a long-lasting inflammatory arthritis. This disease was clinically and histologically similar to human rheumatoid arthritis. The synovium of joints and some tendons was hypertrophied, with thickening of the synovial cell layer and infiltration by polymorphonuclear and mononuclear leukocytes. In some cases, synovial pannus formation led to destructive damage of articular cartilage and bone. Rheumatoid factor and antinuclear, anti-DNA, and anti-type II collagen (CII) antibodies were often found in the sera of both arthritic mice and clinically normal littermates. The presence of CII autoantibodies in this line of mice suggests that a potentially harmful anti-CII T cell autoimmunity can also develop spontaneously and lead to joint damage. Moreover, HI mice are also susceptible to collagen-induced arthritis, while a closely related mouse line (HII) is resistant to both diseases. These data support the hypothesis that collagen-induced arthritis is pathogenetically related both to this spontaneous arthritis and to rheumatoid arthritis.

Polymorphism of Tcrb and Tcrg genes in Biozzi mice: segregation analysis of a new Tcrg haplotype with antibody responsiveness.

Tcrb and Tcrg gene polymorphism was investigated in high (H) and low (L) responder Biozzi mice from selection I, II, and GS by Southern blot analysis with appropriate V and C probes. No polymorphism of the Tcrb haplotype was detected between H and L mice in all selections which were all found to be of the BALB/c type. The H-I and H-II g genotype was of BALB/c and DBA/2 type, respectively. In contrast, a new Tcrg haplotype shared by L-I and L-II mice was identified and characterized by C gamma 1, 2, 3, C gamma 4, V gamma 1, 2, 3, V gamma 5, and V gamma 6 restriction fragment length polymorphisms (RFLPs). Tcrg genotypes were not fixed in the GS selection and two additional new haplotypes were identified in two L-GS mice. An attempt was made to correlate the L-I g genotype with the low responder status by analyzing g haplotypes among highest and lowest responder (H-I X L-I)F2 hybrids immunized with sheep red blood cells (SRBC). No correlation was found in this segregation study, whereas a highly significant one was established with the H-2 haplotype, a locus already known to participate in the genetic control of H-I/L-I difference. The lack of correlation between SRBC response and the Tcrg genotype was consistent with the heterogenous g haplotypes found in mice of the GS selection. Together, the present results suggest that H and L mice have the same Tcrab potential repertoire and that T-cell receptor (Tcr) genes cannot be considered as immune response genes in this model. Our results also indicate that the F2 segregation analysis, given a polymorphic gene, is suitable for an investigation of its immune response functions.

Salmonella typhimurium infection in high and low antibody responder mice: inverse correlation between antibody responsiveness and resistance to infection.

Susceptibility to Salmonella typhimurium infection was compared in H (high Ab responder) and L (low Ab responder) mice obtained by several selective breeding experiments (Selections I, II, III, IV and IV A). H mice were always much more susceptible to infection than their L mice counterparts within a continuous LD 50 variation range. In three of the selections (I, II and IV A) the low responsiveness character is known to result mainly from rapid Ag degradation in L mice macrophages. It was hypothesized that resistance to multiplication of intracellular pathogens could be related to an increased catabolic activity towards Ag. This was actually demonstrated, in F2 segregant hybrids of selection IV A, by the significant inverse correlation between capacity for Ab production and resistance to infection.

Evaluation of T helper function in lines of mice selected for high or low antibody production: quantitative inhibition of immune responses by anti-L3T4+ monoclonal antibody.

The potentialities of T helper cell compartment were compared in lines of mice selected for high or low Ab production against heterologous erythrocytes (H1 and L1 mice) by investigating the "in vivo" and "in vitro" modulation of immune responses by GK 1-5 anti-L3T4+ mAb. FACS analysis showed a frequency of L3T4+ cells similar in non-immunized mice of both lines. However, LI mice were more susceptible to inhibition of the IgG Ab response to SE when injected GK 1-5 prior to immunization. The "in vitro" proliferation of T cells specific for HEL was higher in HI than in LI LN cultures, and a higher GK 1-5 mAb concentration had to be applied to HI cultures in order to produce the inhibitory effect. In contrast, during MLR, the capacity for T proliferation was similar in HI and LI cultures stimulated by a common F1 target. This allo-Ag-induced proliferation was inhibited at similar GK 1-5 mAb concentrations in the two lines. These results demonstrate that there is no intrinsic L3T4+ cell deficiency in LI mice. Differences in GK 1-5 mAb required for inhibition of responses to Ag (other than allo-Ag) in HI and LI mice are mainly due to different macrophage T cell triggering.

Collagen-induced arthritis in Biozzi mice. Joint involvement is not correlated with collagen II IgG2a autoantibodies nor restricted to only H-2q and H-2r.

High (H) and low (L) immune responder "Biozzi" mice, obtained by four different selections, were investigated for their ability to develop collagen-induced arthritis. Both LI and LII lines--characterized by their low antibody responses to a wide variety of Ag--developed arthritis though they do not bear the susceptible H-2q and H-2r haplotypes. Out of the two lines (HI and HII) selected for their high antibody responses and bearing H-2q, only one (HI) developed arthritis. Both the lines with amplified high or low antibody responses (HG and LG), and the lines differing in the levels of cell-mediated immunity (Hpha and Lpha), failed to develop arthritis. Collagen II autoantibodies were found in all the lines: the responses being high (HI and HG), low (LI, LII and LG), or intermediate (HII, Hpha and Lpha). The level of IgG2a autoantibodies, presumed to be the most pathogenic, was low in two (HI and LII) of the three arthritic lines, and was high in the unaffected HG line. These results show that this arthritis is not solely restricted to H-2q and H-2r haplotypes, and argue against a correlation between collagen autoantibody levels and disease incidence.

Inheritance of immune responsiveness, life span, and disease incidence in interline crosses of mice selected for high or low multispecific antibody production.

High (H) and low (L) antibody responder lines of mice separated by selective breeding present a maximal interline difference in antibody (Ab) response to Ag of different specificities (general genetic regulation). The analysis of SRBC agglutinin response in H line, L line, F1 hybrids, F2, and backcross segregants demonstrates that Ab responsiveness is a polygenic trait regulated by the additive interaction of 5 to 7 independent loci, with an incomplete dominance (44% +/- 7%) of the high response character, and a 30% +/- 10% impact of the environmental factors. The life span of H, L, F1, F2, and backcross populations is correlated positively with 2-ME-resistant agglutinin response (r = 0.97, p less than 0.001) and negatively with 2-ME-sensitive agglutinin response (r = 0.95, p = 0.01) (interpopulation correlation). Similar correlations are also observed in individuals of the various populations, especially in F1 x L backcross, in which the largest phenotypic variance is found. The positive correlation between Ab responsiveness and life span was confirmed by ELISA titration for distinct IgG isotypes (intrapopulation correlation). Malignant lymphomas and chronic nephritis were the two most common diseases observed. The age-adjusted incidence of such diseases, which is largely affected by environmental factors, accounts for the longer life span of H, as compared with L, mouse populations. The longevity of the 30% or less survivors, chiefly determined by the rate of physiologic aging, is a polygenic character regulated by the cumulative interaction of 3 to 7 independent loci, with a complete dominance of the long life trait and an impact of the environmental factors of about 60%. Thus we have grounds for regarding general Ab responsiveness and life span as polygenic traits regulated by a small number of identical or closely linked gene loci, and immune responsiveness as a defense mechanism against neoplastic and inflammatory diseases.

Genetic regulation of multispecific antibody responsiveness: improvement of "high" and "low" characters.

The five selections carried out in the mouse for high or low antibody responsiveness to various multideterminant immunogens were successful. In all cases the large interline difference was shown to result from the additive effects of several independently segregating loci (polygenic regulation). However, important peculiarities were demonstrated in these original selections concerning either the cellular mechanisms operating or the effect of the selected genes on antibody responses to antigens unrelated with those used for the selection (multi-specific effect). In an attempt to improve and generalize the effect of selection, the 5 high and the 5 low lines were inter-crossed to obtain populations with a balanced proportion of the 5 genomes. These two populations were then submitted to selective breedings in which the phenotypic character was the weighted responses to pluri-antigen immunization. The data obtained in 16 consecutive generations of two selective breedings (general-primary, GP and general-secondary, GS, responses) carried out from these populations are reported. The genetic parameters of the response to GP and GS selections are compared with those obtained in the original selections. The final result of both GP and GS selections demonstrate a marked improvement of the high and low antibody production traits, both quantitatively (interline divergence) and qualitatively (multi-specific effect). The success of GP and GS selections agrees with the concept that distinct groups of genes are preferentially affected by selection according to the nature of the selection antigen and the immunization procedure.

Effect of silica on the genetic regulation of antibody responsiveness.

The high (H) and low (L) antibody responder lines of mice produced by selective breeding are characterized by different modifications in immunocompetent cell potentialities, according to the immunization procedure used for the selection process. In selections I and II, the difference in antibody responsiveness between H and L lines was clearly shown to depend mainly on macrophage function: the more rapid catabolism of antigens in L mice was the main cause of the low antibody production. In contrast, up to now, no difference has been observed between H and L mice of selections III and IV in terms of the macrophage accessory role. The administration of silica particles has a well known impairment effect on macrophage activity. Therefore, the effect of silica injection on the kinetics of antibody responses to selection antigens was compared in H and L mice of the four selections. Silica was given either intravenously or locally in one hind footpad 6 or 24 h before immunization by the same route. Silica treatment consistently improved antibody responsiveness in the L mice of selections I and II, but had no effect in the L mice of selections III and IV. The antibody responses of the H lines of the four selections were not substantially modified by silica injections. Therefore, the silica treatment reduced the interline difference in antibody responses in selections I and II only, by interfering with the expression of the genetic modification of macrophage activity. However, a similar effect was not obtained with other substances known to affect macrophages, including dextran sulphate or carrageenan. The results reported here are in agreement with the above-mentioned statement that the genetic modification of macrophage function plays a major role in the interline difference in selections I and II and is not involved in selections III and IV.

Basal immunoglobulin serum concentration and isotype distribution in relation to the polygenic control of antibody responsiveness in mice.

Serum Ig concentration and isotype distribution were determined in the high (H) and low (L) responder lines selected for antibody response to complex immunogens. Data were recorded in normal and postimmunization sera from the H and L lines produced by five independent selective breedings (selections I, II, III, IV, and V). Ig levels were much higher in H than in L mice of all the selections. In four selections this interline difference increased further after immunization with the selection antigens. This is in agreement with the general effect of the polygenic control of antibody responses operating in H and L lines. The Ig isotype profiles of normal sera were different in each line; however, similitudes were noticed between H and L lines in selections I and II. In contrast, in selections III, IV, and V a similar interline difference was observed: the lack of IgG2a isotype in L lines only. After immunization there were minor alterations of the isotype profiles except in the H lines of selections III and IV, in which a clear inverse modification of IgG1 and IgG2a proportions occurred. The characteristic pattern of each selection may be partially dependent on isotype-restricted regulatory effects in relation to the immunization procedure used for selective breeding.