Genetic control of mast cell development in bone marrow cultures. Strain-dependent variation in cultures from inbred mice.

A comparison was made of the capacity of bone marrow cells (BM) from genetically distinct strains of mice to develop into mast cells under defined conditions of in vitro culture. In the presence of conditioned media derived from ConA treated spleen cells from normal or Trichinella spiralis-infected mice, mast cell development occurred readily. After 21 days of culture mast cells comprised more than 90% of the total cell population. BM taken from certain strains of mice (SWR and NIH) produced large numbers of mast cells, total cell numbers increasing between 2 and 5 fold; other strains (C57BL/10 [B10] B10 congenics) produced relatively few mast cells, total cell numbers not increasing above the starting concentration or declining during culture. The genetic factors determining the strain-response phenotype (no. of mast cells in culture) were predominantly associated with the background genome. No significant differences in response were noted between the B10 congenic strains B10 [H-2b], B10.G [H-2q] or B10.BR [H-2k], which differ only at the MHC, whereas major differences were seen between B10.G and the other H-2q strains [SWR and NIH]. Response phenotype was not inherited as a simple dominant trait; F1 progeny of high x low responder strains were intermediate between the parental values. The expression of genetic influences upon mast cell response phenotype appears to be at both the level of mast cell precursor cells, as determined from limiting dilution assays of BM from high, low and F1 (high x low) strains, and at the level of mast cell proliferation, as determined by repeated sub-culture of mast cells from these strains.

Medium conditioned by spleen cells of Nematospiroides dubius-infected mice does not support development of cultured mast cells.

The ability of different conditioned media to support mast cell development from precursors in normal bone marrow was evaluated. Many mast cells developed in bone marrow cultures containing medium conditioned by concanavalin-A-stimulated spleen cells of normal mice, Trichinella spiralis-infected mice, or mice infected for 6 days by Nematospiroides dubius. By contrast, medium conditioned by concanavalin-A-stimulated spleen cells of mice having 18-day infections of the nematode N. dubius failed to support mast cell development. The difference between medium conditioned by spleen cells of mice having 6-day versus 18-day infections of N. dubius may be due to the life cycle stage of the parasite, larval or adult, present at those times. These results from cultures are consistent with those from in vivo studies in which mice given primary infections of N. dubius failed to develop the intestinal mastocytosis characteristic of nematode infections.

Systemic anaphylaxis in mast-cell-deficient mice of W/Wv and Sl/Sld genotypes.

Active systemic anaphylaxis was induced in mast-cell-deficient mice of W/Wv and Sl/Sld genotypes. The mast-cell-deficient mice were successfully sensitized either by an intraperitoneal injection of chicken gamma-globulin (C gamma G) mixed with adjuvant, alum and saline extract of Bordetella pertussis, or by an intravenous injection of C gamma G alone. Sensitized mice showed signs of systemic anaphylaxis and died after one or more intravenous challenge injections of C gamma G. These studies show that active systemic anaphylaxis can occur in mast-cell-deficient mice and suggest that cells other than mast cells may release adequate mediators to support systemic anaphylaxis.

Differential recovery of antibody production potential after sublethal, whole-body irradiation of mice.

Mice were given single injections of sheep erythrocytes (SE) or polyvinylpyrrolidone (PVP) at various times after sublethal, whole-body irradiation (550 rad 60CO) and direct, antigen-specific, plaque-forming cell (PFC) responses were quantified. Irradiated mice did not respond to SE or PVP when immunized 15 d postirradiation (PI); by day 30 PI, the responses by irradiated mice were 40-126% of normal to SE and 3-38% of normal to PVP. The impaired recovery after irradiation of immune responses to PVP was not due to altered antigen dose requirements or altered time of peak PFC response and occurred after irradiation of mice by doses as low as 200 rad. Both athymic and euthymic mice had impaired responses to PVP after whole-body irradiation. The impaired response of irradiated mice to PVP was repaired by adoptive transfer of normal bone marrow, fetal liver, or spleen cells and also by spleen cell preparations enriched in Ig+ cells but not by spleen cell preparations enriched in Thy.1+ or Ig- cells. With the aid of additional antigens it was observed that by day 30 PI, mice had recovered ability to respond to the T-cell-dependent antigen SE and the T-cell-independent type-1 antigens 2,4,6-trinitrophenyl-Brucella abortus and butanol-extracted bacterial lipopolysaccharide, but at that time they gave impaired responses to the T-cell-independent type-2 antigens PVP, type III pneumococcal polysaccharide, and phenol-extracted bacterial lipopolysaccharide; they had an immune response pattern similar to that of CBA/N mice having an X-linked immunodeficiency.

Immune response potential of mast cell-deficient W/Wv mice.

Immune responses of mast cell-deficient WBB6F1-W/Wv mice and their mast cell-sufficient littermates (LM: WBB6F1-W/+, Wv/+ and +/+) were compared. After a single intravenous injection of sheep erythrocytes (SE), polyvinylpyrrolidone or bacterial lipopolysaccharide, the antigen-specific IgM plaque-forming cell (PFC) response of W/Wv mice was similar to or greater than the response of LM mice. When both primary and secondary injections of SE or chicken gamma-globulin were given to mice and antigen-specific IgG PFC responses quantified, the response of W/Wv again was similar to or greater than that of LM mice. Serum titers of antigen-specific IgE were higher in W/Wv than in LM mice after injections of ovalbumin in alum or infections of Nippostrongylus brasiliensis. Ovalbumin-sensitized W/Wv and LM mice developed active systemic anaphylaxis after ovalbumin challenge. The ability of W/Wv mice to be sensitized for and elicit contact sensitivity (CS) reactions was studied using picryl chloride or dinitrofluorobenzene as sensitizing and challenge agents and quantifying 24-hour reactions by change in ear thickness. SE or methylated bovine serum albumin was used to sensitize and challenge mice for delayed-type hypersensitivity (DTH) reactions which were quantified at 24 h by change in foot pad or ear thickness. CS and DTH reactions of W/Wv and LM mice were similar. No evidence of immune deficiency of W/Wv mice was found.

Bone marrow origin of mucosal mast cells.

Infection with the intestinal parasite Nippostrongylus brasiliensis stimulates an accumulation of mucosal mast cells (MMC) in the villi of the small intestine of normal but not athymic or W/Wv anemic mice. W/Wv mice are congenitally deficient in both MMC and skin and connective tissue mast cells (CTMC). Athymic mice have normal or elevated numbers of CTMC but are severely deficient in MMC. CTMC derive from the bone marrow. To determine the origin of MMC, athymic and W/Wv mice were given various hematopoietic or lymphoid tissues from normal littermate or beige mice and the MMC response to N. brasiliensis infection was evaluated. The MMC defect in athymic mice was repaired by grafts of thymus cells, thymus gland, or spleen cells, but not by bone marrow cells or anti-Thy 1-treated bone marrow or spleen cells. The MMC and CTMC defects of W/Wv mice were repaired by grafts of bone marrow, spleen cells, or anti-Thy 1-treated bone marrow or spleen cells. Neither the MMC nor the CTMC defect in W/Wv mice was repaired by grafts of thymus cells or thymus glands. These results indicate the following, MMC, like CTMC, derive from the bone marrow and not from the thymus. MMC require a thymic influence for development. Athymic mice possess bone marrow precursors for both MMC and CTMC but lack a thymus-dependent component necessary for MMC development. W/Wv mice lack both MMC and CTMC mast cell precursors but possess the thymus-dependent component required for MMC development.

Immunosuppression in mouse trypanosomiasis: inhibition of secondary antibody responses is dependent on the time of antigen priming.

Trypanosoma musculi-induced immunosuppression was examined in both congenitally athymic (nude) mice and their phenotypically normal, euthymic littermates using T-independent (polyvinylpyrrolidone = PVP) and T-dependent (sheep erythrocyte = SE) antigens. T. musculi-infected nude mice had significantly diminished direct (IgM) plaque-forming cell (PFC) responses to PVP. In euthymic mice, T. musculi parasitemia significantly inhibited the direct PFC response to both PVP and SE. Trypanosoma musculi-infected euthymic mice had significantly diminished indirect (IgG) SE-specific PFC responses if priming occurred during parasitemia; however, T. musculi parasitemia did not significantly decrease indirect SE-specific PFC responses in euthymic mice if the mice were primed before infection.

Delayed expulsion of adult Trichinella spiralis by mast cell-deficient W/Wv mice.

Mast cell-deficient W/Wv mice and their mast cell-sufficient littermates were given infections of Trichinella spiralis. W/Wv mice were slower than their littermates to expel adult T. spiralis. Repair of the mast cell deficiency of W/Wv mice by bone marrow grafting was accompanied by accelerated expulsion of T. spiralis.

Rejection of the intestinal parasite Nippostrongylus brasiliensis by mast cell-deficient W/Wv anemic mice.

The ability of W/Wv anemic mice to accumulate mucosal mast cells and to reject the intestinal parasite Nippostrongylus brasiliensis was examined. W/Wv mice did not accumulate mucosal mast cells in response to infections with N. brasiliensis. They eliminated a primary infection more slowly than did their normal littermate controls but were as refractory as controls to second and third infections. W/Wv mice had higher serum titers of worm-specific immunoglobulin E than did controls. These results indicate that mucosal mast cells are not an absolute requirement for N. brasiliensis rejection.

Absorption of ablastic activity from mouse serum by using a Trypanosoma musculi population rich in dividing forms.

An in vivo assay of ablastin activity was developed by passive transfer of ablastic serum into nude mice. Preparations of Trypanosoma musculi that contained 35 to 50% dividing forms removed ablastic activity from mouse serum, but absorption of serum with a parasite preparation consisting of less than 5% dividing forms did not appreciably alter ablastic activity. These data suggest that ablastin is an antibody that can be absorbed onto homologous trypanosomes.

Immune and mitogenic responses by BALB/c, C3H/HeJ, and nude mice to Brucella abortus bacterin and lipopolysaccharide.

The immunogenic and mitogenic properties of Brucella abortus 1119-3 bacterin (BA) and biologically active B. abortus lipopolysaccharide (BA-LPS) were studied using normal and athymic (nude) BALB/c and C3H/HeJ mice. Although BA stimulated 2-mercaptoethanol-sensitive (2-ME-S) primary and secondary antibody responses in all mice, nude mice, in contrast to normal BALB/c and C3H/HeJ mice, did not make substantial 2-mercaptoethanol-resistant (2-ME-R) antibody responses. Similarly, all mice injected with BA-LPS made 2-ME-S primary responses, and the secondary response of thymus-bearing mice contained a substantial 2-ME-R component. Collectively, these observations suggest that although both BA and BA-LPS can stimulate thymus-independent 2-ME-S antibody synthesis, thymus-derived cells are required for optimal immune responses containing a 2-ME-R component. The antibody responses of normal BALB/c and C3H/HeJ mice to BA and BA-LPS were qualitatively and quantitatively similar. Both BA and BA-LPS were mitogenic for spleen cells from normal and nude BALB/c and C3H/HeJ mice but not for thymus cells from normal BALB/c or C3H/HeJ mice, suggesting that both preparations are B-cell mitogens.

Effect of concanavalin A on lymphocyte interactions involved in the antibody response to type III pneumococcal polysaccharide. II. Ability of suppressor T cells to act on both B cells and amplified T cells to limit the magnitude of the antibody response.

When administered 2 days after immunization with 0.5 microgram Type III pneumococcal polysaccharide (SSS-III), the T lymphocyte mitogen concanavalin A (Con A) stimulates a 2.6-to 7-fold enhancement of the plaque-forming cells (PFC) response to SSS-III in vivo. This enhancement requires the presence of amplified T cells, which act by driving PFC or their precursors to extra rounds of proliferation. The extra proliferation that can be stimulated by Con A is not seen in the normal primary response to SSS-III; but treatment with anti-lymphocyte serum (ALS) to remove suppressor T cells will permit the additional proliferation to occur. This indicates that in the primary response to SSS-III, suppressor T cells act on amplifier T cells to limit the magnitude of the antibody response. Only suppression of B cells can account for the further suppression induced by Con A given at the time of immunization or by low-dose paralysis of the SSS-III response. The relatively late development of amplified activity compared to suppressor activity appears to account for the absence of amplifier activity after primary immunization with SSS-III. It is apparent that one can explain the regulatory effects observed during the development of an immune response to SSS-III only by considering both T cell- B cell and T cell- T cell interactions, together with the temporal relationships involved in those interactions.

Expulsion of Nippostrongylus brasiliensis from mice lacking antibody production potential.

Expulsion of the intestinal nematode of rodents, Nippostrongylus brasiliensis, was assessed in mice experiencing the immunosuppressive effects of anti-micron antibodies. Anti-micron treatment resulted in complete elimination of IgM and severe reduction of IgG1, IgG2 and IgA serum immunoglobulin levels. Specific antibody responses to sheep erythrocytes were virtually eliminated in anti-micron-treated mice as determined by direct and indirect plaque-forming-cell responses, haemagglutination and haemolytic assays. Using passive cutaneous anaphylaxis (PCA) and indirect haemagglutination (IHA) assays, antibodies against N. brasiliensis were not detectable in sera of anti-micron-treated mice; control mice, however, generated strong PCA and IHA responses. The kinetics of worm egg production coupled with adult worm recoveries at necropsy indicate that anti-micron treatment of mice had little or no effect on the capacity of mice to expel N. brasiliensis even though antibody production potential had been eliminated in these mice. Thus, anti-worm antibodies may not be requisite in the mechanism of N. brasiliensis expulsion from mice.

Immunological responses of mice to lipopolysaccharide: lack of secondary responsiveness by C3H/HeJ mice.

Mice of the C3H/HeJ strain, which were unresponsive to the biological effects of bacterial lipopolysaccharide (LPS), could not be induced to make specific secondary immunological responses to LPS; they responded to two doses of LPS with a primary response. This lack of secondary responsiveness by C3H/HeJ mice was due to a defect in a single, autosomal, dominant gene. Thus, further evidence was provided that an intact second immunological signal and responsiveness thereto were required to trigger secondary antibody responses in primed animals.

The course of Hymenolepis nana infections in thymus-deficient mice.

The kinetics of infection with Hymenolepis nana was examined in normal thymus-deficient mice. Following inoculation with 5 cyticercoids, the number of adult lumen-dwelling H. nana in congenitally thymus-deficient (nude) mice ultimately was at least 75 times the maximum infection intensity of normal thymus-bearing mice or thymus-reconstituted nude mice. Similar results were obtained following inoculation of H. nana eggs into nude mice; although thymus-bearing mice eliminated their infections, nude mice harbored more than 1,000 adult worms throughout the 7 weeks of the experiments. These data show that in mice, immunity is not generated against H. nana in the absence of thymus function. The data also confirm and establish the requirement of cysticercoid development in the intestinal mucosa for stimulation of a protective immune response against H. nana.

Development of a localized hemolysis-in-gel assay for Vi antigen: characterization of the Vi-specific PFC response of nude and normal mice.

An assay to detect specific plaque-forming cells (PFC) to Vi antigen (Vi) was developed and the optimal conditions for sensitization of sheep erythrocytes (SE) and plaque development were determined. Using PFC and passive hemagglutination (PHA) assays, Vi-specific immune responses of athymic (nude) and normal mice were characterized. Vi was found to elicit only IgM PFC. No discernable secondary response was detected following a second injection of antigen. Nude and normal mice responded in a quantitatively similar manner to all doses of Vi tested and responded similarly on varying days following immunization. Also, both nude and normal mice produced the greatest number of Vi-specific PFC 4 days following immunization with an optimally immunogenic dose of Vi (1.0 microng/mouse). These results indicate that functional thymus-derived cells are not necessary to elicit an immune response against Vi antigen.