The regulation of expression of MHC products in mice. Factors determining the level of expression in kidneys of normal mice.

Cytokines, especially IFN, have been shown to increase MHC expression above basal levels in many immunologic processes, but their contribution to normal MHC expression is unknown. Environmental stimuli, such as bacterial LPS, may provoke the secretion of cytokines which could influence MHC expression in the normal host. We therefore studied the factors influencing the normal expression of MHC products in mice, by using kidney as a representative nonlymphoid tissue. Considerable variation in MHC expression was observed between otherwise normal individuals by using indirect immunoperoxidase staining and a radiolabeled antibody binding assay. An intact T cell compartment was not necessary for normal levels of expression; in athymic nude mice and mice with severe combined immunodeficiency disease, MHC expression was similar to or greater than that of controls. Neither a course of broad spectrum antibiotics nor genetically determined resistance to LPS influenced the level of normal expression. Mice raised under germ-free conditions, on a chemically defined diet also had similar levels of MHC expression when compared with mice maintained under conventional conditions. However, when germfree mice were placed in a conventional colony, induction of both class I and II MHC products occurred. Thus, although exposure to bacterial flora or other sources of endotoxin was not required for normal MHC expression, a change in flora can up-regulate expression, probably by inducing the secretion of cytokines from non-T cells. Treatment with cyclosporine or anti-IFN antibodies produced, at most, a small reduction in the level of renal MHC expression. Therefore in some mice, a small component of MHC expression may reflect the secretion of cytokines in response to stimuli such as bacterial LPS. However, most MHC expression in nonlymphoid tissues is constitutive and independent of a physiologic IFN response.

Serum immunoglobulin levels and naturally occurring antibodies against carbohydrate antigens in germ-free BALB/c mice fed chemically defined ultrafiltered diet.

This study investigates the influence of exogenous antigenic stimulation on the serum immunoglobulin levels and the levels of circulating natural antibodies against carbohydrate antigens. Thus, BALB/c mice, raised in a germ-free environment and fed a chemically defined, ultrafiltered diet (GF-CD), were employed. These mice had normal serum IgM levels, but IgG and IgA levels were approximately 5% of conventionally reared littermates. The concentrations of all four IgG isotypes were equally low. The variable part of the heavy chains of naturally occurring BALB/c antibodies against a number of carbohydrate antigens, including 3-fucosyllactosamine (3-FL), levan and dextran, are encoded by VH441, and these antibodies express cross-reactive idiotopes recognized by the monoclonal antibodies 6C4 and 6B1. Antibodies against levan and dextran were lower in GF-CD than in conventional mice, but levels of anti-3FL antibodies, and 6C4 and 6B1 idiotopes, were comparable to those in conventional animals. Peptidoglycan polysaccharide complexes (PPC) are carbohydrate antigens of bacterial origin, like levan and galactan. Naturally occurring antibodies against PPC were found in the serum of conventional mice, but were severely reduced in GF-CD mice. The results indicate that most naturally occurring antibodies against carbohydrate antigens of bacterial origin found in conventional mice are caused by exogenous stimulation.

The influence of exogenous antigenic stimulation on the specificity repertoire of background immunoglobulin-secreting cells of different isotypes.

The total number of spontaneously occurring ("background") IgM-, IgG-, and IgA-secreting cells and the frequency of antigen-specific IgM-, IgG-, and IgA-secreting cells were determined in germ-free BALB/c mice fed a chemically defined ultrafiltered diet (GF-CD), in specific pathogen-free BALB/c mice fed an autoclaved natural ingredient diet (SPF-NI), and in conventional BALB/c mice fed nonautoclaved natural ingredients (CV-NI). This was done by means of the ELISA-plaque assay. The results did not show differences among the various groups of mice with regard to the total numbers of IgM-secreting cells in the various lymphoid organs. Also the frequencies of IgM-secreting cells specific for DNP27-BSA and the anti-idiotypic monoclonal antibodies Ac38 and Ac146 did not differ significantly among GF-CD, SPF-NI, and CV-NI mice. GF-CD mice, however, did show substantially decreased numbers of IgG- and IgA-secreting cells in their lymphoid organs. Furthermore, there were striking differences in the frequencies of antigen-specific IgG- and IgA-secreting cells between GF-CD mice and the two other groups of mice. These results indicate that exogenous antigenic stimulation has a great effect on both the total numbers and the specificity repertoires of background IgG- and IgA-secreting cells. Such an influence could not be detected with regard to the background IgM-secreting cells. This suggests two distinct compartments of background Ig-secreting cells: a very stable, endogenously regulated compartment consisting mainly of IgM-secreting cells, and another compartment, consisting mainly of IgG- and IgA-secreting cells, whose numbers and specificity repertoire appeared to be influenced by exogenous antigenic stimulation.

Early development of Ig-secreting cells in young of germ-free BALB/c mice fed a chemically defined ultrafiltered diet.

The influence of antigenic stimulation on the early development of the "spontaneously" occurring ("background") IgM-, IgG-, and IgA-secreting cells has been studied in mice. To evaluate the effect of such exogenous stimulation by an evolving microbial microflora, the young of BALB/c mice that were kept under germ-free conditions and fed a low molecular weight chemically defined synthetic diet (GF-CD) were compared with the young of conventional BALB/c mice fed natural ingredients (CV-NI). The young were first suckling maternal milk and between Days 15 and 18 changed to the same diet as their parents. Background Ig-secreting cells in the spleen were enumerated in the protein A plaque assay. The specificity repertoire of the IgM-secreting cells was determined with plaque assays specific for sheep red blood cells (SRBC) that were haptenized with different concentrations of nitroiodophenyl (NIP), 4-hydroxy-3.5-dinitrophenyl (NNP), and 2,4,6-trinitrophenyl (TNP). The results show that during the first few weeks of life the numbers of background IgM-, IgG-, and IgA-secreting cells in the spleen develop faster in CV-NI mice than in GF-CD mice. At 4 weeks of age equal numbers of IgM- and IgG-secreting cells were found in both groups of mice, but the number of IgA-secreting cells remained reduced in GF-CD mice during the whole period of observation. The frequencies of IgM-secreting cells specific for the differently haptenized SRBC were the same in both groups of mice during the observation period of 10 weeks. This suggests that the ontogenetic appearance of IgM-, IgG-, and IgA-secreting cells in the spleen, and the specificity repertoire of the IgM-secreting cells, as far as was tested in our panel, is independent of exogenous antigenic and/or mitogenic stimulation. However, during neonatal development the rate of development of the background Ig synthesis is enhanced by environmental antigenic stimulation.

'Background' Ig-secreting cells in pregnant germfree mice fed a chemically defined ultrafiltered diet.

During syngeneic pregnancy the numbers of 'spontaneously' occurring ('background') Ig-secreting cells were determined in the spleen, bone marrow (BM) and mesenteric lymph nodes (MLN) of BALB/c mice that were kept under germfree conditions and fed a low molecular weight synthetic diet (GF-CD), SPF BALB/c mice fed autoclaved natural ingredient (SPF-NI) and conventional BALB/c mice fed natural ingredient (CV-NI). 'Background' Ig-secreting cells were enumerated in the protein A plaque assay and the specificity repertoire of the IgM-secreting cells was determined with plaque assays specific for differently haptenized sheep red blood cells (SRBC). The numbers of 'background' Ig-secreting cells, especially the IgG- and IgA-secreting cells, are very much reduced in the BM and MLN of GF-CD mice as compared to SPF-NI and CV-NI mice. During pregnancy the total number of Ig-secreting cells increased in all lymphoid organs tested, but the proportional increase was most prominent for the IgG- and IgA-secreting cells in the BM and MLN of the GF-CD mice. This increase could only be due to their pregnant state since all environmental antigenic influences are excluded in GF-CD mice. No changes were found in the background specificity repertoire of the IgM-secreting cells during pregnancy. This suggests a polyclonal activation of the Ig-secreting cells during pregnancy. The reason for this activation remains obscure, but it has to be endogenous. Pregnancy apparently induces a new steady state of the immune system, which can be most properly investigated in GF-CD mice.

Adequacy of chemically defined, water-soluble diet for germfree BALB/c mice through successive generations and litters.

To determine dietary adequacy, germfree BALB/cAnN mice were fed ad libitum an ultrafiltered solution of chemically defined, water-soluble, low-molecular-weight nutrients. They received a measured daily supplement of membrane-filtered, distillation-purified soy oil containing vitamins A, D, E and K. Mice were kept on ash-free filter paper bedding, which they freely consumed. On this regimen, germfree BALB/c mice reproduced through nine generations, and through eight litters in one generation. Average number born per litter was 4.1, compared with 5.1 in control BALB/c mice, which had a conventional microflora and were fed a natural ingredient diet. From 21 to 32 d of age, the experimental mice gained more slowly than controls. After 32 d, experimental mice gained more rapidly than controls; their weights tended to be lower than controls at 45 d and equal to controls at 56 d. Intake-limiting effects of the diet appeared responsible for reduced growth rates and litter size. Experimental females showed a low incidence of deaths from cecal volvulus. Experimental males experienced a high incidence of deaths from colonic impaction of cecally-formed trichobezoars; this site of formation appeared to be unique to BALB/c males on the experimental regimen. These losses were judged to be unrelated to nutritional deficiency. No overt signs of nutritional deficiency developed in female mice which were fed CD diet up to 18 mo of age.

Frequency analysis of functional immunoglobulin C- and V-gene expression by mitogen-reactive B cells in germfree mice fed chemically defined ultra-filtered "antigen-free" diet.

The frequencies of lipopolysaccharide (LPS)-reactive B cells and their antibody specificity repertoire have been determined in the spleen and bone marrow (BM) of conventional (CV) and "antigen-free" C3H/HeCr mice of various ages. The antigen-free mice were germfree (GF)-raised and were fed an ultrafiltered solution of chemically defined (CD) low m.w. nutrients, and were thus devoid of exogenous antigenic stimulation. Spleen and BM cells were grown in a limiting dilution culture system that allows the growth and development of every newly formed LPS-reactive B cell into a clone of IgM-secreting cells which are capable of switching to other immunoglobulin (Ig) heavy chain isotypes (C-gene expression). The secretion of IgM and IgG1 was determined in the protein A plaque assay, whereas specific IgM antibody-secreting cells (V-gene expression) were detected in plaque assays specific for various heterologous erythrocytes and sheep red blood cells (SRBC) coupled with a number of different haptens. The absolute frequency of LPS-reactive B cells and their capacity to switch to IgG1-secretion was not significantly different in 8- to 12-wk-old and 52-wk-old GF-CD mice and their age-matched CV controls. Moreover, no differences were observed in the frequencies of antigen-specific B cells within the pool of LPS reactive B cells. These frequencies ranged from 1 in 20 to 1 in 50 for NIP4-SRBC and NNP2-SRBC, from 1 in 100 to 1 in 150 for NIP0.4-SRBC, from 1 in 50 to 1 in 100 for TNP30-SRBC, and from 1 in 1000 to 1 in 2000 for SRBC and horse red blood cells. Within the limitations of having determined the switching capacity of IgM to IgG1 only and having assessed only a minor fraction of the total B cell antibody-specificity repertoire, the data indicate that young and old GF-CD mice, although devoid of exogenous antigenic and/or mitogenic stimulation, generate B cells with a similar switching capacity and a similar IgM antibody specificity repertoire as CV mice.

Isotypes and specificities of immunoglobulins produced by germ-free mice fed chemically defined ultrafiltered "antigen-free" diet.

The regulation of the "spontaneously" occurring ("background") Ig synthesis of mice has been studied by determining the numbers of IgM-, IgG- and IgA-secreting cells and a part of the IgM antibody-specificity repertoire in spleen, bone marrow (BM) and mesenteric lymph nodes (MLN) of conventional and "antigen-free" mice. These antigen-free mice were germ-free raised and fed an ultrafiltered solution of chemically defined low molecular weight nutrients, and thus devoid of exogenous antigenic stimulation. The secretion of IgM, IgG and IgA by spleen, BM and MLN cells was assessed in the protein A plaque assay, while specific IgM antibody-secreting cells were detected by plaque assays specific for differently haptenated sheep red blood cells. In general, antigen-free and conventional mice were found to have roughly equal numbers of IgM-secreting cells in spleen and BM. The number of IgG-secreting cells in the spleen of antigen-free mice was the same as in the spleen of conventional mice, but in the BM their number was 3-5-fold decreased. About one half of the antigen-free mice did not have MLN, and in the half which did, 5 times less IgM- and more than 100-fold less IgG-secreting cells were found as compared with conventional mice. The number of IgA-secreting cells in antigen-free mice was drastically decreased in all three organs tested. The antibody-specificity repertoire of the "background" IgM-secreting cells in the spleen and BM of the antigen-free and conventional mice was much alike. This indicates that in antigen-free mice the available antibody repertoires are established independently of exogenous and/or mitogenic stimulation.

The effect of microbial flora, diet, and age on the tumoricidal activity of natural killer cells.

This study examined the effects of diet (chemically defined vs natural-ingredient), age, and microbial flora on the tumoricidal activity of natural killer (NK) cells from the spleens of mice. Results from a 4-h 51Cr-release assay indicate the following: Germfree C3H/HeCr mice raised on a chemically defined diet had significantly greater NK cell activity than their germfree or "clean-conventional" (i.e., barrier-maintained) counterparts who were raised on a sterilized natural-ingredient diet. The NK activity of germfree mice was dramatically increased after their alimentary tract was colonized with a complex intestinal flora. Conventional mice raised under clean (barrier) conditions had significantly less NK cell activity than nonbarrier-maintained mice. Switching germfree mice from a chemically defined diet to a sterile natural-ingredient diet did not enhance NK cell activity. No significant differences in NK activity were evident with C3H/HeCr mice of different (6-10 wk vs 29-36 wk) ages. These results indicate that diet and microbial flora can modulate the NK cell activity of mice.

Oxygen consumption and thyroid hormones in germfree mice fed glucose-amino acid liquid diet.

Germfree rats and mice fed solid diets have previously been shown to consume less oxygen than their conventional counterparts. Young adult germfree C3H/He mice how serum T3 concentrations comparable to those of conventional mice, but their serum T4 values are 10-15% lower. Fed chemically defined liquid glucose-amino acid diets, germfree C3H mice use 27% more oxygen than comparable germfree mice fed a solid natural ingredient diet, and 12% more than conventional mice fed the solid diet. Like germfree rats, the germfree mice fed natural ingredient diet had smaller hearts and livers than those of conventional mice. However, the chemically defined diet-fed germfree C3H mice showed larger hearts than conventional C3H mice fed solid diets, and livers of comparable size. Serum T3 levels were similar in the two germfree groups at both 3 and 8 months of age, while serum T4 levels were again slightly but significantly higher in the germfree chemically defined diet group at both ages. Both T3 and T4 decline with age in conventional mice, but not in either germfree group, similar to findings in germfree rats. The data suggest that germfree mice fed chemically-defined diet require additional energy expenditure to effectively utilize its major dietary components.

Administration of exogenous queuine is essential for the biosynthesis of the queuosine-containing transfer RNAs in the mouse.

1. Normal mouse liver contains predominantly tRNA that contains queuosine in the first position of the anticodon ((Q+)tRNA). 2. Germ-free mice fed a chemically defined diet devoid of queuine for 1 year have no queuine in all four of the tRNAs that respond to the NAUC codons. 3. The synthesis of (Q+)tRNAs can be induced by injecting queuine, feeding free queuine, or by feeding (Q+)tRNA. 4. When mice that have no (Q+)tRNA are titrated with exogenous queuine, tRNAAsp is modified to the (Q+) state before tRNAHis.