Biomedical subjects
Publications and source records attributed to L S Wicker.
The immunosuppressants FK506 and FR 900520 were desmethylated by Actinoplanes sp. ATCC 53771 to yield various O-desmethylated products. The products were isolated and purified by solvent extraction and HPLC chromatography, and identified by NMR and MS spectroscopy.
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Human type 1 (insulin-dependent) diabetes is a common auto-immune disease of the insulin-producing beta cells of the pancreas which is caused by both genetic and environmental factors. Several features of the genetics and immunopathology of diabetes in nonobese diabetic (NOD) mice are shared with the human disease. Of the three diabetes-susceptibility genes, Idd-1 -3 and -4 that have been mapped in mice to date, only in the case of Idd-1 is there any evidence for the identity of the gene product: allelic variation within the murine immune response I-A beta gene and its human homologue HLA-DQB1 correlates with susceptibility, implying that I-A beta is a component of Idd-1. We report here the mapping of Idd-5 to the proximal region of mouse chromosome 1. This region contains at least two candidate susceptibility genes, the interleukin-1 receptor gene and Lsh/Ity/Bcg, which encodes resistance to bacterial and parasitic infections and affects the function of macrophages.
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The effects of the immunosuppressants cyclosporin A (CsA), FK506 and rapamycin have been compared using murine B cells activated with a variety of mitogens. FK506 is a macrolide antibiotic that has been recently shown to inhibit T cell activation by a mechanism that appears similar to that of CsA. Rapamycin is a macrolide structurally related to FK506 whose mechanism of T cell suppression appears to be distinct from that of FK506 and CsA. While CsA and FK506 were found to preferentially inhibit B cell activation caused by stimuli which induce a rise in intracellular calcium, rapamycin partially inhibited activation by all stimuli tested, including those which are not associated with a calcium flux. All three compounds were found to inhibit cell cycle progression within the G1 phase; however, the rapamycin-sensitive event within G1 was completed earlier than the G1 events inhibited by CsA and FK506. In addition, inhibition of anti-IgM-activated B cells with CsA and FK506, but not with rapamycin, resulted in cell death. These data suggest that although CsA, FK506 and rapamycin are all inhibitors of B cell activation, the inhibitory activity of rapamycin can be clearly distinguished from that of CsA and FK506. Although the suppressive effects of CsA and FK506 on B cell proliferation were nearly identical in this study, their biological activities were distinguishable since FK506, but not CsA, could antagonize rapamycin-mediated suppression.
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A potent cytolytic pore-forming protein (perforin or cytolysin) has previously been found to be associated with the cytoplasmic granules of CTL and NK cells. Inasmuch as all previous studies on perforin have been conducted with cultured CTL and NK cell lines, it is not clear whether perforin may play a role in the cytotoxicity mediated by CTL that have been primed in vivo. In this study, we investigated the presence of perforin in pancreata from nonobese diabetic (NOD) mice, which have been studied as a model of autoimmune, insulin-dependent (type I) diabetes mellitus. Whereas adult NOD mice spontaneously develop diabetes, it is possible to induce diabetes in young, irradiated NOD mice by adoptive transfer of splenocytes obtained from diabetic donors. By means of immunohistochemical analysis, we were able to detect perforin Ag in a small subpopulation of CD8+/Thy-1+/asialo GM1-/CD4- lymphocytes in the pancreatic islets of animals undergoing both spontaneous and adoptive transfer-mediated insulitis. Perforin+/CD8+ lymphocytes were found in small clusters and were observed to display the morphology of large granular lymphocytes. These observations show for the first time the presence of perforin-containing CD8+ lymphocytes in tissues of animals undergoing autoimmune disease.
The development of autoimmune type 1 diabetes mellitus in man and the nonobese diabetic (NOD) mouse is greatly influenced by a gene linked to the MHC. Although homozygosity at the NOD MHC is required for a high prevalence of disease, during backcross studies we have found a small number of diabetic H-2nod/b MHC heterozygotes. These diabetic heterozygotes could either represent a crossover event between the MHC and a putative MHC-linked diabetogenic gene or, alternatively, they could indicate that there is a dominant MHC-linked diabetic gene that has low penetrance in the heterozygous state. Pedigree analysis of a diabetic H-2nod/b MHC heterozygote favors the latter hypothesis.
The development of autoimmune diabetes in the nonobese diabetic (NOD) mouse is controlled by at least three recessive loci, including one linked to the MHC. To determine whether any of these genetic loci exert their effects via the immune system, radiation bone marrow chimeras were constructed in which (NOD X B10)F1-irradiated recipients were reconstituted with NOD bone marrow cells. Unmanipulated (NOD X B10)F1 mice, or irradiated F1 mice reconstituted with F1 or B10 bone marrow, did not display insulitis or diabetes. In contrast, insulitis was observed in a majority of the NOD----F1 chimeras and diabetes developed in 21% of the mice. These data demonstrate that expression of the diabetic phenotype in the NOD mouse is dependent on NOD-derived hematopoietic stem cells. Diabetogenic genes in the NOD mouse do not appear to function at the level of the insulin-producing beta cells since NOD----F1 chimeras not only developed insulitis and diabetes but also rejected beta cells within pancreas transplants from newborn B10 mice. These data suggest that the beta cells of the NOD mouse do not express a unique antigenic determinant that is the target of the autoimmune response.
It has been shown that 8-bromoguanosine (8-BrGuo) activates T and B cells, but the underlying mechanism of this effect is not clear. We found that it also activates NK cells and macrophages by induction of IFN production. Culturing spleen cells with 8-BrGuo for 16 to 18 h induced cytotoxic activity to YAC cells. The cytotoxic cells expressed Qa-5, asialo-Gm-1, and NK-1.1 Ag. Similarly, preincubation of peptone-elicited peritoneal macrophages with 8-BrGuo induced macrophage cytolytic activity to P815 cells in an 18-h assay. Anti-IFN antibodies to IFN-alpha -beta and -gamma abolished these inductive events. Furthermore, elimination of asialo-Gm-1+ cells reduced the responses of NK and macrophages to 8-BrGuo, indicating that these cells possibly produce the IFN. We conclude from these observations that one biologic effect of 8-BrGuo is due to IFN production.
The nonobese diabetic mouse is a model of spontaneous type I diabetes mellitus. It is possible to induce diabetes in young, irradiated nonobese diabetic mice by using adoptive transfer of splenocytes or splenic T cells obtained from diabetic donors. This study demonstrates that the induction of diabetes in the adoptive transfer system is dependent on both the L3T4+ and Lyt-2+ subsets of T cells. Neither of these T cell subsets alone mediates the development of severe insulitis or diabetes when adoptively transferred to young, irradiated recipients. In addition, we show that both the L3T4+ and Lyt-2+ subsets must be obtained from diabetic donors in order to transfer diabetes; neither subset can be replaced with cells obtained from young, nondiabetic donors.
5-Halo-6-phenyl pyrimidinones, represented by 2-amino-5-bromo-6-phenyl-4(3H)-pyrimidinone (ABPP) and 2-amino-5-iodo-6-phenyl-4(3H)-pyrimidinone (AIPP), and 8-substituted guanosines, represented by 8-bromoguanosine (8-BrGuo) and 8-mercaptoguanosine (8-MGuo), are well-documented biological response modifiers. We have found that these substituted pyrimidinones and guanosines are very similar in their abilities to activate B cells. ABPP, AIPP, 8-BrGuo, and 8-MGuo induced murine B cells to polyclonally proliferate and differentiate in vitro. The maximal B-cell response levels and the kinetics of the responses elicited with both classes of compounds were comparable; however, ABPP and AIPP were approximately 10-fold more potent than 8-BrGuo and 8-MGuo. An additional similarity observed between the two classes was that polyclonal activation of B cells by ABPP, AIPP, 8-BrGuo, and 8-MGuo was limited to large B cells which had probably been activated previously in vivo. This is in contrast to lipopolysaccharide which is capable of inducing both large, activated B cells and small, resting B cells to proliferate and differentiate. Although substituted pyrimidinones and guanosines were not able to induce new DNA synthesis or antibody production in small B cells, both classes of compounds increased the expression of Ia antigens on the surface of both small and large B cells. These data, together with the recent observations that 8-BrGuo, like ABPP and AIPP, can stimulate NK and cytotoxic macrophage activity via the induction of interferon, strongly suggest that 5-halo-6-phenyl pyrimidinones and 8-substituted guanosines belong to the same structural class of biological response modifiers. Thus, the residues held in common by these two classes of stimulators may interact with the same cellular constituent in the target cells.
Genetic analysis of the development of diabetes and insulitis has been performed in the nonobese diabetic (NOD) mouse strain, a model of insulin-dependent (type I) diabetes mellitus. (NOD X C57BL/10)F1, F2, and (F1 X NOD) first-, second-, and third-backcross generations were studied. The data obtained were consistent with the hypothesis that diabetes is controlled by at least three functionally recessive diabetogenic genes, or gene complexes, one of which is linked to the MHC of the NOD. In contrast, pancreatic inflammation leading to insulitis was found to be controlled by a single incompletely dominant gene. One of the two diabetogenic loci that is not linked to the MHC appears to be essential for the development of severe insulitis. This diabetogenic gene may be identical to the gene that controls the initiation of the autoimmune response that progresses to insulitis. Although this gene appears to be functionally recessive in its control of diabetes, it is incompletely dominant in its control of insulitis. The MHC-linked diabetogenic gene, although not required for the development of insulitis, apparently influences the progression of the autoimmune response since NOD MHC homozygotes in the backcross generations displayed the highest incidence and most severe cases of insulitis. Interestingly, we have found two MHC heterozygous backcross females that have become diabetic, suggesting that either the MHC-linked diabetogenic gene is not strictly recessive or that a recombination event has occurred between the diabetogenic gene and the K or I-A regions of the MHC. The third diabetogenic locus appears to influence the progression of severe insulitis to overt diabetes. In animals homozygous at this locus, diabetes may result from a decreased ability to develop a protective suppressor response to the autoimmune process.
Previous studies have documented the ability of 8-bromoguanosine (8-BrGuo) and 8-mercaptoguanosine (8-MGuo) to induce polyclonal proliferation and differentiation of B cells from a variety of mouse strains. In the present study, we have defined the cellular target of this mitogenic activity. Using B cells fractionated according to size, we have found that large B cells are responsive to 8-BrGuo- and 8-MGuo-induced proliferation and differentiation whereas small, resting B cells are relatively unresponsive to these compounds. Addition of splenic adherent cells to the small B-cell fraction partially restored the proliferative but not the differentiative responses to 8-BrGuo and 8-MGuo. Although small B cells alone did not proliferate or differentiate in response to 8-BrGuo and 8-MGuo, cell surface expression of Ia antigens increased following incubation with these compounds. Thus, the biological activity of 8-BrGuo and 8-MGuo appears to be dictated by the cell type upon which it is acting. Small B cells are activated as evidenced by increased levels of surface Ia whereas large B cells are not only activated but are also induced to proliferate and differentiate.
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We have examined the requirement for major histocompatibility complex (MHC)-restricted T-cell help in the secondary in vivo antibody response to phosphocholine (PC). The memory response to PC has been demonstrated previously to be comprised of T15-dominant IgM and IgG3 plaque-forming cells (PFC) derived primarily from the Lyb-5+ B-cell subset, and IgG1 and IgG2 PFC, few of which bear the T15 idiotype and are predominantly derived from the Lyb-5- B-cell subset. Using carrier-primed (A X B)F1 T cells which have matured in a parentA chimeric environment so that "self" recognition is of the MHC determinants of parentA but not parentB, we have found that parentA PC-primed B cells, but not parentB PC-primed B cells, are activated. Even in the presence of an ongoing parentA anti-PC response, parentB PC-primed B cells were not activated, indicating that the restriction was between the helper T cell and the B cell, not between T-helper and accessory cells. MHC-restricted T-cell help was required by B cells producing T15+ and T15- IgM, IgG3, IgG1, and IgG2 responses.
The nonobese diabetic (NOD) mouse, a model of human type I diabetes, develops insulitis beginning at 4-6 wk of age. By 30 wk of age, 72% of females and 39% of males develop spontaneous diabetes, apparently because of an overwhelming autoimmune response to the insulin-producing beta-cells within the islets. To identify the immune mechanism responsible for destruction of beta-cells in the NOD mouse, we developed an adoptive transfer protocol that induces diabetes in NOD mice at an age when spontaneous diabetes is rarely observed. Splenocytes from overtly diabetic NOD mice were unable to transfer diabetes to very young (less than or equal to 6 wk) irradiated NOD mice but effectively transferred diabetes to irradiated NOD mice greater than 6 wk of age. In such transfers, overt diabetes was induced within 12-22 days in greater than 95% (79/82) of the recipients. Thus, transfer of splenocytes to young mice induces them to become diabetic at a higher frequency and at a younger age than their untreated littermates. Equally successful transfers with as few as 5 X 10(6) spleen cells have been performed in male and female NOD mice, even though males display a lower spontaneous incidence of diabetes than females. Splenocytes obtained from diabetic mice maintained on insulin for up to 2 mo also transferred diabetes. Because NOD mice display increasing levels of insulitis with age, spleen cells obtained from nondiabetic NOD mice of different ages were tested for their ability to transfer diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)