SumrlrlaryA number of distinct functional abnormalities have been observed in B ceils derived from pS0/ NF-KB or c-rel knockout mice. RelB, another member of the NF-KB/Rel family of transcription factors, is expressed during the latter stages of B cell maturation and can bind to regulatory sites within the Ig heavy chain locus. Therefore, we tested the ability of B ceils from relB knockout mice (relB -/-) to proliferate, undergo maturation to IgM secretion, and switch to the expression of downstream Ig isotypes in response to distinct activators including LPS, anti-CD40 mAb or CD40 ligand, and/or dextran anti-IgD antibodies in combination with various cytokines, including IL-4, IL-5, IFN-3', and TGF-13. B cells lacking RelB showed up to 4-fold reductions in DNA synthesis in response to LPS, CD40, and membrane Ig-dependent activation relative to controls. However, relB -/-B cells were comparable to control B cells in their ability to undergo maturation to IgM secretion and switch to the expression of IgG3, IgG1, IgG2b, IgG2a, IgE, and/or IgA under all activation conditions tested. Thus, RelB, like c-Rel and p50/NF-KB, plays a role in B cell proliferation. However, in contrast to c-Rel and p50/ NF-KB, it is not critically involved in maturation to Ig secretion or expression oflg isotypes.
The c-rel protooncogene encodes a member of the Rel/nuclear factor (NF)-κB family of transcriptional factors. To assess the role of the transcriptional activation domain of c-Rel in vivo, we generated mice expressing a truncated c-Rel (Δc-Rel) that lacks the COOH-terminal region, but retains a functional Rel homology domain. Mice with an homozygous mutation in the c-rel region encoding the COOH terminus of c-Rel (c-relΔCT/ΔCT) display marked defects in proliferative and immune functions. c-relΔCT/ΔCT animals present histopathological alterations of hemopoietic tissues, such as an enlarged spleen due to lymphoid hyperplasia, extramedullary hematopoiesis, and bone marrow hypoplasia. In older c-relΔCT/ΔCT mice, lymphoid hyperplasia was also detected in lymph nodes, liver, lung, and stomach. These animals present a more severe phenotype than mice lacking the entire c-Rel protein. Thus, in c-relΔCT/ΔCT mice, the lack of c-Rel activity is less efficiently compensated by other NF-κB proteins.
IFN-gamma has been shown to either stimulate or inhibit Ig secretion. No studies have yet addressed the basis for these seemingly conflicting properties nor whether IFN-gamma acted directly at the level of the B cell to mediate its effects. Thus, we studied the ability of IFN-gamma to regulate Ig secretion in sort-purified, resting murine B cells that were >99% Ig+, activated either through membrane Ig using unconjugated or dextran-conjugated anti-IgD antibodies (alphadelta-dex) or through CD40 using soluble or membrane CD40 ligand (CD40L). B cells activated with alphadelta-dex proliferated but do not secrete Ig, even in the presence of IL-1 + IL-2. We demonstrate that IFN-gamma only when added subsequent to B cell stimulation with alphadelta-dex, but not unconjugated anti-IfD antibody, plus IL-1 + IL-2 induces up to 100-fold enhancements in Ig secretion and in the numbers of Ig-secreting cells. The predominant Ig isotype secreted is IgM, with IgG3 and IgG2a comprising the majority of non-IgM antibody. IFN-gamma must act in concert with IL-2 for stimulation of Ig secretion. Further, IFN-gamma synergizes with IL-3 + granulocyte-macrophage colony stimulating factor for induction of Ig synthesis. IFN-gamma also enhances IgA syntheses by transforming growth factor-beta-induced membrane IgA+ cells. By contrast, 125IIFN-gamma fails to stimulate Ig secretion in B cells activated with CD40L in the presence or absence of IL-1 + IL-2 or IL-4. However, the combination of CD40L and alphabeta-dex is strongly synergistic for IFN-gamma-induced Ig secretion. Thus, these data establish that IFN-gamma can act directly on the B cell to induce Ig synthesis without the participation of any other cell and demonstrates that the mode of activation of the B cell plays an important role in directing the action of IFN-gamma.
Resting B cells stimulated with dextran-conjugated anti-immunoglobulin D (anti-IgD) antibodies (anti-Igdex), a model for B-cell activation in response to polysaccharide antigens, proliferate but secrete little if any Ig, unless additional stimuli are present. In order to elucidate the parameters which costimulate T-cellindependent antipolysaccharide antibody responses during bacterial infections, we tested the capacities of highly purified porin proteins from Neisseria meningitidis and Neisseria gonorrhoeae to augment in vitro proliferation and induce Ig secretion by anti-Ig-dex-activated B cells. Resting B cells, from lipopolysaccharide (LPS)-nonresponsive C3H/HeJ mice, proliferated and secreted IgM in response to each of three distinct porins acting alone. Further, porins, even at concentrations that were minimally inductive when acting alone, were strongly synergistic with anti-Ig-dex for proliferation and Ig secretion. Similar synergistic effects of porins with CD40-ligand were also observed. These effects of porins were shown to occur directly at the level of the B cell. The predominant Ig isotype elicited in response to porins plus anti-Ig-dex or CD40-ligand was IgM (>97%), with the remainder comprising IgG. Surprisingly, picogram-per-milliliter amounts of neisserial LPS were also found to be highly synergistic with anti-Ig-dex for induction of IgM secretion by LPS-responsive C3H/HeN, but not C3H/HeJ, B cells. Thus, these data suggest that porins, as well as LPS, may provide critical second signals for T-cell-independent induction of polysaccharide-specific Ig in response to neisserial and other gramnegative porin-expressing bacterial pathogens, without a requirement for the participation of non-B cell types. These data may also help to explain the potent immunopotentiating effects of porins for polysaccharidespecific, as well as protein-specific, humoral responses in vivo.
Cell therapy and bioengineering hold great promise as therapeutic approaches using cells and cell-derived factors to treat various pathologic or trauma-induced states. One possible application is the transplantation of cells into wounded tissue to help regulate tissue repair. Cells engineered for optimal wound healing may help to minimize scarring following surgery or to enhance the rate of healing of chronic wounds. The purpose of the current study was to determine the effect of a viral insert, the LacZ-bearing, first generation adenovirus AdRGD, on the survival of dermally transplanted murine skin allogenic fibroblasts. The LacZ insert facilitated quantitation of both cell survival and gene expression and was used here to measure viable cell number. In addition to bearing the LacZ marker, the AdRGD vector is capable of carrying therapeutic gene inserts, so this study tested the feasibility of gene therapy for wound healing. Murine skeletal muscle PP6 (i.e., Pre-Plate 6) myogenic stem cells served as an alternate donor cell type. Cells were labeled with the LacZ-bearing AdRGD adenovirus vector and injected (50,000 cells/site) into the dorsal skin of adult normal, immunocompetent mice as well as in immunodeficient SCID mice. Skin biopsies were taken on days 0, 1, 2, 3, and 7 post-transplant, and assayed for LacZ expression. Soon after transplant (day 1), cell numbers underwent a transient decrease, but by day 2 post-transplant they were present in appreciable numbers. Between days 2-7 post-transplant, both allogenic fibroblasts and PP6 myogenic stem cells maintained survivability in similar numbers. Further, survival of transplanted cell types was similar in both normal, immunocompetent as well as SCID mice during this time period. There were no signs of acute inflammation or rejection in any of the samples. This study shows that AdRGD-transduced cells are not immunogenic in the mouse skin model and the cells show similar survival for the first 7 days post-transplantation independent of the cell type or immunocompetence of the host.
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