Terri L. Towers

The molecular basis for the anti-inflammatory property of intravenous gamma globulin (IVIG) was investigated in a murine model of immune thrombocytopenia. Administration of clinically protective doses of intact antibody or monomeric Fc fragments to wild-type or Fcgamma receptor-humanized mice prevented platelet consumption triggered by a pathogenic autoantibody. The inhibitory Fc receptor, FcgammaRIIB, was required for protection, because disruption either by genetic deletion or with a blocking monoclonal antibody reversed the therapeutic effect of IVIG. Protection was associated with the ability of IVIG administration to induce surface expression of FcgammaRIIB on splenic macrophages. Modulation of inhibitory signaling is thus a potent therapeutic strategy for attenuating autoantibody-triggered inflammatory diseases.

T-lymphocyte proliferation is suppressed by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the active metabolite of vitamin D3, and is associated with a decrease in interleukin 2 (IL-2), gamma interferon, and granulocyte-macrophage colony-stimulating factor mRNA levels. We report here that 1,25(OH)2D3-mediated repression in Jurkat cells is cycloheximide resistant, suggesting that it is a direct transcriptional repressive effect on IL-2 expression by the vitamin D3 receptor (VDR). We therefore examined vitamin D3-mediated repression of activated IL-2 expression by cotransfecting Jurkat cells with IL-2 promoter/reporter constructs and a VDR overexpression vector and by DNA binding. We delineated an element conferring both DNA binding by the receptor in vitro and 1,25(OH)2D3-mediated repression in vivo to a short 40-bp region encompassing an important positive regulatory element, NF-AT-1, which is bound by a T-cell-specific transcription factor, NFATp, as well as by AP-1. VDR DNA-binding mutants were unable to either bind to this element in vitro or repress in vivo; the VDR DNA-binding domain alone, however, bound the element but also could not repress IL-2 expression. These results indicate that DNA binding by VDR is necessary but not sufficient to mediate IL-2 repression. By combining partially purified proteins in vitro, we observed the loss of the bound NFATp/AP-1-DNA complex upon inclusion of VDR or VDR-retinoid X receptor. Order of addition and off-rate experiments indicate that the VDR-retinoid X receptor heterodimer blocks NFATp/AP-1 complex formation and then stably associates with the NF-AT-1 element. This direct inhibition by a nuclear hormone receptor of transcriptional activators of the IL-2 gene may provide a mechanistic explanation of how vitamin derivatives can act as potent immunosuppressive agents.

The 1,25-dihydroxyvitamin D3 receptor, like other members of the nuclear receptor superfamily, forms dimers in solution that are probably stabilized by a dyad symmetrical interface formed by the ligand-binding domain. This receptor, however, recognizes DNA targets that are not dyad symmetric but rather are organized as direct repeats of a hexameric sequence with a characteristic 3-bp spacing. Using molecular modeling and site-directed mutagenesis, we have identified regions within the vitamin D3 receptor zinc finger region that confer selectivity for direct repeats with appropriate spacing. Reflecting the organization of the DNA target, these regions, mapping to the tip of the first zinc finger module and the N and C termini of the second finger module, direct asymmetrical protein-protein contacts. A stereochemical model is proposed for these interactions.

The primary function of activated T lymphocytes is to produce various cytokines necessary to elicit an immune response; these cytokines include interleukin-2 (IL-2), interleukin-4, and granulocyte-macrophage colony-stimulating factor (GMCSF). Steroid hormones and vitamin A and D3 metabolites act to repress the expression of cytokines. 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) down-modulates activated IL-2 expression at the level transcription, through direct antagonism of the transactivating complex NFAT-1/AP-1 by the vitamin D3 receptor (VDR). We report here that GMCSF transcription in Jurkat T cells is also directly repressed by 1, 25-(OH)2D3 and VDR. Among four NFAT/AP-1 elements in the GMCSF enhancer, we have focused on one such element that when multimerized, is sufficient in mediating both activation by NFAT-1 and AP-1 and repression in response to 1,25-(OH)2D3. Although this element does not contain any recognizable vitamin D response elements (VDREs), high affinity DNA binding by recombinant VDR is observed. In contrast to VDR interactions with positive VDREs, this binding is independent of VDR's heterodimeric partner, the retinoid X receptor. Moreover, VDR appears to bind the GMCSF element as an apparent monomer in vitro. Protease digestion patterns of bound VDR, and receptor mutations affecting DNA binding and dimerization, demonstrate that the receptor binds to the negative site in a distinct conformation relative to a positive VDRE, suggesting that the DNA element itself acts as an allosteric effector of VDR function. This altered conformation may account for VDR's action as a repressing rather than activating factor at this locus.