Inhibition of IL-12 production by 1,25-dihydroxyvitamin D3. Involvement of NF-kappaB downregulation in transcriptional repression of the p40 gene.

Abstract

Interleukin 12 (IL-12), produced by myelomonocytic cells, plays a pivotal role in the development of T helper 1 (Th1) cells, which are involved in the pathogenesis of chronic inflammatory autoimmune disorders. 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] inhibits IL-12 production by activated macrophages and dendritic cells, thus providing a novel interpretation to its immunosuppressive properties. 1,25(OH)2D3 significantly inhibits mRNA expression for both IL-12 p35 and p40 subunits acting at the transcriptional level. The effect of 1,25(OH)2D3 on p40 promoter activation was analyzed by cotransfecting monocytic RAW264.7 cells with p40 promoter/reporter constructs and expression vectors for vitamin D3 receptor (VDR) and/or retinoid X receptor (RXRalpha). We observed transcriptional repression of the p40 gene by 1,25(OH)2D3, which required coexpression of VDR with RXR and an intact VDR DNA-binding domain. The repressive effect maps to a region in the p40 promoter containing a binding site for NF-kappaB (p40-kappaB). Deletion of the p40-kappaB site abrogates part of the inhibitory effect on the p40 promoter, confirming the functional relevance of this site. Activation of monocytic THP-1 cells in the presence of 1,25(OH)2D3 results in reduced binding to the p40-kappaB site. Thus, 1,25(OH)2D3 may negatively regulate IL-12 production by downregulation of NF-kappaB activation and binding to the p40-kappaB sequence.

Differential Regulation of Vitamin D Receptor and Its Ligand in Human Monocyte-Derived Dendritic Cells

“…Quantitative RT-PCR analysis of 1␣-OHase, VDR, and 25(OH)D 3 ase) mRNA expression 1␣OHase, VDR, and 24(OH)ase mRNA levels were analyzed using an ABI 7700 sequence detection system (PE Biosystems, Warring, U.K.) using previously reported protocols (12). RNA was extracted from cell pellets using the StrataPrep total RNA miniprep kit (Stratagene, Amsterdam, The Netherlands) and was reverse-transcribed using AMV reverse transcriptase (Promega, Madison, WI) and random hexamers in 40-l reaction volumes according to manufacturer's instructions.…”

“…C hanges in dendritic cell (DC) 3 function represent the net cellular response to a large repertoire of microenvironmental positive and negative stimuli (1). An understanding of how developing or terminally differentiated DCs integrate these signals is of crucial importance in terms of defining regulatory pathways that contribute to the homeostatic control of their functions.…”

“…One important regulatory mechanism involves the actions of the secosteroid hormone, 1␣25-dihydroxyvitamin D 3 (1␣25(OH) 2 D 3 ), the biologically active metabolite of vitamin D 3 , which influences the immune response at multiple levels (2), including at the level of the APC. For example, 1␣25(OH) 2 D 3 inhibits the release of inflammatory cytokines such as IL-12 (3) or TNF-␣ by macrophages (4).…”

“…The ligand-VDR complex heterodimerizes with the 9-cis-retinoic acid liganded retinoid X receptor, before binding as a transregulatory complex to vitamin D 3 -responsive cis elements in target gene promoters (9). These genes include IL-12p35 and p40 (3), that are expressed upon CD40-mediated licensing of DCs and consequently the repressive effect of 1␣25(OH) 2 D 3 upon IL-12 secretion is lost in DCs derived from VDR knockout mice (7). Indeed, the lymph nodes in VDR knockout mice are increased in size as compared with wild type and the proportion of mature DCs within them is greater, suggesting that 1␣25(OH) 2 D 3 exerts an essential negative regulatory function in vivo that limits the degree to which DCs undergo spontaneous maturation (7).…”

The phenotype and function of murine bone marrow-derived dendritic cells is not affected by the absence of VDR or its ability to bind 1α,25-dihydroxyvitamin D3

“…For example, in the current study, we observed an up-regulation of il-12p40 and tnf-a in VDR null DCs, as compared to WT and VDR DAF-2 DCs. The ligand-VDR complex is known to interfere with the signaling of transcription factors such as the nuclear factor of activated T cells (NFAT) and nuclear factor kB (NF-kB) [25,26], via obstruction of its activation and genomic binding, which results in the 1a,25 (OH) 2 D 3 -mediated down-regulation of il-12p40 [27,28]. In addition, reduced levels of the inhibitor of NF-kB were observed in VDR null cells [29].…”