The role of gamma-interferon, vitamin D3 metabolites and tumour necrosis factor in the pathogenesis of tuberculosis.

Endotoxin (LPS)-triggered release of tumour necrosis factor (TNF) from human monocytes is high for the first 3 days in culture, and then falls to a trough between Days 4 and 6. This trough is less deep if the cells are cultured in the presence of indomethacin. If monocytes are cultured in the presence of either recombinant gamma-interferon or 1,25-(OH)2 vitamin D3, their capacity for LPS-triggered TNF release is increased. Live virulent Mycobacterium tuberculosis can substitute for the LPS, and is markedly more effective as a trigger than several strains of BCG prepared in an identical manner. Since secretion of IFN-gamma and conversion of circulating 25-(OH) vitamin D3 to the active dihydroxy metabolite by interferon-activated macrophages probably occur in tuberculosis, we suggest that triggering of TNF release from the resulting activated macrophage populations might explain much of the weight loss and tissue damage that characterize this disease. IFN-gamma does not activate effective anti-mycobacterial mechanisms in human monocytes, so its role in tuberculosis may be immunopathological rather than protective.     

1,25-Dihydroxyvitamin D3 down-regulation of HLA-DR on human peripheral blood monocytes.

The regulatory activity of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on human leucocyte antigen (HLA)-DR (MHC class II) antigen expression in monocytes from normal human peripheral blood was examined. Using forward light and side scatter by flow cytometry most cells within the discrete monocyte area expressed high levels of HLA-DR antigens following 4-day culture in medium alone (culture-enhanced HLA-DR) and expression was further up-regulated in the presence of interferon-gamma (IFN-gamma) (IFN-gamma-enhanced HLA-DR). Treatment with 1,25-(OH)2D3 markedly inhibited both culture and IFN-gamma-enhanced HLA-DR but not HLA-ABC (MHC class I). This 1,25-(OH)2D3 inhibition was as effective as PGE2 and hydrocortisone. To ascertain if HLA-DR was specifically down-regulated on monocytes, the effect of vitamin D3 analogues in CD33+ cells was examined. Incubation of the CD33+ cells with 1,25-(OH)2D3, 24-25-(OH)2D3 and 25-OHD3 resulted in dose-dependent inhibition of culture-enhanced HLA-DR paralleling the vitamin D3-receptor affinities of these compounds. Northern analysis also demonstrated that 1,25-(OH)2D3 treatment markedly decreased both expression of culture-enhanced and IFN-gamma-enhanced HLA-DR beta chain messenger RNA (mRNA) in monocyte-enriched populations. In total, our findings are consistent with the proposal that vitamin D3 analogues can contribute to down-regulating immune responses as a consequence of inhibiting class II expression.     

Modulation of Mycobacterium bovis-specific responses of bovine peripheral blood mononuclear cells by 1,25-dihydroxyvitamin D(3).

Historically, administration of vitamin D has been considered beneficial in the treatment of tuberculosis. The interaction of this vitamin [i.e., 1,25-dihdroxyvitamin D(3) [1,25(OH)(2)D(3)]] with the antitubercular immune response, however, is not clear. In the present study, in vitro recall responses of peripheral blood mononuclear cells (PBMC) from cattle infected with Mycobacterium bovis were used to study the immune-modulatory effects of 1,25(OH)(2)D(3) on M. bovis-specific responses in vitro. Addition of 1 or 10 nM 1,25(OH)(2)D(3) inhibited M. bovis-specific proliferative responses of PBMC from M. bovis-infected cattle, affecting predominantly the CD4(+) cell subset. In addition, 1,25(OH)(2)D(3) inhibited M. bovis-specific gamma interferon (IFN-gamma) production yet enhanced M. bovis-specific nitric oxide (NO) production. Lymphocyte apoptosis, measured by flow cytometry using annexin-V staining, was diminished by addition of 1,25(OH)(2)D(3) to PBMC cultures. These findings support the current hypothesis that 1,25(OH)(2)D(3) enhances mycobacterial killing by increasing NO production, a potent antimicrobial mechanism of activated macrophages, and suggest that 1,25(OH)(2)D(3) limits host damage by decreasing M. bovis-induced IFN-gamma production.     

Effects of vitamin D metabolites and bisphosphonates on fibronectin release from monocyte-derived macrophages.

Fibronectin release from cultured macrophages, derived from human blood monocytes, was measured during incubation of the cells with increasing concentrations of vitamin D3 metabolites or of aminobutane bisphosphonate (AHButBP) or dichloromethylene bisphosphonate (Cl2MBP), two powerful inhibitors of bone resorption. The bisphosphonates significantly inhibited fibronectin release at 10(-8) M concentration and this inhibition was almost complete at 10(-5) M concentration. Opposite results were observed when the cells were incubated with vitamin D3 metabolites: the stimulation of fibronectin release was specific for 1,25-dihydroxyvitamin D3 relative to other vitamin D3 metabolites (1,25-dihydroxyvitamin D3 greater than 25-hydroxyvitamin D3 greater than 24,25-dihydroxyvitamin D3).     

1,25-Dihydroxyvitamin D3-Differentiated Human Promyelocytic Leukaemia Cells (HL-60) Can Kill Antibody-Coated Tumour Cells (K562)

The active metabolite of vitamin D3, 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3), was tested for the ability to differentiate promyelocytic leukaemia cells (HL-60) and histiocytic lymphoma cells (U937) into cytotoxic effector cells against K562 leukaemia cells. A concentration of 10(-6) M 1,25(OH)2D3 differentiated HL-60 cells into substrate-adherent monocyte-like cells with cytolytic activity against antibody-coated K562 cells. These differentiated HL-60 cells were not able to lyse uncoated K562 cells in an 18-h Cr-release assay. Similar treatment of the U937 cells with 1,25(OH)2D3 did not make them cytolytic towards K562 cells. These data indicate that 1,25(OH)2D3 can differentiate HL-60 cells to mature monocytes with cytolytic activity against antibody-coated leukaemia cells.     

1,25-Dihydroxyvitamin D3, but not retinoic acid, induces the differentiation of U937 cells.

We have examined the effects of vitamin D3 metabolites and retinoic acid on the myelomonocyte cell line U937. Inhibition of proliferation, measured by incorporation of 125iodo-deoxyuridine was seen at 72 h with 1,25-(OH)2D3 but not 25(OH)D3 or 24, 25(OH)2D3 metabolites. CD14 molecules, not normally present on U937 cells, were induced on the cell surface. However, Class II major histocompatibility complex (MHC) molecules were not induced and Class I MHC molecules not increased in density as determined by flow cytometry. Retinoic acid inhibited proliferation but failed to induce CD14 molecules. These data suggest that both 1,25(OH)2D3 and retinoic acid act as an antiproliferation signal to U937 cells; only 1,25-(OH)2D3 induces the differentiation towards a more mature phenotype.     

Vitamin D differentially regulates B7.1 and B7.2 expression on human peripheral blood monocytes.

The hormonal active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1, 25(OH)2D3), inhibits (through an unknown mechanism) the ability of monocytes/macrophages to induce T-cell activation. For T cells to be optimally activated, recognition of antigen/major histocompatibility complexes (MHC) by the T-cell receptor (TCR) must be accompanied by a second costimulatory signal. Considerable experimental data now suggest that this costimulatory signal is predominantly generated by B7.1 and/or B7.2 molecules, expressed on antigen-presenting cells (APC), when engaged to their counter-receptor, CD28, present on T cells. To determine whether the inhibitory effect of 1,25(OH)2D3 on monocytes/macrophages might involve modulation of the expression of B7.1 and B7.2 molecules, we analysed (by flow cytometry) the influence of 1,25(OH)2D3 and an analogue, KH 1060, on the expression of these two molecules at the surface of resting human peripheral blood monocytes. In parallel, we tested the effect of these two agents on human monocyte expression of cell-surface markers (CD14 and CD4) and antigen-presenting molecules (MHC class I and MHC class II). Our results showed that both 1,25(OH)2D3 and KH 1060 inhibited the basal expression of B7.2 in a dose- and time-dependent manner, without affecting B7.1. Moreover, these two compounds increased CD14 and reduced MHC class II and CD4 expression. Furthermore, the effect of 1,25(OH)2D3 on B7 molecule expression in combination with lipopolysaccharide (LPS) or cytokines, including interleukin-10 (IL-10), interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha), was studied. The 1,25(OH)2D3-induced B7.2 down-regulation was still detectable when monocytes were activated by IL-10, IFN-gamma and TNF-alpha but not with LPS. Moreover, the induction of B7.1 by TNF-alpha was inhibited by addition of 1, 25(OH)2D3. We conclude that the ability of 1,25(OH)2D3 to decrease B7.2 expression on human monocytes might contribute to its inhibitory effect on APC-dependent T-cell activation and to its immunosuppressive properties observed in autoimmune diseases and organ transplantation.     

Killing of Mycobacterium tuberculosis within human monocytes: activation by cytokines and calcitriol.

Human monocytes were isolated and their ability to harbour growth of virulent tubercle bacilli was assessed, in the presence or absence of various immunomodulators. Calcitriol (1,25(OH2), vitamin D3) alone, at doses of 10(-7)-10(-9) M endowed human monocytes with a significant ability to restrict intracellular growth of the tubercle bacilli. Crude immune lymphokines as well as recombinant interferon-gamma (IFN-gamma) endowed monocytes with no tuberculostatic activity. Similarly, other recombinant cytokines tested, notably colony-stimulating factor-1 (CSF-1), interleukin-1 (IL-1), interleukin-3 (IL-3) and interleukin-6 (IL-6) all failed to stimulate anti-tuberculous properties, and even increased growth of the tubercle bacilli in monocytes, in the case of CSF-1. Conversely, incubation of crude lymphokines in combination with calcitriol led to total stasis of the growth of M. tuberculosis. Experiments with recombinant cytokines and immunologically active vitamins showed that a combination of IFN-gamma tumour necrosis factor-alpha and calcitriol induced a significant amount of intramonocyte killing of M. tuberculosis. Addition of this cocktail of factors to already infected monocytes led to substantial killing of tubercle bacilli. These sets of experiments establish clearly that combinations of recombinant cytokines and vitamins may induce substantial intramonocyte killing of M. tuberculosis. The mechanism involved in this killing activity was not clarified.     

Human neutrophils express messenger RNA of vitamin D receptor and respond to 1alpha,25-dihydroxyvitamin D3

1Alpha,25-Dihydroxyvitamin D3 (1,25(OH)2D3) has been shown to modulate the production of various cytokines or the expression of certain differentiation markers in human T cells or monocytes. Its effects on neutrophils, however, are poorly understood. In this paper, we show several lines of evidence indicating that neutrophils express functional vitamin D receptors (VDR). Sort-purified neutrophils from human peripheral blood expressed VDR mRNA at a level comparable to that of monocytes. As reported to occur in monocytes, protein expression of CD14 on the cell surface of neutrophils was augmented when the cells were incubated with 1,25(OH)2D3. To investigate the physiological roles for VDR in neutrophils, we investigated possible modulating effects of 1,25(OH)2D3 on the expression of several genes in lipopolysaccharide-stimulated neutrophils by using differential display analysis. Of the genes we identified, trappin-2/elafin/SKALP, which was originally reported to be an inhibitor of elastase, was induced in neutrophils by lipopolysaccharide, but was suppressed significantly in the presence of 1,25(OH)2D3. Under the same conditions, interleukin-1beta expression was also inhibited. These findings suggest that 1,25(OH)2D3 has a potential to affect the inflammatory process by modulating the expression of neutrophil genes.     

Influence of vitamin D3 metabolites on the production of interleukins 1,2 and 3

We investigated the effect of vitamin D3 metabolites on the release of the three interleukins (IL) IL 1, IL 2 and IL 3 by mononuclear cells. Models for the production of these mediators were the release of IL 1 by the murine macrophage cell line P388D1, of IL2 by rat spleen cells, and of IL 3 by the murine WEHI-3 cell line. IL 1 production was significantly increased with 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) at 10(-10)M and above. 1,25(OH)2D3 did not stimulate cell proliferation as assessed with [methyl-3H]thymidine (3H-TdR) incorporation. 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) and 25-hydroxyvitamin D3 (25(OH)D3) were about 1000 times less effective than 1,25(OH)2D3. IL 2 production, by cultured rat spleen cells stimulated with Concanavalin A, was decreased by increasing concentrations of 1,25(OH)2D3. The minimal effective dose varied between experiments and ranged from 10(-11) to 10(-8) M. Moreover, proliferation (3H-TdR incorporation) of mouse thymocytes treated with phytohemagglutinin and IL 1 was decreased in a dose-dependent fashion by 1,25(OH)2D3 starting at 10-11) M. This effect might be secondary to a decrease of endogenous IL 2 production. IL 3 release by WEHI-3 cells was significantly increased with 10(-11)-10(-9) M 1,25(OH)2D3, whereas higher concentrations were less effective or decreased IL 3 production. These results show that 1,25(OH)2D3 and, to a lesser extent, 24,25(OH)2D3 and 25(OH)D3 have selective effects on lymphokine production. It is tempting to speculate that the actions of 1,25(OH)2D3 on bone might in part be mediated by lymphokines. Moreover, we suggest that 1,25(OH)2D3 might not be an immunoregulator per se, but makes use of the immune system to exert its influence on one of its classical targets, namely the bone, and possibly on other connective tissues.     

Cholecalciferol Supplementation in HIV-Infected Youth with Vitamin D Insufficiency: Effects on Vitamin D Status and T-Cell Phenotype: A Randomized Controlled Trial

Abstract

Objectives: In addition to its known effects on bone metabolism, vitamin D may regulate immune function. Design: We performed a randomized controlled trial (RCT) to test whether cholecalciferol supplementation can improve vitamin D status and affect the T-cell phenotype in HIV-infected youth with vitamin D insufficiency. Methods: Fifty-two HIV-infected patients aged 8 to 26 years and with serum 25(OH) D <30 ng/mL were randomized to receive orally vitamin D3 100,000 IU or placebo every 3 months for 4 doses. Serum 25(OH)D, 1,25(OH)₂D, PTH, and CD4+ T cells were assessed 3 months before baseline and at 0, 3, 6, 9, and 12 months, while Th1-, Th2-, Th17-, and Treg-subsets and T-lymphocyte vitamin D receptor were assessed at 0, 3, and 12 months. Results: Forty-eight subjects (25 receiving vitamin D and 23 receiving placebo) completed the RCT. Cholecalciferol supplementation produced an early (3 months) decrease in PTH, a concomitant increase in 25(OH)D, and a later (6 months) increase in 1,25(OH)₂D levels, all persisting at 12 months. The frequency of vitamin D insufficiency at 12 months was 20% versus 60% in the intervention versus placebo group (P = .007). Cholecalciferol supplementation had no effect on CD4+ T-cell counts but was associated with a decreased Th17:Treg ratio at 3 months. Conclusions: In our cohort of HIV-infected youth, a 12-month cholecalciferol supplementation increased 25(OH)D and 1-25(OH)₂D and decreased PTH levels but had no effect on CD4+ T-cells. However, it was associated with changes in CD4+ T-cell phenotype, warranting further investigation.     

Comparative abilities of various metabolites of vitamin D to protect cultured human macrophages against tubercle bacilli

Vitamin D metabolites have several biologic functions besides the best-known one of controlling mineral metabolism, one of which may be protection against tuberculosis. The chemical structures of the metabolites govern their functions. The most potent metabolite for regulating calcium metabolism is 1,25(OH)2-vitamin D3 (1,25-D3). This metabolite also is able to protect cultured human monocytes and macrophages (MP) against tubercle bacilli (TB). To determine whether these two functions are connected, 14 other analogs or metabolites of vitamin D were compared with 1,25-D3 for ability to protect cultured MP against TB. Blood-derived MP from 18 different donors were used in 70 experiments. The MP were infected with TB, then incubated for 7 days in medium containing 4 micrograms/ml of metabolite or synthesized analog. Growth of TB in the MP was measured by colony-forming unit counts from samples of lysed MP at 0, 4, and 7 days after infection. The metabolites, none of which inhibited TB in the absence of MP, varied from unprotective to bacteriostatic in the MP. Four of them were nearly as protective as 1,25-D3, and the metabolite 25S, 26(OH)2-vitamin D3 was consistently more protective. An analog synthetically designed for maximum ability to promote cell differentiation was unprotective. There was no correlation between metabolite ability to protect and known ability to stimulate calcium mobilization. These results suggest that antituberculosis protection of human MP by vitamin D metabolites or analogs can be separated from their functions of inducing cell differentiation and controlling mineral metabolism.     

Regulation of human tonsillar T-cell proliferation by the active metabolite of vitamin D3.

We have examined the effects of 1,25(OH)2D3 on T-cell populations isolated by buoyant density and E rosetting from human tonsils. Cell proliferation was assessed by measuring the incorporation of 125iododeoxyuridine; interleukin-2 (IL-2) production was measured using an IL-2-dependent cell line, and the number of 1,25(OH)2D3 receptors was measured by whole-cell nuclear association assay. At a concentration of 10(-7) M, 1,25(OH)2D3 inhibited mitogen-induced T-cell proliferation in all E+ T-cell populations. This effect was more pronounced in the cells from the intermediate and high density layers and was reflected both in cell proliferative responses and in relative IL-2 synthesis. By adding the 1,25(OH)2D3 during the course of the mitogen assay, we demonstrated that activation of the T cell precedes the 1,25(OH)2D3-mediated inhibition. Cells that had been preincubated with mitogen in the presence of the 1,25(OH)2D3 were refractory to further stimulation by mitogens. Receptors for 1,25(OH)2D3 could not be detected in unstimulated T cells. However, activation led to the expression of high-affinity receptors for 1,25(OH)2D3. Co-incubation of the cells with mitogen and 1,25(OH)2D3 increased the number of receptors compared with mitogen alone. The effects provide further evidence for the hypothesis that 1,25(OH)2D3 is an important potential modulator of the immune system through its action on T cells. Taking our observations in conjunction with the known capacity of monocytes to hydroxylate the precursor metabolite (and thus synthesize the active form of cholecalciferol), the results support the suggestion that 1,25(OH)2D3 plays a role as a local mediator of mononuclear phagocyte-T cell interaction in human lymphomedullary tissues.     

Mycobacterium bovis infection of vitamin D-deficient NOS2-/- mice

Vitamin D deficiency is associated with an increased risk for tuberculosis infection. Studies using in vitro systems indicate that 1,25-dihydroxyvitamin D(3) [i.e. 1,25(OH)(2)D(3)], the most active form of the vitamin, enhances mycobacterial killing by increasing nitric oxide (NO) production. To evaluate concurrently the role of 1,25(OH)(2)D(3) and NO on the host response to tuberculosis infection, mice deficient in NO synthase 2 (NOS2(-/-)) and/or vitamin D were aerosol-challenged with Mycobacterium bovis and subsequently evaluated for mycobacterial colonization and lesion formation. Infected NOS2(-/-) mice developed severe necrotizing pyogranulomatous inflammation of the lungs with heavy M. bovis colonization and systemic dissemination of the bacillus. Colonization and lung lesion area of NOS2(-/-) mice exceeded that of NOS2(+/+) mice. Additionally, disease progression was more rapid in NOS2(-/-) mice than in NOS2(+/+) mice. Lung colonization and lesion area of vitamin D deficient mice exceeded that of vitamin D replete mice, regardless of NOS2 phenotype. However, effects of vitamin D on colonization, but not lesion area, were more pronounced in NOS2(+/+) mice than in NOS2(-/-) mice. These findings are consistent with the current hypothesis that 1,25(OH)(2)D(3) enhances mycobacterial killing through a NO-dependent mechanism. As responses of NOS2(-/-) mice were affected by 1,25(OH)(2)D(3) deficiency, albeit to a lesser extent than were those of NOS2(+/+) mice, NO-independent actions of 1,25(OH)(2)D(3) also likely exist.     

1,25-dihydroxyvitamin D3 stimulates interleukin-2 production by a T cell lymphoma line (MLA-144) cultured in vitamin D-deficient rat serum

A lymphocyte T cell line (MLA-144), which constitutively secretes interleukin-2 (IL-2), was shown to express receptors for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). The proliferation of an IL-2-dependent cell line (HT-2) in response to supernatants from MLA-144 cells was employed as an index of IL-2 production by MLA-144 cells. IL-2 production was two fold higher from MLA-144 cells cultured in 2% vitamin D-deficient rat serum compared to 10% fetal calf serum (FCS). The addition of 1,25(OH)2D3 at 10(-15) M or 10(-11) M augmented IL-2 production by MLA-144 cells in vitamin D-deficient rat serum, but not in fetal calf serum. At 10(-7) M 1,25(OH)2D3 there was inhibition of IL-2 production by MLA-144 cells in either vitamin D-deficient serum or FCS. There was no effect of 1,25(OH)2D3 added directly to HT-2 cells. Monoclonal antibody to the IL-2 receptor competitively inhibited the proliferation of HT-2 cells in response to MLA-144 supernatants, suggesting that it was IL-2 from the MLA-144 supernatants which influenced HT-2 proliferation. Our findings demonstrate biphasic dose effects of 1,25(OH)2D3 on lymphokine secretion. The use of vitamin D-deficient rat serum allowed us to demonstrate the effects of 1,25(OH)2D3 in the physiologic and subphysiologic range.     

Inhibition of tropoelastin expression by 1,25-dihydroxyvitamin D3.

Elastin production is modulated by steroid hormones and is dependent on calcium. Because vitamin D3 is involved in the regulation of calcium metabolism and influences the expression of various extracellular matrix proteins, we investigated whether vitamin D3 influences tropoelastin expression. Three elastin-producing, bovine cell types, auricular chondroblasts, nuchal ligament fibroblasts and arterial smooth muscle cells, were treated with the principal active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25[OH]2D3), and with 24,25 dihydroxyvitamin D3 (24,25[OH]2D3). Tropoelastin levels in culture media and cell layers, as measured by an enzyme-linked immunoassay, decreased in a dose and exposure dependent manner after treatment with 1,25(OH)2D3; 24,25(OH)2D3 had no effect on tropoelastin production relative to solvent-treated controls. The maximal effective dose of 1,25(OH)2D3 was 10(-7) M for 48 hr, which resulted in a severalfold reduction of tropoelastin production, and decreased tropoelastin levels were detected at 8 hr after treatment. Reduction of tropoelastin protein production was paralleled by a decrease of equal magnitude in the steady-state levels of tropoelastin mRNA. Vitamin D3 metabolites had no effect on DNA or total protein synthesis. These results suggest that vitamin D3 may be an important modulator of elastin expression.     

The role of monocytes in the suppression of PHA-induced proliferation and IL 2 production of human mononuclear cells by 1,25-dihydroxyvitamin D3

1,25-dihydroxyvitamin D3 [1,25(OH)2D3] inhibited phytohemagglutinin (PHA)-induced proliferation of human blood mononuclear cells (MNC) at concentrations of 10(-11) M or more. Interleukin 2 (IL 2) production of T cells activated with PHA was also inhibited by 1,25(OH)2D3. Furthermore, 1,25(OH)2D3 suppressed interleukin 1 (IL 1) production of monocytes (Mo), and the agent-treated Mo were unable to promote IL 2 production of non-adherent cells (NAC). Thus, the reduction of proliferative response of MNC to PHA by 1,25(OH)2D3 appeared to have resulted from the inhibitory effects of the agent on both IL 2 and IL 1 production. From these data, 1,25(OH)2D3 appears to play an important role in the immunoregulatory system.     

Effects and interactions of 24R,25(OH)2D3 and 1,25(OH)2D3 on bone

The effects of various combinations of therapy with 1 alpha,25-dihydroxycholecalciferol (1,25(OH)2D3) and 24R,25-dihydroxycholecalciferol (24R,25(OH)2D3) on structural and dynamic parameters of bone were evaluated in 40 chicks raised on a vitamin D-deficient diet from time of hatching and supplemented with the dihydroxylated metabolites. The results showed that: 1) the maintenance of volumetric density of bone is dependent on the presence of 1,25(OH)2D3, 2) lack of 1,25(OH)2D3 is associated with an increase in the number of osteocytes per unit volume of bone, most probably due to decreased amounts of bone formed by each osteoblast before becoming an osteocyte, 3) adequate quantities of either 24R,25(OH)2D3 or 1,25(OH)2D3 are needed to prevent accumulation of osteoid or the production of endosteal fibrosis, and 4) maintenance of normal tetracycline label width requires both hydroxylated compounds with one of them in sufficient amounts. The data of this study demonstrate that the integrity of certain parameters of bone structure could be maintained only with 1,25(OH)2D3, others with either dihydroxylated metabolites, and still others with a combination of both. These data underscore the biological activity of 24R,25(OH)2D3 by demonstrating its effectiveness on bone.     

1 alpha,25-Dihydroxyvitamin D3 and a novel vitamin D analogue MC 903 are potent inhibitors of human interleukin 1 in vitro

1 alpha,25-dihydroxycholecalciferol (1,25(OH)2D3) inhibits the lymphocyte growth hormone, interleukin 2. Since its production is dependent upon interleukin 1 (IL-1) produced by antigen-presenting cells, we tested five vitamin D3 analogues for effects on the production and function of human natural and recombinant IL-1. The production was not affected, but 1,25(OH)2D3 (greater than 10(-11) M) and a synthetic derivative MC 903 (greater than = 10(-10) M) inhibited the proliferation of mouse thymocytes to IL-1. The vitamins failed to affect the cytotoxic activity of tumor necrosis factor. 1,25(OH)2D3 may play a physiological immunomodulatory role as a selective inhibitor of the function of IL-1, and MC 903 may prove clinically useful in this regard because of its limited calcium metabolic activity.     

1,25-Dihydroxyvitamin D3 in teeth of rats and humans: receptors and nuclear localization

Autoradiographic and biochemical studies were used to demonstrate 1,25 (OH)2 vitamin D3 target cells in teeth. Incisor pulp of rats and molar pulp of humans were incubated in vitro with 3H-1,25 (OH)2 vitamin D3. Subsequent frozen-section autoradiography revealed a large population of cells in the pulp of both incisors and molars which selectively concentrated radioactivity in their nuclei. Extracts of incisor pulp from mature rats were found to bind 3H-1,25 (OH)2 vitamin D3 and this binding was displaceable with excess 1,25 (OH)2 vitamin D3. Sucrose density analysis revealed that the protein in tooth pulp which binds 1,25 (OH)2 vitamin D3 sediments at 3.2-3.5S. The 1,25 (OH)2 vitamin D3 receptor of intestine and kidney also sediments in this region, indicating that the 1,25 (OH)2 vitamin D3 binding protein of tooth pulp is similar to that found in other target organs. These autoradiographic and biochemical data indicate that pulpal cells of mature rat and human teeth contain receptors for 1,25 (OH)2 vitamin D3.