Inhibition of human T lymphocyte proliferation and cytokine production by 1,25-dihydroxyvitamin D3. Differential effects on CD45RA+ and CD45R0+ cells.

1,25-dihydroxyvitamin D3 (1,25-(OH)2 D3), the biologically active form of vitamin D3, has been shown to modulate lymphocyte functions in vitro. These effects are exerted through binding to specific receptors that are expressed in activated, but not in resting lymphocytes. 1,25-(OH)2 D3 inhibits lymphocyte proliferation, immunoglobulin production and the release of cytokines including interleukin-2 (IL-2) and interferon gamma (IFN gamma) by mitogen driven blood mononuclear cells (MNC). A distinction between CD45RA+ and CD45R0+ subsets of T cells has, however, proven extremely relevant in terms of immunoactivation and immunopathology. The present study was undertaken to evaluate effects of 1,25-(OH)2 D3 on proliferation and cytokine production by purified CD45RA+ and CD45R0+ T cells. 1,25-(OH)2 D3 caused a dose- and time-dependent reduction in phytohemagglutinin-(PHA) and poke-weed mitogen (PWM)-driven proliferation of purified CD45R0+ T cells. In contrast, proliferation of the CD45RA+ subset was unaffected by this treatment. Comparable levels of lymphotoxin (LT), IFN gamma and IL-2 were obtained in cultures of both subsets. 1,25-(OH)2 D3 reduced these levels, but the suppressive effect of the hormone was delayed in cultures of CD45RA+ T cells. The results suggest that the CD45R0+ subset is relatively more sensitive than CD45RA+ subset to the inhibitory effects of 1,25-(OH)2 D3. This finding may be of pharmacological interest, because the CD45R0+ subset plays a key role in immune activation and because these cells have been associated with the pathogenesis of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis.     

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.     

1,25(OH)2D3 regulates c-myc mRNA levels in tonsillar T lymphocytes.

The effects of 1,25(OH)2D3 on proliferation, c-myc mRNA levels and 1,25(OH)2D3 receptor expression in activated tonsillar T lymphocytes were studied. Activation of resting T cells with phytohaemagglutinin (PHA) for 72 hr led to an increase in proliferation, c-myc mRNA levels and to induction of 1,25(OH)2D3 receptor expression. However, when activation was carried out in the presence of 1,25(OH)2D3, there was inhibition of PHA-stimulated proliferation and c-myc mRNA levels. Increased cell proliferation, c-myc mRNA expression and 1,25(OH)2D3 receptor number were also observed, albeit to a lesser extent, when T cells were stimulated by phorbol myristate acetate (PMA), anti-CD3 antibody or A23187. However, in these cases 1,25(OH)2D3 was unable to prevent increased proliferation or c-myc mRNA expression. PMA and anti-CD3 used in combination produced similar or greater changes in proliferation, c-myc mRNA levels, 1,25(OH)2D3 receptor expression and responsiveness to the hormone when compared to PHA alone. Thus the inhibition of c-myc expression in activated T lymphocytes by 1,25(OH)2D3 can be related to its anti-proliferative effects. Moreover this inhibition seems to be dependent on the level of 1,25(OH)2D3 receptor expression, which in turn appears to be related to the degree of cell activation.     

Signal-dependent pleiotropic regulation of lymphocyte proliferation and cytokine production by 1,25-dihydroxyvitamin D3: potent modulation of the hormonal effects by phorbol esters.

The influence of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the proliferation of lymphocytes and on the production of interleukin-6 (IL-6), interleukin-1 beta (IL-1 beta) and interferon-gamma (IFN-gamma) was examined in normal human peripheral blood mononuclear cells (PBMC) activated in vitro either by phytohaemagglutinin (PHA) or by the monoclonal antibody to the T-cell receptor OKT3, or by the combination of each of these two stimuli with phorbol myristate acetate (PMA). 1,25(OH)2D3 inhibited the proliferative response of PBMC to PHA; this effect, however, was abrogated by the addition of PMA (1.6 nM), and it was reversed from inhibition to stimulation by higher concentrations of the phorbol ester. In contrast to the PHA-activated cells, 1,25(OH)2D3 had no effect on the proliferative response of PBMC to OKT3. Further, 1,25(OH)2D3 inhibited the release of IL-6 in cultures of PHA-activated PBMC, whereas it stimulated IL-6 with the addition of PMA in these cultures. In contrast to the PHA-activated cells, 1,25(OH)2D3 increased IL-6 release in OKT3-activated cells. IL-1 beta production was not affected in either PHA- or OKT3-activated cells by the presence of the hormone, but it was stimulated by 1,25(OH)2D3 when PMA was used as a co-stimulus with either PHA or OKT3. Finally, 1,25(OH)2D3 inhibited IFN-gamma in both PHA- and OKT3-activated cells, but these effects were attenuated in the presence of PMA. These findings demonstrate that the in vitro effects of 1,25(OH)2D3 on lymphocyte proliferation and cytokine production by PBMC are pleiotropic, and that such pleiotropism depends upon the mode of PBMC activation and presumably the signals that are generated in response to the specific agents used to activate these cells.     

1,25-Dihydroxyvitamin D3-mediated suppression of T lymphocyte functions and failure of T cell-activating cytokines to restore proliferation.

1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) has been shown to be a potent inhibitor of lymphocyte proliferation in vitro. The present study was undertaken to determine if this is caused by a direct effect on the lymphocytes, and to evaluate to what degree this suppression may be restored by the addition of cytokines. 1,25-(OH)2D3, > or = 10(-10) M, significantly inhibited the proliferation of pokeweed mitogen (PWM)-driven human peripheral blood mononuclear cells (MNC). Depletion of monocytes did not alter the response to 1,25-(OH)2D3. The antiproliferative effect was preceded by decreased production of interleukin (IL)-1 alpha and lymphotoxin (LT), both of which are crucially involved in T cell activation. However, the suppressive effect of 1,25-(OH)2D3, seen in MNC cultures stimulated with PWM alone, was of the same magnitude as the effect seen in MNC cultures stimulated with a combination of PWM and recombinant (r)IL-1 alpha, rIL-6, recombinant tumour necrosis factor (rTNF) alpha, rIL-2 or rLT, as well as PWM plus conditioned medium. Although pretreatment of monocytes for 2 h with 1,25-(OH)2D3 caused significant reduction in the release of IL-1 alpha and TNF alpha, reconstitution of monocyte-depleted cultures with similarly treated monocytes had no inhibitory effect on the proliferative response. In conclusion, even though it cannot be excluded that a low but critical number of monocytes are essential for the suppressive effect of 1,25-(OH)2D3-mediated inhibition of MNC proliferation, the inhibition is most likely the result of a direct effect on the lymphocytes and independent of monocytes and exogenously added cytokines.(ABSTRACT TRUNCATED AT 250 WORDS)     

1,25-Dihydroxyvitamin D3-mediated inhibition of human B cell differentiation.

We have examined the mechanisms of 1,25-dihydroxyvitamin D3 (D3)-mediated inhibition of human B cell differentiation to immunoglobulin (Ig) secreting cells. B lymphocytes were purified from human tonsils and peripheral blood mononuclear cells. Mononuclear cells were separated into adherent cells and nonadherent cells. Cells were stimulated with Staphylococcus aureus Cowen I (SAC) or pokeweed mitogen (PWM) for 7 days and immunoglobulin production was measured by ELISA assay, 1,25-dihydroxyvitamin D3 was added at different times during cultures. 1,25-Dihydroxyvitamin D3, in a dose-dependent manner, inhibited both SAC and PWM-induced Ig production by mononuclear cells. The maximum inhibition was observed when 1,25-dihydroxyvitamin D3 was added at the beginning of culture, but inhibition could still be observed when 1,25-dihydroxyvitamin D3 was added on day 4 of cultures. The inhibitory effect of 1,25-dihydroxyvitamin D3 on Ig production was significantly greater on mononuclear cells than on nonadherent cells. Addition of in vitro purified IL-1 to nonadherent cells enhanced 1,25-dihydroxyvitamin D3-induced inhibition of Ig production. 1,25-Dihydroxyvitamin D3 also inhibited the expression of IL-2 receptors on B cells activated with SAC. 1,25-dihydroxyvitamin D3 did not inhibit Ig production by SAC preactivated B cell blasts in response to T cell supernatants. These data suggest that vitamin D3 inhibits Ig production by inhibiting IL-2 receptor expression on B cells and via its effect on adherent macrophages. Vitamin D3 does not influence the effect of differentiation factors on activated B cells that have already expressed growth/differentiation factor receptors.     

Exogenous interleukin-2 does not reverse the immunoinhibitory effects of 1,25-dihydroxyvitamin D3 on human peripheral blood lymphocyte immunoglobulin production

1,25-Dihydroxyvitamin D3 (1,25-D3) is known to have potent inhibitory effects on human peripheral blood mononuclear cell (PBMC) functions. Previous experiments suggest that addition of interleukin-2 (IL-2) to cell cultures can reverse the antiproliferative action of 1,25-D3. Previous studies have also shown that the CD4+ T-cell subset is more sensitive to the antiproliferative actions of 1,25-D3 than are the CD8+ T-cells. The objective of this study was to determine whether exogenous IL-2 could reverse the antiproliferative and immunoinhibitory action (inhibition of Ig production) in mitogen-activated PBMC cultures and in fluorescein-activated cell sorting (FACS) experiments where CD8+ T-cells were removed from PBMCs before mitogen stimulation with/without exogenous IL-2 added. In these studies, addition of IL-2 to mitogen-activated, 1,25-D3-treated PBMCs allowed the cells to overcome the 1,25-D3 suppressive effect on cell proliferation. However, exogenous IL-2 did not overcome the 1,25-D3-mediated inhibitory effect on PBMC Ig production. Using FACS lymphocyte populations (CD4+, CD8+ and B-cells), we showed that CD4+ T-cell-directed Ig synthesis in co-culture with autologous B-cells was inhibitable by incubation of cells with 1,25-D3, but Ig synthesis was restored to near-normal levels by addition of exogenous IL-2. This clearly contrasts with the inability of Il-2 to reverse the 1,25-D3 inhibitory effect on Ig synthesis in PBMCs. In other experiments, when CD8+ cells were removed from mitogen-stimulated, 1,25-D3-treated PBMCs, addition of exogenous IL-2 resulted in a full reversal of the 1,25-D3-mediated Ig inhibition. These data suggest that the inability of IL-2 to reverse the inhibitory effects of 1,25-D3 on PBMC Ig production is probably a result of a lack of sensitivity of CD8+ T-cells to the antiproliferative and immunoregulatory actions of 1,25-D3. This is possibly because of a differential expression of 1,25-D3 receptors on CD4+ and CD8+ T-cells.     

维生素D3受体mRNA在肝细胞增殖和肝癌发展中的作用

目的：探讨维生素D3受体mRNA在肝细胞增生和肝癌发展中的作用。方法：体外培养肝癌细胞株SMMC－7721和HCC－T细胞，培养时添加1000nmol/L、100nmol/L、10nmol/L 1，25-(OH)2D3作用1、3、6天后，用四唑盐比色试验（MTT）检测细胞的存活和生长；用反转录PCR（RT－PCR）检测维生素D3受体mRNA的表达。结果：1.25-(OH)2D3可以抑制维生素D3受体mRNA表达阳性的SMMC－7721细胞增生并且有剂量效应关系；对维生素D3受体mRNA表达阴性的HCC－T细胞没有抑制作用。9例肝癌组织标本维生素D3受体mRNA表达均为阳性。结论：1，25－（OH）2D3对于人肝癌细胞株SMMC－7721的增殖具有显著的抑制作用，其机械可能是通过维生素D3受体来实现的。     

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.     

Inhibition of Human Natural Killer Cell and Lymphokine-Activated Killer Cell Cytotoxicity and Differentiation by Vitamin D3

Recent data suggest that vitamin D3 may be capable of immunoregulation after it is converted to an active metabolite, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). The effect of vitamin D3 and 1,25(OH)2D3 on human natural killer (NK) cells and their activation by interferon (IFN) and interleukin 2 (IL-2) was investigated. Vitamin D3 and 1,25(OH)2D3 inhibited NK cytotoxicity in a dose-dependent manner. Pretreatment of non-adherent (NA) cells at 37 degrees C for 18 h with the vitamins also led to inhibition of NK activity. Both the inhibition of NK lysis and pretreatment of NA cells were dependent on the concentrations of fetal calf serum (FCS) in the medium. The inhibition of NK activity was less effective in the presence of 10% FCS than with 1% FCS. Vitamin D3 inhibited both IFN and IL-2 activation of NK activity. However, increasing doses of IL-2 were able to abrogate the inhibition caused by vitamin D3. Vitamin D3 was able to inhibit NK activity of phytohaemagglutinin and IL-2-activated cells, and also inhibit the proliferation and lymphokine-activated killer activity induced by IL-2. NA cells pretreated with vitamin D3 did not respond well to IL-2. NA cells pretreated with low doses of IL-2 were sensitive to inhibition by vitamin D3 while those pretreated with high doses of IL-2 were not. The data presented suggest that vitamin D3 and 1,25(OH)2D3 inhibit NK activity and LAK cellular differentiation.     

Inhibition of production and function of interleukin-6 by 1,25-dihydroxyvitamin D3

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) has been shown to interfere with immunoglobulin production and lymphocyte proliferation in vitro. These lymphocyte functions are influenced by interleukin (IL)-6 produced by antigen presenting cells. Hence, the ability of 1,25-(OH)2D3 to interfere with the production and function of IL-6 was investigated. 1,25-(OH)2D3 and the analogue MC 903 inhibited IL-6 production by LPS-stimulated human mononuclear cells. The precursor 25-OH D3 was ineffective. Likewise, 1,25-(OH)2D3 but not 25-OH D3 inhibited rIL-6-driven as well as rIL-1 alpha/beta-driven proliferation of murine thymocytes. This effect of 1,25-(OH)2D3 was partially or totally overcome by larger concentrations of rIL-6 as well as by rIL-2 and ionomycin. Consistently, the production of IL-6 and IL-2 in rIL-1 driven thymocyte cultures were found to be reduced by 1,25-(OH)2D3. Inhibition of production and function of IL-6 may therefore be involved in 1,25-(OH)2D3-mediated regulation of lymphocyte functions in vitro.     

Dendritic cells from human tissues express receptors for the immunoregulatory vitamin D3 metabolite, dihydroxycholecalciferol.

Dendritic cells have been isolated from human tonsillar tissue and shown to act as accessory cells in a mitogenic response. The dendritic cells will induced receptors for the active metabolite of vitamin D3, 1,25(OH)2D3, in the responder E+ T cells. The dendritic cells themselves constitutively express receptors for the metabolite, and this distinguishes them from other non-T cells in lymphomedullary tissue. Expression of the 1,25(OH)2D3 receptor may be a dendritic cell property that facilitates their accessory cell role within the tissue microenvironment.     

1,25-Dihydroxyvitamin D3 acts directly on the T lymphocyte vitamin D receptor to inhibit experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is an incurable autoimmune neurodegenerative disease. Environmental factors may be key to MS prevention and treatment. MS prevalence and severity decrease with increasing sunlight exposure and vitamin D(3) supplies, supporting our hypothesis that the sunlight-dependent hormone, 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2) D(3) ), inhibits autoimmune T-cell responses in MS. Moreover, 1,25-(OH)(2) D(3) inhibits and reverses experimental autoimmune encephalomyelitis (EAE), an MS model. Here, we investigated whether 1,25-(OH)(2) D(3) inhibits EAE via the vitamin D receptor (VDR) in T lymphocytes. Using bone marrow chimeric mice with a disrupted VDR only in radio-sensitive hematopoietic cells or radio-resistant non-hematopoietic cells, we found that hematopoietic cell VDR function was necessary for 1,25-(OH)(2) D(3) to inhibit EAE. Furthermore, conditional targeting experiments showed that VDR function in T cells was necessary. Neither 1,25-(OH)(2) D(3) nor T-cell-specific VDR targeting influenced CD4(+) Foxp3(+) T-cell proportions in the periphery or the CNS in these studies. These data support a model wherein 1,25-(OH)(2) D(3) acts directly on pathogenic CD4(+) T cells to inhibit EAE.     

1,25-二羟维生素D3抑制被动致敏人气道平滑肌细胞的增殖

目的: 检测1,25-二羟维生素D₃ 对被动致敏的人气道平滑肌细胞(HASMCs)增殖的调节作用,探讨其对哮喘气道重塑的可能作用。方法: 离体培养HASMCs,并将细胞分为对照组、哮喘组及1,25-(OH)₂D₃组。MTT法检测细胞增殖活力并确定1,25-(OH)₂D₃最佳作用浓度;用最佳作用浓度刺激HASMCs 48 h后以流式细胞术测定细胞周期,免疫细胞化学染色(SABC法)检测增殖细胞核抗原(PCNA)的表达。结果: 1,25-(OH)₂D₃在10^-9-10^-7 mol/L范围内能显著抑制哮喘血清被动致敏的HASMCs增殖(P<0.05),且10^-7 mol/L时抑制作用最强;流式细胞术检测显示这一最佳作用浓度的1,25-(OH)₂D₃能显著抑制被动致敏HASMCs由G₀/G₁期向S期的转化;此外,1,25-(OH)₂D₃能显著抑制被动致敏HASMCs中PCNA的表达。结论: 1,25-(OH)₂D₃能抑制哮喘血清被动致敏的HASMCs增殖,有助于哮喘气道重塑的防治。     

Inhibitory versus stimulatory effects of natural human interferon-alpha on proliferation of lymphocyte subpopulations.

Highly purified natural human interferon-alpha (IFN-alpha) inhibited in a dose-dependent manner the proliferation of human peripheral blood lymphocytes (PBL) stimulated with T-cell mitogen concanavalin A (Con A) or with interleukin-2 (IL-2). Contrary to this inhibitory effect, IFN-alpha at the same concentrations significantly increased proliferation of PBL stimulated with B-cell mitogen bacterial lipopolysaccharide (LPS) or with IL-3, and even spontaneous proliferation of PBL was enhanced by IFN-alpha. Proliferation of Con A-stimulated PBL depleted of CD8+ cells was sensitive to the inhibitory action of IFN-alpha, while proliferation of the Con A-stimulated CD4+ cell-depleted PBL was not affected by IFN-alpha. The inhibitory effect of IFN-alpha on PBL proliferation was due to neither inhibition of IL-2 receptor (IL-2R) expression, activation of suppressor cells, nor inhibition of lymphokine production. Rather, IFN-alpha augmented production of IL-1 and IL-2 by PBL. These results show that the suppressive effect of natural IFN-alpha on Con A-induced proliferation of PBL is due to a direct growth-inhibitory effect on CD4+ T cells, and that IFN-alpha simultaneously augments production of lymphokines. This could in turn lead to the increased proliferation of IFN-alpha-resistant cell populations.     

1,25-Dihydroxyvitamin D3 inhibits proliferation but not the suppressive function of regulatory T cells in the absence of antigen-presenting cells

Vitamin D₃ is known to induce regulatory T (Treg) cells by rendering antigen-presenting cells tolerogenic, its direct effect on human naturally occurring Treg cells is unclear. Here, we investigated if and how 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] can directly affect the proliferation and function of human naturally occurring Treg cells in vitro. First, we demonstrated that these Treg cells express vitamin D receptors that were up-regulated following anti-CD3/CD28-bead stimulation. 1,25(OH)₂D₃ inhibited proliferation of Treg cells even when exogenous interleukin-2 was provided. Treg cells were more susceptible to the inhibitory effect of 1,25(OH)₂D₃ than conventional T cells_. 1,25(OH)₂D₃ neither affected the anergic state nor the suppressive function of Treg cells but induced a subtle increase in interleukin-10-secreting cells. The cell-division-inhibiting effect of 1,25(OH)₂D₃ on Treg cells was also demonstrated in vivo by supplementing vitamin D-deficient HIV-1-infected patients with 2000 IU cholecalciferol (vitamin D₃). Increased serum 1,25(OH)₂D₃ levels were associated with a drop in the number and percentage of Treg cells, which may be attributed to a decrease in the proliferating Foxp3⁺ Treg cell population. In conclusion, 1,25(OH)₂D₃ directly affects Treg cell growth and promotes interleukin-10 production without apparent effects on activation status and suppressive phenotype whereas in vivo, high serum 1,25(OH)₂D₃ levels are associated with reduced Treg cell proliferation and a reduced number of Treg cells.     

1,25-Dihydroxyvitamin-D3 regulation of interleukin-2 and interleukin-2 receptor levels and gene expression in human T cells

1,25-Dihydroxyvitamin-D3 (1,25-D3) is known to inhibit DNA synthesis, immunoglobulin and lymphokine production [interleukin-2 (IL-2), gamma interferon (G-IFN), and granulocyte-monocyte colony-stimulating factor (GM-CSF)] by mitogen-stimulated human peripheral blood mononuclear cells (PBMCs). Recent data suggest these inhibitory effects are mediated at the gene level through inhibition of mRNA accumulation of specific lymphokines in the activated cells. In previous studies, we have demonstrated the CD8+ T cell population was less sensitive to the anti-proliferative actions of 1,25-D3 than CD4+ T cells. The purpose of this investigation was to further assess ability of 1,25-D3 to regulate CD4+ and CD8+ T cell functions. Initial experiments showed that 1,25-D3 inhibited both IL-2 production and mRNA accumulation in mitogen-stimulated PBMC. However, IL-2 receptor (IL-2R) expression and mRNA accumulation in stimulated PBMC was not affected by 1,25-D3. Both FACS sorted CD4+ and CD8+ T cells expressed IL-2R equally upon stimulation and neither showed an inhibitory effect on this expression by 1,25-D3. Human CD4+ and CD8+ T cells showed a stimulus-specific production of IL-2. CD4+ cells stimulated with mitogen and HLA-DR positive accessory cells produced measurable levels of IL-2 that were completely inhibited by 1,25-D3. CD8+ T cells did not generate measurable amounts of IL-2 in this system. However, CD4+ and CD8+ T cells produced large amounts of IL-2 when stimulated with mitogen and a protein kinase C activator, phorbol myristate acetate (PMA). Under these circumstances, both CD4+ and CD8+ T cell IL-2 production was inhibited completely by 1,25-D3. These data suggest that IL-2R expression in PBMCs and T cell subsets is equal and unaffected by 1,25-D3 while IL-2 production in T cell subsets is stimulus-specific and completely inhibited by 1,25-D3.     

1alpha,25-dihydroxyvitamin D3 or analogue treated dendritic cells modulate human autoreactive T cells via the selective induction of apoptosis

Epidemiological evidence indicates that the vitamin D status after birth modulates the risk for development of type 1 diabetes mellitus (T1DM). We previously demonstrated that the biologically active form of vitamin D, 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3), as well as its analogue TX527 permanently alter the morphology and T cell stimulatory function of human dendritic cells (DC). Here, we studied the mechanism of T cell modulation by 1,25(OH)2D3 or analogue treated DC. By using CFSE-labelled autoreactive T cells, we observed that T cell proliferation is hampered upon coculture with modulated DCs, i.e. T cells underwent fewer cycles of cell divisions when compared to T cells stimulated by nontreated DCs. Moreover, 1,25(OH)2D3 or analogue modulated DCs induced significantly higher numbers of early apoptotic (annexin V+/PI-) and/or late apoptotic (annexin V+/PI+) T cells. Apoptosis was selectively induced in T cells activated by modulated DC, since other T cells present in the same cultures, either resting or activated by control untreated DC, were unaffected. Thus, in vitro preconditioning of DC with 1,25(OH)2D3 or analogue yields regulatory DC that may interfere with ongoing autoimmunity in vivo without affecting T cells with other specificities.     

Surface roughness mediates its effects on osteoblasts via protein kinase A and phospholipase A2

Earlier studies have shown that implant surface roughness influences osteoblast proliferation, differentiation, matrix synthesis and local factor production. Moreover, the responsiveness of osteoblasts to systemic hormones, such as 1,25-(OH)2D3, at the implant surface is also influenced by surface roughness and this effect is mediated by changes in prostaglandins. At present, it is not known which signaling pathways are involved in mediating cell response to surface roughness and how 1,25-(OH)2D3 treatment alters the activation of these pathways. This paper reviews a series of studies that have addressed this question. MG63 osteoblast-like cells were cultured on commercially pure titanium (cpTi) surfaces of two different roughnesses (Ra 0.54 and 4.92 microm) in the presence of control media or media containing 1,25-(OH)2D3 or 1,25-(OH)2D3 plus H8 (a protein kinase A inhibitor) or quinacrine (a phospholipase A2 inhibitor). At harvest, the effect of these treatments on cell number and alkaline phosphatase specific activity was measured. Compared to cultures grown on the smooth surface, cell number was reduced on the rough surface. 1,25-(OH)2D3 inhibited cell number on both surfaces and inhibition of protein kinase A in the presence of 1,25-(OH)2D3 restored cell number to that seen in the control cultures. Inhibition of phospholipase A2 in the presence of 1,25-(OH)2D3 caused a further reduction in cell number on the smooth surface, and partially reversed the inhibitory effects of 1,25-(OH)2D3 on the rough surface. Alkaline phosphatase specific activity was increased in cultures grown on the rough surface compared with those grown on the smooth surface; 1,25-(OH)2D3 treatment increased enzyme specific activity on both surfaces. Cultures treated with H8 and 1,25-(OH)2D3 displayed enzyme specific activity that approximated that seen in control cultures. Inhibition of phospholipase A2 also inhibited the 1,25-(OH)2D3-dependent effect on the smooth surface, but on the rough surface there was an inhibition of the 1,25-(OH)2D3 effect as well as a partial inhibition of the surface roughness-dependent effect. The results indicate that surface roughness and 1,25-(OH)2 D3 mediate their effects through phospholipase A2, which catalyzes one of the rate-limiting steps in prostaglandin E2 production. Further downstream, prostaglandin E2 activates protein kinase A.