Differential effects of 1,25-dihydroxyvitamin D3 on human lymphocytes and monocyte/macrophages: inhibition of interleukin-2 and augmentation of interleukin-1 production

Human peripheral blood monocytes and activated, but not resting, lymphocytes possess specific intracellular receptors for the active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). The effects of 1,25-(OH)2D3 on the function of these cells was therefore examined. The addition of physiologic concentrations of the hormone (0.001-0.1 nM) to lectin- or antigen-activated lymphocytes resulted in inhibition of lymphocyte proliferation. Supernatants from lectin-activated lymphocytes incubated with 1,25-(OH)2D3 had reduced interleukin-2 (IL-2) activity. The immediate biological precursor of 1,25-(OH)2D3, 25-hydroxyvitamin D3, did not affect function of lymphocytes or monocytes. The ability of exogenous recombinant IL-2 to reverse the inhibitory effects of the hormone on lymphocyte proliferation suggest that 1,25-(OH)2D3 does not alter the generation of IL-2 receptors. In contrast to its effects on IL-2 production, 1,25-(OH)2D3 caused a dose-dependent increase in the production of interleukin-1 (IL-1) by monocyte/macrophages. These results suggest that immune cells and their products can be regulated in a specific but diverse fashion by the vitamin D3-endocrine system.     

1,25-Dihydroxyvitamin D3 enhancement of concanavalin-A induced bovine lymphocyte proliferation: requirement of monocytes

The effect of 1,25-(OH)2D3 on Concanavalin-A (ConA) mediated bovine lymphocyte proliferation was dependent on the magnitude of the proliferative response. Lymphocytes from cows with high inherent proliferative responses (134,904 +/- 15,488 cpm/well) were unaffected by 1,25-(OH)2D3; whereas, lymphocytes from cows with low inherent proliferative responses (46,317 +/- 6000 cpm/well) were stimulated 75% (P less than 0.01) by 10(-10)M 1,25-(OH)2D3. At low initial proliferation rates, seen with low density cultures enriched for lymphocytes, the effect of 1,25-(OH)2D3 on proliferation changed from inhibition in the absence of added monocytes to stimulation at 18-23% monocyte addition. These results suggest that 1,25-(OH)2D3 possesses both stimulatory and inhibitory effects on ConA-induced proliferation that are monocyte dependent.     

Augmentation of monocyte chemotaxis by 1 alpha,25-dihydroxyvitamin D3. Stimulation of defective migration of AIDS patients

Preincubation for 20 h with 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) markedly augmented the chemotactic responsiveness of human blood monocytes to the classical chemoattractant, FMLP. A modest enhancement of monocyte spontaneous locomotion in the absence of chemoattractants was also observed. Maximal increase of monocyte migration was observed after pretreatment with 10(-9) M 1,25(OH)2D3 and was detectable at FMLP concentrations ranging from 10(-10) to 10(-7) M. Pretreatment with 1,25(OH)2D3 augmented the number of monocyte high affinity FMLP receptors (1500 +/- 220 and 3800 +/- 300 sites per cell for untreated and 1,25(OH)2D3-pretreated cells, respectively) with no significant changes in Kd values (2 +/- 0.5 nM and 4 +/- 1 nM, for untreated and 1,25(OH)2D3-pretreated monocytes). Enhanced chemotaxis was not restricted to FMLP, because 1,25(OH)2D3-treated monocytes showed enhanced migration also in response to activated C components and chemotactic cytokines. In agreement with previous observations, monocytes from AIDS patients showed defective migration capacity. In vitro exposure to 1,25(OH)2D3 stimulated monocyte migration in all 10 patients examined with considerable quantitative differences among individuals. Regulating the responsiveness of mature monocytes to chemo-attractants, 1,25(OH)2D3, produced systemically or in situ by immunocompetent cells, could play a role in the regulation of the recruitment of monocytes at sites of inflammation, cell-mediated immunity, or bone resorption. The potential of 1,25(OH)2D3 as a restorative agent under conditions of defective phagocyte recruitment deserves further exploration.     

Vitamin D and the immune system

The investigation of the potential influence of 1,25-(OH)2D3 on immune cells has expanded our understanding of hormone-cytokine interactions. 1,25-(OH)2D3 stimulates phenotypic and function changes in immature monocytes, alters protein synthesis, increases adherence, and augments interleukin-1 secretion. T lymphocyte proliferation and B cell immunoglobulin production are inhibited by the hormone. 1,25-(OH)2D3 decreases IL 2 and IFN-gamma synthesis by activated T lymphocytes in association with decreases in mRNA for these proteins. The step from the investigation of in vitro interactions to an understanding of in vivo effects of 1,25-(OH)2D3 on immune cells requires further study. On the basis of information at hand, such as the potential for macrophage conversion of 25-OH-D3 to 1,25-(OH)2D3, decreased or increased macrophage function in association with vitamin D3 status in vitro and in vivo, as well as altered T cell subset ratios and proliferative responses with administration of the hormone, it is tempting to speculate that 1,25-(OH)2D3 exerts an influence on immune cell function in concert with other recognized soluble mediators of monocyte and lymphocyte origin. The primary influence of 1,25-(OH)2D3 may vary with the tissue site. Systemic levels of hormone may aid in maintaining tonic immunosuppression and thus prevent trivial antigenic stimuli from initiating an immune response. Upon initiation of an immune response to a significant antigenic challenge 1,25-(OH)2D3 may, in concert with other suppressor mechanisms, limit the extent of the host response by inhibition of IL 2 and IFN-gamma production. At local sites of chronic inflammation concentrations of 1,25-(OH)2D3 may be elevated and may act in an autocrine or paracrine fashion to alter the immune response, for example, by increasing IL 1 production and antigen presentation by tissue monocyte/macrophages. The activation of T cells is associated with the synthesis of 1,25-(OH)2D3 receptors, thus potentially limiting T cell proliferation in the presence of the hormone. Other biological actions of IL 1, however, including effects on cells in bone, joint, and brain may be augmented. Thus, the end result of the opposing effects of 1,25-(OH)2D3 on immune cells and their secretory products may vary with the specific cells involved, their state of maturation and activation, and the local concentrations of the hormone. Studies to date support the concept of an expanded role for 1,25-(OH)2D3 in immune cell biology.     

Interferon-gamma-induced IA expression in WEHI-3 cells is enhanced by the presence of 1,25-dihydroxyvitamin D3

It has been suggested recently that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is involved in the regulation of the immune functions of lymphocytes and in the differentiation of monocytic cells. This report examined the possibility that 1,25(OH)2D3 influences immune functions mediated by monocytic cells by studying its effect on the murine myelomonocytic line WEHI-3. We found that WEHI-3 cells possess 3.3S receptor proteins with high affinity (Kd = 3.3 X 10(-10) M) for 1,25(OH)2D3 that are capable of binding to DNA. Also we found that 1,25(OH)2D3 enhances the interferon-gamma (IFN-gamma)-induced expression of the class II major histocompatibility complex antigens (Ia molecules), and such enhancement leads to increased capacity of the WEHI-3 cells to stimulate antigen-specific Ia-restricted T cell activation. Finally, 1,25(OH)2D3 inhibits the proliferation of WEHI-3 cells, and this inhibition is enhanced in the presence of IFN-gamma. The 1,25(OH)2D3 modulation of IFN-gamma induction of Ia antigens suggests that the hormone might promote monocytes to function more efficiently as antigen-presenting cells.     

1 alpha,25-Dihydroxyvitamin D3-binding macromolecules in human B lymphocytes: effects on immunoglobulin production

Previous studies have indicated that upon in vitro activation with mitogenic lectins, human peripheral blood T lymphocytes express receptors for the steroid hormone 1 alpha, 25-dihydroxyvitamin D3(1,25(OH)2D3). Furthermore, the hormone can inhibit interleukin 2 production by the activated cells. In this investigation, we report that human peripheral B lymphocytes activated in vitro with the B lymphotropic Epstein-Barr virus (EBV) also express 1,25(OH)2D3 receptor-like macromolecules. These receptors are localized in the cell nucleus and exhibit properties similar to those found in classical target tissues for 1,25(OH)2D3. They sediment on sucrose gradients at 3.3 S, display a dissociation constant (Kd) of 4 X 10(-10) M, and can bind to DNA. In addition to the 1,25(OH)2D3 receptors, however, EBV-activated lymphocytes express a second class of 1,25(OH)2D3-binding proteins that appear to occur mainly in the cell cytosol and exhibit distinct biochemical properties from the receptor, including higher sedimentation coefficients (3.7 S to 4 S) and the lack of ability to bind to DNA. The addition of 1,25(OH)2D3 to cultures of EBV-infected cells inhibited the production of IgM and IgG by the B cells. The vitamin D3 analog 24,25(OH)2D3 did not inhibit Ig production, thus suggesting that the effect is probably mediated through the high affinity receptor macromolecule localized in the nucleus. Because the EBV-induced Ig production is independent of T cell participation, the data also suggest that the effects of 1,25(OH)2D3 are exerted directly on the B cell. The present results add to the evidence of the importance of 1,25(OH)2D3 as an immunoregulatory hormone.     

1,25-Dihydroxyvitamin D3-induced differentiation in a human promyelocytic leukemia cell line (HL-60): receptor-mediated maturation to macrophage-like cells

The human-derived promyelocytic leukemia cell line, HL-60, is known to differentiate into mature myeloid cells in the presence of 1,25-dihydroxyvitamin D3 (1,25[OH]2D3). We investigated differentiation by monitoring 1,25(OH)2D3-exposed HL-60 cells for phagocytic activity, ability to reduce nitroblue tetrazolium, binding of the chemotaxin N-formyl-methionyl-leucyl-[3H]phenylalanine, development of nonspecific acid esterase activity, and morphological maturation of Wright-Giemsa-stained cells. 1,25(OH)2D3 concentrations as low as 10(-10) M caused significant development of phagocytosis, nitroblue tetrazolium reduction, and the emergence of differentiated myeloid cells that had morphological characteristics of both metamyelocytes and monocytes. These cells were conclusively identified as monocytes/macrophages based upon their adherence to the plastic flasks and their content of the macrophage-characteristic nonspecific acid esterase enzyme. The estimated ED50 for 1,25(OH)2D3-induced differentiation based upon nitroblue tetrazolium reduction and N-formyl-methionyl-leucyl-[3H]phenylalanine binding was 5.7 X 10(-9) M. HL-60 cells exhibited a complex growth response with various levels of 1,25(OH)2D3: less than or equal to 10(-10) M had no detectable effect, 10(-9) M stimulated growth, and greater than or equal to 10(-8) M sharply inhibited proliferation. We also detected and quantitated the specific receptor for 1,25(OH)2D3 in HL-60 and HL-60 Blast, a sub-clone resistant to the growth and differentiation effects of 1,25(OH)2D3. The receptor in both lines was characterized as a DNA-binding protein that migrated at 3.3S on high-salt sucrose gradients. Unequivocal identification was provided by selective dissociation of the 1,25(OH)2D3-receptor complex with the mercurial reagent, p-chloromercuribenzenesulfonic acid, and by a shift in its sedimentation position upon complexing with anti-receptor monoclonal antibody. On the basis of labeling of whole cells with 1,25(OH)2[3H]D3 in culture, we found that HL-60 contains approximately 4,000 1,25(OH)2D3 receptor molecules per cell, while the nonresponsive HL-60 Blast is endowed with approximately 8% of that number. The concentration of 1,25(OH)2D3 (5 X 10(-9) M) in complete culture medium, which facilitates the saturation of receptors in HL-60 cells, is virtually identical to the ED50 for the sterol's induction of differentiation. This correspondence, plus the resistance of the relatively receptor-poor HL-60 Blast, indicates that 1,25(OH)2D3-induced differentiation of HL-60 cells to monocytes/macrophages is occurring via receptor-mediated events.     

1,25-Dihydroxyvitamin D3 increases the toxicity of hydrogen peroxide in the human monocytic line U937: the role of calcium and heat shock

1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) increases synthesis of heat shock proteins in monocytes and U937 cells and protects these cells from thermal injury. We examined whether 1,25-(OH)2D3 would also modulate the susceptibility of U937 cells to H2O2-induced oxidative stress. Cell viability was assessed by trypan blue exclusion and [3H]thymidine incorporation into DNA. Prior incubation for 24 h with 1,25-(OH)2D3 (25 pM or higher) unexpectedly increased H2O2 toxicity. Since cellular Ca2+ may be a mediator of cell injury we investigated effects of altering extracellular Ca2+ ([Ca2+]e) on 1,25-(OH)2D3-enhanced H2O2 toxicity as well as effects of 1,25-(OH)2D3 and H2O2 on cytosolic free Ca2+ concentration ([Ca2+]f). Basal [Ca2+]f in medium containing 1.5 mM Ca as determined by fura-2 fluorescence was higher in 1,25-(OH)2D3-pretreated cells than control cells (137 versus 112 nM, P less than 0.005). H2O2 induced a rapid increase in [Ca2+]f (to greater than 300 nM) in both 1,25-(OH)2D3-treated and control cells, which was prevented by a reduction in [Ca2+]e to less than basal [Ca2+]f. The 1,25(OH)2D3-induced increase in H2O2 toxicity was also prevented by preincubation with 1,25-(OH)2D3 in Ca2+-free medium or by exposing the cells to H2O2 in the presence of EGTA. Preexposure of cells to 45 degrees C for 20 min, 4 h earlier, partially prevented the toxic effects of H2O2 particularly in 1,25-(OH)2D3-treated cells, even in the presence of physiological levels of [Ca2+]e. Thus 1,25-(OH)2D3 potentiates H2O2-induced injury probably by increasing cellular Ca2+ stores. The 1,25-(OH)2D3-induced amplification of the heat shock response likely represents a mechanism for counteracting the Ca2+-associated enhanced susceptibility to oxidative injury due to 1,25-(OH)2D3.     

The selenoprotein thioredoxin reductase is expressed in peripheral blood monocytes and THP1 human myeloid leukemia cells--regulation by 1,25-dihydroxyvitamin D3 and selenite

1,25(OH)2 Vitamin D3 (1,25(OH)2D3) and adhesion propagate monocyte differentiation. We identified the selenoprotein thioredoxin reductase (TrxR) as a new molecular target for 1,25(OH)2D3 in monocytes during this process. In THP1 monocytic leukemia cells 1,25(OH)2D3 stimulated TrxR mRNA levels 2-4-fold by 4-8 h and enhanced TrxR activity (60%) (as measured by the dithionitrobenzole-assay) after 24 h, which declined below baseline after 96 h. The addition of 100 nM selenite enhanced (approx. 50%) basal and stimulated enzyme activity in THP1 cells. The relative stimulation by 1,25(OH)2D3 was very similar but peak levels were sustained in THP1 cells up to 48 h. Human peripheral blood monocytes (PBM) of different donors showed very low basal TrxR steady state mRNA levels which were markedly enhanced (as analyzed by Northern blotting) after 4 h of adherence to culture dishes. 1,25(OH)2D3 (100 nM) further stimulated TrxR mRNA expression (4 h, 3-fold). TrxR enzyme activity mirrored the mRNA changes. Basal activity was stimulated approx. 25% by adhesion in culture alone and was further stimulated (approximately 15%) by 1,25(OH)2D3 after 4 h. By 24 h similar results were achieved but the effect of 1,25(OH)2D3 could be seen in the presence of 100 nM selenium only. The expression of TrxR and its regulation by 1,25(OH)2D3 and selenite in monocytes might be important for their induction of differentiation and maintenance of function.     

Suppressive effect of 1alpha,25-dihydroxyvitamin D3 on type I IFN-mediated monocyte differentiation into dendritic cells: impairment of functional activities and chemotaxis

Dendritic cells (DCs) generated by a single-step exposure of human monocytes to type I IFN and GM-CSF (IFN-DCs) are endowed with potent immunostimulatory activities and a distinctive migratory response to specific chemokines. In this study, we evaluated the effects of 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), the biologically active metabolite of vitamin D(3), on the DC differentiation/activation induced by type I IFN. We found that 1,25(OH)(2)D(3) prevented the generation of IFN-DCs when added to freshly isolated monocytes, and was capable of redirecting already differentiated IFN-DCs toward a more immature stage, as revealed by their immunophenotype, reduced allostimulatory activity, and impaired LPS-induced production of Th1-polarizing cytokines. Control and 1,25(OH)(2)D(3)-treated IFN-DCs exhibited a similar expression of vitamin D receptor, as well as comparable cell death rates. Furthermore, the chemotactic response of IFN-DCs to CCL4 and CCL19 was markedly reduced or completely abrogated by 1,25(OH)(2)D(3). Despite these changes in the IFN-DC migratory behavior, the expression of CCR5 and CCR7 and the calcium fluxes triggered by CCL4 and CCL19 were not affected. These findings indicate that, in this innovative single-step DC generation model from monocytes, the suppressive effect of 1,25(OH)(2)D(3) is associated with a potent impairment of DC migration in response to inflammatory and lymph node-homing chemokines, thus unraveling a novel mechanism involved in 1,25(OH)(2)D(3)-mediated immunomodulation.     

Biological activity of fluorinated vitamin D analogs at C-26 and C-27 on human promyelocytic leukemia cells, HL-60

Vitamin D compounds added to the culture medium induce HL-60 cells to differentiate into macrophage/monocytes via a receptor mechanism. This system provides a biologically relevant assay for the study of biopotency of vitamin D analogs. Using this system, the biological activity of various fluorinated derivatives of vitamin D3 was compared with that of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). As assessed by cell morphology, nitroblue tetrazolium reduction and nonspecific esterase activity, 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3 (26,27-F6-1,25-(OH)2D3) and 26,26,26,27,27,27-hexafluoro-1,24-dihydroxyvitamin D3 (26,27-F6-1,24-(OH)2D3) were about 10 times as potent as 1,25-(OH)2D3 in suppressing HL-60 cell proliferation and inducing cell differentiation. The biological activity of 26,26,26,27,27,27-hexafluoro-1-hydroxyvitamin D3 (26,27-F6-1-OH-D3) was equal to that of 1,25-(OH)2D3 in this system. 1,25-(OH)2D3 and its fluorinated analogs exerted their effects on HL-60 cells in a dose-dependent manner. HL-60 cells have a specific receptor for 1,25-(OH)2D3 with an apparent Kd of 0.25 nM, identical with that of chick intestinal receptor. While the binding affinities of 26,27-F6-1,25-(OH)2D3 and 26,27-F6-1,24-(OH)2D3 for chick intestinal receptor were lower than that of 1,25-(OH)2D3 by factors of 3 and 1.5, respectively, they were as competent as 1,25-(OH)2D3 in binding to HL-60 cell receptor. The ability of 26,27-F6-1-OH-D3 to compete for receptor protein from HL-60 cells and chick intestine was about 1/70 that of 1,25-(OH)2D3. These results indicate that trifluorination of carbons 26 and 27 of vitamin D3 can markedly enhance the effect on HL-60 cells.     

1,25-Dihydroxyvitamin D3 activates secretion of hydrogen peroxide by human monocytes

Human blood monocytes cultured in the presence of 1,25(OH)2D3 developed enhanced competence for secretion of H2O2 relative to cells suspended in media. This effect was maximal at a concentration of 10(-8) M 1,25(OH)2D3. After 3 days of incubation, monocyte-derived macrophages (MDM) exposed to 1,25(OH)2D3 demonstrated competence for secretion of H2O2 equivalent to cells exposed to recombinant IFN-gamma. Both IFN-gamma and 1,25(OH)2D3 offset decay of this function among cells in culture after 7 days. Simultaneous exposure of cells to 1,25(OH)2D3 and IFN-gamma did not activate competence for H2O2 secretion more than either agent alone. 24,25(OH)2D3 and 25(OH)2D3 activated MDM but at higher concentration than required for 1,25(OH)2D3. Progesterone did not affect H2O2 production. Incubation of MDM with a monoclonal antibody directed against IFN-gamma inhibited activation induced by lymphokine, and to a lesser extent by cells activated with IFN-gamma; this antibody had an insignificant effect on cells treated with 1,25(OH)2D3. These results suggest that 1,25(OH)2D3 exerts a receptor-mediated effect on monocyte function that results in cellular activation as manifested by enhanced competence for secretion of H2O2. It is possible that smaller concentrations of 1,25(OH)2D3 present in serum are permissive for macrophage activation, or that monocytic phagocytes are exposed to high concentrations of vitamin D metabolites under some clinical circumstances.     

Regulation of Mycobacterium-specific mononuclear cell responses by 25-hydroxyvitamin D3

The active vitamin D metabolite, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), has been shown to be an important regulator of innate and adaptive immune function. In addition, synthesis of 1,25(OH)(2)D(3) from 25-hydroxyvitamin D(3) (25(OH)D(3)) by the enzyme 1α-hydroxylase in monocytes upon activation by TLR signaling has been found to regulate innate immune responses of monocytes in an intracrine fashion. In this study we wanted to determine what cells expressed 1α-hydroxylase in stimulated peripheral blood mononuclear cell (PBMC) cultures and if conversion of 25(OH)D(3) to 1,25(OH)(2)D(3) in PBMC cultures regulated antigen-specific immune responses. Initially, we found that stimulation of PBMCs from animals vaccinated with Mycobacterium bovis (M. bovis) BCG with purified protein derivative of M. bovis (M. bovis PPD) induced 1α-hydroxylase gene expression and that treatment with a physiological concentration of 25(OH)D(3) down-regulated IFN-γ and IL-17F gene expression. Next, we stimulated PBMCs from M. bovis BCG-vaccinated and non-vaccinated cattle with M. bovis PPD and sorted them by FACS according to surface markers for monocytes/macrophages (CD14), B cells (IgM), and T cells (CD3). Sorting the PBMCs revealed that 1α-hydroxylase expression was induced in the monocytes and B cells, but not in the T cells. Furthermore, treatment of stimulated PBMCs with 25(OH)D(3) down-regulated antigen-specific IFN-γ and IL-17F responses in the T cells, even though 1α-hydroxylase expression was not induced in the T cells. Based on evidence of no T cell 1α-hydroxylase we hypothesize that activated monocytes and B cells synthesize 1,25(OH)(2)D(3) and that 1,25(OH)(2)D(3) down-regulates antigen-specific expression of IFN-γ and IL-17F in T cells in a paracrine fashion.     

Differential protein pathways in 1,25-dihydroxyvitamin d(3) and dexamethasone modulated tolerogenic human dendritic cells

Tolerogenic dendritic cells (DC) that are maturation-resistant and locked in a semimature state are promising tools in clinical applications for tolerance induction. Different immunomodulatory agents have been shown to induce a tolerogenic DC phenotype, such as the biologically active form of vitamin D (1,25(OH)(2)D(3)), glucocorticoids, and a synergistic combination of both. In this study, we aimed to characterize the protein profile, function and phenotype of DCs obtained in vitro in the presence of 1,25(OH)(2)D(3), dexamethasone (DEX), and a combination of both compounds (combi). Human CD14(+) monocytes were differentiated toward mature DCs, in the presence or absence of 1,25(OH)(2)D(3) and/or DEX. Cells were prefractionated into cytoplasmic and microsomal fractions and protein samples were separated in two different pH ranges (pH 3-7NL and 6-9), analyzed by 2D-DIGE and differentially expressed spots (p < 0.05) were identified after MALDI-TOF/TOF analysis. In parallel, morphological and phenotypical analyses were performed, revealing that 1,25(OH)(2)D(3)- and combi-mDCs are closer related to each other than DEX-mDCs. This was translated in their protein profile, indicating that 1,25(OH)(2)D(3) is more potent than DEX in inducing a tolerogenic profile on human DCs. Moreover, we demonstrate that combining 1,25(OH)(2)D(3) with DEX induces a unique protein expression pattern with major imprinting of the 1,25(OH)(2)D(3) effect. Finally, protein interaction networks and pathway analysis suggest that 1,25(OH)(2)D(3), rather than DEX treatment, has a severe impact on metabolic pathways involving lipids, glucose, and oxidative phosphorylation, which may affect the production of or the response to ROS generation. These findings provide new insights on the molecular basis of DC tolerogenicity induced by 1,25(OH)(2)D(3) and/or DEX, which may lead to the discovery of new pathways involved in DC immunomodulation.     

1,25-Dihydroxycholecalciferol-induced differentiation of myelomonocytic leukemic cells unresponsive to colony stimulating factors and phorbol esters

The murine myelomonocytic leukemia cell line WEHI-3B D+, which differentiates in response to granulocyte colony stimulating factor (G-CSF), can also be induced to differentiate into monocyte-macrophages by phorbol myristate acetate (PMA) treatment, whereas the WEHI-3B D- subline, which is unresponsive to G-CSF and PMA, can be induced to differentiate to granulocytes as well as monocytes by 1,25-dihydroxycholecalciferol [1,25-(OH)2 D3], the biologically active metabolite of vitamin D3. A newly developed variant of the WEHI-3B D+ line, named WEHI-3B D+ G, which was responsive to G-CSF but not to PMA, was also differentiated to granulocytes by 1,25-(OH)2 D3. Although vitamin D3 has been reported to induce macrophage differentiation in responsive tumor cells, this is the first demonstration that 1,25-(OH)2 D3 can induce granulocyte differentiation. In both differentiation pathways, cessation of cellular proliferation accompanies changes in morphologic and cytochemical properties of the cells. This suggests that leukemic cell lines unresponsive to differentiation agents acting at the cell surface retain their ability to differentiate in response to agents that do not act via the plasma membrane such as 1,25-(OH)2 D3, which has cytosolic/nuclear receptors. Vitamin D3 could act through different cellular pathways inducing differentiation or by bypassing only the first step of a common differentiation cascade used by agents with cell surface receptors such as CSF. These results suggest that low doses of 1,25-(OH)2 D3 may be useful in combination with hemopoietic growth factors (CSFs) as therapeutic agent to induce leukemic cell differentiation in vivo.     

1 alpha,25-dihydroxyvitamin D3 inhibits productive infection of human monocytes by HIV-1.

Pretreatment of freshly isolated human peripheral blood monocytes with the steroid hormone, 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)D), markedly reduced (by 95%) productive infection of human monocytes by HIV-1. Equivalent concentrations (10nM) of 25-hydroxyvitamin D3 (25(OH)D), the biologic precursor of 1,25(OH)D, were ineffective at reducing either CD4 expression or HIV-1 production. Pretreatment was required for modulation of HIV-1 infection by 1,25(OH)D. Interestingly, 1,25(OH)D-mediated decreases in p24 antigen production were observed prior to any observed reduction in CD4 expression, suggesting that 1,25(OH)D treatment may modulate HIV-1 infection of monocytes through additional factors besides decreased HIV-1 binding. These data raise the possibility that 1,25(OH)D compounds may be important in host resistance to HIV-1.     

Dibutyryl cAMP enhances the effect of 1,25-dihydroxyvitamin D3 on a human promyelocytic leukemia cell, HL-60, at both the receptor and the postreceptor steps.

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) induces differentiation of a human promyelocytic leukemia cell line, HL-60, into monocytes/macrophages, and 25-hydroxyvitamin D3- and 1,25-(OH)2D3-24-hydroxylase activities in HL-60 mitochondria via a steroid-hormone receptor mechanism. Dibutyryl cyclic adenosine monophosphate (dbcAMP), a granulocyte inducer, significantly augmented the differentiation-inducing effect of 1,25-(OH)2D3 along the monocyte/macrophage pathway. Furthermore, dbcAMP significantly potentiated the effect of 1,25-(OH)2D3 on HL-60 cells to hydroxylate 1,25-(OH)2[26,27-3H]D3 to form 1,24,25-(OH)3[26,27-3H]D3. DbcAMP seemed to augment the effect of 1,25-(OH)2D3 in part through upregulation of the 1,25-(OH)2D3 receptor, because 10(-7) M dbcAMP increased 1,25-(OH)2D3 receptor levels approximately 2.3-fold, which was similar to a 1.9-fold augmentation by the same concentrations of dbcAMP of 1,25-(OH)2D3-induced cell characteristics to hydroxylate C-24 of 1,25-(OH)2[26,27-3H]D3. However, dbcAMP is also known to enhance HL-60 cell differentiation caused by other differentiation inducers. We have established another HL-60 clone which acquires resistance to 1,25-(OH)2D3 in the induction of cell differentiation by a defect at the postreceptor step, as reflected by resistance to other differentiation inducers, such as retinoic acid and dimethyl sulfoxide. Even in this resistant clone, dbcAMP significantly enhanced the differentiation-inducing effect of 1,25-(OH)2D3. Of interest, this clone showed resistance to dbcAMP in the induction of cell differentiation. Furthermore, we have demonstrated that intracellular cAMP levels were significantly lower in uremic serum-treated cells than in cells treated with normal human serum and that a significant positive correlation was found between intracellular cAMP levels and 1,25-(OH)2D3-induced cell differentiation. These data indicated that the intracellular cAMP level is one of the major determinants of 1,25-(OH)2D3-induced HL-60 cell differentiation and that dbcAMP could enhance the effects of 1,25-(OH)2D3 on HL-60 cells not only by increasing 1,25-(OH)2D3 receptor levels but also at the postreceptor step.     

Synthesis in vitro of 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 by interferon-gamma-stimulated normal human bone marrow and alveolar macrophages

Cultured human macrophages from normal donors were examined for their capability to metabolize 25-hydroxyvitamin D3 (25-(OH)D3). Upon exposure to recombinant human interferon-gamma (IFN-gamma) both bone marrow-derived macrophages (BMM) and pulmonary alveolar macrophages (PAM) produced a polar 25-(OH)D3 metabolite which was purified from conditioned media and unequivocally identified as 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) by UV-absorbance spectrophotometry and mass spectrometry. The BMM and PAM also synthesized a second 25-(OH)D3 metabolite which was structurally identified as 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3). The time course of 25-(OH)D3 metabolism by macrophages suggested that the production of 24,25-(OH)2D3 was stimulated by high intracellular levels of 1,25-(OH)2D3 and not by IFN-gamma. The 1,25-(OH)2D3 obtained from BMM and PAM promoted macrophage-like differentiation of promyelocytic HL-60 leukemia cells and inhibited IFN-gamma production by normal human lymphocytes. Our data suggest that locally high levels of 1,25-(OH)2D3 in the microenvironment of IFN-gamma-stimulated BMM and PAM may modulate the function of hormone-responsive cells.     

1α,25-Dihydroxyvitamin D3 inhibits CD40L-induced pro-inflammatory and immunomodulatory activity in Human Monocytes

CD40 ligand (CD40L) stimulation induces proinflammatory and immunomodulatory activity in monocytes. Here, we report on the effects of the steroid hormone 1α,25-dihydroxyvitamin D3 (1,25D3) on human blood monocytes that have been stimulated with the CD40L ligand. Co-treatment of CD40L-stimulated monocytes with 1,25D3 resulted in reduced production and secretion of tumor necrosis factor (TNF)-α and interleukin (IL)-1β, as well as in reduced expression of the surface co-stimulatory molecules CD80 and CD86. In addition, costimulation of CD4+ T lymphocytes by monocytes co-treated with CD40L and 1,25D3 resulted in reduced cell proliferation and diminished interferon (IFN)-γ but enhanced IL-10 production by CD4+ T cells. Finally, 1,25D3 interfered with the ability of CD40L to rescue monocytes from apoptosis induced by serum withdrawal. These findings suggest that 1,25D3 may regulate the interaction of monocytes with T cells or other cell types that express CD40L, thus influencing the outcome of the immune or inflammatory response.