1,25-Dihydroxyvitamin D3 stimulates odontoblastic differentiation of human dental pulp-stem cells in vitro

Background: 1,25-Dihydroxyvitamin D₃ (1,25-OH D₃) plays an important role in mineralized tissue metabolism, including teeth. However, few studies have addressed its role in odontoblastic differentiation of human dental pulp-stem cells (hDPSCs). Aim: This study aimed to understand the influence of various concentrations of 1,25-OH D₃ on the proliferation capacity and early dentinogenesis responses of hDPSCs. Materials and Methods: hDPSCs were obtained from the impacted third molar teeth. Monolayer cultured cells were incubated with a differentiation medium containing different concentrations of 1,25-OH D₃ (0.001, 0.01, and 0.1 µM). All groups were evaluated by S-phase rate [immunohistochemical (IHC) bromodeoxyuridine (BrdU) staining], STRO-1 and dentin sialoprotein (DSP)+ levels (IHC), and alkaline phosphatase (ALP, enzyme-linked immunosorbent assay (ELISA)) levels. Results: The number of cells that entered the S-phase was determined to be the highest and lowest in the control and 0.001 µM 1,25-OH D₃ groups, respectively. The 0.1 µM vitamin D₃ group had the highest increase in DSP+ levels. The highest Stro-1 levels were detected in the control and 0.1 µM 1,25-OH D₃ groups, respectively. The 0.1 µM 1,25-OH D₃ induced a mild increase in ALP activity. Conclusions: This study demonstrated that 1,25-OH D₃ stimulated odontoblastic differentiation of hDPSCs in vitro in a dose-dependent manner. The high DSP + cell number and a mild increase in ALP activity suggest that DPSCs treated with 0.1 μM 1,25-OH D₃ are in the later stage of odontoblastic differentiation. The results confirm that 1,25-OH D₃-added cocktail medium provides a sufficient microenvironment for the odontoblastic differentiation of hDPSCs in vitro.     

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.     

1,25-Dihydroxyvitamin D3 (calcitriol) suppresses concanavalin A-stimulated human T cell proliferation through monocytes

1,25-Dihydroxyvitamin D3 (calcitriol) inhibits mitogen-stimulated T cell proliferation by blocking the production of interleukin 2 (IL-2). The present study was initiated to determine the role of monocytes (Mo) in this process. Either Mo or T cells were preincubated with calcitriol for 24 or 48 hr and were thoroughly washed prior to concanavalin A (Con A)-stimulated coculture period. The inhibitory effect of calcitriol was Mo mediated; pretreatment of Mo resulted in inhibition of T cell proliferation, whereas pretreatment of T cells lacked the suppressive effect. Similar results were obtained by using 7- to 20-day-old IL-2-dependent human cultured T cells instead of fresh T cells. Calcitriol did not diminish the IL-1 secretion or intracellular IL-1 production of fresh Mo and actually increased the secretion of 24-hr-old Mo. Furthermore, addition of recombinant IL-1 to Mo-T cell coculture failed to reconstitute proliferative defect. Thus, these experiments clearly demonstrate that the suppression of Con A-driven T cell proliferation by calcitriol is Mo mediated and works by a non-IL-1 mechanism. At least, under these experimental conditions of Con A-driven T cell proliferation in which Mo play an obligatory role, antiproliferative action of calcitriol is Mo dependent.     

Toll-like receptor 2-mediated human B cell differentiation

Human B cells likely have a major role in the adjuvant activity of Toll-like receptor (TLR) 9 agonists by enhancing innate and adaptive immune responses. As several TLR2 ligands are promising vaccine adjuvant candidates, our aim was to characterize the effects of TLR2 stimulation on human B cell activation and differentiation using cells derived from healthy peripheral blood (PB), spleen, and diseased tonsils. We found a subset of partially differentiated TLR2+ PB and splenic B cells which responds to TLR2 agonists by mediating events involved in germinal center formation, such as upregulating CD77 and secreting chemokines. Furthermore, we show that TLR2-activated monocytes induce B cells to secrete significant quantities of IgM. Finally, activated TLR2+ B cells from tonsils are induced to secrete IgM directly by TLR2 ligands. Thus, TLR2 is likely involved in specific B cell-mediated functions and may be a viable vaccine adjuvant target in humans.     

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, 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.     

1,25-Dihydroxyvitamin D3 and Development of Tuberculosis in Cattle

This report describes the presence and activity of 1,25-dihydroxyvitamin D₃ (1,25-D₃) in experimental bovine tuberculosis. Animals that went on to develop tuberculous lesions exhibited a rapid transient increase in serum 1,25-D₃ within the first 2 weeks following infection with Mycobacterium bovis. 1,25-D₃-positive mononuclear cells were later identified in all tuberculous granulomas by immunohistochemical staining of postmortem lymph node tissue. These results suggest a role for 1,25-D₃ both at the onset of infection and in the development of the granuloma in these infected animals. Using a monoclonal antibody to the vitamin D receptor (VDR) as a VDR agonist, we confirmed that activation of the vitamin D pathway profoundly depresses antigen-specific, but not mitogenic, bovine peripheral blood T-cell responses (proliferation and gamma interferon production). Investigation of the mechanism of this suppression showed that the VDR antibody modified the expression of CD80 by accessory cells, such that a significant positive correlation between T-cell proliferation and accessory cell CD80 emerged.     

1,25-Dihydroxyvitamin D3 induces monocytic differentiation of the PLB-985 leukemic line and promotes c-fgr mRNA expression.

1,25-dihydroxyvitamin D3 [1,25(OH)2D] is known to stimulate maturation of the human promyelocytic line HL-60 and murine bone marrow precursor cells along a monocyte/macrophage pathway. In this study, the steroid's effects on the PLB-985 leukemic line were examined. We found that 1,25(OH)2D induces monocytic differentiation of PLB-985 as manifested by morphological appearance, histochemical staining, and changes in cell surface antigen expression. Additionally, there was acquisition of functional monocyte characteristics including phagocytic activity and superoxide anion production via the respiratory burst pathway. Steady state levels of mRNA derived from the leukocyte-specific proto-oncogene c-fgr were also increased by the steroid. Thus, 1,25(OH2)D effectively differentiates PLB-985 cells along a monocytic pathway, providing a useful model of macrophage differentiation.     

1,25-Dihydroxyvitamin D3 modulates T cell differentiation and impacts on the production of cytokines from Chinese Han patients with early rheumatoid arthritis

To study the effects of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) on the differentiation of T cells and the levels of cytokines in patients with early rheumatoid arthritis (eRA). The levels of Th1, Th2, Th17, and Treg cells were detected with BDFACS Calibur flow cytometer. The expression of IFN-ɤ, TNF-α, IL-2, IL-4, IL-6, IL-10, IL-17, and IL-22 was examined in 54 patients with eRA using a cytometric bead array (CBA). After 72 h of incubation of PBMCs from eRA patients with 1,25(OH)₂D₃, the levels of IFN-ɤ, TNF-α, IL-2, IL-6, and IL-17 significantly decreased compared to those of the control. 1,25(OH)₂D₃ had no significantly impact on the levels of IL-4, IL-10, and IL-22. The proportion of Th17 and the ratio of Th17/Treg significantly decreased in 1,25(OH)₂D₃-treated groups compared to those of the control. 1,25(OH)₂D₃ had no significantly impact on the proportion of Th1, Th2, Treg, and the ratio of Th1/Th2. Although no statistically significant difference was observed, proportion of Th1 was decreased after 1,25(OH)₂D₃ treatment compared with anti-CD3/CD28 only. The present study demonstrated that 1,25(OH)₂D₃ inhibited the synthesis of specific cytokines: Th1 (IFN-ɤ) and Th17 (IL-17, IL-22, IL-6, TNF-α) might upregulated Th2 cytokine (IL-4), which indicated the possible immunoregulatory roles and bone-sparing effects of 1,25(OH)₂D₃ in eRA through modulation of the Th1 and Th17 cytokine balance.

     

1,25-Dihydroxyvitamin D3 and phorbol esters (TPA) may induce select in vitro differentiation pathways in the HL60 promyelocytic cell line

Monocytic features can be induced in the myeloid cell line HL60 in order to provide a suitable in vitro model for the investigation of in vitro activity in mononuclear phagocytes. 1,25-Dihydroxyvitamin D3 (calcitriol) induced the HL60 cell line to express the monocytic differentiation antigen Leu M3 in about 30-50% of the cells along with an increase (up to 20%) in the expression of HLA-DR but not HLA-DQ class II antigen. Functional investigation showed that calcitriol-treated cells formed rosettes with sheep erythrocytes coated with an anti-sheep erythrocyte-specific IgG2a mouse MoAb and readily ingested them. In addition, these same sensitized erythrocytes were lysed in an 18-hr antibody-dependent cellular cytotoxicity (ADCC) assay. All together these data indicate the presence of functionally active Fc-IgG receptors (FcR). Sorting experiments demonstrated that only Leu M3+ HLA-DR+ cells contained the effector cell population; such was also the case for blood monocytes. This phenotypic profile was, however, not predictive per se of FcR presence and function, as 12,O-tetradecanoylphorbol-13-acetate (TPA)-induced HL60 cells neither formed rosettes nor phagocytosed nor exhibited ADCC activity, although they express Leu M3 and HLA-DR (as well as HLA-DQ) antigens. These results suggest that calcitriol and TPA cause the differentiation of HL60 cells along distinct pathways. On the other hand, different subpopulations with given predetermined differentiation capabilities may coexist in HL60 cell line. This hypothesis gains support by the observation that when TPA and calcitriol were added together to the undifferentiated cells, the resulting phenotypic pattern was representative of the different activities of both of the inducers as they were used separately.     

1,25-Dihydroxyvitamin D3 (calcitriol) suppresses IL-2 induced murine thymocyte proliferation

1,25-dihydroxyvitamin D3 (Calcitriol) inhibits mitogen stimulated T cell proliferation by blocking the production of Interleukin-2 (IL-2). The present study was initiated to determine whether the action of calcitriol was limited only to inhibition of IL-2 production, or if it influenced other events as well. To avoid the use of lectins, thymocytes from CD-I Swiss mice were chosen, which proliferate in response to Interleukin-I (IL-I) or IL-2 without the addition of lectins. Calcitriol inhibited (80-90%) IL-I induced CD-I mouse thymocyte proliferation, whereas 25-OH-D3 was ineffective. Further addition of IL-I failed to overcome this suppression. Surprisingly, calcitriol also inhibited IL-2 induced thymocyte proliferation (60-80%). Further addition of IL-2, in the presence of calcitriol, was ineffective in enhancing thymocyte proliferation. Similar results were obtained with C57BL/10 mouse thymocytes. Additional studies excluded the possibilities that calcitriol mediated inhibition was due to calcitriol IL-2 binding or damage to thymocytes by calcitriol. Thus, calcitriol not only blocks IL-2 production, but these results strongly suggest, that it also interferes with IL-2-thymocyte interaction, which in turn results in inhibition of thymocyte proliferation.     

1,25-Dihydroxyvitamin D3 induces a myelomonocytic phenotype with enhanced effector cell function in the HL-60 promyelocytic leukemia cell line

Human promyelocytic leukemia (HL-60) cells were induced by 1,25-dihydroxyvitamin D3 (calcitriol) to differentiate and examined using a panel of monoclonal antibodies (MoAbs) and functional assays. Although morphologically and histochemically these cells appeared to be of the monocyte-macrophage phenotype, there was a decline in Fc receptors for IgGl and no induction of class II HLA antigens. There was, however, dramatic induction of the antigen detected by the myeloid-specific MoAb AML-2-23. These data suggest that the phenotypic changes induced by calcitriol in HL-60 cells are consistent with myelomonocytic differentiation in that the resultant cells possess characteristics of both monocytes (morphology, non-specific esterase staining, high levels of AML-2-23 reactivity) and granulocytes (PMN 29 binding, decreased Fc receptors for IgGl, absence of class II HLA antigens). Perhaps more important, the ability of calcitriol-treated cells to perform antibody-dependent cellular cytotoxicity and phagocytosis was markedly augmented. Lysis of antibody-coated erythrocytes by HL-60 cells increased from 5% in controls to 30-35% with calcitriol treatment for 4 days. This enhanced effector cell function was seen despite a decline in Fc receptors measured by cytofluorography. These data suggest that calcitriol may be involved in both differential and functional activation of myeloid cells.     

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.     

Synergistic effects of 1,25-Dihydroxyvitamin D3 and TGF-beta1 on the production of insulin-like growth factor binding protein 3 in human bone marrow stromal cell cultures

1,25-Dihydroxyvitamin D3 (calcitriol), transforming growth factor-beta (TGF-beta), and insulin-like growth factors (IGFs) are all important bone regulatory factors known to affect proliferation and differentiation of human bone-forming cells (osteoblasts). We have previously shown that TGF-beta1 increased IGF-I and IGF-binding protein (IGFBP)-3 production in human bone marrow stromal (hMS) osteoblast progenitors and calcitriol stimulated IGFBP-3 and IGFBP-4 production. As interaction between signaling pathways of these factors has been reported, the present study aimed at examining the concerted actions on components of the IGF-system. We report that co-treatment with TGF-beta1 and calcitriol resulted in a synergistic increase in IGFBP-3 production, thereby suggesting that the effects of these factors on hMS osteoblast differentiation may involve the observed increase in IGFBP-3.     

Short-Term Effects of Vitamin D3 and 1,25-Dihydroxyvitamin D3 on Osteomalacia in Uremic Rats Fed a Low-Calcium-Low-Phosphorus Diet

The objectives of this study were to evaluate the effects of vitamin D₃ (D₃) and 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) on uremic bone disease independent of their action on the intestine. The histomorphology of tibial metaphyses in uremic (5/6 nephrectomized [5/6 Nx]) rats fed a low-calcium-low-phosphorus (LCLP) diet was compared with sham-operated (SO) rats fed an LCLP diet and 5/6 Nx rats fed an LCLP diet and given 15,000 IU D₃ or 5 units (135 ng) 1,25-(OH)₂D₃ daily for 7 days. A marked osteomalacia characterized by an increased percentage of active and inactive trabecular osteoid surface and thickened growth plates developed in proximal tibial metaphyses in 5/6 Nx rats given the placebo, compared with SO rats. These bone changes were associated with relative hypophosphatemia, hypophosphaturia, and hypercalciuria in 5/6 Nx rats. In 5/6 Nx rats treated with D₃ or 1,25-(OH)₂D₃ the growth plates had undergone mineralization and vascular invasion and were markedly reduced in thickness. Other parameters of osteomalacia in trabecular bone were not different from 5/6 Nx rats given the placebo. There was a significant decrease in osteoclasts per millimeter of trabecular surface perimeter in D₃- and 1,25-(OH)₂D₃-treated rats. These bone changes were associated with hypercalcemia, hyperphosphatemia, and hyperphosphaturia, compared with 5/6 Nx rats given the placebo. It was concluded that in uremic rats fed the LCLP diet, shortterm treatment with either pharmacologic levels of D₃ or 1,25-(OH)₂D₃ corrected only lesions in the growth plate. Osteoid seams were not reduced in treated rats, although the serum calcium-phosphorus product was elevated. The 5/6 Nx rat fed the LCLP diet appears to be a useful model for the rapid induction of uremic osteomalacia in adult animals.     

Modulation of Mycobacterium bovis-Specific Responses of Bovine Peripheral Blood Mononuclear Cells by 1,25-Dihydroxyvitamin D3

Historically, administration of vitamin D has been considered beneficial in the treatment of tuberculosis. The interaction of this vitamin {i.e., 1,25-dihdroxyvitamin D₃ [1,25(OH)₂D₃]} 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)₂D₃ on M. bovis-specific responses in vitro. Addition of 1 or 10 nM 1,25(OH)₂D₃ inhibited M. bovis-specific proliferative responses of PBMC from M. bovis-infected cattle, affecting predominately the CD4⁺ cell subset. In addition, 1,25(OH)₂D₃ inhibited M. bovis-specific gamma interferon (IFN-γ) 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)₂D₃ to PBMC cultures. These findings support the current hypothesis that 1,25(OH)₂D₃ enhances mycobacterial killing by increasing NO production, a potent antimicrobial mechanism of activated macrophages, and suggest that 1,25(OH)₂D₃ limits host damage by decreasing M. bovis-induced IFN-γ production.