Mycobacterium bovis bacille Calmette-Guerin vaccination of cattle: activation of bovine CD4+ and gamma delta TCR+ cells and modulation by 1,25-dihydroxyvitamin D3

SETTING: 1,25-dihydroxyvitamin D3 (1,25(OH)(2)D(3)) is a potent modulator of immune responses and may be beneficial in the treatment of tuberculosis. Recent evidence suggest that 1,25(OH)(2)D(3) may affect T-dependent responses in cattle; however, mechanisms by which this vitamin modulates activation of bovine T cells are unclear. OBJECTIVE: Determine the effects of 1,25(OH)(2)D(3) on the expression of CD25, CD44, and CD62L by bovine T cell subsets proliferating in response to antigen stimulation. DESIGN: Antigen-specific recall responses of Mycobacterium bovis bacille Calmette-Guerin (BCG) vaccinated cattle were used as a model system to evaluate effects of 1,25(OH)(2)D(3) on the proliferation and activation of bovine T cell subsets. RESULTS: CD4(+) and gamma delta TCR(+) cells were the predominant T cell subsets responding to soluble crude M. bovis-derived antigens (i.e., purified protein derivative and a BCG whole cell sonicate) by proliferation and activation-induced alterations in phenotype. These subsets exhibited increased CD25 and CD44 mean fluorescence intensity (mfi) and decreased CD62L mfi upon antigen stimulation. Addition of 1,25(OH)(2)D(3) inhibited proliferation of CD4(+) cells and decreased the expression of CD44 on responding (i.e., proliferating) CD4(+) and gamma delta TCR(+) cells. CONCLUSION: These findings suggest that the production of 1,25(OH)(2)D(3) by macrophages within tuberculous lesions would inhibit proliferation and CD44 expression by co-localized CD4(+) and gamma delta TCR(+) cells.     

1,25-Dihydroxyvitamin D3 reversibly blocks the progression of relapsing encephalomyelitis, a model of multiple sclerosis.

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease believed to be a model for the human disease multiple sclerosis (MS). Induced by immunizing B10.PL mice with myelin basic protein (MBP), EAE was completely prevented by the administration of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. 1,25-(OH)2D3 could also prevent the progression of EAE when administered at the appearance of the first disability symptoms. Withdrawal of 1,25-(OH)2D3 resulted in a resumption of the progression of EAE. Thus, the block by 1,25-(OH)2D3 is reversible. A deficiency of vitamin D resulted in an increased susceptibility to EAE. Thus, 1,25-(OH)2D3 or its analogs are potentially important for treatment of MS.     

Effects of the administration of phosphate on nuclear 1,25-dihydroxyvitamin D3 uptake by duodenal mucosal cells of Hyp mice

Effects of the administration of phosphate on nuclear 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] uptake by duodenal mucosal cells of Hyp mice were investigated. In Hyp mice fed a high phosphate diet (1.1% Ca and 2.0% phosphate) for 2 weeks, maximal nuclear 1,25-(OH)2D3 binding by duodenal mucosal cells is significantly increased from 5.01 +/- 0.49 x 10(3) to 8.23 +/- 1.10 x 10(3) sites/cell (P less than 0.05). No significant change was observed in normal mice fed the same diet. The serum phosphate concentration of Hyp mice increased significantly (P less than 0.01), whereas no significant change was found in normal mice. On this regimen, serum calcium, urinary cAMP to creatinine ratio, and cytosolic 1,25-(OH)2D3 receptor number in Hyp mice were not changed significantly. On the basis of these data, we speculate that the recovery of serum phosphate in Hyp mice fed a high phosphate diet affects the recovery of nuclear 1,25-(OH)2D3 uptake by duodenal mucosal cells. The mechanism for this recovery is not related to either the secondary hyperparathyroidism or the change in cytosolic 1,25-(OH)2D3 receptor content but, rather, to increased binding of 1,25-(OH)2D3-receptor complex to nuclei. Hypophosphatemia, therefore, appears to play a role in the vitamin D resistance in Hyp mice.     

Intestinal resistance to 1,25 dihydroxyvitamin D in mice heterozygous for the vitamin D receptor knockout allele

We tested the hypothesis that low vitamin D receptor (VDR) level causes intestinal vitamin D resistance and intestinal calcium (Ca) malabsorption. To do so, we examined vitamin D regulated duodenal Ca absorption and gene expression [transient receptor potential channel, vallinoid subfamily member 6 (TRPV6), 24-hydroxylase, calbindin D(9k) (CaBP) mRNA, and CaBP protein] in wild-type mice and mice with reduced tissue VDR levels [i.e. heterozygotes for the VDR gene knockout (HT)]. Induction of 24-hydroxylase mRNA levels by 1,25 dihydroxyvitamin D(3) [1,25(OH)(2) D(3)] injection was significantly reduced in the duodenum and kidney of HT mice in both time-course and dose-response experiments. TRPV6 and CaBP mRNA levels in duodenum were significantly induced after 1,25(OH)(2) D(3) injection, but there was no difference in response between wild-type and HT mice. Feeding a low-calcium diet for 1 wk increased plasma PTH, renal 1alpha-hydroxylase (CYP27B1) mRNA level, and plasma 1,25(OH)(2) D(3), and this response was greater in HT mice (by 88, 55, and 37% higher, respectively). In contrast, duodenal TRPV6 and CaBP mRNA were not higher in HT mice fed the low-calcium diet. However, the response of duodenal Ca absorption and CaBP protein to increasing 1,25(OH)(2) D(3) levels was blunted by 40% in HT mice. Our data show that low VDR levels lead to resistance of intestinal Ca absorption to 1,25(OH)(2) D(3), and this resistance may be due to a role for the VDR (and VDR level) in the translation of CaBP.     

Development of experimental autoimmune encephalomyelitis (EAE) in mice requires vitamin D and the vitamin D receptor

The development of experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, has been studied in mice that were (i) vitamin D-deficient, (ii) minus the vitamin D receptor, (iii) minus a vitamin D 25-hydroxylase, and (iv) minus the vitamin D 25-hydroxyvitamin D-1α-hydroxylase. EAE development was markedly suppressed in mice lacking the vitamin D receptor and partially suppressed in vitamin D-insufficient mice. However, the absence of either of the two key hydroxylases (i.e., 25-hydroxylase and 1α-hydroxylase) neither inhibits nor enhances the development of EAE. These results indicate that vitamin D and the vitamin D receptor are required for the development of EAE. The results also suggest that 1,25-dihydroxyvitamin D(3) may not play a role in this autoimmune response.     

Regulation of vitamin D-1alpha-hydroxylase and -24-hydroxylase expression by dexamethasone in mouse kidney

We investigated the effects of dexamethasone on vitamin D-1alpha-hydroxylase and -24-hydroxylase expression and on vitamin D receptor (VDR) content in the kidneys of mice fed either a normal (NCD) diet or a calcium- and vitamin D-deficient (LCD) diet for 2 weeks. For the last 5 days mice received either vehicle or dexamethasone (2 mg/kg per day s.c.). Dexamethasone significantly increased plasma calcium concentrations without changing plasma concentrations of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) in both NCD and LCD groups. Northern blot and enzyme activity analyses in NCD mice revealed that dexamethasone increased renal VDR mRNA expression modestly and greatly increased 24-hydroxylase mRNA abundance and enzyme activity, but did not affect 1alpha-hydroxylase mRNA abundance and enzyme activity. In mice fed an LCD diet, dexamethasone increased renal VDR mRNA expression 1.5-fold, decreased 1alpha-hydroxylase mRNA abundance (52%) and activity (34%), and markedly increased 24-hydroxylase mRNA abundance (16-fold) and enzyme activity (9-fold). Dexamethasone treatment did not alter functional VDR number (B(max) 125-141 fmol/mg protein) or ligand affinity (K(d) 0.13-0.10 nM) in LCD mice. Subcutaneous injections of 1,25(OH)(2)D(3) (0.24 nmol/kg per day for 5 days) into NCD mice strongly increased renal 24-hydroxylase mRNA abundance and enzyme activity, while there was no effect of dexamethasone on renal 24-hydroxylase expression in these mice. This may be due to overwhelming induction of 24-hydroxylase by 1,25(OH)(2)D(3). These findings suggest that glucocorticoid-induced osteoporosis is caused by direct action of the steroids on bone, and the regulatory effect of glucocorticoids on renal 25-hydroxyvitamin D(3) metabolism may be less implicated in the initiation and progression of the disease.     

Contrasting effects of exogenous 1,25-dihydroxyvitamin D [1,25-(OH)2D] versus endogenous 1,25-(OH)2D, induced by dietary calcium restriction, on vitamin D receptors

The biological actions of 1,25-dihydroxyvitamin D [1,25-(OH)2D] are mediated by specific binding of the hormone with an intracellular vitamin D receptor, which ultimately regulates expression of genes within the target tissues. The quantity of vitamin D receptors varies between target tissues and within target tissues, depending on the physiological state of the animal. One factor that can modulate tissue vitamin D receptor content is 1,25-(OH)2D. In the present study performed in male rats, exogenous administration of 36 ng 1,25-(OH)2D3/day for 7 days increased plasma 1,25-(OH)2D concentrations 5-fold above those in control rats (to 261 +/- 17 pg/ml). Compared with those in control rats, 1,25-(OH)2D3 treatment resulted in a 1.5-fold increase in duodenal vitamin D receptor content (351 +/- 16 vs. 520 +/- 21 fmol/mg protein) and a 3-fold increase in renal vitamin D receptor content (60.3 +/- 5.2 vs. 193.8 +/- 22.7 fmol/mg protein). The effects of endogenously produced 1,25-(OH)2D on tissue vitamin D receptor content were studied by feeding rats either a 0.02% or 1% calcium diet for 2, 7, 14, or 21 days. Rats fed the low calcium diet exhibited plasma 1,25-(OH)2D concentrations similar to (day 7) or exceeding (days 14 and 21) those achieved by exogenous administration of 1,25-(OH)2D3, yet duodenal vitamin D receptor content was not up-regulated by dietary calcium restriction at any time point. The renal vitamin D receptor content of calcium restricted rats was 20-38% lower (P less than 0.05) than that in rats fed a calcium-replete diet 7, 14, and 21 days after initiation of the dietary treatments. These data suggest that under physiological conditions, increased plasma concentrations of 1,25-(OH)2D do not result in up-regulation of tissue vitamin D receptor concentrations, and that dietary calcium restriction must induce some factor(s) that results in down-regulation of vitamin D receptors in the kidney.     

Control of autoimmune diseases by the vitamin D endocrine system

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], the biologically active form of vitamin D(3), is a secosteroid hormone essential for bone and mineral homeostasis. It regulates the growth and differentiation of multiple cell types, and displays immunoregulatory and anti-inflammatory properties. Cells involved in innate and adaptive immune responses--including macrophages, dendritic cells, T cells and B cells--express the vitamin D receptor (VDR), and can both produce and respond to 1,25(OH)(2)D(3). The net effect of the vitamin D system on the immune response is an enhancement of innate immunity coupled with multifaceted regulation of adaptive immunity. Epidemiological evidence indicates a significant association between vitamin D deficiency and an increased incidence of several autoimmune diseases, and clarification of the physiological role of endogenous VDR agonists in the regulation of autoimmune responses will guide the development of pharmacological VDR agonists for use in the clinic. The antiproliferative, prodifferentiative, antibacterial, immunomodulatory and anti-inflammatory properties of synthetic VDR agonists could be exploited to treat a variety of autoimmune diseases, from rheumatoid arthritis to systemic lupus erythematosus, and possibly also multiple sclerosis, type 1 diabetes, inflammatory bowel diseases, and autoimmune prostatitis.     

Hereditary 1,25-dihydroxyvitamin D resistant rickets due to a mutation causing multiple defects in vitamin D receptor function

Hereditary vitamin D-resistant rickets (HVDRR) is an autosomal recessive disease caused by mutations in the vitamin D receptor (VDR). We studied a young Saudi Arabian girl who exhibited the typical clinical features of HVDRR, but without alopecia. Analysis of her VDR gene revealed a homozygous T to C mutation in exon 7 that changed isoleucine to threonine at amino acid 268 (I268T). From crystallographic studies of the VDR ligand-binding domain, I268 directly interacts with 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and is involved in the hydrophobic stabilization of helix H12. We recreated the I268T mutation and analyzed its effects on VDR function. In ligand binding assays, the I268T mutant VDR exhibited an approximately 5- to 10-fold lower affinity for [(3)H]1,25(OH)(2)D(3) compared with the wild-type (WT) VDR. The I268T mutant required approximately a 65-fold higher concentration of 1,25(OH)(2)D(3) to be equipotent in gene transactivation. Both retinoid X receptor heterodimerization and coactivator binding were reduced in the I268T mutant. Analogs of 1,25(OH)(2)D(3) have been proposed as potential therapeutics for patients with HVDRR. Interestingly, in protease sensitivity assays, treatment with the potent vitamin D analog, 20-epi-1,25(OH)(2)D(3), stabilized I268T mutant proteolytic fragments better than 1,25(OH)(2)D(3). Moreover, 20-epi-1,25(OH)(2)D(3) restored transactivation of the I268T mutant to levels exhibited by WT VDR treated with 1,25(OH)(2)D(3). In conclusion, we describe a novel mutation, I268T, in the VDR ligand-binding domain that alters ligand binding, retinoid X receptor heterodimerization, and coactivator binding. These combined defects in VDR function cause resistance to 1,25(OH)(2)D(3) action and result in the syndrome of HVDRR.     

Regulation of the murine renal vitamin D receptor by 1,25-dihydroxyvitamin D3 and calcium

Renal vitamin D receptor (VDR) is required for 1,25-dihydroxyvitamin D3-[1,25(OH)2D3]-induced renal reabsorption of calcium and for 1,25(OH)2D3-induced 1,25(OH)2D3 24-hydroxylase. The long-term effect of vitamin D and dietary calcium on the expression of renal VDR was examined in the nonobese diabetic mouse. Vitamin D-deficient and vitamin D-replete mice were maintained on diets containing 0.02%, 0.25%, 0.47%, and 1.20% calcium with or without 50 ng of 1,25(OH)2D3 per day. Vitamin D-replete mice on a 1.20% calcium diet had renal VDR levels of 165 fmol/mg protein. Calcium restriction caused renal VDR levels to decrease to <30 fmol/mg protein in vitamin D-deficient mice and to approximately 80 fmol/mg protein in vitamin D-replete mice. When dietary calcium was present, 50 ng of 1,25(OH)2D3 elevated the VDR levels 2- to 10-fold, depending on vitamin D status and the level of calcium. In the absence of either vitamin D or calcium, the VDR mRNA was expressed at a basal level. 1,25(OH)2D3 supplementation caused relative VDR mRNA to increase 8- to 10-fold in the vitamin D-deficient mouse when dietary calcium was available. This increase was completely absent in the calcium-restricted mice. This in vivo study demonstrates that 1,25(OH)2D3 and calcium are both required for renal VDR mRNA expression above a basal level, furthering our understanding of the complex regulation of renal VDR by 1,25(OH)2D3 and calcium.     

Antiproliferative effects of 1alpha,25-dihydroxyvitamin D(3) and vitamin D analogs on tumor-derived endothelial cells

Although there is abundant evidence that 1alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] inhibits the growth of several cancer cell types, inhibition of angiogenesis may also play a role in mediating the antitumor effects of 1,25-(OH)(2)D(3.) We examined the ability of 1,25-(OH)(2)D(3) to inhibit the growth of tumor-derived endothelial cells (TDECs) and normal endothelial cells and to modulate angiogenic signaling. 1,25-(OH)(2)D(3) inhibited the growth of TDECs from two tumor models at nanomolar concentrations, but was less potent against normal aortic or yolk sac endothelial cells. The vitamin D analogs Ro-25-6760, EB1089, and ILX23-7553 were also potent inhibitors of TDEC proliferation. Furthermore, the combination of 1,25-(OH)(2)D(3) and dexamethasone had greater activity than either agent alone. 1,25-(OH)(2)D(3) increased vitamin D receptor and p27(Kip1) protein levels in TDECs, whereas phospho-ERK1/2 and phospho-Akt levels were reduced. These changes were not observed in normal aortic endothelial cells. In squamous cell carcinoma and radiation-induced fibrosarcoma-1 cells, 1,25-(OH)(2)D(3) treatment caused a reduction in the angiogenic signaling molecule, angiopoietin-2. In conclusion, 1,25-(OH)(2)D(3) and its analogs directly inhibit TDEC proliferation at concentrations comparable to those required to inhibit tumor cells. Further, 1,25-(OH)(2)D(3) modulates cell cycle and survival signaling in TDECs and affects angiogenic signaling in cancer cells. Thus, our work supports the hypothesis that angiogenesis inhibition plays a role in the antitumor effects of 1,25-(OH)(2)D(3).     

1,25-Dihydroxyvitamin D3 selectively and reversibly impairs T helper-cell CNS localization

Pharmacologic targeting of T helper (T_H) cell trafficking poses an attractive opportunity for amelioration of autoimmune diseases such as multiple sclerosis (MS). MS risk is associated with vitamin D deficiency, and its bioactive form, 1,25-dihydroxyvitamin D3 [1,25(OH)₂D₃], has been shown to prevent experimental autoimmune encephalomyelitis, a mouse model of MS, via an incompletely understood mechanism. Herein, we systematically examined 1,25(OH)₂D₃ effects on T_H cells during their migration from the lymph nodes to the CNS. Our data demonstrate that myelin-reactive T_H cells are successfully generated in the presence of 1,25(OH)₂D₃, secrete proinflammatory cytokines, and do not preferentially differentiate into suppressor T cells. These cells are able to leave the lymph node, enter the peripheral circulation, and migrate to the s.c. immunization sites. However, T_H cells from 1,25(OH)₂D₃-treated mice are unable to enter the CNS parenchyma but are instead maintained in the periphery. Upon treatment cessation, mice rapidly develop experimental autoimmune encephalomyelitis, demonstrating that 1,25(OH)₂D₃ prevents the disease only temporarily likely by halting T_H cell migration into the CNS.Multiple sclerosis (MS) is an immune-mediated demyelinating disorder of the central nervous system (CNS). Whereas its etiology is unknown, a number of environmental and genetic factors contribute to the risk of developing MS (1). Epidemiologic studies demonstrate a strong correlation between MS risk and vitamin D deficiency (2). Higher levels of circulating 25-hydroxyvitamin D₃ have been recently shown to correlate with a reduction in the number of new T2 and gadolinium-enhancing lesions (3). Moreover, the bioactive form of vitamin D, 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], can completely prevent and treat established experimental autoimmune encephalomyelitis (EAE) (4), a murine MS model. Nonetheless, the mechanism of 1,25(OH)₂D₃^’s in vivo action remains incompletely understood.The pathogeneses of MS and EAE require localization of self-reactive T cells to the CNS. Myelin-specific effector T cells are primed in the lymph nodes (LNs) and subsequently travel a complex route and arrive at the CNS barriers. There, a sequential interaction with a multitude of endothelial adhesion molecules and chemokines leads to T helper (T_H) cell migration into the CNS parenchyma. Several ligand/receptor pairs have been implicated in the pathogeneses of MS and EAE (5, 6). Therefore, manipulation of molecules involved in T-cell trafficking poses an attractive opportunity for therapeutic interventions. Recent studies have highlighted a role for vitamin D in immune cell migration (7–10), but whether 1,25(OH)₂D₃ affects trafficking of lymphocytes in MS and/or EAE has not been investigated.A recent study using conditional deletion of the vitamin D receptor (VDR) has demonstrated that 1,25(OH)₂D₃ prevents EAE by acting directly on the encephalitogenic T_H cells (11). We hypothesized that 1,25(OH)₂D₃ could be inhibiting encephalitogenic T cells via regulation of molecules involved in their trafficking into the CNS. Herein, we provide unique evidence that 1,25(OH)₂D₃ prevents EAE by modulating the migratory phenotype of the pathogenic T_H cells without affecting their priming or effector functions. Our findings further emphasize that 1,25(OH)₂D₃ is a reversible immune modulator, as treatment cessation leads to rapid disease onset. These findings may have important implications for the use of vitamin D as an adjunct treatment of immune-mediated diseases such as MS.     

Functional vitamin D receptor (VDR) in the t-tubules of cardiac myocytes: VDR knockout cardiomyocyte contractility

We have previously shown that the active form of vitamin D, 1,25 dihydroxyvitamin D3 [1,25(OH)(2)D(3)], has both genomic and rapid nongenomic effects in heart cells; however, the subcellular localization of the vitamin D receptor (VDR) in heart has not been studied. Here we show that in adult rat cardiac myocytes the VDR is primarily localized to the t-tubule. Using immunofluorescence and Western blot analysis, we show that the VDR is closely associated with known t-tubule proteins. Radioligand binding assays using (3)H-labeled 1,25(OH)(2)D(3) demonstrate that a t-tubule membrane fraction isolated from homogenized rat ventricles contains a 1,25(OH)(2)D(3)-binding activity similar to the classic VDR. For the first time, we show that cardiac myocytes isolated from VDR knockout mice show accelerated rates of contraction and relaxation as compared with wild type and that 1,25(OH)(2)D(3) directly affects contractility in the wild-type but not the knockout cardiac myocyte. Moreover, we observed that acute (5 min) exposure to 1,25(OH)(2)D(3) altered the rate of relaxation. A receptor localized to t-tubules in the heart is ideally positioned to exert an immediate effect on signal transduction mediators and ion channels. This novel discovery is fundamentally important in understanding 1,25(OH)(2)D(3) signal transduction in heart cells and provides further evidence that the VDR plays a role in heart structure and function.     

Noncalcemic actions of vitamin D receptor ligands

1alpha,25-Dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], the active metabolite of vitamin D(3), is known for the maintenance of mineral homeostasis and normal skeletal architecture. However, apart from these traditional calcium-related actions, 1,25-(OH)(2)D(3) and its synthetic analogs are being increasingly recognized for their potent antiproliferative, prodifferentiative, and immunomodulatory activities. These actions of 1,25-(OH)(2)D(3) are mediated through vitamin D receptor (VDR), which belongs to the superfamily of steroid/thyroid hormone nuclear receptors. Physiological and pharmacological actions of 1,25-(OH)(2)D(3) in various systems, along with the detection of VDR in target cells, have indicated potential therapeutic applications of VDR ligands in inflammation (rheumatoid arthritis, psoriatic arthritis), dermatological indications (psoriasis, actinic keratosis, seborrheic dermatitis, photoaging), osteoporosis (postmenopausal and steroid-induced osteoporosis), cancers (prostate, colon, breast, myelodysplasia, leukemia, head and neck squamous cell carcinoma, and basal cell carcinoma), secondary hyperparathyroidism, and autoimmune diseases (systemic lupus erythematosus, type I diabetes, multiple sclerosis, and organ transplantation). As a result, VDR ligands have been developed for the treatment of psoriasis, osteoporosis, and secondary hyperparathyroidism. Furthermore, encouraging results have been obtained with VDR ligands in clinical trials of prostate cancer and hepatocellular carcinoma. This review deals with the molecular aspects of noncalcemic actions of vitamin D analogs that account for the efficacy of VDR ligands in the above-mentioned indications.     

维生素D与自身免疫性疾病

1,25-二羟维生素D3[1,25-dihydroxyvitamin D3,1,25(OH)2-D3]是维生素D3的活性形式,自从发现细胞内表达维生素D受体和激活的树突状细胞产生维生素D后,1,25(OH)2-D3的免疫调节作用开始被证实。其主要通过特定的维生素D受体对靶细胞,如T细胞、抗原呈递细胞发挥效应进行调节。本文就1,25(OH)2-D3免疫调节机制,特别是对自身免疫性疾病的免疫调节作用作一综述。