Epigenetic reduction in invariant NKT cells following in utero vitamin D deficiency in mice

Vitamin D status changes with season, but the effect of these changes on immune function is not clear. In this study, we show that in utero vitamin D deficiency in mice results in a significant reduction in invariant NKT (iNKT) cell numbers that could not be corrected by later intervention with vitamin D or 1,25-dihydroxy vitamin D(3) (active form of the vitamin). Furthermore, this was intrinsic to hematopoietic cells, as vitamin D-deficient bone marrow is specifically defective in generating iNKT cells in wild-type recipients. This vitamin D deficiency-induced reduction in iNKT cells is due to increased apoptosis of early iNKT cell precursors in the thymus. Whereas both the vitamin D receptor and vitamin D regulate iNKT cells, the vitamin D receptor is required for both iNKT cell function and number, and vitamin D (the ligand) only controls the number of iNKT cells. Given the importance of proper iNKT cell function in health and disease, this prenatal requirement for vitamin D suggests that in humans, the amount of vitamin D available in the environment during prenatal development may dictate the number of iNKT cells and potential risk of autoimmunity.     

Effect of intermittent oral 1,25(OH)2D3 therapy on bone Gla protein in dialysis patients.

Serum Bone Gla Protein (BGP) levels were measured by both immunoradiometric assay (IRMA) and radioimmunoassay (RIA) to investigate the effect of intermittent 1,25(OH)2D3 administration to dialysis patients who could not tolerate an increase in an active vitamin D3 dose and/or calcium to control secondary hyperparathyroidism due to hypercalcemia. The administration of active vitamin D3 gradually increased the serum BGP to more than 3 times the original level by the 8th week. At the 12th week after starting the active vitamin D3 therapy, mean BGP was about twice the original level, which was about half the maximum level at the 8th week. The BGP (IRMA)/BGP (RIA) ratio was increased significantly at 4th and 8th weeks compared to the original level. During this period, serum calcium, phosphorous, or intact molecule PTH (I-PTH) levels showed insignificant changes, with a slight reduction in the mid molecule PTH (m-PTH) level, and a significant reduction in ALP. Serum BUN and creatinine levels were not changed significantly. These data suggest that BGP was increased through direct stimulation of osteoblasts by the active vitamin D3, and the increase was not due to deterioration of secondary hyperparathyroidism. The reduction of the increase in the BGP level at the 12th week with insignificant biochemical changes suggest that activation of osteoblasts by vitamin D3 may be transient. In conclusion, intermittent active vitamin D3 increases serum BGP, without deterioration of major biochemical changes even in patients with moderate to severe secondary hyperparathyroidism, although the increase may be transient. These facts suggest that the serum BGP of hemodialysis patients is controlled at least in part by active vitamin D3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Skin calcium-binding protein: effect of vitamin D deficiency and vitamin D treatment

The amount of skin calcium-binding protein, evaluated using a sensitive radioimmunoassay and indirect immunofluorescence, was decreased in vitamin-D deficient rats and increased after one week vitamin D3 or 1 alpha-hydroxyvitamin D3 treatment. In vitamin D replete and in vitamin D-deficient animals, skin calcium-binding protein was not sensitive to changes in dietary and/or serum calcium concentrations. These results indicate that this protein is different from other calcium-binding proteins such as parvalbumin and calmodulin which are not vitamin D-dependent, and also different from intestinal calcium-binding protein which, in D replete animals, is sensitive to changes in dietary and serum calcium concentrations. Skin calcium-binding protein may, therefore, represent a new class of vitamin D-dependent protein.     

The paradoxical effects of vitamin D on type 1 mediated immunity

Low vitamin D status is associated with an increased risk of Th1 mediated autoimmune diseases like inflammatory bowel disease. 1,25(OH)(2)D(3) treatments have been shown to suppress Th1 mediated immunity and protect animals from experimental autoimmunity. Th1 mediated immunity is important for clearance of a number of different infectious diseases. For tuberculosis 1,25(OH)(2)D(3) treatment is associated with decreased Th1 mediated immunity but increased bactericidal activity. Systemic candidiasis is unaffected by 1,25(OH)(2)D(3) treatment. The seemingly paradoxical effects of 1,25(OH)(2)D(3) and vitamin D on Th1 mediated autoimmunity versus infectious immunity point to a broad array of vitamin D targets in the immune system. The interplay of these vitamin D targets and their impact on the host-immune response then dictate the outcome.     

The influence of vitamin D deficiency on cancers and autoimmune diseases development

There is a growing number of diseases which prevalence can be associated with vitamin D deficiency. The link between low cholecalciferol concentration and bone diseases is well established, however there is also data suggesting that it may influence development and progression of different cancers and autoimmune diseases. The in vitro studies proved that the active vitamin D metabolite--1,25(OH)(2)D(3) may arrest the cell cycle progression, induce apoptosis as well as regulate T cells and antigen presenting cells function. Results of the in vivo experiments suggest that vitamin D deficiency accelerates development of autoimmune diseases and cancers in animals. Epidemiological studies imply that the vitamin D deficiency is also associated with the increased incidence of autoimmune diseases and cancers in people. The main determinant of vitamin D serum concentration in a human body is skin synthesis. The changes in the lifestyle, air pollution as well as a common use of sun screens caused that the contemporary European receives little sunlight compared to his ancestors. According to the recent epidemiological studies, the vitamin D concentrations in serum of people who live in high latitudes (above 34 degrees N/S), including Poland, is far from being sufficient. This paper reviews results of the recent studies concerning the potential role of the vitamin D in the development of cancers and autoimmune diseases, as well as provides guidelines for vitamin D supplementation.     

Dietary calcium deficiency increases Ca2+ uptake and Ca2+ extrusion mechanisms in chick enterocytes

Ca2+ uptake and Ca2+ extrusion mechanisms were studied in enterocytes with different degree of differentiation from chicks adapted to a low Ca2+ diet as compared to animals fed a normal diet. Chicks adapted to a low Ca2+ diet presented hypocalcemia, hypophosphatemia and increased serum 1,25(OH)2D3 and Ca2+ absorption. Low Ca2+ diet increased the alkaline phosphatase (AP) activity, independently of the cellular maturation, but it did not alter gamma-glutamyl-transpeptidase activity. Ca2+ uptake, Ca2+-ATPase and Na(+)/Ca2+ exchanger activities and expressions were increased by the mineral-deficient diet either in mature or immature enterocytes. Western blots analysis shows that vitamin D receptor (VDR) expression was much higher in crypt cells than in mature cells. Low Ca2+ diet decreased the number of vitamin D receptor units in both kinds of cells. In conclusion, changes in Ca2+ uptake and Ca2+ extrusion mechanisms in the enterocytes by a low Ca2+ diet appear to be a result of enhanced serum levels of 1,25(OH)2D3, which would promote cellular differentiation producing cells more efficient to express vitamin D dependent genes required for Ca2+ absorption.     

The effect of vitamin D on the structural crosslinks and maturation of chick bone collagen.

The quantitative relationships were determined between the structural crosslinks, dihydroxylysinonorleucine (Lys(OH)2-Nle) and hydroxylysinonorleucine (Lys (OH) -Nle) in NaB 3H4-reduced diaphyseal bone collagen from 1-, 2-, 3- and 4-week-old chicks fed either a vitamin D-deficient diet, a normal-vitamin D diet or a high-, but non toxic, vitamin D diet from time of hatching. Chicks fed the normal diet showed a progressive decrease in the ratio of Lys(OH)2-Nle/Lys(OH)-Nle with age. This decrease was accelerated in chicks receiving the High-vitamin D diet. In the vitamin D-deficient group, the ratio was higher than controls at 1 and 2 weeks and increased further at 3 and 4 weeks. Similar changes in Lys(OH)2-Nle/Lys(OH)-Nle ratio did not occur in skin collagen. Compared to Control-vitamin D animals, the increased crosslink ratios in the vitamin D-deficient bone collagen occurred prior to changes in growth rate and could not be correlated with lysine hydroxylation or the hypocalcemia seen in this group. These results suggest that the type of crosslink analysis used in this study provides one of the earliest and most sensitive indications of a bone disturbance due to vitamin D deficiency and that vitamin D specifically acts to increase the rate of maturation of bone collagen.     

Vitamin D3 and androgen receptors in testis and epididymal region of roosters (Gallus domesticus) as affected by epididymal lithiasis

Epididymal lithiasis is a dysfunction characterized by formation of calcium-rich stones in the epididymal region of roosters, associated with decreased serum testosterone and loss of fertility. The segment most affected by the lithiasis is the efferent ductules, which, in birds, are responsible for reabsorption of calcium and luminal fluid. Therefore, we postulated that epididymal lithiasis could result from local impairment of calcium or fluid homeostasis, culminating in initiation of stone formation. Transepithelial calcium transport depends on vitamin D3 and vitamin D3 receptor (VDR). Based on the fact that VDR are present in efferent ductules, possible changes in the pattern of VDR in roosters affected by the epididymal lithiasis was investigated, to start to gain an understanding of the molecular mechanisms involved in the development of calcium stones. To evaluate the potential impact of androgen reduction, changes in androgen receptor (AR) were also investigated. Both VDR and AR were increased in specific segments of the epididymal region, whereas no alterations were found in the testes of affected animals. The increase in VDR was most likely due to an increase in the number of VDR-positive mononuclear leukocyte infiltrates found in the connective tissue followed by an increase in epithelial receptors. The AR were increased, however, mainly in the epididymal duct epithelium. These results suggest that the vitamin D3 and androgen responsive system may be directly/indirectly involved in the development of the disease.     

Vitamin D Status in Rheumatoid Arthritis: Inflammation,Arterial Stiffness and Circulating Progenitor Cell Number

Background and Aims

Suboptimal vitamin D status was recently acknowledged as an independent predictor of cardiovascular diseases and all-cause mortality in several clinical settings, and its serum levels are commonly reduced in Rheumatoid Arthritis (RA). Patients affected by RA present accelerated atherosclerosis and increased cardiovascular morbidity and mortality with respect to the general population. In RA, it has been reported an impairment of the number and the activity of circulating proangiogenic haematopoietic cells (PHCs), including CD34+, that may play a role in endothelial homeostasis. The purpose of the study is to investigate the association between vitamin D levels and PHCs, inflammatory markers, and arterial stiffening in patients with RA.

Methods and Results

CD34+ cells were isolated from 27 RA patients and 41 controls. Vitamin D levels, C-reactive protein (CRP), fibrinogen, pulse wave velocity (PWV), and carotid intima-media thickness (cIMT) were also evaluated. CD34+ count and vitamin D levels were lower in RA patients as compared to controls, while fibrinogen, CRP, PWV and cIMT were higher in RA patients. CD34+ cell number appeared to be associated with vitamin D levels, and negatively correlated to fibrinogen and early atherosclerosis markers (PWV and cIMT); vitamin D levels appear also to be inversely associated to fibrinogen.

Conclusions

RA patients with moderate disease activity presented with low vitamin D levels, low CD34+ cell count, increased PWV and cIMT; we found that vitamin D deficiency is associated to CD34+ cell reduction in peripheral blood, and with fibrinogen levels. This suggests that vitamin D might contribute to endothelial homeostasis in patients with RA.     

Effect of vitamin D(3) treatment in the neonatal or adolescent age (hormonal imprinting) on the thymic glucocorticoid receptor of the adult male rat

Single neonatal treatment with 25 microg vitamin D(3) significantly decreased the thymic glucocorticoid receptor density (B(max)) of 6-week-old male rats. In females, a similar treatment did not cause any changes. Single vitamin D(3) treatment (50 microg) during adolescence (i.e. 6-week-old animals) significantly increased the glucocorticoid receptor density in adult (10-week-old) males. No significant changes in receptor affinity (K(d)) could be observed. Considering that in earlier experiments similar neonatal treatments influenced bone mineral mass and sexual behavior, the hormonal imprinting effect of vitamin D(3) and its harmful effect on the development of other members of the steroid receptor superfamily, seems to be unquestionable.     

Respiratory epithelial cells convert inactive vitamin D to its active form: potential effects on host defense

The role of vitamin D in innate immunity is increasingly recognized. Recent work has identified a number of tissues that express the enzyme 1alpha-hydroxylase and are able to activate vitamin D. This locally produced vitamin D is believed to have important immunomodulatory effects. In this paper, we show that primary lung epithelial cells express high baseline levels of activating 1alpha-hydroxylase and low levels of inactivating 24-hydroxylase. The result of this enzyme expression is that airway epithelial cells constitutively convert inactive 25-dihydroxyvitamin D(3) to the active 1,25-dihydroxyvitamin D(3). Active vitamin D that is generated by lung epithelium leads to increased expression of vitamin D-regulated genes with important innate immune functions. These include the cathelicidin antimicrobial peptide gene and the TLR coreceptor CD14. dsRNA increases the expression of 1alpha-hydroxylase, augments the production of active vitamin D, and synergizes with vitamin D to increase expression of cathelicidin. In contrast to induction of the antimicrobial peptide, vitamin D attenuates dsRNA-induced expression of the NF-kappaB-driven gene IL-8. We conclude that primary epithelial cells generate active vitamin D, which then influences the expression of vitamin D-driven genes that play a major role in host defense. Furthermore, the presence of vitamin D alters induction of antimicrobial peptides and inflammatory cytokines in response to viruses. These observations suggest a novel mechanism by which local conversion of inactive to active vitamin D alters immune function in the lung.     

The role of vitamin E in metabolism and reception of vitamin D

It was found that calcium exchange disturbances under vitamin E deficiency is due to changes in the metabolism of vitamin D. In vitamin E-deficient rats the serum blood levels of hydroxyvitamin D (25-OHD) showed no significant changes, whereas the concentration of the hormonal form of 1.25-hydroxyvitamin D [1.25(OH)2D], decreased by 40%. In vitro studies showed that the 25-hydroxylase D3 activity in the livers of rats with E-avitaminosis had a tendency to decrease (by 22%), whereas that of 24-hydroxylase dropped drastically (by 52%). The serum blood levels of the parathyroid hormone (PTH) and kidney levels of cAMP under E-avitaminosis were significantly lowered. Preincubation of kidney slices with the adenylate cyclase activator, forskolin, increased the activity of 1-OHase in about the same degree as that in vitamin E-rich rats. The free radical scavenger, BHT, added to kidney slices suppressed the activity of the both enzymes; this finding testifies to the low O2-binding affinity of these monooxygenases. The content of 1.25(OH)2D3 receptors occupied in vivo in the kidneys of vitamin E-deficient rats decreased 2.5-fold; however, the binding of 1.25(OH)2D3-receptor complexes to heterologous DNA was unaffected thereby. The vitamin deficiency in vivo results in the inhibition of vitamin D metabolism in the liver and kidney concomitant with the formation of active metabolites and decreases the concentration of hormone-receptor complexes in target tissues.     

Hepatic accumulation of vitamin D3 and 25-hydroxyvitamin D3.

Concomitant intravenous administration of 25-hydroxycholecalciferol and [3H] vitamin D3 to vitamin D-depleted rats did not affect the conversion of [3H] vitamin D3 to 25-OH-[3H] vitamin D3 as indicated by a serum 25-OH-[3H] vitamin D3 to content at 3 and 24 h identical to those observed in animals receiving [3H] vitamin D3 alone. Similarly, pre-dosing with 25-OH vitamin D3 24 h earlier did not affect the conversion. Co-administration to vitamin D depleted rats of vitamin D2 or D3, at 200-fold higher doses than a control group receiving tracer [3H] vitamin D3 alone, resulted in serum 25-OH vitamin D levels that were 15-20 fold higher than the control, indicating a similar metabolic fate for synthetic and natural vitamin D in rats and the ability of increased substrate to overwhelm hepatic constraints on 25-OH vitamin D production. Following intravenous administration of 25-OH-[3H] vitamin D3 to vitamin D depleted rats, hepatic 3H content decreased in parallel with serum radioactivity. Hepatic accumulation of intravenously administered vitamin D3 ([14C] vitamin D3) alone or with 25-OH-[3H] vitamin D3, by vitamin D-depleted rats revealed a marked preference for vitamin D3; the hepatic accumulation of [14C] vitamin D3 increased to 35% of the dose by 45 min, at which time 25-OH-[3H] vitamin D3 hepatic content was 7-fold less, and decreasing. Chromatography of extracts of hepatic subcellular fractions revealed more [14C] vitamin D3 than 25-OH-[3H] vitamin D3 in the microsomes, the reported site of calciferol 25-hydroxylase. Circulating 25-OH vitamin D, therefore, has comparatively minimal potential for hepatic accumulation. Product inhibition of the calciferol 25-hydroxylase must, therefore, result from recently synthesized hepatic 25-OH vitamin D, and is not affected by exogenous 25-OH vitamin D3.     

Incubation of Metanephroi with Vitamin D3 Increases Numbers of Glomeruli

To characterize actions of vitamin D3 on metanephroi transplanted from rat embryos to adult recipients, we incubated metanephroi with or without 0.01, 0.1 or 1 ug/ml vitamin D3, 25-hydroxyvitamin D₃ [25(OH)D₃] or 1, 25-hydroxyvitamin D₃ [1,25(OH)2D₃] prior to implantation. The number of glomeruli in developed metanephroi three weeks post-transplantation that had been incubated with 1.0 ug/ml vitamin D₃ was increased relative to the number in metanephroi that were not incubated with vitamin D₃ (control), an effect that was not recapitulated by administration of vitamin D₃ directly to hosts at the time of transplantation. Incubation of metanephroi with 1.0 ug/ml vitamin D₃ also enhanced inulin clearances of metanephroi measured at 12 weeks post-transplantation. The hydroxylated derivative of vitamin D₃, 25(OH)D₃, increased glomerulus number when applied at 0.01 ug/ml but not at higher concentrations, while the twice-hydroxylated derivative 1,25(OH)₂D₃, failed to increase glomerulus number at any concentration tested. We conclude that incubation with vitamin D₃ prior to implantation enhances inulin clearance possibly by increasing the number of glomeruli that develop post-transplantation.

Our findings suggest the vitamin D₃ effect is mediated locally.     

Incubation of Metanephroi with Vitamin D3 Increases Numbers of Glomeruli

To characterize actions of vitamin D3 on metanephroi transplanted from rat embryos to adult recipients, we incubated metanephroi with or without 0.01, 0.1 or 1 ug/ml vitamin D3, 25-hydroxyvitamin D₃ [25(OH)D₃] or 1, 25-hydroxyvitamin D₃ [1,25(OH)2D₃] prior to implantation. The number of glomeruli in developed metanephroi three weeks post-transplantation that had been incubated with 1.0 ug/ml vitamin D₃ was increased relative to the number in metanephroi that were not incubated with vitamin D₃ (control), an effect that was not recapitulated by administration of vitamin D₃ directly to hosts at the time of transplantation. Incubation of metanephroi with 1.0 ug/ml vitamin D₃ also enhanced inulin clearances of metanephroi measured at 12 weeks post-transplantation. The hydroxylated derivative of vitamin D₃, 25(OH)D₃, increased glomerulus number when applied at 0.01 ug/ml but not at higher concentrations, while the twice-hydroxylated derivative 1,25(OH)₂D₃, failed to increase glomerulus number at any concentration tested. We conclude that incubation with vitamin D₃ prior to implantation enhances inulin clearance possibly by increasing the number of glomeruli that develop post-transplantation.Our findings suggest the vitamin D₃ effect is mediated locally.Key Words: kidney, organogenesis, transplantation     

Binding properties of duodenal 1,25-dihydroxyvitamin D3 receptors as affected by phosphorus depletion in lactating goats

1. Capacity and affinity of duodenal 1,25(OH)2D3 receptors were measured in P depleted goats and in control animals kept on an adequate P supply. Plasma concentrations of Pi, Ca and vitamin D3 metabolites and activity of plasma alkaline phosphatase were measured to characterize the effects of P depletion. 2. During P depletion plasma Pi concentrations decreased significantly whereas plasma Ca and alkaline phosphatase activity increased. No changes were recorded for plasma vitamin D3, 25OHD3 and 1,25(OH)2D3 concentrations. 3. P depletion resulted in a significant decrease of the equilibrium dissociation constant Kd of duodenal 1,25(OH)2D3 receptors without affecting the maximum binding capacity.     

1,25-Dihydroxyvitamin D3 stimulated increase of 7,8-didehydrocholesterol levels in rat skin

A convenient, accurate assay was developed for determining skin cholesta-5,7-dien-3 beta-ol (7,8-didehydrocholesterol) concentrations. Ultraviolet spectrophotometry provided quantitation of the sterol from rat skins following saponification and chromatography on Lipidex and high-performance liquid chromatography. Correction for recoveries was accomplished by using 7,8-didehydro[3 alpha-3H]cholesterol as an internal standard. Chronic dosing of vitamin D-deficient rats with 1,25-dihydroxyvitamin D3 caused a 4-fold increase in skin 7-dehydrocholesterol content. This rise was not the result of changes in food consumption, body weight, or plasma calcium. Cholesterol concentrations were not significantly elevated although some of the other nonsaponifiable lipid components found in the high-performance liquid chromatogram appeared to be increased by the treatment. These results suggest that the vitamin D hormone 1,25-(OH)2D3 may exert a positive feedback regulation on the production of vitamin D3 in skin.     

Stimulation of 1,25-dihydroxyvitamin D3 production by 1,25-dihydroxyvitamin D3 in the hypocalcaemic rat.

Serum 1,25-dihydroxyvitamin D3 concentration and renal 25-hydroxyvitamin D 1 alpha-hydroxylase activity were measured in rats fed various levels of calcium, phosphorus and vitamin D3. Both calcium deprivation and phosphorus deprivation greatly increased circulating levels of 1,25-dihydroxyvitamin D3. The circulating level of 1,25-dihydroxyvitamin D3 in rats on a low-calcium diet increased with increasing doses of vitamin D3, whereas it did not change in rats on a low-phosphorus diet given increasing doses of vitamin D3. In concert with these results, the 25-hydroxyvitamin D 1 alpha-hydroxylase activity was markedly increased by vitamin D3 administration to rats on a low-calcium diet, whereas the same treatment of rats on a low-phosphorus diet had no effect and actually suppressed the 1 alpha-hydroxylase in rats fed an adequate-calcium/adequate-phosphorus diet. The administration of 1,25-dihydroxyvitamin D3 to vitamin D-deficient rats on a low-calcium diet also increased the renal 25-hydroxy-vitamin D 1 alpha-hydroxylase activity. These results demonstrate that the regulatory action of 1,25-dihydroxyvitamin D3 on the renal 25-hydroxyvitamin D3 1 alpha-hydroxylase is complex and not simply a suppressant of this system.