Quantifying division of aortal smooth muscle cells in culture stimulated by 1,25(OH)2D3

Inhibitory effect of 1α,25dihydroxycholecalciferol (1,25D₃ = calcitriol) in different cell type is well recognized but its promoting effect on vascular smooth muscle cells (SMCs) is poor established. Therefore, the aim of this study was to determine stimulatory effect of calcitriol on aortal SMCs proliferation in culture. We used the cell division analysis procedure based on the quantitative sequential halving of the stably incorporating fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE). This technique allowed the visualization of cycles of SMCs division by flow cytometry. Rat aortal SMCs were labeled with CFSE and cultured for up to 10 days with defined concentration of calcitriol in medium. Proliferative activity as the percentage of SMCs in different phases of the cell cycle using propidium iodide was determined. Apoptosis was assessed using Annexin-V/CFDA method. The results suggest that low concentrations of an active form of vitamin D—1α,25dihydroxycholecalciferol applied in supraphysiological concentration of 10 nmol/l is a mitogenic factor for aortal SMCs. None of the applied concentrations of calcitriol caused apoptosis. The findings well support our morphological (LM) and ultrastructural (TEM and SEM) observations.     

In vitro study of effects of 1,25 dihydroxyvitamin D3 on the morphology of human breast cancer cell line BT.20

The morphological effect induced by 1,25 dihydroxycholecalciferol - 1,25(OH)2VitD3 - on the malignant human breast cell line BT.20 was studied. This cell line is devoid of oestrogen (ER) and progesterone (PGR) receptors. This effect, which requires treatment for at least 3 days, was evidenced by an increase in the cell projection surface, assessed on scanning electron microscopy (SEM) and by quantimetric analysis, for optimal final concentrations of 1.25(OH)2VitD3 in the medium of the order of 10(-11)M. The cells became more spread out and rounded with many junctional systems; there was occlusion of the intercellular space, but hardly any cells overlapped. The cytoskeleton was considerably developed, with microtubules running parallel to the cell surfaces associated with microfilaments. This positive action on cell differentiation was very similar to that noted regarding the reduction in growth under 1,25(OH)2VitD3 treatment. The two actions were, however, observed at different times and with different concentrations of 1,25(OH)2VitD3 in the medium.     

Potentiation of cell killing by fractionated radiation and suppression of proliferative recovery in MCF-7 breast tumor cells by the Vitamin D3 analog EB 1089

A senescence-like growth arrest succeeded by recovery of proliferative capacity was observed in MCF-7 breast tumor cells exposed to fractionated radiation, 5 × 2 Gy. Exposure to EB 1089, an analog of the steroid hormone 1, 25 dihydroxycholecalciferol (1, 25 dihydroxy Vitamin D₃; calcitriol), prior to irradiation promoted cell death and delayed both the development of a senescent phenotype and the recovery of proliferative capacity. EB 1089 also reduced clonogenic survival over and above that produced by fractionated radiation alone and further conferred susceptibility to apoptosis in MCF-7 cells exposed to radiation. In contrast, EB 1089 failed to enhance the response to radiation (or to promote apoptosis) in normal breast epithelial cells or BJ fibroblast cells. EB 1089 treatment and fractionated radiation additively promoted ceramide generation and suppressed expression of polo-like kinase 1. Taken together, these data indicate that EB 1089 (and 1, 25 dihydroxycholecalciferol or its analogs) could selectively enhance breast tumor cell sensitivity to radiation through the promotion of cell death, in part through the generation of ceramide and the suppression of polo-like kinase.     

1,25-Dihydroxycholecalciferol enhances butyrate-induced p21(Waf1/Cip1) expression

Butyrate, a short-chain fatty acid produced in the colon, as well as its prodrug tributyrin, reduce proliferation and increase differentiation of colon cancer cells. p21(Waf1/Cip1) and p27(Kip1) are negative regulators of cell cycle and are thought to have a key function in the differentiation of various cell lines. We studied the effects of butyrate on differentiation, VDR expression, as well as on p21(Waf1/Cip1) and p27(Kip1) expression in human colon cancer cells (Caco-2). Butyrate induced cell differentiation, which was further enhanced after addition of 1,25-dihydroxycholecalciferol. Synergistic effect of butyrate and dihydroxycholecalciferol in Caco-2 cells was due to butyrate-induced overexpression of VDR. While butyrate as well as dihydroxycholecalciferol increased p21(Waf1/Cip1) and p27(Kip1) expression, in contrast combined exposure of butyrate and dihydroxycholecalciferol resulted in a synergistic amplification of p21(Waf1/Cip1), but not of p27(Kip1) expression. These data imply that butyrate selectively increases p21(Waf1/Cip1) expression via upregulation of VDR in Caco-2 cells.     

Characterization of Vitamin D insensitive prostate cancer cells

The antitumor effects of 1,25-dihydroxyvitamin D₃ (calcitriol) are being exploited for prevention and treatment of prostate cancer (CaP). These studies examined the antiproliferative effects of calcitriol in primary cell cultures derived from transgenic adenocarcinoma of mouse prostate (TRAMP) mice chronically treated with calcitriol (20 μg/kg) or vehicle 3×/week from 4 weeks-of-age until palpable tumors developed. This is a report on the response of two representative control (Vitamin D naïve, naïve) and calcitriol-treated (Vitamin D insensitive, VDI) cells to calcitriol. VDI cells were less sensitive to calcitriol based on less cell growth inhibition and less inhibition of DNA synthesis as measured by MTT and BrdU incorporation assays. Similarly, VDI cells were less sensitive to growth inhibition by the vitamin analog, 19-nor-1,25-dihydroxyvitamin D₂ (paricalcitol). There was no change in apoptosis following treatment of naïve and VDI cells with calcitriol. Vitamin D receptor (VDR) expression was up-regulated by calcitriol in both naïve and VDI cells. In addition, calcitriol induced the Vitamin D metabolizing enzyme, 24-hydroxylase (cyp24) mRNA and enzyme activity similarly in naïve and VDI cells as measured by RT-PCR and HPLC, respectively. In summary, VDI cells are less responsive to the antiproliferative effects of calcitriol. Understanding Vitamin D insensitivity will further clinical development of Vitamin D compounds for prevention and treatment of CaP.     

Falls are associated with decreased renal function and insufficient calcitriol production by the kidney

In a double blind placebo controlled 3-year osteoporosis study in elderly women, we collected prospective data on falls. The study population comprised 489 normal elderly women aged 65–77 years randomized to four groups: placebo, calcitriol 0.25 mcg b.i.d., conjugated equine estrogens (0.625 mg/day) and calcitriol + estrogen. Falls occurred in 57% of all women.

Using a Poisson regression model, the placebo group with low GFR-creatinine clearance (CrCl < 60 ml/min) had 60% more falls compared to the group with CrCl ≥ 60 ml/min. Further sub group analyses showed that there is no increased risk of falls with CrCl 60–70, 70–80 and >90 ml/min. Calcitriol treatment significantly reduced the number of falls by 50% (OR = 0.5, 95% CI: 0.4–0.9, p = 0.010) compared to placebo in the low CrCl group.

The group with lower CrCl had lower calcium absorption (p < 0.001), lower serum 1,25-dihydroxyvitamin D (1,25(OH)₂D) (p < 0.001) and normal serum 25OHD suggesting that there is decreased conversion of 25OHD to 1,25(OH)₂D by the aging kidney. It is postulated that the decrease in falls on calcitriol therapy is related to an increase in serum 1,25(OH)₂D, upregulation of VDR and improvement in muscle strength although one cannot exclude an effect on the central nervous system.     

IL-21 dependent IgE production in human and mouse in vitro culture systems is cell density and cell division dependent and is augmented by IL-10

IL-21 is known to enhance immunoglobulin production using human in vitro models. Using either PBMC or purified tonsilar B cells both stimulated with anti-CD40, IL-4+/-IL-21, this enhancement was shown to correlate with increased cell division especially for IgE and to a lesser extent for IgM and total IgG. Cell division was monitored by CFSE staining and maximum cell division was found at low initial cell plating densities. A correlation between increased cell division and IL-10-mediated enhancement of IgE production was also seen; however, increased cell division plays a smaller role with IL-10 than IL-21. This is further emphasized in that when IL-10 and IL-21 were added together there was a further synergistic increase in IgE seen, but no accompanying further increase in cell division. The mouse system was also examined for IL-21 effects as a function of cell concentration, and as in humans, IL-21 added to murine cells increased IgE production over IL-4/CD40 stimulated cells at lower cell concentrations; however, IL-21 significantly reduced IgE at higher plated cell concentrations.     

Modeling T Cell Proliferation and Death in Vitro Based on Labeling Data: Generalizations of the Smith–Martin Cell Cycle Model

The fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) classifies proliferating cell populations into groups according to the number of divisions each cell has undergone (i.e., its division class). The pulse labeling of cells with radioactive thymidine provides a means to determine the distribution of times of entry into the first cell division. We derive in analytic form the number of cells in each division class as a function of time based on the distribution of times to the first division. Choosing the distribution of time to the first division to fit thymidine labeling data for T cells stimulated in vitro under different concentrations of IL-2, we fit CFSE data to determine the dependence of T cell kinetic parameters on the concentration of IL-2. As the concentration of IL-2 increases, the average cell cycle time is shortened, the death rate of cells is decreased, and a higher fraction of cells is recruited into division. We also find that if the average cell cycle time increases with division class then the qualify of our fit to the data improves.     

Effects of 1,25(OH)2D3, 25OHD3, and EB1089 on cell growth and Vitamin D receptor mRNA and 1alpha-hydroxylase mRNA expression in primary cultures of the canine prostate

The aim of this study was to investigate effects of 1,25(OH)(2)D(3) (calcitriol), 25OHD(3), and EB1089 on cell growth and on Vitamin D receptor (VDR) mRNA and 1alpha-hydroxylase (1alpha-OHase) mRNA expression in normal canine prostatic primary cultures. Canine prostatic epithelial cells were isolated, cultured, and treated with vehicle (ethanol), calcitriol, 25OHD(3), and EB1089 at 10(-9) and 10(-7)M. The VDR was present in epithelial and stromal cells of the canine prostate gland. 1,25(OH)(2)D(3), 25OHD(3), and EB1089 inhibited epithelial cell growth at 10(-7)M compared to vehicle-treated controls [calcitriol (P < 0.01), EB1089 (P < 0.01), and 25OHD(3) (P < 0.05)]. Epithelial cells treated with calcitriol and EB1089 at 10(-7)M had slightly increased VDR mRNA expression (0.2-0.3-fold) at 6 and 12h compared to controls. There was no difference in 1alpha-OHase mRNA expression in epithelial cells treated with these three compounds. 1,25(OH)(2)D(3) and its analogs may be effective antiproliferative agents of epithelial cells in certain types of prostate cancer.     

The involvement of phospholipases C and D in the asymmetric division of subsidiary cell mother cells of Zea mays

In the present study, the involvement of phospholipase C and D (PLC and PLD) pathways in the asymmetric divisions that produce the stomatal complexes of Zea mays was investigated. In particular, the polar organization of microtubules (MTs) and actin filaments (AFs) and the process of asymmetric division were studied in subsidiary cell mother cells (SMCs) treated with PLC and PLD modulators. In SMCs treated with butanol-1 (but-1), which blocks phosphatidic acid (PA) production via PLDs, AF-patch formation laterally to the inducing guard cell mother cell (GMC) and the subsequent asymmetric division were inhibited. In these SMCs, cell division plane determination, as expressed by MT preprophase band (MT-PPB) formation, was not disturbed. Exogenously applied PA partially relieved the but-1 effects on SMCs. In contrast to SMCs, but-1 did not affect the symmetric GMC division. Inhibition of the PLC catalytic activity by neomycin or U73122 resulted in inhibition of asymmetric SMC division, while AF-patch and MT-PPB were organized as in control SMCs. These data show that the PLC and PLD signaling pathways are involved in the transduction and/or perception of the inductive stimulus that is emitted by the GMCs and induces the polar AF organization and asymmetric SMC division. In contrast, division plane determination in SMCs, as expressed by MT-PPB formation, does not depend on PLC and PLD signaling pathways.     

Perturbations in factors that modulate osteoblast functions in vitamin B6 deficiency

It was hypothesized that the widespread structural defect of collagen in connective tissue of vitamin B6 deficient-animals and the consequent alteration in bone biomechanical properties cause an additional stress to their inflamed swollen tibiotarsometatarsal joints. The present study showed a 32% elevation (P < 0.02) in mean plasma free cortisol concentration. Vitamin D metabolism was impaired but without changing plasma calcium homeostasis and bone mineral content. Mean plasma calcitriol [1,25(OH)2D] concentration was significantly reduced (P < 0.001). Because plasma calcidiol concentration did not change, we speculated that either renal 25-hydroxycalciferol-1alpha-hydroxylase activity was reduced or 1,25(OH)2D turnover was increased. Plasma osteocalcin, an index of osteoblast function related to bone formation, was significantly decreased (P < 0.05). This adverse effect on osteoblasts was consistent with the reduction of bone specific alkaline phosphatase activity (another index of bone formation) found in a previous study. The excess of cortisol may have impaired these bone cells functions directly and (or) indirectly via the decline in calcitriol synthesis. Plasma hydroxyproline concentrations in B6-deficient animals were found to be significantly reduced (P < 0.001), suggesting that cortisol in excess had also a suppressive effect on another hydroxylase, namely tissue (mainly bone and liver) prolyl hydroxylase. The bone uncoupling (in formation and resorption) associated with vitamin B6 deficiency seems to be due to secondary hypercortisolism and (or) another unknown factors but not related to a change in bone modulators such as IGF-1 and eicosanoids.     

Caffeine decreases vitamin D receptor protein expression and 1,25(OH)2D3 stimulated alkaline phosphatase activity in human osteoblast cells

Of the various risk factors contributing to osteoporosis, dietary/lifestyle factors are important. In a clinical study we reported that women with caffeine intakes >300 mg/day had higher bone loss and women with vitamin D receptor (VDR) variant, tt were at a greater risk for this deleterious effect of caffeine. However, the mechanism of how caffeine effects bone metabolism is not clear. 1,25-Dihydroxy vitamin D₃ (1,25(OH)₂D₃) plays a critical role in regulating bone metabolism. The receptor for 1,25(OH)₂D₃, VDR has been demonstrated in osteoblast cells and it belongs to the superfamily of nuclear hormone receptors. To understand the molecular mechanism of the role of caffeine in relation to bone, we tested the effect of caffeine on VDR expression and 1,25(OH)₂D₃ mediated actions in bone. We therefore examined the effect of different doses of caffeine (0.2, 0.5, 1.0 and 10 mM) on 1,25(OH)₂D₃ induced VDR protein expression in human osteoblast cells. We also tested the effect of different doses of caffeine on 1,25(OH)₂D₃ induced alkaline phosphatase (ALP) activity, a widely used marker of osteoblastic activity. Caffeine dose dependently decreased the 1,25(OH)₂D₃ induced VDR expression and at concentrations of 1 and 10 mM, VDR expression was decreased by about 50–70%, respectively. In addition, the 1,25(OH)₂D₃ induced alkaline phosphatase activity was also reduced at similar doses thus affecting the osteoblastic function. The basal ALP activity was not affected with increasing doses of caffeine. Overall, our results suggest that caffeine affects 1,25(OH)₂D₃ stimulated VDR protein expression and 1,25(OH)₂D₃ mediated actions in human osteoblast cells.     

Steroid inhibition of calcitriol-induced prolactin production in GH4C1 cells. Specificity and sensitivity.

The induction of prolactin (PRL)-gene expression by calcitriol (1,25-dihydroxyvitamin D3, 1,25-dihydroxycholecalciferol) in clonal rat pituitary tumour (GH4C1) cells was selectively inhibited by cortisol [IC50 (concentration causing 50% inhibition) = 3.2-4.1 nM]. The steroid specificity of this effect was investigated and various steroids were found to inhibit calcitriol-stimulated PRL production with the following relative potencies: cortisol, 1; dexamethasone, 8; 11-deoxycortisol, 0.5; corticosterone, 0.4; aldosterone, 0.07; testosterone and oestradiol, less than 0.003. The steroid antagonist RU 38486 did not affect basal or calcitriol-stimulated PRL production, but antagonized the effect of 10 nM-cortisol in a concentration-dependent manner. Neither progesterone nor 11-deoxycortisol antagonized the effect of 10 nM-cortisol. Calcitriol-induced PRL production was 14 times more sensitive to dexamethasone inhibition than was non-stimulated PRL production. Growth-hormone production was stimulated by dexamethasone, in the presence or absence of calcitriol, with a concentration-dependence similar to that of dexamethasone inhibition of basal PRL production. These data indicate that steroid inhibition of calcitriol-stimulated PRL production is a specific glucocorticoid effect. The sensitivity of calcitriol-stimulated PRL production to dexamethasone was 14-26-fold greater than that of other measured responses in the same cells. Two of the possible explanations for this selectively increased sensitivity to glucocorticoids are: amplification of the glucocorticoid effect via an induced mediator; and the presence of very-high-affinity glucocorticoid-receptor-binding sites on DNA.     

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies¹. To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia^2-8. The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma^2,7,8. The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds⁹. The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division¹⁰. CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity². This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry². The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.     

Calcitriol Modulates the CD46 Pathway in T Cells

The complement regulator CD46 is a costimulatory molecule for human T cells that induces a regulatory Tr1 phenotype, characterized by large amounts of IL-10 secretion. Secretion of IL-10 upon CD46 costimulation is largely impaired in T cells from patients with multiple sclerosis (MS). Vitamin D can exert a direct effect on T cells, and may be beneficial in several pathologies, including MS. In this pilot study, we examined whether active vitamin D (1,25(OH)₂D₃ or calcitriol) could modulate the CD46 pathway and restore IL-10 production by CD46-costimulated CD4+ T cells from patients with MS. In healthy T cells, calcitriol profoundly affects the phenotype of CD46-costimulated CD4+ T cells, by increasing the expression of CD28, CD25, CTLA-4 and Foxp3 while it concomitantly decreased CD46 expression. Similar trends were observed in MS CD4+ T cells except for CD25 for which a striking opposite effect was observed: while CD25 was normally induced on MS T cells by CD46 costimulation, addition of calcitriol consistently inhibited its induction. Despite the aberrant effect on CD25 expression, calcitriol increased the IL-10:IFNγ ratio, characteristic of the CD46-induced Tr1 phenotype, in both T cells from healthy donors and patients with MS. Hence, we show that calcitriol affects the CD46 pathway, and that it promotes anti-inflammatory responses mediated by CD46. Moreover, it might be beneficial for T cell responses in MS.     

Estimating Lymphocyte Division and Death Rates from CFSE Data

The division tracking dye, carboxyfluorescin diacetate succinimidyl ester (CFSE) is currently the most informative labeling technique for characterizing the division history of cells in the immune system. Gett and Hodgkin [Nat. Immunol. 1:239–244, 2000] have pioneered the quantitative analysis of CFSE data. We confirm and extend their data analysis approach using simple mathematical models. We employ the extended Gett and Hodgkin [Nat. Immunol. 1:239–244, 2000] method to estimate the time to first division, the fraction of cells recruited into division, the cell cycle time, and the average death rate from CFSE data on T cells stimulated under different concentrations of IL-2. The same data is also fitted with a simple mathematical model that we derived by reformulating the numerical model of Deenick et al. [J. Immunol. 170:4963–4972, 2003]. By a non-linear fitting procedure we estimate parameter values and confidence intervals to identify the parameters that are influenced by the IL-2 concentration. We obtain a significantly better fit to the data when we assume that the T cell death rate depends on the number of divisions cells have completed. We provide an outlook on future work that involves extending the Deenick et al. [J. Immunol. 170:4963–4972, 2003] model into the classical smith-martin model, and into a model with arbitrary probability distributions for death and division through subsequent divisions.     

Role of monocytes in the inhibitory effect of calcitriol on PHA-stimulated lymphocytes

The possible role played by monocytes in the inhibitory effect of calcitriol on phytohemagglutinin (PHA)-stimulated lymphocyte proliferation was assessed by testing the effect of this sterol under different cell culture conditions. Calcitriol had a dose-dependent inhibitory effect on lymphocyte proliferation in concentrations ranging from 10(-10) up to 10(-8) M. The effect of 10(-9) M calcitriol was almost completely abolished by: a) monocyte depletion, b) inhibition of prostaglandin (PG) synthesis by indomethacin, and c) addition of exogenous interleukin-2 (IL-2). These results suggested that the inhibitory effect of calcitriol was mediated through monocytes. This possibility was substantiated by the following observations: a) the calcitriol inhibitory effect was restored when autologous adherent cells were added to monocyte-depleted PBM cells; b) the supernatant of adherent cells cultured for 24 hours in the presence of calcitriol exerted a marked inhibitory effect on lymphocyte proliferation; and c) this effect was not longer evident when adherent cells were cultured in the presence of calcitriol plus indomethacin. These data support the hypothesis that calcitriol acts, at least partially, through the monocytes, inducing an increased release of PG, with subsequent inhibition of IL-2 synthesis, then resulting in a decreased lymphocyte proliferation.     

1,25-(OH)2-16ene-23yne-D3 reduces secondary hyperparathyroidism in uremic rats with little calcemic effect

OBJECTIVES: To compare the effects of vitamin D analogs versus calcitriol on serum levels of Ca, P and parathyroid hormone (PTH). A compound better than calcitriol should increase the Ca x P product less than calcitriol for an equivalent decrease in PTH levels. METHODS: Biological activity of 4 vitamin D analogs, 1,25-(OH)(2)-16ene- D(3) (RO(1)), 1,25-(OH)(2)-16ene-23yne-D(3) (RO(2)), 1,25-(OH)(2)-26,27-hexafluoro-16ene-23yne-D(3) (RO(3)) and 1,25-(OH)(2)-16ene-23yne-26,27-hexafluoro-19nor-D(3) (RO(4)) was tested vs. calcitriol in parathyroidectomized rats. In a second set of experiments, the effects of RO(2), RO(4) and calcitriol were studied in 5/6 nephrectomized rats with secondary hyperparathyroidism. RESULTS: In parathyroidectomized rats, all analogs (250 pmol/day) led calcemia to rise after 7 days. In uremic rats, all treatments reduced PTH levels. RO(4) revealed toxicity. RO(2) was as effective as calcitriol in suppressing PTH in a dose dependent manner. Mean plasma ionized calcium did not change from baseline to day 14 and day 28 on RO(2) (250 or 500 pmol/day) whereas it increased significantly on RO(2) (1,000 pmol/day) and calcitriol (125 or 250 pmol/day). Increasing the dose of calcitriol led Ca x P to rise more dramatically than increasing the dose of RO(2), which appears to have a wider therapeutic window than calcitriol. CONCLUSION: 1,25-(OH)(2)-16ene-23yne-D(3) (RO(2)) may represent a novel candidate for the treatment of renal osteodystrophy in humans.     

1,25-Dihydroxyvitamin D3 enhances the generation of nonspecific suppressor cells while inhibiting the induction of cytotoxic cells in a human MLR.

The active vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25-D3), has been shown in both in vitro and in vivo experiments to be immunoregulatory. We analyzed the effects of the hormone on the human mixed lymphocyte reaction (MLR), the in vitro model of allograft response. Suppressor-cell activity of MLR-generated effector cells was enhanced by calcitriol (10(-10) to 10(-8) M). This suppressor activity was nonspecific since calcitriol-generated effector cells could suppress a primary MLR with stimulators and/or responders heterologous to the effector-generating MLR. Calcitriol (10(-9) to 10(-7) M) was also effective in preventing the generation of cytotoxic T cells when tested in a 51Cr release assay. While no differences were observed in the phenotypic analyses of the MLR-generated effector cells between 1,25-D3-treated cells and control cells, a significant reduction of class II antigen expression was observed in the presence of the hormone. The effects of calcitriol on human MLR are similar to those observed with cyclosporine.