Helmut Reichel

γ-interferon stimulates production of 1,25-dihydroxyvitamin D3 by normal human macrophages

We show for the first time that normal human pulmonary alveolar macrophages (PAM) markedly enhance their basal rate of the production of [3H]-1,25(OH)2D3 when cultured in the presence of recombinant gamma-interferon (gamma-IFN). The rate of conversion of [3H]-25(OH)D3 to [3H]-1,25(OH)2D3 was dose-dependent in a linear fashion. A maximal production of 1,25(OH)2D3 by PAM occurred after exposure of PAM to gamma-IFN for one day. This maximum plateau-level was sustained for at least five days. The authenticity of the putative 1,25(OH)2D3 obtained from PAM was tested by demonstrating the exact comigration of [3H]-1,25(OH)2D3 with chemically synthesized 1,25(OH)2D3 in four different HPLC-systems.

Lymphocyte cell lines from vitamin D-dependent rickets type II show functional defects in the 1α,25-dihydroxyvitamin D3 receptor

Lymphocyte cell lines were established from five patients with vitamin D-dependent rickets, type II (VDDR-II). These lines were established by infection with human T-lymphotrophic virus type I (HTLV-I). Binding of [3H]1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) to its receptor in these cell lines was compared to binding studies using a T-lymphocyte cell line (S-LB1) from a normal individual. The 1,25(OH)2D3 receptor of S-LB1 was comparable to the well-characterized chick intestinal 1,25(OH)2D3 receptor in terms of its ligand binding affinity and capacity, its mobility on 5-20% sucrose gradients, and its adsorption to and elution properties from DNA-cellulose. Three cell lines established from patients with VDDR-II (Rh-VDR, Sh-VDR, and Ab-VDR) showed no specific binding of 1,25(OH)2D3 to a receptor and treatment of the cultured cells with 1,25(OH)2D3 did not stimulate production of 24,25-dihydroxy-vitamin D3 (24,25(OH)2D3), a response which is diagnostic of the presence of a functional 1,25(OH)2D3 receptor. In a fourth cell line, A1-VDR, the receptor for 1,25(OH)2D3 had a low binding capacity and 25(OH)D3-24-hydroxylase activity was not detectable. Induction of 24,25-(OH)2D3 synthesis by 1,25(OH)2D3 was observed in the fifth cell line, designated Ro-VDR, although the sensitivity to hormone treatment was lower than in the control cell line from a normal donor. The capacity of the receptor for 1,25(OH)2D3 was low in Ro-VDR. In all cell lines where 1,25(OH)2D3 binding to a receptor was detectable, the receptor had the typical sedimentation coefficient of 3.7 S on sucrose density gradient analysis. Binding and elution properties to DNA-cellulose, however, differed from normal in both Ro-VDR and A1-VDR cells where elution from DNA-cellulose occurred at a lower salt concentration than is typical of the 1,25(OH)2D3 receptor. While Ro-VDR cells showed typical nuclear localization of the unoccupied 1,25(OH)2D3 receptor, neither the unoccupied nor the occupied receptor from A1-VDR cells was completely localized in the nucleus. In a series of functional studies we found that modulation of the level of the mRNAs coding for both the c-myc oncogene and the growth factor known as granulocyte-monocyte colony stimulating activity by 1,25(OH)2D3 correlated with the 1,25(OH)2D3 receptor status of these cells. Use of these cell lines will facilitate further study of the molecular defect(s) in the receptor for 1,25(OH)2D3 in vitamin D-dependent rickets type II and will allow a correlation with impairment of cellular functions.

Regulation of 25-hydroxyvitamin D3 metabolism in a human promyelocytic leukemia cell line (HL-60): 1,25-dihydroxyvitamin D3 stimulates the synthesis of 24,25-dihydroxyvitamin D3.

The human promyelocytic leukemia cell line HL-60 undergoes macrophage-like differentiation after exposure to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the biologically active metabolite of vitamin D3. In the current study, we demonstrate that 1,25(OH)2D3 also regulates 25-hydroxyvitamin D3 [25(OH)D3] metabolism in HL-60 cells. The presence of 1,25(OH)2D3 in the culture medium of HL-60 cells stimulated the conversion of 7-10% of the substrate [25(OH)D3] to a more polar metabolite, which was identified as 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] from the elution positions on sequential HPLC systems and the sensitivity to periodate treatment. The HL-60 subclone HL-60 blast, which is unresponsive to 1,25(OH)2D3 in terms of differentiation, also responded to 1,25(OH)2D3 treatment with the production of 24,25(OH)2D3. Maximal stimulation of 24,25(OH)2D3-synthesis (approximately 7 pmol/5 X 10(6) cells) in HL-60 cells was noted with a 12-h exposure to 10(-9) M 1,25(OH)2D3. The ability of vitamin D3 metabolites other than 1,25(OH)2D3 to induce the synthesis of 24,25(OH)2D3 in HL-60 cells was, with the exception of 1 alpha-hydroxyvitamin D3, in correlation with their reported affinities for the specific 1,25(OH)2D3 receptor which is present in HL-60 cells. Treatment of HL-60 cells with phorbol diesters abolished the 1,25(OH)2D3 responsiveness, while treatment with dimethylsulfoxide and interferon gamma did not markedly alter the 25(OH)D3 metabolism of HL-60 cells. Small amounts (approximately 1% of substrate) of two 25(OH)D3 metabolites, which comigrated with 5(E)- and 5(Z)-19-nor-10-keto-25-hydroxyvitamin D3 on two HPLC solvent systems, were synthesized by HL-60 cells, independently from 1,25(OH)2D3 treatment or stage of cell differentiation. Our results indicate that 1,25(OH)2D3 influences 25(OH)D3 metabolism of HL-60 cells independently from its effects on cell differentiation.

Evidence for 1,25-dihydroxyvitamin D3 production by cultured porcine alveolar macrophages.

Previous studies have demonstrated that human alveolar and bone marrow macrophages when activated in vitro can metabolize 25-hydroxyvitamin[3H]D3 to 1 alpha,25-dihydroxyvitamin[3H]D3; however, to date no animal models to study this system have been available. In the present study, cultured porcine pulmonary alveolar macrophages from two animals were assayed for their capability for metabolism of 25-hydroxyvitamin[3H]D3. The porcine alveolar macrophages constitutively produced a metabolite of 25-hydroxyvitamin[3H]D3 which was identified as 1 alpha,25-dihydroxyvitamin[3H]D3 by high performance liquid chromatography. The apparent KM was in the range of 300 nM. Unlike human macrophages, treatment of porcine alveolar macrophages with lipopolysaccharide did not stimulate 1 alpha,25-dihydroxyvitamin[3H]D3 production. Addition of 1 alpha,25-dihydroxyvitamin-D3 to macrophages cultures led to a sensitive proportional inhibition of 1 alpha,25-dihydroxyvitamin[3H]D3 synthesis.

Synthesis and biological activity of 1α,23,25,26-tetrahydroxyvitamin D3

Abstract The metabolic pathway from 1α,25-dihydroxyvitamin D3 [1α,25-(OH)2D3] to 1α,25-dihydroxyvitamin D3-26,23-lactone includes the formation of 1α,23,25,26-tetrahydroxyvitamin D3 [1α,23,25,26-(OH)4D3]. The aim of the current study was to explore the as yet unknown biological properties of this vitamin D3 sterol. The four diastereoisomers of 1α,23,25,26-(OH)3D3 were chemically synthesized. They were compared to 1α,25-(OH)2D3 in terms of their affinity for the chick intestinal 1α,25-(OH)2D3 receptor and their biologic activity in vivo (stimulation of intestinal calcium absorption and mobilization of calcium from bone in vitamin D-deficient rats). The 1,25-(OH)2D3 receptor binding affinities of 1α,23(R)25(R)26-(OH)4D3, 1α,23(S)25(S)26-(OH)4D3, 1α,23(S)25(R)26-(OH)4D3, and 1α,23(R)25(S)26-(OH)4D3 were 11,100,216, and 443 times weaker than the binding affinity of 1α,25-(OH)2D3, respectively. Compared to 1α,25-(OH)2D3, the relative capacities of the 1α,23,25,26-(OH)4D3 compounds to stimulate intestinal calcium absorption were 1 4 for 1α,23(R)25(R)26-(OH)4D3; 1 19 for 1α,23(S)25(S)26-(OH)4D3; 1 90 for 1α,23(S)25(R)26-(OH)4D3; and 1 136 for 1α,23(R)25(S)26-(OH)4D3. Maximal stimulation of intestinal calcium transport occurred 8 h after administration of vitamin D3 metabolites. Mobilization of calcium from bone was quantitated by serum calcium concentration measurements. The activities of 1α,23(R)25(R)26-(OH)4D3, 1α,23(S)25(S)26-(OH)4D3, 1α,23(S)25(R)26-(OH)4D3, and 1α,23(R)25(S)26-(OH)4D3 to increase serum calcium were estimated to be 4, 13, 43, and 69 times weaker than that of 1α,25-(OH)2D3, respectively. These results illustrate the stereospecificity of the chicken intestine 1α,25-(OH)2D3 receptor for binding of 1α,23,25,26-(OH)4D3 and suggest that the 1α,23,25,26-(OH)4D3 exerts its biological activity in the rat through an interaction with 1,25-(OH)2D3 receptors. In summary, the 1α,23,25,26-(OH)4D3 had a markedly lower biological activity than 1α,25-(OH)2D3.

Interaction of 1,25 Dihydroxyvitamin D3 with Normal and Abnormal Hematopoiesis

A diagram of the human hematopoietic system with their stem cells is shown on Figure 1. In vitro clonogenic studies in the murine and human systems have shown that several lymphokines, known as colony stimulating factors (CSF), induce proliferation and differentiation of hematopoietic stem cells. The myeloid stem cell known as the granulocyte-monocyte colony forming cell (GM-CFC) can differentiate either to macrophage colonies when grown in the presence of macrophage or granulocyte-macrophage CSF or differentiate to granulocyte colonies in the presence of granulocyte or granulocyte-macrophage CSF. Few studies have examined the ability of other physiological substances to influence differentiation of myeloid stem cells. Recently 1,25 dihydroxyvitamin D3[1,25(OH)2D3] was found to induce cells from both a murine myeloid leukemia line known as Ml (1) and a human promyelocytic leukemia line (HL-60) (2–5) to differentiate to monocyte-macrophage like cells (Fig. 2, Table 1).