In vitro effects of everolimus and intravenous immunoglobulin on cell proliferation and apoptosis induction in the mixed lymphocyte reaction.

Targeting multiple pathways in the activation of alloimmune responses by multi-drug immunosuppressive regimens with complementary mechanisms of action enhances allograft survival and improves quality of life, owing to the reduction of adverse drug effects. In this report we investigated the effect of the combination of everolimus and intraveneous immunoglobulin (IVIG) on cell proliferation and apoptosis induction in human two-way mixed lymphocyte reaction (MLR). Everolimus alone (0.1-50 ng/ml) and IVIG (1-10 mg/ml) alone inhibited cell proliferation in a dose-dependent manner (16.4-67.2% and 12.1-66.3% inhibition, respectively). The inhibition by everolimus was not enhanced in the presence of 1 mg/ml IVIG. Addition of 10 and 50 ng/ml everolimus increased the inhibitory effect of 5 and 10 mg/ml IVIG, but only by 10-27%. Addition of 0.1 and 1 ng/ml everolimus did not increase IVIG's inhibitory effects. Apoptosis was significantly higher in IVIG (5 mg/ml)-treated CD19+ cells and less so in CD3+ cells as assessed by Annexin V and TUNEL assays. However, everolimus (0.1-50 ng/ml) did not induced apoptosis or alter apoptosis induced by IVIG. These results suggest that everolimus is a potent inhibitor of immune cell proliferation but does not act additively or synergistically with IVIG when analyzed in this in vitro system.

Substrate and enzyme trafficking as a means of regulating 1,25-dihydroxyvitamin D synthesis and action: the human innate immune response.

Tissue availability of the active vitamin D metabolite, 1,25-dihydroxyvitamin D [1,25(OH)(2)D] is dependent on expression of the activating enzyme 1alpha-hydroxylase (CYP27b1) and its catabolic counterpart 24-hydroxylase (CYP24). The activity of these two enzymes is in turn controlled by factors including affinity of the serum vitamin D-binding protein (DBP) for 25-hydroxyvitamin D [25(OH)D]; the availability of enzyme cofactors; and the relative amount of hydroxylase gene product expressed. In recent years, it has become clear that directed trafficking of substrate and enzyme is also a pivotal component of the regulated process of hormone synthesis by both renal and extrarenal tissues expressing the CYP27b1 and CYP24 genes. Extracellular regulatory trafficking events are defined by the quantity of substrate 25(OH)D entering the circulatory pool. Entry into some target cells in vivo, such as the macrophage and proximal renal tubular epithelial cells, requires 25(OH)D binding to serum DBP, followed by recognition, internalization, and intracellular release. The "released" intracellular substrate is moved to specific intracellular destinations (i.e., the mitochondrial CYP enzymes and the vitamin D receptor [VDR]) by the hsc70 family of chaperone proteins. Synthesis of 1,25(OH)(2)D is also regulated by CYP24 and its metabolically inactive splice variant CYP24-SV. Finally, initiation of transcription of 1,25(OH)(2)D-regulated genes, such as the CYP24, requires movement of the CYP27b1 product, 1,25(OH)(2)D, to the VDR in the same cell for intracrine action or export to another cell for paracrine action. In either case, the 1,25(OH)(2)D ligand is required for the VDR to heterodimerize with the retinoid x receptor and compete away the dominant-negative acting, heterogeneous nuclear ribonucleoprotein (hnRNP)-related, vitamin D response element-binding proteins that inhibit hormone-directed transactivation of genes. In this review, we use vitamin D-directed events in the human innate immune response to Mycobacterium tuberculosis as a physiologically relevant model system in which to highlight the importance of these intracellular traffic patterns.

Adenosine 5'-triphosphate-dependent vitamin D sterol binding to heat shock protein-70 chaperones.

Chaperone proteins in the heat shock protein-70 family possess endogenous ATP binding and ATPase activity and interact with intracellular protein substrates in an ATP-dependent manner; the hydrolysis of ATP to ADP results in an increase in the affinity of the chaperone for protein substrates. Heat shock protein-70s can also specifically interact with 25-hydroxylated vitamin D metabolites. Using constitutively expressed heat shock protein-70 (hsc70) as chaperone, here we demonstrate that vitamin D metabolite binding to hsc70 is also ATP dependent. Transient overexpression of an hsc70-green fluorescent protein chimeric construct in primate kidney cells resulted in a 6-fold increase in specific, extractable 25-hydroxyvitamin D(3) binding. When ATPase capability of hsc70 was disabled, this increase was completely blocked. In solution, the binding of 25-hydroxylated vitamin D metabolites to hsc70 was significantly increased (P < 0.01) in the presence of ATP and a nonmetabolizable ATP analog. The ATP-directed increase in specific binding resulted from an increase in the abundance of relatively high-affinity hormone-binding sites (K(d), approximately 0.24 nM). These results suggest that ATP hydrolysis to ADP would favor the release of vitamin D from a donor hsc70 molecule at a time when an hsc70-bound acceptor protein substrate is anchored to the chaperone with relative avidity. We theorize that the endogenous ATPase activity of hsc70 promotes the transfer of vitamin D sterols to other intracellular vitamin D binding proteins, such as the vitamin D receptor and vitamin D hydroxylases, to which hsc70 is known to bind.

Response element binding proteins and intracellular vitamin D binding proteins: novel regulators of vitamin D trafficking, action and metabolism.

Using vitamin D-resistant New World primates as model of natural diversity for sterol/steroid action and metabolism, two families of novel intracellular vitamin D regulatory proteins have been discovered and their human homologs elucidated. The first family of proteins, heterogeneous nuclear ribonucleoproteins (hnRNPs), initially considered to function only as pre-mRNA-interacting proteins, have been demonstrated to be potent cis-acting, trans-dominant regulators of vitamin D hormone-driven gene transactivation. The second group of proteins bind 25-hydroxylated vitamin D metabolites. Their overexpression increases vitamin D receptor (VDR)-directed target gene expression. We found that these intracellular vitamin D binding proteins (IDBPs) are homologous to proteins in the heat shock protein-70 family. Our ongoing studies indicate directly or indirectly through a series of protein interactions that the IDBPs interact with hydroxylated vitamin D metabolites and facilitate their intracellular targeting.

Regulation of 1,25-dihydroxyvitamin d synthesis by intracellular vitamin d binding protein-1.

Control of 125-dihydroxyvitamin D (1,25-(OH)2D) synthesis is believed to be primarily at the level of expression of the vitamin D-1-hydroxylase (CYP1alpha; CYP1alpha) gene. Once transcribed, generation of product, as catalyzed by 1-hydroxylase, depends upon the availability of various co-factors, molecular oxygen, electrons as well as substrate to the enzyme. Here we provide evidence that the quantity of product 1,25-(OH)2D generated also relies on the presence and level of expression of the intracellular vitamin D binding protein-1 (IDBP-1) and its capacity to promote 24-hydroxylase (CYP24) gene expression. Stable transfection of the IDBP-1 cDNA increased 1,25-(OH)2D synthesis up to 700% (p < 0.001) in cells devoid of 24-hydroxylating potential but only 70% (p = 0.018) in cells in which the CYP24 gene is expressed. IDBP-1-mediated increase in 1,25-(OH)2D production was independent of any change in CYP1alpha expression but highly dependent on the ability of exogenously-added or endogenously-synthesized 1,25-(OH)2D to stimulate CYP24 gene expression. These data suggest that IDBP-1 is capable of controlling 1,25-(OH)2D production by modulating the delivery of 1) substrate 25-OHD to in the mitochondrial CYP1alpha gene product and 2) CYP1alpha product 1,25-(OH)2D to the vitamin D receptor for upregulation of expression of the catabolic CYP24 gene.

Absence of thyroid hormone resistance in vitamin D-;resistant new world primates.

New World primates express a form of sterol/steroid resistance resulting from the presence of a 58-60 kDa intracellular protein (IDBP) which competes with nuclear receptor proteins for binding of vitamin D metabolites and sex steroids. As the thyroid hormone receptor is a recognized member of the steroid/retinoid/vitamin D receptor gene superfamily, we sought to confirm previous data describing the existence of thyronine resistance in New World primate species and to determine whether IDBP was capable of binding ligands which confer transcriptional regulatory potential on thyroid hormone-retinoid heterodimeric complexes. Circulating thyroid hormone levels were compared between vitamin D-resistant New World primates and nonresistant Old World primates. Total triiodothyronine (T3), free T3 index, and free thyroxine were not different between New World and Old World primates. Thyroxine was significantly lower (P < 0.03) in New World than in Old World primates. T3 (1 pM-1µM), 9-cis retinoic acid (100 nM) and all-trans retinoic acid (100 nM) were incapable of displacing 2 nM [3H]25-hydroxyvitamin D3 from IDBP extracted from B95-8 cells, a B-lymphoblastoid cell line derived from the vitamin D-resistant New World primate Callithrix jacchus. We conclude that the sterol/steroid-resistant state characteristic of many genera of New World primates does not extend to thyroid hormones and that the IDBP responsible for vitamin D resistance does not bind T3, 9-cis retinoic acid, and all-trans retinoic acid. © 1994 Wiley-Liss, Inc.

Influence of ultraviolet B radiation on vitamin D3 metabolism in vitamin D3-resistant New World primates.

We investigated the occurrence of rickets in adolescent tamarins (Saguinus imperator) residing at the Los Angeles Zoo. Compared to tamarins in the same colony without clinical evidence of bone disease (N = 6), rachitic platyrrhines (N = 3) had a decrease in their serum calcium concentration (P < .05). The affected tamarins also had lower serum 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) levels than did nonaffected colony mates, but 2-10-fold higher concentrations than in Old World primates of a comparable developmental stage. New World primates in many different genera are known to exhibit target organ resistance to the active vitamin D3 metabolite, 1,25-(OH)2D3, compensated by maintenance of high circulating concentrations of 1,25-(OH)2D3. The relatively low serum 1,25-(OH)2D3 concentration in rachitic tamarins and ultraviolet B radiation deficient environment of these primates suggested that bone disease may be linked to a deficiency in substrate for 1,25-(OH)2D3, 25 hydroxyvtamin D3 (25-OHD3). Chronic exposure of platyrrhines in three different vitamin D resistant genera to an artificial UVB source resulted in 1) a significant increase in the mean serum 25-OHD3 (P < .001) and 1,25-(OH)2D3 (P < .02) level over that encountered in platyrrhines not exposed to UVB; and 2) prevention of rachitic bone disease in irradiated individuals. These data further show that the serum 25-OHD3 and 1,25-OH2D3 levels are positively correlated in vitamin D-resistant platyrrhines (r = 0.64; P= .0014) and suggest that a compromise in cutaneous vitamin D3 production by means of UVB deprivation may limit necessary 1,25-(OH)2D3 production. © 1992 Wiley-Liss, Inc.

Immunoreactive parathyroid hormone levels in platyrrhini and catarrhini: A comparative analysis with three different assays.

Serum concentrations of the hormonal form of vitamin D3-1,25-dihydroxy-vitamin D3 [1,25-(OH)2-D3]-are elevated in many genera of platyrrhines when compared to catarrhines; this elevation is presumed to result from a decrease in the ability of the target cell receptor effectively to recognize 1,25-(OH)2-D3. The activity of the renal 25-hydroxyvitumin D3-1α-hydroxylase, the mammalian enzyme which synthesizes the majority of the circulating 1,25-(OH)2-D3, is accelerated by parathyroid hormone (PTH). In order to determine whether the elevated serum concentrations of 1,25-(OH)2-D3 in platyrrhines were the result of relative hyperparathyroidism, we measured serum levels of immunoreactive parathyroid hormone (iPTH) in normocalcemic platyrrhines, catarrhines, and human subjects with assays that recognize different domains of the human PTH molecule. Antisera directed against the biologically active, aminoterminus of PTH yielded comparable mean values for iPTH among three test groups. The mean concentration of iPTH as assessed by a "proximal" midregion assay was significantly reduced in platyrrhine serum when compared to either human or catarrhine serum. A "distal" midregion assay yielded a reduced mean value for iPTH in both platyrrhine and catarrhine serum when compared to human serum. These data suggest that 1) high circulating levels of 1,25-(OH)2-D3 in New World primates are not the result of hyperparathyroidism; and 2) structural homology between human and primate PTH diminishes progressively as one moves toward the carboxyterminus of the molecule and is lost more rapidly in the platyrrhine than in the catarrhine hormone.

Serum concentrations of 1,25-dihydroxyvitamin D3 in Platyrrhini and Catarrhini: A phylogenetic appraisal.

We measured the serum concentration of 25-hydroxyvitamin D3 (25-OH-D3) and 1,25-dihydroxyvitamin D3 (1,25-[OH]2-D3) in 23 different Platyrrhines from four different genera and in 21 Catarrhines from six different genera in residence at the Los Angeles Zoo. The mean (±S.E.) serum concentration of 1,25-(OH)2-D3 was significantly greater in Platyrrhines (810 ± 119 pg/ml) than in Catarrhines (61 ± 5 pg/ml), suggesting that high circulating concentrations of the active vitamin D hormone were a characteristic of New World primates in both the Cebidae and Callitrichidae family. This increase in the serum concentration of 1,25-(OH)2-D3 is probably an adaptational response on the part of Platyrrhini to offset a relative decrease in the concentration of specific receptor for 1,25-(OH)2-D3 in target tissues for the hormone.