Immunomodulatory effects of vitamin D in peripheral blood monocyte-derived macrophages from patients with rheumatoid arthritis

1,25-Dihydroxyvitamin D (1,25(OH)₂D₃), the active form of vitamin D, modulates both innate and adaptive immune responses. Emerging epidemiological data has also demonstrated disease-modifying and immunomodulatory effects of vitamin D in a wide range of human autoimmune diseases, including rheumatoid arthritis (RA). To evaluate in vitro effects of 1,25(OH) ₂D₃ in primary cultures of peripheral blood monocyte-derived macrophages of RA patients, monocyte/macrophages, isolated from peripheral blood mononuclear cells of RA patients and healthy subjects by exploiting their ability to adhere to plastic, were treated with increasing concentrations of 1,25(OH)₂D₃ for 48 h. TNF-α, IL-1 α, IL-1β, IL-6 and RANKL production was determined by ELISA and nitric oxide (NO) release using the Griess method. Immunocytochemistry analysis was also performed to evaluate alterations in transmembrane TNF-α expression after 1,25(OH) ₂D₃ treatment. A significant dose-dependent decrease in TNF-α and RANKL production by cultured RA macrophages after 1,25(OH)₂D₃ treatment was found, whereas a significant reduction in normal cells was observed only at higher concentrations. IL-1 α, IL-1β and IL-6 levels were reduced by 1,25(OH) ₂D₃ at higher concentrations in all cell populations. TNF-α immunostaining was less intense in treated cells compared with untreated. 1,25(OH) ₂D₃ significantly reduced NO levels regardless of the concentration used. Vitamin D downregulated proinflammatory mediators in monocyte-derived macrophages, and RA cells appeared more sensitive than normal cells. These effects further provide a rationale for the therapeutic value of vitamin D supplementation in the treatment for RA.     

1,25OH-Vitamin D3 and IL-17 Inhibition Modulate Pro-Fibrotic Cytokines Production in Peripheral Blood Mononuclear Cells of Patients with Systemic Sclerosis

Objectives: IL-17 modulates the synthesis of several molecules involved in the pathogenesis of Systemic Sclerosis (SSc). Vitamin D (1,25(OH)₂D3) shows anti-fibrotic properties and it is able to affect the IL-17 production in several experimental conditions.The aim of this study is to assess the production of IL-17A and pro-fibrotic cytokines in peripheral blood mononuclear cells (PBMCs) from subjects with SSc in basal conditions and after treatment with 1,25(OH)2D3 and IL-17A neutralizing antibodies.Methods: The production of IL-17A and pro-fibrotic cytokines (TGFβ, CTGF and FGF2) in PBMCs obtained from 51 SSc patients and 31 healthy subjects was assessed both in basal conditions and in presence of anti-IL17A antibodies and several concentrations of 1,25(OH)₂D3. The association of cytokines production with clinical disease characteristics and the in vitro effect of 1,25(OH)₂D3 and IL-17A inhibition were assessed.Results: PBMCs from SSc subjects produced higher amount IL-17A, TGFβ, CTGF and FGF2 compared to healthy controls. IL17, TGFβ, CTGF and FGF2 levels were higher in SSc patients with interstitial lung disease and digital ulcers, whereas IL-17A production was lower in patients with PAH. IL- 17A inhibition reduced the production of FGF2, whereas enhanced the synthesis of TGFβ and CTGF. 1,25(OH)₂D3 decreased the production of IL17A and pro-fibrotic cytokines in a dose- dependent manner.Conclusions: IL-17A is involved in the regulation of fibrogenesis in SSc, and could represent an intriguing potential therapeutic target, even if its role remains controversial. 1,25(OH)₂D3 inhibits both IL-17A and pro-fibrotic cytokines, confirming its potential anti-fibrotic effect.     

Ex vivo all-trans retinoic acid modulates NO production and regulates IL-6 effect during rheumatoid arthritis: a study in Algerian patients

Rheumatoid arthritis (RA) is a chronic autoimmune disease. The pathophysiology of RA implicates several mediators such as nitric oxide (NO) and cytokines such as interleukin-6 (IL-6), which is deeply involved in the main characteristics of RA. Furthermore, all-trans retinoic acid (ATRA) is an active vitamin A derivative well-known to have diverse immunomodulatory actions. In our study, we investigated first, the ex vivo immunomodulatory potential of ATRA on NO pathway by peripheral blood mononuclear cells (PBMCs) from Algerian RA patients. Then, we assessed the possible regulatory effect of ATRA on NO production induced by IL-6. PBMCs isolated from active and inactive RA patients and healthy controls were cultured with different concentrations of IL-6 or/with ATRA. NO production was assessed using the Griess method. Inducible nitric oxide synthase expression and NF-κB activity were analyzed by immunofluorescence test. Our results revealed a high NO production during active RA. We noticed that while IL-6 induced a high NO production and iNOS expression, ATRA downregulated both. ATRA also inhibited nuclear NF-κB translocation. Interestingly, it seems that NO production mediated by IL-6 on PBMCs of RA patients is downregulated by ATRA. Taken together, our results highlight the immunomodulatory effect of ATRA on NO pathway in RA patients and its possible role in regulating IL-6-mediated NO production. All these findings suggest its potential therapeutic role during RA.     

The D‐vitamin metabolite 1,25(OH)2D in serum is associated with disease activity and Anti‐Citrullinated Protein Antibodies in active and treatment naïve,early Rheumatoid Arthritis Patients

Rationale

Sufficient levels of vitamin D seem to be essential for proper immune function, and low levels might be associated to disease activity in Rheumatoid Arthritis (RA ). Most studies investigate only 25OHD and not the physiologically active vitamin D metabolite, 1,25(OH )₂D.

Objective

To investigate associations between serum level of vitamin D metabolites and disease activity parameters in 160 inflammatory active and treatment naïve early RA patients. Serum level of vitamin D metabolites (25OHD ₂, 25OHD ₃ and 1,25(OH )₂D) was measured by isotope dilution mass spectrometry and radio‐immunoassays at baseline. Disease characteristics were gender, number of tender joints, number of swollen joints, DAS 28‐CRP , HAQ , VAS ‐scores, CRP , erosive status (Total Sharp Score; TSS ), ACPA and IgM‐RF ‐status. Associations were evaluated using Spearman's and Wilcoxon rank‐sum tests. The study was registered in clinical trials; trial registration number: NCT 00209859.

Findings

Statistically significant inverse associations were found between the active metabolite 1,25(OH )₂D and DAS 28‐CRP (P  = 0.004, rho = −0.23), HAQ (P  = 0.005, rho = −0.22), CRP (P  = 0.001, rho = −0.25), VAS _{patient‐pain} (P  = 0.008, rho = −0.21), and a positive association was found to ACPA ‐status (P  = 0.04).

Conclusion

The vitamin D metabolite 1,25(OH )₂D was inversely associated with disease activity and positively associated with ACPA in treatment naïve and inflammatory active early RA . The results indicate that in RA , both the degree of inflammatory activity, and the diagnostic sensitivity and specificity might affect—or might be affected by the level of vitamin 1,25(OH )₂D.

     

Decreased immunosuppressive actions of 1α, 25-dihydroxyvitamin D3 in patients with immune thrombocytopenia

Primary immune thrombocytopenia (ITP) is an acquired autoimmune disease. 1α, 25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] and vitamin D receptor (VDR) play important immune-suppressive roles in immune system. It has been reported that serum 1,25(OH)₂D₃ were lower in ITP patients. In this study, we evaluated local 1,25(OH)₂D₃ level and VDR mRNA expression further, and determined whether 1,25(OH)₂D₃/VDR were correlated with T cell dysfunction in ITP patients. We found that 1,25(OH)₂D₃/VDR levels were decreased in active ITP patients, and 1,25(OH)₂D₃ had significant anti-inflammatory effects on ITP patients, including both anti-proliferation of peripheral blood mononuclear cells (PBMCs) and reversing the abnormal T cells polarization. 1,25(OH)₂D₃ inhibited the differentiation of T helper (Th)1 and Tc1 cells but induced the differentiation of Th2, Tc2 and T regulatory (Treg) cells in ITP patients. However, the percentage of Th17 cells were not affected obviously with 1,25(OH)₂D₃. In addition, 1,25(OH)₂D₃ also suppressed pro-inflammatory cytokines (INF-γ and IL-17A) but promoted anti-inflammatory cytokine (IL-10) secretion in ITP patients. In conclusion, decreased 1,25(OH)₂D₃/VDR might participate in the pathogenesis of ITP, and appropriate supplement of 1,25(OH)₂D₃ may be a promising treatment.     

Vitamin D status is not associated with inflammatory cytokine levels during experimental human endotoxaemia

Vitamin D has been shown to modulate innate immune responses in vitro and ex vivo; however, human in‐vivo data are lacking. At high latitudes, seasonal vitamin D deficiency is common due to alternating ultraviolet (UV)‐B radiation exposure. In the present study, we investigated whether levels of 25 hydroxyvitamin D₃ [25(OH)D₃] and its active metabolite 1,25 dihydroxyvitamin D₃ [1,25(OH)₂D₃] are subject to seasonal variation and whether plasma levels of these vitamin D metabolites correlate with the in‐vivo cytokine response during experimental human endotoxaemia [administration of lipopolysaccharide (LPS) in healthy volunteers]. Plasma levels of 25(OH)D₃ and 1,25(OH)₂D₃ were determined in samples obtained just prior to administration of an intravenous bolus of 2 ng/kg LPS (derived from Escherichia coli O:113) in 112 healthy male volunteers. In the same subjects, plasma levels of the inflammatory cytokines tumour necrosis factor (TNF)‐α, interleukin (IL)‐6 and IL‐10 were analysed serially after endotoxin administration. Plasma levels of 1,25(OH)₂D₃, but not 25(OH)D₃, were subject to significant seasonal variation, with lower levels in autumn and winter. 25(OH)D₃ and 1,25(OH)₂D₃ levels did not correlate with plasma cytokine responses. Furthermore, 25(OH)D₃ deficient subjects (< 50 nmol/l) displayed an identical cytokine response compared with sufficient subjects. In conclusion, plasma levels of vitamin D are not correlated with the LPS‐induced TNF, IL‐6 and IL‐10 cytokine response in humans in vivo. These findings question the direct role of vitamin D in modulation of the innate immune response.     

Effect of protein kinase inhibitors on IL-8/NAP-1 release from human umbilical vein endothelial cells

Several protein kinase inhibitors (PKIs) were investigated for their effects on IL-1β, TNFα and PMA-induced IL-8 production from human umbilical vein endothelial cells (HUVEC). IL-1β (ED₅₀ 0.07 ng/ml), TNFα (ED₅₀ 100 ng/ml) and PMA (ED₅₀ 20 ng/ml) induced IL-8 production that could be detected as early as 2 h following stimulation. Staurosporine, a potent but non-specific inhibitor of protein kinases, inhibited PMA-induced (IC₅₀ 2 nM) but not IL-1β or TNFα (IC50>200 nM) induced IL-8 production. Neither the cAMP-dependent PKI, KT5720, nor the tyrosine PKIs, genistein, tyrphostin (1–100 μM) or lavendustin A (0.0001–1 μM), inhibited IL-8 production elicited by IL-1β. However, the macrolide protein kinase inhibitor geldanamycin (IC₅₀=30 nM), but not the closely related analog herbimycin A (5–500 nM), inhibited IL-8 production by 60%. Northern blot analysis of IL-8 mRNA revealed that staurosporin suppressed mRNA increase following stimulation by PMA but not by IL-1. It is proposed that a novel protein kinase susceptible to geldanamycin inhibition may be involved in IL-1-mediated signal transduction.

     

Methotrexate specifically modulates cytokine production by T cells and macrophages in murine collagen-induced arthritis (CIA): a mechanism for methotrexate-mediated immunosuppression

Immunosuppressive therapy with methotrexate (MTX) has been established as effective treatment for patients with rheumatoid arthritis. To analyse the therapeutic potential and mechanisms of action of MTX, we determined serum cytokine levels and cytokine production by splenic T cells and macrophages in untreated and MTX-treated mice. Furthermore, we assessed the role of MTX in a murine model of experimental arthritis induced by collagen type II (CIA). MTX reduced spontaneous and IL-15-induced tumour necrosis factor (TNF) production by splenic T cells but not by macrophages from healthy mice in vitro in a dose-dependent manner. In contrast, interferon-gamma (IFN-γ) production was less strikingly reduced and IL-4 production was virtually unaffected. In addition, treatment of healthy mice with MTX in vivo led to reduced TNF serum levels and diminished TNF production by splenic T cells and macrophages. Intraperitoneal administration of MTX prior to the onset of arthritis completely prevented clinical and pathological signs of CIA. This was associated with a striking reduction of TNF production by spleen cells from MTX-treated mice. The role of TNF in MTX-mediated effects on cytokine production was further underlined by the finding that MTX effects on IFN-γ production were augmented in TNF-transgenic mice but abrogated in mice in which the TNF-α gene had been inactivated by homologous recombination. Thus, MTX specifically modulates spontaneous and IL-15-induced TNF-α production in mice and prevents experimental murine CIA. These data suggest that TNF production by T cells is an important target of MTX and may serve as a basis to understand and further analyse MTX-mediated mechanisms of immunosuppression in patients with RA.     

1α,25‐dihydroxyvitamin D3 acts via transforming growth factor‐β to up‐regulate expression of immunosuppressive CD73 on human CD4+ Foxp3– T cells

Vitamin D deficiency is associated with increased incidence and severity of various immune‐mediated diseases. Active vitamin D (1α,25‐dihydroxyvitamin D3; 1,25(OH)₂D3) up‐regulates CD4⁺ T‐cell expression of the purine ectonucleotidase CD39, a molecule that is associated with the generation of anti‐inflammatory adenosine. Here we aimed to investigate the direct impact of 1,25(OH)₂D3 on expression of the downstream ecto‐5′‐nucleotidase CD73 by human CD4 T cells, and components of the transforming growth factor‐β (TGF‐β) pathway, which have been implicated in the modulation of CD73 by murine T cells. At 10^?8 to 10^?7 m , 1,25(OH)₂D3 significantly increased expression of CD73 on peripheral human CD4⁺ T cells. Although 1,25(OH)₂D3 did not affect the mRNA expression of latent TGF‐β₁, 1,25(OH)₂D3 did up‐regulate expression of TGF‐β‐associated molecules [latency‐associated peptide (LAP), glycophorin A repetitions predominant (GARP), GP96, neuropilin‐1, thrombospondin‐1 and α_v integrin] which is likely to have contributed to the observed enhancement in TGF‐β bioactivity. CD73 was highly co‐expressed with LAP and GARP following 1,25(OH)₂D3 treatment, but unexpectedly, each of these cell surface molecules was expressed primarily on CD4⁺ Foxp3^– T cells, rather than CD4⁺ Foxp3⁺ T cells. Notably, neutralization of TGF‐β significantly impaired 1,25(OH)₂D3‐mediated induction of CD73. Collectively, we show that 1,25(OH)₂D3 enhances expression of CD73 on CD4⁺ Foxp3^– T cells in a process that is at least partially TGF‐β‐dependent. These data reveal an additional contributing mechanism by which vitamin D may be protective in immune‐mediated disease.     

Enhancement of 1,25-Dihydroxyvitamin D3– and all-Trans Retinoic Acid-Induced Differentiation of Human Leukemia HL-60 Cells by Phyllostachys nigra var. henonis

Human myeloid leukemia HL-60 cells are differentiated into monocytic or granulocytic lineage when treated with 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] or all-trans retinoic acid (RA), respectively. In this study, the effect of acetone fraction prepared from bamboo leaf on cell differentiation was investigated in a HL-60 cell culture system. Treatment of HL-60 cells with 50–400 μg/ml acetone fraction of bamboo leaf for 72 hr inhibited cell proliferation and induced a little increase in cell differentiation, as demonstrated by the MTT and nitroblue tetrazolium reduction assay. Interestingly, synergistic induction of HL-60 cell differentiation was observed when the acetone fraction of bamboo leaf was combined with either 5 nM 1,25-(OH)₂D₃ or 50 nM all-trans RA. Flow cytometric analysis indicated that combinations of 1,25-(OH)₂D₃ and the acetone fraction of bamboo leaf stimulated differentiation predominantly to monocytes, whereas combinations of all-trans RA and the acetone fraction of bamboo leaf stimulated differentiation predominantly to granulocytes. These results suggest that the acetone fraction of bamboo leaf enhanced leukemia cell differentiation and suggest a possibility of bamboo in the treatment of leukemia.     

1,25‐Dihydroxy‐vitamin D3 regulates NK‐cell cytotoxicity,cytokine secretion,and degranulation in women with recurrent pregnancy losses

Vitamin D has a pivotal role in regulating immune responses by promoting Th2 immune responses and suppressing Th1 responses. Propensities to a Th1 immune response and increased NK‐cell levels and cytotoxicity have been reported in women with recurrent pregnancy losses (RPL). In women with RPL, vitamin D deficiency is prevalent; however, the effect of vitamin D on NK cells is largely unknown. In this study, we demonstrated that CD69⁺ activating receptor expression on NK cells was significantly decreased by incubation with 1,25(OH)₂D₃ in a dose‐dependent manner, while CD158a and CD158b inhibitory receptor expression was upregulated. The degranulation marker CD107a was significantly downregulated on NK cells following incubation with 1,25(OH)₂D₃. NK‐cell conjugation with K562 target cells was not affected by 1,25(OH)₂D₃; however, depolarization of perforin granules in conjugated NK cells was significantly increased. TLR4 expression on NK cells was significantly decreased and TNF‐α and IFN‐γ production was significantly reduced by 1,25(OH)₂D₃ through interference with NF‐κB. Our results suggest 1,25(OH)₂D₃ has immune regulatory effects on NK cell cytotoxicity, cytokine secretion and degranulation process as well as TLR4 expression. Potential therapeutic application of 1,25(OH)₂D₃ for dysregulated NK‐cell immunity should be explored in the future.     

Effects of 1,25 dihydroxyvitamin D on osteoclast number and cytochemistry in normal and osteopetrotic (os) rabbits

Osteoclast-mediated bone resorption is increased in response to 1,25 dihydroxyvitamin D (1,25[OH]₂D or calcitriol). Osteopetrosis is a metabolic bone disease characterized by defective, osteoclast-mediated bone resorption, which co-exists with elevated serum 1,25-(OH)₂D levels in some osteopetrotic children and animals. We examined the effects of high doses of calcitriol on osteoclast number and cytochemistry in both normal and osteopetrotic (os) rabbits. Calcitriol was continuously infused at doses of 0.5, 2.5, or 25 μg/kg/day via subcutaneously implanted osmotic minipumps for a period of 7 days. Following treatment, the proximal tibial metaphyses were processed for histomorphometric and cytochemical analyses. Sections were stained for tartrate-resistant acid phosphatase (TrAP) or acid ATPase (TraATPase). Osteoclasts were significantly reduced in untreated os rabbits compared with age-matched normal littermates between birth and 3 weeks of age (41–46% of normal). Whereas most normal osteoclasts (85%) stained heavily for TrAP or TraATPase, less than half of os osteoclasts were heavily stained for these acid hydrolases. Infusions of 1,25(OH)₂D resulted in elevations of osteoclast numbers in both normal and os rabbits, but the number of osteoclasts remained significantly lower in mutants than in normal littermates at any given dose. Calcitriol infusions also resulted in a significant increase in the percentage of os osteoclasts staining heavily for TrAP and TraATPase. These results suggest that in response to 1,25(OH)₂D normal osteoclasts increase their production of acid hydrolases before increasing cell numbers and that, in spite of high levels of endogenous calcitriol, os rabbits can respond to exogenous 1,25(OH)₂D as evidenced by increased osteoclast number and cytochemical staining, even though these osteoclasts fail to resorb the excess skeletal matrix. Thus the defect in the mutant skeleton appears to be in the initial cytodifferentiation of osteoclasts, not in their responsiveness to endogenous regulators of bone resorption.     

Vitamin D and the vitamin D receptor are critical for control of the innate immune response to colonic injury

Background

The active form of vitamin D (1,25(OH)₂D₃) has been shown to inhibit development of inflammatory bowel disease (IBD) in IL-10 KO mice. Here, the role of the vitamin D receptor (VDR) and 1,25(OH)₂D₃in acute experimental IBD was probed.

Results

VDR KO mice were extremely sensitive to dextran sodium sulfate (DSS) and there was increased mortality of the VDR KO mice at doses of DSS that only caused a mild form of colitis in wildtype (WT) mice. DSS colitis in the VDR KO mice was accompanied by high colonic expression of TNF-α, IL-1 α, IL-1β, IL-12, IFN-γ, IL-10, MIP-1α and KC. DSS concentrations as low as 0.5% were enough to induce bleeding, ulceration and weight loss in VDR KO mice. VDR KO mice failed to recover following the removal of DSS, while WT mice showed signs of recovery within 5 days of DSS removal. The early mortality of DSS treated VDR KO mice was likely due to perforation of the bowel and resulting endotoxemia. VDR KO mice were hyper-responsive to exogenously injected LPS and cultures of the peritoneal exudates of moribund DSS treated VDR KO mice were positive for bacterial growth. 1,25(OH)₂D₃in the diet or rectally decreased the severity and extent of DSS-induced inflammation in WT mice.

Conclusion

The data point to a critical role for the VDR and 1,25(OH)₂D₃in control of innate immunity and the response of the colon to chemical injury.     

Differential effects of gangliosides on human IgE and IgG4 production

The effects of gangliosides on human IgE and IgG4 production were studied. Of the various gangliosides tested, only GM2 and GM3 inhibited the IgE and IgG4 production induced by interleukin (IL)-4 plus hydrocortisone (HC), or that induced by IL-13 plus HC, in human surface IgE- and IgG4-negative (sIgE^?, sIgG4^?) B cells without affecting the production of IgG1, IgG2, IgG3, IgM, IgA1 or IgA2. In contrast, GM1, GD1a, GD1b, GD3, GT1b and GQ1b were without effects. The GM2- and GM3-mediated inhibition was specific, since each was blocked by a corresponding antibody. Of the various factors tested, IL-6, IL-10, and tumor necrosis factor (TNF)-α enhanced the IgE and IgG4 production induced by IL-4 plus HC or by IL-13 plus HC, while IL-8 and transforming growth factor (TGF)-β inhibited these responses. However, only TNF-α counteracted the GM2- and GM3-mediated inhibition of IgE and IgG4 production, while IL-6, IL-10, anti-IL-8 monoclonal antibody and anti-TGF-β antibody failed to do so. Anti-TNF-α monoclonal antibody, but not control IgG1, not only inhibited IgE and IgG4 production in the absence of TNF-α but also blocked the counteraction of inhibition by TNF-α. In cultures containing IL-4 plus HC or IL-13 plus HC. GM2 and GM3 specifically inhibited TNF-α production without affecting TNF-α receptors, IL-6 production or IL-6 receptors. These results indicate that GM2 and GM3 inhibit IgE and IgG4 production by inhibiting endogenous TNF-α production.     

1,25-Dihydroxyvitamin D3 modulates T cell differentiation and impacts on the production of cytokines from Chinese Han patients with early rheumatoid arthritis

To study the effects of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) on the differentiation of T cells and the levels of cytokines in patients with early rheumatoid arthritis (eRA). The levels of Th1, Th2, Th17, and Treg cells were detected with BDFACS Calibur flow cytometer. The expression of IFN-ɤ, TNF-α, IL-2, IL-4, IL-6, IL-10, IL-17, and IL-22 was examined in 54 patients with eRA using a cytometric bead array (CBA). After 72 h of incubation of PBMCs from eRA patients with 1,25(OH)₂D₃, the levels of IFN-ɤ, TNF-α, IL-2, IL-6, and IL-17 significantly decreased compared to those of the control. 1,25(OH)₂D₃ had no significantly impact on the levels of IL-4, IL-10, and IL-22. The proportion of Th17 and the ratio of Th17/Treg significantly decreased in 1,25(OH)₂D₃-treated groups compared to those of the control. 1,25(OH)₂D₃ had no significantly impact on the proportion of Th1, Th2, Treg, and the ratio of Th1/Th2. Although no statistically significant difference was observed, proportion of Th1 was decreased after 1,25(OH)₂D₃ treatment compared with anti-CD3/CD28 only. The present study demonstrated that 1,25(OH)₂D₃ inhibited the synthesis of specific cytokines: Th1 (IFN-ɤ) and Th17 (IL-17, IL-22, IL-6, TNF-α) might upregulated Th2 cytokine (IL-4), which indicated the possible immunoregulatory roles and bone-sparing effects of 1,25(OH)₂D₃ in eRA through modulation of the Th1 and Th17 cytokine balance.

     

25-hydroxyvitamin D, 24, 25-dihydroxyvitamin D and 1,25-dihydroxyvitamin D in human cerebrospinal fluid

Summary Samples of CSF and plasma were obtained simultaneously from 46 adult patients who had no endocrine disorders and were undergoing routine diagnostic lumbar puncture because of suspected or proved prolapse of a disc. Concentrations of 25-OHD, 24,25(OH)₂D and 1,25(OH)₂D were measured. The samples were purified by column chromatography and fractionated by HPLC. In the appropriate fractions the vitamin D metabolites were measured by PBA, and cytoreceptor assay. The results were as follows (median, range in brackets): 25-OHD in CSF 8.3 ng/ml (2.0–24.8), in plasma 14.5 ng/ml (7.0–36.0). 24,25(OH)₂D in CSF 1.8 ng/ml (0.3–4.6) and 2.5 ng/ml (0.4–4.7) in plasma. 1.25(OH)₂ D in CSF 25.0 pg/ml (2.2–39.0) and 31.0 pg/ml (10.1–55.0) in plasma. The correlations between plasma and CSF concentrations were as follows: 25-OHDr=0.479 (P<0.001); 24,25(OH)₂Dr=0.815 (P<0.001) and for 1.25(OH)₂Dr=0.497 (P<0.001).Our findings showed vitamin D metabolites to be present in human CSF.Abbreviations Ca Calcium - CSF Cerebrospinal fluid - Vitamin D₃ Cholecalciferol - CPM Counts per min - 24, 25 (OH)₂D 24, 25-dihydroxyvitamin D₃ - 1,25(OH)₂D 1,25-dihydroxyvitamin D₃ - Vitamin D₂ Ergocalciferol - HPLC High-pressure liquid chromatography - 25OHD 25-hydroxyvitamin D₃ - PTH Parathyroid hormone - PBA Protein binding assay - RIA Radioimmunoassay - D-CaBP Vitamin D dependent calcium-binding protein     

Inhibition of human T lymphocyte proliferation and cytokine production by 1,25-dihydroxyvitamin D3. Differential effects on CD45RA+ and CD45R0+ cells.

1,25-dihydroxyvitamin D3 (1,25-(OH)2 D3), the biologically active form of vitamin D3, has been shown to modulate lymphocyte functions in vitro. These effects are exerted through binding to specific receptors that are expressed in activated, but not in resting lymphocytes. 1,25-(OH)2 D3 inhibits lymphocyte proliferation, immunoglobulin production and the release of cytokines including interleukin-2 (IL-2) and interferon gamma (IFN gamma) by mitogen driven blood mononuclear cells (MNC). A distinction between CD45RA+ and CD45R0+ subsets of T cells has, however, proven extremely relevant in terms of immunoactivation and immunopathology. The present study was undertaken to evaluate effects of 1,25-(OH)2 D3 on proliferation and cytokine production by purified CD45RA+ and CD45R0+ T cells. 1,25-(OH)2 D3 caused a dose- and time-dependent reduction in phytohemagglutinin-(PHA) and poke-weed mitogen (PWM)-driven proliferation of purified CD45R0+ T cells. In contrast, proliferation of the CD45RA+ subset was unaffected by this treatment. Comparable levels of lymphotoxin (LT), IFN gamma and IL-2 were obtained in cultures of both subsets. 1,25-(OH)2 D3 reduced these levels, but the suppressive effect of the hormone was delayed in cultures of CD45RA+ T cells. The results suggest that the CD45R0+ subset is relatively more sensitive than CD45RA+ subset to the inhibitory effects of 1,25-(OH)2 D3. This finding may be of pharmacological interest, because the CD45R0+ subset plays a key role in immune activation and because these cells have been associated with the pathogenesis of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis.     

The effect of interleukin-6 and soluble interleukin-6 receptor protein on the bone resorptive activity of human osteoclasts generated in vitro

The role of interleukin-6 (IL-6) in the regulation of bone resorption is and has not been studied using human tissue in vitro. This study exploits a recently described in vitro model, whereby osteoclasts, defined as cells that resorb bone, can be generated from human bone marrow, and investigated the effect of IL-6 and its soluble receptor on bone resorption, in the presence of 1,25-dihydroxyvitamin D₃[1,25(OH)₂ vitamin D₃]. Human bone marrow was cultured to form a confluent stroma, sedimented onto devitalized bone slices, and recharged with non-adherent bone marrow cells. 1,25(OH)₂ vitamin D₃ increased bone resorption, whereas IL-6 failed to induce a similar stimulatory effect. Both IL-6 at 100 ng/ml and soluble IL-6 receptor protein in the absence of exogenous IL-6 inhibited the stimulatory effect of 1,25(OH)₂ vitamin D₃. Bone resorption was never observed when non-adherent haemopoietic cells were cultured in the absence of stroma but in the presence of IL-6, which indicates that IL-6 cannot replace the stromal factor(s) required for the formation of cells capable of resorbing bone. These results suggest that IL-6 at high concentrations is not a critical cytokine in stimulating osteoclastic bone resorption.