Human dendritic cell antigen presentation and chemotaxis are inhibited by intrinsic 25-hydroxy vitamin D activation

https://doi.org/10.1016/j.intimp.2010.05.003Get rights and content

Abstract

The immunomodulatory effects of vitamin D have primarily been investigated using the biologically active form 1,25-dihydroxy vitamin D3 (1,25-D3). It was recently demonstrated that dendritic cells (DC) are able to convert the inactive 25-hydroxy vitamin D3 (25-D3) into the active form via 1α-hydroxylase. In this study, we set out to examine the possible consequences of this conversion on adaptive immune functions. Human monocyte-derived DC were matured by lipopolysaccharide (LPS) in the presence or absence of 25-D3. Subsequently, the conversion of 25-D3 into 1,25-D3, and the effects on surface marker expression, cytokine production, antigen-presenting capacity and chemotaxis of the DC were examined. 25-D3 was clearly converted into 1,25-D3 in the DC cultures and the process was accompanied by a reduced expression of CD80 (p < 0.01), CD83 (p < 0.01), CD86 (p = 0.02), and HLA-DR (p = 0.02). Also, the levels of the pro-inflammatory cytokines tumour necrosis factor (TNF) α (p = 0.02) and interleukin (IL) 12 (p < 0.01) were reduced. Interestingly, however, the CD14 expression (p < 0.01) and the production of IL-1β (p < 0.01) and IL-6 (p < 0.01) increased. Thus, 25-D3 affected the delicate interplay between anti- and pro-inflammatory cytokines produced by the DC. Concurrently, 25-D3 reduced DC capacity to induce proliferation of antigen-specific T cells and DC chemotaxis towards chemokine (CC) ligand 21. This indicates that 25-D3 has a regulating function following intrinsic 1α-hydroxylation, a mechanism that potentially has an immunomodulatory effect in vivo.

Introduction

Vitamin D is a fat-soluble vitamin with well-known effects on calcium metabolism. Additionally, vitamin D supplementation reduces the risk of several cancer forms and cardiovascular disease, thus reducing overall mortality rates, but we have little understanding of the underlying mechanisms [1]. Vitamin D seems to control adaptive immune responses, and deficiency of this vitamin is believed to play a role in diseases with an autoimmune aspect such as diabetes mellitus, rheumatoid arthritis, multiple sclerosis, and Crohn's disease [2].

Modulation of adaptive immune functions has been studied mainly by using the active form of vitamin D, 1,25-dihydroxy vitamin D3 (1,25-D3). Responsiveness to 1,25-D3 depends on the intracellular vitamin D receptor (VDR), which is present in most immune cells and has a high affinity for the 1,25-D3 form, but not for the inactive 25-hydroxy vitamin D3 (25-D3) [3], [4].

Dendritic cells (DC) are among the cell types affected by 1,25-D3. Widespread in body tissues, DC can instantly respond to intruders through pattern-recognition receptors, take up antigens, and migrate to lymphoid tissues, where they effectively activate antigen-specific T cells [5]. DC matured in vitro under the influence of 1,25-D3 exhibit a reduced expression of MHC II and the co-stimulatory molecules CD80, CD83 and CD86 [6], [7], [8]. Furthermore, the DC release of pro-inflammatory cytokines, such as interleukin (IL) 12 and tumour necrosis factor (TNF) α, is inhibited, while IL-10 production is increased by 1,25-D3 [7], [8]. 1,25-D3-conditioned DC show a reduced ability to activate T cells [6], [8], [9], and they stimulate an increase in the number of regulatory T cells [10], [11]. 1,25-D3 also directly affects CD4+ T cells and induces a regulatory cytokine profile with increased IL-10 and reduced TNF-α and interferon (IFN) γ levels [12], [13]. Finally, DC trafficking is also affected by 1,25-D3. Human myeloid DC which are exposed to 1,25-D3 reduce the expression of chemokine receptor 7 (CCR7) [14]. In mice, a similar decrease in CCR7 expression is observed by 1,25-D3 exposure, which leads to DC bypassing draining lymph nodes [15]. Stimulation with 1,25-D3 decreases the chemotaxis of Langerhans cells towards chemokine ligand 21 (CCL21), probably owing to an inhibitory effect on the CCR7 expression [16].

Serum 1,25-D3 levels are very low in comparison with 25-D3 levels, and not sufficiently high to have biological effects in DC [17]. Due to that VDR has low or no affinity to 25-D3, 1α-hydroxylation is essential to obtain biological effects of vitamin D3. While this hydroxylation traditionally is thought to take place in the kidneys, the process is now also known to occur in DC [18], [19]. Local conversion of 25-D3 into 1,25-D3 may therefore be an important faction in immune reactions in vivo.

Intrigued by the outlined possibility, we aimed at further elucidating the interplay between DC and 25-D3. Initially, we aimed to confirm that DC convert 25-D3 into 1,25-D3 in quantities sufficient to affect surface marker expression and IL-12 production. We further hypothesized that other cytokines like TNF-α, IL-10, and IL-6 were affected as well, and that the 1,25-D3 quantities were sufficient to affect the DC function in terms of antigen presentation and chemotaxis.

Section snippets

Biological material

The Central Denmark Regional Committee on Biomedical Research Ethics approved the study (j. no. 1998/4330).

After written informed consent was obtained, blood was drawn from healthy volunteers, nine men and six women (age range 27 to 61 years). Six of the volunteers had recently had a tetanus vaccination.

Peripheral blood mononuclear cells were collected and separated by Ficoll-Paque PLUS (GE Healthcare Bio-Science AB, Uppsala, Sweden) density gradient separation. Cells were cryopreserved in 50%

Monocytes differentiated into DC

Cells were harvested from cultures of adherent peripheral blood mononuclear cells after 7 days in the presence of IL-4 and GM-CSF. A flow cytometric analysis was performed to establish cell phenotype. The cells were identified as DC by their low CD14 expression (results not shown) and a high HLA-DR and CD86 expression (Fig. 1). If DC were exposed to LPS after 6 days of culture, they matured as reflected in an increased surface expression of CD80, HLA-DR and CD86 (Fig. 1).

Vitamin D did not affect cell number

To exclude a toxic effect

Discussion

The present study reaffirmed that 25-D3 added to LPS-stimulated DC cultures gave rise to 1,25-D3 levels sufficiently high to change DC expression of MHC II and co-stimulatory surface molecules. For the first time, we showed that DC cytokine production, antigen presentation, and chemotaxis were also changed by 25-D3 exposure.

The observed effects in our study were probably caused by intrinsic DC 1-α-hydroxylase activity since 1,25-D3 production was reduced by the 1α-hydroxylase inhibitor

Conflict of interest

None.

Acknowledgements

We thank laboratory technicians Brita Holst and Rikke Andersen for excellent laboratory assistance. Nordjyske Lægekredsforenings Forskningsfond, The A.P. Møller Foundation for the Advancement of Medical Science, Desiree og Niels Ydes Fond, The Danish Medical Association Research Fund/Højmosegård-Legatet, Aase og Ejnar Danielsens Fond, and the Karen Elise Jensen Foundation all supported this study.

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