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Keywords:

  • 1,25-dihydroxyvitamin D3;
  • immune cells;
  • 1alpha-hydroxylase

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. VITAMIN D ACTIONS
  5. VITAMIN D IN THE IMMUNE SYSTEM
  6. REGULATION OF VITAMIN D METABOLISM
  7. CONCLUSION
  8. Acknowledgments
  9. REFERENCES

Vitamin D homeostasis in the immune system is the focus of this review. The production of both the activating (25- and 1α-hydroxylase) and the metabolizing (24-hydroxylase) enzymes by cells of the immune system itself, indicates that 1,25(OH)2D3 can be produced locally in immune reaction sites. Moreover, the strict regulation of these enzymes by immune signals is highly suggestive for an autocrine/paracrine role in the immune system, and opens new treatment possibilities.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. VITAMIN D ACTIONS
  5. VITAMIN D IN THE IMMUNE SYSTEM
  6. REGULATION OF VITAMIN D METABOLISM
  7. CONCLUSION
  8. Acknowledgments
  9. REFERENCES

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3), the biologically active form of vitamin D3, is well known for its effects on mineral homeostasis and bone metabolism. This secosteroid hormone can be obtained by nutritional uptake (e.g., fortified dairy products and fatty fish and their liver oils); however, UVB-mediated photosynthesis in the skin serves as the main source of vitamin D3.1 To become biologically active, two hydroxylation steps of vitamin D3 are necessary. The first hydroxylation, 25-hydroxylation, takes place mainly in the liver with 25-hydroxylases (CYP27A1, CYP2R1, CYP3A4, and CYP2J3) and 25-hydroxyvitamin D3[25(OH)D3] is formed. This is the main circulating form of vitamin D3 in the blood. The second hydroxylation takes place mainly in the proximal tubule cells of the kidney, but it also occurs in other tissues such as the skin, bone and cartilage, prostate, and macrophages.2–4 In this case, 25(OH)D3 is hydroxylated by 1α-hydroxylase (CYP27B1) and 1,25(OH)2D3 is formed. For transport, vitamin D compounds are complexed to the vitamin D binding protein (DBP). For uptake of the DBP-25(OH)D3-complex by the kidney cells, the presence of both megalin and cubilin, members of the LDL-receptor family, is necessary. Mice lacking megalin, as well as humans with mutations in cubilin, exhibit vitamin D deficiency as a consequence of impaired renal uptake of the DBP-25(OH)D3-complex and are unable to form 1,25(OH)2D3. The megalin-DBP-25(OH)D3-complex is internalized by invagination. A schematic overview of these events is shown in Figure 1. Recently, intracellular vitamin D binding proteins have been described.5,6 These proteins are members of the hsp-70 family and may be involved in the intracellular trafficking of vitamin D metabolites to specific intracellular destinations. 1,25(OH)2D3 is inactivated by 24-hydroxylase (CYP24A1), which is expressed in nearly all cell types.1,7 Hereby, the inactive 1,24,25(OH)3D3 is formed, which is further processed to the excretion product calcitroic acid. 1,25(OH)2D3 strongly induces expression of 24-hydroxylase, and thus induces its own inactivation.

image

Figure 1. Overview of vitamin D3 metabolism. Vitamin D3 is mainly synthesized from 7-dehydrocholesterol when skin is exposed to sunlight, while a small amount is derived from the diet. To become biologically active, two hydroxylation steps are necessary, the first hydroxylation, 25-hydroxylation, mainly takes place in the liver and 25-hydroxyvitamin D3[25(OH)D3] is formed. For transport in serum, vitamin D3 is complexed to the vitamin D binding protein (DBP). 25(OH)D3 is 1α-hydroxylated by the 1α-hydroxylase and 1,25(OH)2D3 is formed. This second hydroxylation takes place primarily in the proximal tubule cells of the kidney, but it also occurs in other tissues such as skin, intestine, macrophage, and bone.

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VITAMIN D ACTIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. VITAMIN D ACTIONS
  5. VITAMIN D IN THE IMMUNE SYSTEM
  6. REGULATION OF VITAMIN D METABOLISM
  7. CONCLUSION
  8. Acknowledgments
  9. REFERENCES

Vitamin D metabolites are lipophilic molecules that can easily penetrate cell membranes and translocate to the nucleus.8,9 1,25(OH)2D3 binds to the VDR (vitamin D receptor), which is mainly located in the nucleus, thereby inducing heterodimerization of VDR with RXR (retinoid X receptor). This heterodimer binds to VDRE (vitamin D response elements) in the promoter of responsive genes, and induces their transcription after recruitment of several coactivators. Alternatively, when the 1,25(OH)2D3/VDR/RXR-complex binds to an inhibitory VDRE, corepressors are recruited and transcription is inhibited.

The VDR gene, of which transcription is induced by 1,25(OH)2D3 itself, maps to human chromosome 12q13 and has at least 14 exons.10 Multiple polymorphisms of the VDR gene, distributed throughout the complete VDR gene region, have been described, as have associations of these polymorphisms with disorders like cancer and autoimmune diseases.11 The FokI single nucleotide polymorphism of the translation start-site results in a VDR protein shortened by three amino acids12 displaying a stronger biological and transcriptional activity.13,14 Recently, our group demonstrated that this stronger activity is also reflected in the immune system, with increased transcriptional activity of immune-specific transcription factors leading to changes in proliferation and cytokine synthesis of different immune cell types.15 Other polymorphisms of the VDR, of which ApaI, BsmI, and TaqI are the most often investigated, may affect mRNA stability.11 The Cdx2 polymorphism in the promoter region results in a defective binding site of the intestine-specific transcription factor Cdx2, and may affect calcium absorption.16 Additionally, a certain proportion of the VDR molecules are alternatively spliced. In particular, the VDRB1 variant, extended by 50 amino acids and originating from alternative splicing, and the use of a more upstream in-frame start codon can be found in most humans.17

Since the VDR is widely expressed, 1,25(OH)2D3 has effects on a large number of tissues. Its best-known actions are in bone, kidney, and intestine.1,18–20 Furthermore, 1,25(OH)2D3 plays a role in the immune system, and has effects on differentiation and proliferation of various normal as well as malignant cell types. Vitamin D deficiency causes rickets among children, osteoporosis in adults, and has been associated with higher risks of cancer, cardiovascular disease, and autoimmune diseases.21

VITAMIN D IN THE IMMUNE SYSTEM

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. VITAMIN D ACTIONS
  5. VITAMIN D IN THE IMMUNE SYSTEM
  6. REGULATION OF VITAMIN D METABOLISM
  7. CONCLUSION
  8. Acknowledgments
  9. REFERENCES

1,25(OH)2D3 has immunomodulatory effects.22,23 This was already suggested by the expression of VDR in activated T lymphocytes and in APCs (antigen presenting cells). In addition, 1,25(OH)2D3 treatment of T lymphocytes inhibits their activation and proliferation and alters their cytokine expression profile. IFNγ and IL2 production are decreased in CD4+ T cells, although by different mechanisms. IFNγ transcription is inhibited through binding of VDR/RXR to a silencer region in the promoter as well as to the minimal promoter region.24 The latter might impede initiation or progression of transcription. The promoter of IL2 contains a positive regulatory NFAT1 (nuclear factor of activated T cells 1) site, to which a complex consisting of the T cell-specific transcription factor NFATp and AP1 (activator protein 1) can bind.25 1,25(OH)2D3 decreases IL2 transcription through interference of 1,25(OH)2D3/VDR/RXR with NFATp/AP1 complex formation and through occupancy of the NFAT1 binding site. Moreover, 1,25(OH)2D3 directly increases IL4 production, thus contributing to Th2 differentiation.26 Also, GATA3 (GATA binding protein 3), a transcription factor involved in Th2 development, is upregulated by 1,25(OH)2D3.

1,25(OH)2D3 induces monocytic differentiation, as shown in human myeloid leukemia HL60 cells, which is mediated by activation of MAPK (mitogen-activated protein kinase) signaling and by increased C/EBPβ (CAAT/enhancer-binding protein β) expression.27,28 Furthermore, 1,25(OH)2D3 causes increased phagocytic and oxidative burst ability.29 Wang et al.30 have shown that 1,25(OH)2D3 induces expression of antimicrobial peptides in neutrophils and monocytes. During differentiation from monocytes to macrophages, cells obtain an enhanced capability to synthesize 1,25(OH)2D3, through increased 1α-hydroxylase expression, in contrast to reduced VDR and 24-hydroxylase expression.31

The most pronounced effects of 1,25(OH)2D3 on immune cells are observed on dendritic cells (DCs). Treatment of DCs with 1,25(OH)2D3 or analogs dramatically inhibits maturation and differentiation.32,33 Treated DCs display a downregulated expression of the costimulatory molecules CD40, CD80, and CD86. In addition, they have decreased IL12 and increased IL10 production. These characteristics result in decreased T cell activation. Moreover, these tolerogenic DCs are capable of inducing CD4+CD25+ suppressor T cells. In addition, DCs themselves are capable of producing 1,25(OH)2D3, whereas they lose VDR expression during maturation, thereby becoming insensitive to 1,25(OH)2D3.34 Downregulation of IL12 results from interference with the nuclear factor (NF)-κB pathway.35 1,25(OH)2D3 interferes with both activation of NF-κB as well as its binding to the promoter of IL12p40.

These immunomodulatory effects of 1,25(OH)2D3 are exploited in treatments of autoimmune diseases. Treatment of non-obese diabetic mice, the murine model for type 1 diabetes, with 1,25(OH)2D3 prevents the disease, while 1,25(OH)2D3 deficiency in early life aggravates it.36–38 Also, prevention of experimental autoimmune encephalomyelitis, a model for multiple sclerosis, is observed upon treatment with a 1,25(OH)2D3 analog.39 Furthermore, graft rejection after transplantation is reduced upon treatment with 1,25(OH)2D3.40

REGULATION OF VITAMIN D METABOLISM

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. VITAMIN D ACTIONS
  5. VITAMIN D IN THE IMMUNE SYSTEM
  6. REGULATION OF VITAMIN D METABOLISM
  7. CONCLUSION
  8. Acknowledgments
  9. REFERENCES

Tissue availability of 1,25(OH)2D3 depends on dietary intake and sun exposure, but is certainly also influenced by the activity of the hydroxylating enzymes. Hydroxylations of vitamin D3 are performed by the cytochrome P450 (CYP) enzymes, i.e., 25-hydroxylases, 1α-hydroxylase, and 24-hydroxylase.

25-Hydroxylation

The first hydroxylation, 25-hydroxylation, results in the formation of 25(OH)D3. Most enzyme activity is located in the microsomes of the liver, but 25-hydroxylation also occurs in skin and intestine. At least four different CYP enzymes have been described as being responsible for the 25-hydroxylation of vitamin D3 in humans: CYP27A1, CYP3A4, CYP2R1, and CYP2J3. CYP27A1 resides in the mitochondria and displays preferential cholesterol-27-hydroxylation, next to minor activities such as 25-hydroxylation of vitamin D3. CYP27A1 gene knockout mice do not exhibit abnormalities in vitamin D metabolism, suggesting redundancy of this enzyme in 25-hydroxylation of vitamin D3, at least in mice.41 CYP3A4 has a very broad substrate specificity and low affinity for vitamin D3; thus, it is not a very attractive candidate. On the other hand, CYP2R1, which resides abundantly in the microsomes of liver and testis, seems to be the main enzyme responsible for 25-hydroxylation of vitamin D3.42 A mutation in the gene for CYP2R1 results in low 25(OH)D3 levels and vitamin D-dependent rickets.42 25-Hydroxylation is poorly regulated, and 25(OH)D3 levels rise with increases in serum levels of vitamin D3.43 Consequently, 25(OH)D3 is the major circulating form of vitamin D3 and its concentration is commonly used as an indicator of vitamin D status.44

In the immune system, a recent study demonstrated the expression of CYP2R1 in activated T cells and of CYP27A1 and CYP2R1 in DCs.45 Conversion experiments demonstrated that whereas CYP2R1 was not sufficiently expressed in activated T cells, nor was it functional to mediate efficient 25-hydroxylation of vitamin D3, 25-hydroxylation was functional in DCs.

24-Hydroxylation

24-Hydroxylation, performed by the CYP24A1 enzyme, is responsible for catabolism of active 1,25(OH)2D3, which will eventually lead to excretion of the hormone as calcitroic acid.1 Its expression is downregulated by PTH (parathyroid hormone), and upregulated by phosphate.46,47 1,25(OH)2D3 itself is a very strong inducer of 24-hydroxylase expression, and thus induces its own catabolism. This negative feedback loop probably serves as an internal rescue to avoid excessive 1,25(OH)2D3 levels and signaling. CYP24A1 knockout mice show poor viability, hypercalcemia, and intramembranous ossification of bone.48 Two VDREs are present in the promoter of the 24-hydroxylase gene, making 24-hydroxylase highly inducible by 1,25(OH)2D3.49 This 1,25(OH)2D3-mediated induction was shown to be highly dependent on the chromatin acetylation status of the CYP24A1 promoter.50 In addition, a C/EBPβ site was identified. C/EBPβ, which by itself is upregulated by 1,25(OH)2D3, and the VDR cooperate in 24-hydroxylase upregulation.

Besides its high renal expression and extensive negative feedback regulation by 1,25(OH)2D3 itself, monocytes, macrophages, and DCs also express CYP24A1.34,51 In these cells, however, the presence of this feedback mechanism depends on the differentiation/maturation stage of the cells. Undifferentiated monocytes are highly susceptible to 1,25(OH)2D3-mediated 24-hydroxylase induction, whereas differentiated/activated macrophages are resistant. The latter is due to an interplay between IFNγ-mediated and 1,25(OH)2D3-mediated effects: STAT-1α, a transcription factor involved in IFNγ-signalling, interacts with the DNA-binding-domain of the VDR, thereby prohibiting binding of the 1,25(OH)2D3/VDR/RXR-complex to the CYP24A1 promoter and preventing 1,25(OH)2D3-mediated induction of the enzyme.51 These findings may explain the persistent overproduction of 1,25(OH)2D3 in macrophages of patients with granulomatosis diseases.52 Remarkably, 24-hydroxylase-expression in DCs was only observed when the cells underwent their differentiation process in the presence of 1,25(OH)2D334.

1α-Hydroxylation

A third important hydroxylation is performed by the 1α-hydroxylase or CYP27B1 enzyme. In this case, 25(OH)D3 is hydroxylated, resulting in the formation of the active 1,25(OH)2D3 compound. The regulation of this hydroxylation step has been studied extensively in renal as well as in other cell types, including immune cells.

Regulation of 1α-hydroxylase in renal cells

The regulation of CYP27B1 in renal cells is summarized in Figure 2. Calcium-sensing cells in the parathyroid gland secrete PTH upon a decrease in serum calcium levels. PTH then induces the expression of CYP27B1, resulting in a rise of 1,25(OH)2D3 levels. 1,25(OH)2D3 mediates increased intestinal calcium uptake, increased renal calcium reabsorption and bone resorption, resulting in a rise in serum calcium levels. PTH stimulates the transcription of CYP27B1 in the kidney, by a mechanism involving the cAMP-activated protein kinase A pathway.53 More recently, Zierold et al.54 demonstrated that NR4A2, a nuclear orphan receptor, and C/EBPβ control transcriptional regulation of the CYP27B1 enzyme following PTH stimulation in kidney cells.

image

Figure 2. Overview of the regulation of 1α-hydroxylase in renal cells. PTH, calcitonin, low calcium levels, and low phosphate levels induce 1α-hydroxylase expression. 1,25(OH)2D3 is formed, which exerts its effects on kidney, intestine, and bone, leading to increased calcium and phosphate levels. Subsequently, 1,25(OH)2D3 itself as well as high calcium and phosphate levels have negative effects on the 1α-hydroxylase expression, both directly and by affecting PTH levels. Note: only 1,25(OH)2D3 actions are displayed. PTH also has direct effects on calcium and phosphate metabolism in intestine, kidney, and bone, i.e., elevated calcium (re)absorption/release and decreased phosphate reabsorption in kidney. Another hormone involved in calcium metabolism is calcitonin, which is secreted by the thyroid gland in response to high calcium levels. It induces elevated calcium uptake by bone and elevated secretion by the kidney.

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Analysis of the CYP27B1 promoter reveals the presence of a multitude of transcription factor binding sites.55,56 Several putative cAMP-responsive binding sites are present in the promoter, such as CRE, AP1, AP2, and Sp2. In addition, a CCAAT-box binding factor is important for PTH-induced upregulation.53 In distal parts of the nephron, CYP27B1 is also induced by calcitonin.57 1,25(OH)2D3 itself is a negative regulator of CYP27B1 transcription. Recently, two consensus VDRE-responsive sites have been identified in the human CYP27B1 promoter.58 Still, an indirect mechanism interfering with the PTH-induced signaling cascade might play a role.59 Furthermore, low calcium concentrations directly stimulate CYP27B1 synthesis via activation of protein kinase C and MAPK pathways by a calcium-sensing receptor protein.7 In contrast, low phosphate levels appear to stimulate 1,25(OH)2D3 synthesis in an indirect way via growth hormone, insulin-like growth factors, and “phosphatonins”, factors involved in phosphate homeostasis.60,61 Mice with a knockout in the phosphaturic peptide fibroblast growth factor 23 (FGF-23) demonstrated significant hypercalcemia and hyperphosphatemia, diminished renal phosphate excretion, and elevated serum 1,25(OH)2D3 levels, as well as upregulated renal CYP27B1 expression.62 Thus, FGF-23 inhibits CYP27B1 activity and 1,25(OH)2D3 synthesis and reduces intestinal phosphate absorption. Moreover, it is suggested that secreted frizzled related protein 4 is able to inhibit 1,25(OH)2D3 synthesis by inhibiting the activity of the CYP27B1. Extracellular phosphoglycoprotein, however, increases circulating 1,25(OH)2D3 concentrations.63

The Klotho protein, a beta-glucuronidase, has been described to suppress CYP27B1 gene expression.64 Klotho exists in a secreted and a membrane-bound form, and is mainly expressed in distal renal tubules, testes, and brain choroid plexus. Klotho mutant mice display a variety of phenotypes similar to age-related diseases. In addition, elevated calcium, phosphate, and 1,25(OH)2D3 levels occur. Moreover, a recent study has demonstrated that the Klotho protein can bind FGF receptors and can function as a regulator of FGF23 signaling in tubular cells.65 The fact that Klotho knockout mice and FGF23 knockout mice have elevated CYP27B1 activity and 1,25(OH)2D3 synthesis suggests that both Klotho and FGF23 are essential for the negative feedback circuit.62,66

Regulation of 1α-hydroxylase in non-renal cells

In situ hybridization studies and immunostaining assays have demonstrated that CYP27B1 is widely distributed in extra-renal tissues such as skin, bone and cartilage, prostate, colon, pancreas, and immune cells. CYP27B1 activity in non-renal cells delivers 1,25(OH)2D3 for autocrine/paracrine needs. In contrast to the elaborated understanding concerning regulation in renal cells, regulation of CYP27B1 in non-renal cells is less documented. However, it is clear that the extra-renal regulation of CYP27B1 is controlled by environmental signals other than those regulating renal synthesis.

In keratinocytes, CYP27B1 activity is upregulated by vitamin D3 and UVB.67 CYP27B1 activity is greatest in non-differentiated keratinocytes. In this view, differentiating keratinocytes produce their own 1,25(OH)2D3, which drives their differentiation.68 In prostate cells, CYP27B1 expression is induced by epidermal growth factor and inhibited by 1,25(OH)2D3.69 Prostate tumors are characterized by decreased CYP27B1 expression. Moreover, in human prostate cancer cells, the oncoprotein GFI1 (growth factor independent 1) acts as a repressor of the CYP27B1 gene through binding to a repressive region in the promoter.70 GFI1 forms a complex with corepressors that recruit histone deacetylases. Another mechanism whereby CYP27B1 is downregulated in prostate cancer progression is hypermethylation.71 In colon cells, CYP27B1 is not influenced by low calcium levels, and in bone, CYP27B1 levels are independent of circulating 1,25(OH)2D3 levels.72,73 In colon cancer cells, CYP27B1 levels are elevated in early phase, whereas CYP27B1 expression is lost in high-grade undifferentiated tumor cells. This might suggest an autocrine/paracrine antitumor role for 1,25(OH)2D3 in early phase.

Regulation of 1α-hydroxylase in immune cells

CYP27B1 is expressed in APCs, such as monocytes, macrophages, and DCs, suggesting that these immune cells are not only responsive to 1,25(OH)2D3, due to the presence of VDR, they are also able to produce the hormone autonomously.7,8 The 25(OH)D3-hydroxylating enzyme present in macrophages is identical to the renal form, but its expression is regulated in a completely different manner. Indeed, in macrophages CYP27B1 is not regulated by the classical stimuli as described above, i.e., calcium, PTH, 1,25(OH)2D3. This can be observed in patients who suffer from sarcoidosis.52,74 The alveolar macrophages of sarcoidosis patients display a very high level of CYP27B1 activity, leading to high concentrations of 1,25(OH)2D3 and hypercalcemia. High levels of calcium and 1,25(OH)2D3 lead in to the shut-down of CYP27B1 in kidney cells, while macrophages obviously do not respond to these signals. In contrast, CYP27B1 in macrophages has been shown to be regulated by immune signals, such as IFNγ and LPS, or viral infections, whereas the lack of negative feedback by 1,25(OH)2D3 has been confirmed.3,52,75,76 Extensive research has been performed in an attempt to unravel the intracellular pathways involved in this immune regulation. In human peripheral blood monocytes CYP27B1 is synergistically induced by IFNγ in combination with CD14/TLR4 ligation, an induction that was shown to require JAK-STAT, NF-κB, and p38 MAPK pathways. In addition, phosphorylation of C/EBPβ by members of the p38 MAPK pathway, as well as direct binding of C/EBPβ to its recognition sites in the CYP27B1 promoter, is necessary to enable this immune-stimulated upregulation.77 Adding to this, a more recent study in THP1 cells showed again the necessity of a second stimulus for IFNγ-mediated induction of CYP27B1 to occur, such as LPS or PMA, with the latter inducing differentiation to macrophages. Next to the JAK-STAT pathway, we demonstrated the importance of the MAPK ERK1/2 or p38 as other important pathways, dependent on the differentiation and activation state of the cells. These pathways play a role in the phosphorylation of the transcription factors STAT-1α and C/EBPβ. Moreover, both HATs (p300) and HDACs may play a role in the observed upregulation.78

Interestingly, Liu et al.79 recently showed that TLR2/1 activation of human monocytes/macrophages induced expression of the antimicrobial peptide cathelicidin, and was paralleled by increased expression of VDR and 1α-hydroxylase, indicating a role for 1,25(OH)2D3 in innate immunity. This resulted in reduced viability of intracellular Mycobacterium tuberculosis. In addition, they showed that the induced production of cathelicidin was lower in the serum of African Americans than of Caucasians, a difference that could be explained by the lower levels of 25(OH)D3 in the first population. The addition of 25(OH)D3 to African American serum could indeed restore the impaired cathelicidin production. These data provide evidence for a model in which the triggering of TLRs results in the conversion of 25(OH)D3 into active 1,25(OH)2D3, through induction of VDR and 1α-hydroxylase expression, in the induction of cathelicidin, eventually initiating an antimicrobial response.

More recently, CYP27B1 expression has also been observed in DCs and this phenomenon is associated with the p38 MAPK- and NF-κB-dependent maturation of these cells.34 Importantly, and in contrast with CYP27B1 regulation in kidney, neither in macrophages nor in DCs is CYP27B1 activity subjected to negative feedback signals deriving from 1,25(OH)2D3 itself.3,34 This observation explains the massive local production of 1,25(OH)2D3 by disease-associated macrophages that is seen in patients with granulomatous diseases (sarcoidosis and tuberculosis). Since it has been shown that upregulation of CYP27B1 and, therefore, 1,25(OH)2D3 synthesis by APCs occurs only at a later stage of macrophage and DC differentiation and activation, this system may act as a negative feedback loop in order to tone down inflammation.3,31,80

Next to this, a recent study demonstrated the functional expression of CYP27B1 not only in DCs but also in activated T cells, thereby enabling T cells themselves to accomplish the final step in the generation of active 1,25(OH)2D3.45 Antigen-specific immune responses are generated by the formation of DC-T conjugates in extralymphoid tissues such as skin and intestine, sites where vitamin D3 is taken up from food or generated through exposure to sunlight, or in tissue-draining lymph nodes. The ability of DC-T conjugates to generate 1,25(OH)2D3 from vitamin D3 locally, through the presence of 25- and 1α-hydroxylase activity in these cells, may enhance specific T cell programming by providing sustained and concentrated amounts of 1,25(OH)2D3 during the initiation of these antigen-specific responses.

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. VITAMIN D ACTIONS
  5. VITAMIN D IN THE IMMUNE SYSTEM
  6. REGULATION OF VITAMIN D METABOLISM
  7. CONCLUSION
  8. Acknowledgments
  9. REFERENCES

During the last decades it has become evident that 1,25(OH)2D3, the active form of vitamin D, plays an important role in the immune system, apart from its well-known role in calcium and bone homeostasis. Within the immune system, 1,25(OH)2D3 not only targets both APCs and T cells, which express the vitamin D receptor, but also the different enzymes responsible for activation of vitamin D3 and which are expressed by APCs and/or T cells. Indeed, a recent study convincingly showed 25-hydroxylase expression in DCs, suggesting efficient local activation of the sunlight-generated inactive pro-hormone vitamin D3, for instance in the skin. This finding supported a model in which DCs may play a key role in processing vitamin D3 to program “T-cell homing”.45 Next to this, 1α-hydroxylase is also expressed in monocytes, macrophages, DCs, and T cells. Recent findings point to a complex regulation in which a synergistic induction – upon prolonged exposure – of 1α-hydroxylase by IFNγ and TLR4/CD14 activation is crucial in monocytes/macrophages.77,78 This suggests a potential physiological role in the immune system. In the early phase of inflammation, when only macrophages are involved, 1α-hydroxylase is absent or low. Only upon production of the second necessary signal, IFNγ, by recruitment of other immune cells such as lymphocytes or NK cells, will the activated macrophages induce their 1α-hydroxylase levels and 1,25(OH)2D3 will be produced. This 1,25(OH)2D3 can then perform its well-known immunosuppressive action and shut down the ongoing immune reaction, thus preventing unrestricted immune responses. This complex regulation of 1α-hydroxylase also provides a better understanding of the abnormally high levels of 1,25(OH)2D3 observed in the pleural fluid of patients with different granulomatous diseases, such as sarcoidosis and tuberculosis, which are produced by activated pulmonary alveolar macrophages. From early studies in these diseases, it was clear that IFNγ plays a crucial role in the induction of 1,25(OH)2D3 and, thus, disease-associated hypercalcemia.52,81,82 In addition, it is now clear that IFNγ, although important, is not sufficient alone, since other macrophage activators/differentiators, such as LPS, PMA, or TNF-α, need to be present simultaneously to activate the complex network of signaling pathways necessary for 1α-hydroxylase induction. Adding to this, TLR2/1 triggering, as well as TLR4 activation, is able to induce 1α-hydroxylase in human macrophages, which is paralleled by induction of an antimicrobial response.

Finally, the 1,25(OH)2D3 metabolizing enzyme, 24-hydroxylase, is also expressed by APCs. Intriguingly, although highly induced by active 1,25(OH)2D3 itself, this induction is suppressed in the presence of IFNγ, which again points to an increased production of 1,25(OH)2D3 in situations of inflammation (summarized in Figure 3).

image

Figure 3. Overview of vitamin D3 homeostasis in the immune system. The presence of both 25-hydroxylase and 1α-hydroxylase has been demonstrated in APCs, enabling them to produce locally active 1,25(OH)2D3. Under steady-state conditions 24-hydroxylase expression is induced by 1,25(OH)2D3 creating a self-regulatory feedback loop and enabling 1,25(OH)2D3 to fulfill its role in maintaining immune balance. In the immune system, and in contrast with the kidney, 1α-hydroxylase is not incorporated in this self regulatory loop. In conditions of infection or inflammation, upregulation of 24-hydroxylase is hampered by interference of IFNγ-induced STAT1α, giving rise to sustained elevation of 1,25(OH)2D3 levels and, thus, sustained antimicrobial activity. Moreover, inflammatory factors, either derived from pathogens or inflammatory mediators produced by the immune system, also stimulate 1α-hydroxylase activity (through multiple intracellular signaling pathways), thereby also contributing to the rise in 1,25(OH)2D3 levels. When inflammation acquires a chronic character, 1,25(OH)2D3 levels may increase such that spillover in the general circulation is unavoidable, with consequent hypercalcemic side effects.

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In conclusion, the presence of both the activating and the metabolizing enzymes by cells of the immune system itself, indicates that 1,25(OH)2D3 can be produced locally in its active form. Moreover, the strict regulation of these enzymes in immune cells is highly suggestive for an autocrine/paracrine role in the immune system, and opens new treatment possibilities. Indeed, it suggests that vitamin D supplementation may be an adequate strategy for the treatment of immune-associated diseases, such as microbial infections and autoimmune diseases.

Acknowledgments

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. VITAMIN D ACTIONS
  5. VITAMIN D IN THE IMMUNE SYSTEM
  6. REGULATION OF VITAMIN D METABOLISM
  7. CONCLUSION
  8. Acknowledgments
  9. REFERENCES

Funding. This work was supported by the Catholic University of Leuven (GOA 2004/10 and EF/05/007), the Flemish Research Foundation (FWO G.0084.02, G.0233.04, and G.0552.06), and the Belgium Program on Interuniversity Poles of Attraction initiated by the Belgian State (IUAP P5/17 and P6/34). EVE, KS, CG, and CM were supported by a postdoctoral fellowship (Juvenile Diabetes Research Foundation, JDRF), a doctoral fellowship (FWO), a postdoctoral fellowship (FWO) and a clinical research fellowship (FWO), respectively.

Declaration of interest. The authors have no relevant interests to declare.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. VITAMIN D ACTIONS
  5. VITAMIN D IN THE IMMUNE SYSTEM
  6. REGULATION OF VITAMIN D METABOLISM
  7. CONCLUSION
  8. Acknowledgments
  9. REFERENCES