Decreased Anti-Inflammatory Responses to Vitamin D in Neonatal Neutrophils

Neutrophil activity is prolonged in newborns, suggesting decreased exposure and/or responses to immunosuppressive modulators, such as 1,25-hydroxyvitamin D3 (1,25-vit D3). We hypothesized that 1,25-vit D3 suppresses neutrophil activation and that this response is impaired in newborns. Consistent with this, 1,25-vit D3 decreased LPS-induced expression of macrophage inflammatory protein-1β and VEGF in adult, but not neonatal, neutrophils. Expression of vitamin D receptor (VDR) and 25-hydroxyvitamin D3-1α-hydroxylase was reduced in neonatal, relative to adult neutrophils. Moreover, 1,25-vit D3 induced VDR gene expression in activated adult, but not neonatal, neutrophils. 1,25-vit D3 also suppressed expression of cyclooxygenase-2 and induced expression of 5-lipoxygenase in LPS-exposed adult neutrophils, while neonatal cells were not affected. 1,25-vit D3 had no effect on respiratory burst in either adult or neonatal cells. Anti-inflammatory activity of vitamin D is impaired in neonatal neutrophils, and this may be due to decreased expression of VDR and 1α-hydroxylase. Insensitivity to 1,25-vit D3 may contribute to chronic inflammation in neonates.


Introduction
There is a high incidence of vitamin D insufficiency in neonates, particularly those who are exclusively breast fed [1]. After undergoing 25-hydroxylation in the liver, 25hydroxyvitamin D 3 is converted, via 25-hydroxyvitamin D 3 -1α-hydroxylase (1α-hydroxylase), to the active form 1,25vit D 3 in many cell types. The biologic effects of 1,25vit D 3 are primarily mediated via the nuclear transcription factor, vitamin D receptor (VDR), which triggers expression of vitamin D-responsive genes. While the importance of vitamin D in calcium and phosphate homeostasis is well known, recent studies have demonstrated that vitamin D has "nonclassical" effects, including an important role in downregulating immune responses. VDR and 1α-hydroxylase are expressed in monocytes, dendritic cells, T-lymphocytes, and granulocytes, suggesting that 1,25-vit D 3 plays a role in differentiation, biosynthetic activity, and function of leukocytes [2,3].
Circulating neutrophils are the main effector cells in innate immunity but can contribute to pathology if their activity is prolonged [4,5]. Resolution of the neutrophilic inflammatory response is an active process involving downregulation of proinflammatory cytokines, upregulation of anti-inflammatory eicosanoids, decreased generation of reactive oxygen intermediates, and removal of the cells [6]. In this regard, we have previously shown that the clearance of neutrophils by apoptosis is impaired in neonates when compared to adults [7]. Prolonged viability of neonatal neutrophils is thought to play a role in chronic inflammatory disease because these cells release oxygen radicals, hydrolases, and inflammatory cytokines. This results in cytotoxicity, potentially contributing to the high incidence of neutrophilmediated inflammatory diseases in newborns.
Previous studies have suggested that vitamin D downregulates neutrophil function [8]. In these studies, we investigated the hypothesis that 1,25-vit D 3 decreases production of inflammatory mediators and reactive oxygen intermediates in neutrophils and that this activity is impaired in neonatal neutrophils. We further hypothesized that reduced responses in neonates may be related to decreased expression of VDR and 1α-hydroxylase in neonatal neutrophils. Impaired responsiveness to 1,25-vit D 3 may contribute to prolonged viability and activity of these cells, leading to increased susceptibility to chronic inflammatory diseases.

Materials and Methods
2.1. Reagents. RNA purification kits were purchased from Qiagen (Chatsworth, CA). Primers for RT-PCR were obtained from Integrated DNA Technologies (Coralville, IA). Reagents for RT-PCR were from Applied Biosystems (Foster City, CA). Amplex Red and horseradish peroxidase were from Molecular Probes (Carlsbad, CA), DMEM and dextran from Sigma Chemical Co. (St. Louis, MO), and Ficoll-paque from GE Healthcare (Piscataway, NJ). 7-actinomycin D (7-AAD) and cytometric bead array flex sets were from BD Biosciences (San Jose, CA). 1,25-vit D 3 was obtained from Enzo Life Sciences (Plymouth Meeting, PA) and solubilized in ethanol at a stock concentration of 10 −4 M. The final concentration of 10 −7 M in cell culture was based on estimates of tissue concentrations and on previous studies demonstrating biologic effects at this concentration [8].

Subjects and Neutrophil
Isolation. Studies were approved by the Institutional Review Board of Robert Wood Johnson Medical School and informed consent obtained from subjects. Umbilical cord blood was obtained from the placentas of term infants (≥37 wk gestation, n = 27) delivered by cesarean section without labor. Peripheral venous blood collected from healthy adults (n = 25) was used for comparison. Neonatal and adult samples were anticoagulated using heparin and processed immediately in parallel. Subjects were excluded for clinical evidence of infection, history of immunodeficiency, diabetes, pregnancy-induced hypertension, or exposure to tobacco or to medications that affect immune function. While subjects were also to be excluded if there was recent history of vitamin D intoxication (due to unusual dietary supplementation) or deficiency (diagnosed secondary to neonatal hypocalcemia), no prospective subjects met these criteria. Equivalent numbers of adult and neonatal samples were collected concurrently and uniformly throughout a single year, to minimize any potential seasonal effects. Neutrophils were isolated by dextran sedimentation and Ficoll gradient centrifugation, with >95% viability and purity confirmed by trypan staining. In preliminary experiments, 1,25-vit D 3 did not affect neutrophil viability or apoptosis, as determined by flow cytometric measurement of propidium iodide or annexin V binding, respectively.

Inflammatory Proteins.
Neutrophils were incubated with control medium (DMEM + 10% fetal bovine serum), LPS (1 μg/mL), and/or 1,25-vit D 3 (10 −7 M) for 24 hr. Culture supernatants were incubated with premixed flex set beads coated with antibodies to macrophage inflammatory protein-1β (MIP-1β), IL-8, vascular endothelial growth factor (VEGF), and IL-1β in 96-well filtration plates. Premixed phycoerythrin-labeled detection reagent was then added to the wells and incubated in the dark for 2 hr. The bead-protein complexes were analyzed for fluorescence intensity using a BD FACSArray Bioanalyzer. Data were analyzed using BD FCAP software (v 2.0).

Results and Discussion
In order to model bacterial-induced tissue inflammation, we analyzed the responses of LPS-treated adult and neonatal Mediators of Inflammation In further studies, we compared the effects of 1,25vit D 3 , LPS, and LPS + 1,25-vit D 3 on the production of inflammatory cytokines and proteins by adult and neonatal neutrophils. Constitutive production of MIP-1β, IL-8, VEGF, and IL-1β was similar in adult and neonatal neutrophils ( Figure 2). LPS induced production of MIP-1β, IL-8, and IL-1β adult cells, while neonatal cells were not affected. In adult neutrophils, LPS + 1,25-vit D 3 significantly inhibited the generation of MIP-1β and VEGF when compared to LPS alone, and there was a trend toward decreased production of IL-8. In contrast, 1,25-vit D 3 did not significantly affect production of these inflammatory proteins in LPS-treated neonatal neutrophils.
We next compared the effects of 1,25-vit D 3 on production of H 2 O 2 in adult and neonatal neutrophils. Activated neutrophils secrete reactive oxygen intermediates in response 4 In these studies, we demonstrated that 1,25-vit D 3 decreases specific inflammatory responses in mature circulating neutrophils and that this is impaired in neonatal cells. Previous findings on the effects of vitamin D on neutrophils have been inconsistent. For example, vitamin D induces expression of human cathelicidin antimicrobial peptide 18 [11], and vitamin D deficiency decreases TLRinduced cathelicidin expression in monocytes [12]. Therefore, vitamin D appears to play an important role in the integrity of the innate immune response. However, vitamin D also decreases production of IL-1β in neutrophils [8], and increasing evidence suggests that vitamin D can trigger resolution of inflammatory responses. 1,25-vit D 3 inhibits differentiation, proliferation, and synthetic activity in T lymphocytes and induces a tolerogenic state in dendritic cells, suggesting that it suppresses adaptive immunity [13,14]. While 1,25-vit D 3 may promote differentiation and synthetic activity in monocytes and macrophages [15,16], it also decreases proliferation and interleukin-2 production and inhibits production of IL-12 by downregulating NF-κB activation in these cells [17,18]. It also downregulates Toll-like receptor-(TLR-)2 and TLR-4 expression in human monocytes [19]. Our findings that 1,25-vit D 3 decreases neutrophil activity support the idea that vitamin D plays a physiologic role in the suppression of immune responses.

Mediators of Inflammation
Neonates are at particularly high risk for developing neutrophil-mediated inflammatory diseases, suggesting that there are developmental defects in signaling pathways that down regulate neutrophil activity and induce their clearance by macrophages. Consistent with this idea, our laboratory has previously shown that apoptosis is impaired in neutrophils from neonates when compared to adults [7]. In these studies, we have shown that responsiveness to vitamin D is also impaired in neonatal neutrophils. This may contribute to prolonged viability and activity of these cells, possibly contributing to chronic inflammatory disease secondary to the release of reactive oxygen species and inflammatory cytokines. Decreased responsiveness may be due to reduced expression of VDR. We found that adult neutrophils express VDR and that 1,25-vit D 3 induces VDR expression in the presence of LPS. Induction of VDR by 1,25-vit D 3 and LPS has also been observed previously in a human monocytic cell line [20]. In contrast, neonates expressed significantly lower levels of VDR, and this was not affected by vitamin D and LPS. We also found that expression of 1α-hydroxylase is significantly decreased in neonatal neutrophils, when compared to adults. 1α-hydroxylase is required for the generation of 1,25-vit D 3 , the biologically active form that triggers binding to response elements in the promoter regions of vitamin D responsive genes. Decreased expression of this enzyme suggests that activation of vitamin D may be impaired in neonatal leukocytes. In addition, 1,25-vit D 3 induced 5-LOX and suppressed COX-2 gene expression in LPS-treated adult neutrophils, while neonatal cells were not affected. 5-LOX catalyzes the generation of anti-inflammatory eicosanoids such as lipoxins, while COX-2 exerts inflammatory effects via synthesis of prostaglandins [21]. The shift from generation of pro-to anti-inflammatory eicosanoids is thought to play a key role in signaling the resolution of neutrophil activity [22]. Impaired alteration of the ratio of 5-LOX/COX-2 gene expression in response to vitamin D may contribute to prolonged inflammation in neonates.
Previous in vitro and in vivo studies in humans have suggested that vitamin D is associated with decreased inflammatory cytokine production [8,23]. To our knowledge, this is the first study to measure cytokine responses to vitamin D in neonatal neutrophils. We found that while vitamin D suppressed LPS-induced production of MIP-1β and VEGF in adult cells, it had no effect on the production of these mediators in neonatal cells. This is consistent with our finding that expression of VDR is impaired in neonatal neutrophils. Taken together, these data suggest that neonatal neutrophils have decreased capacity to synthesize 1,25-vit D 3 and decreased responsiveness to its immunosuppressive effects.
Surprisingly, vitamin D did not reduce basal or PMAinduced generation of hydrogen peroxide in either adult or neonatal neutrophils. Consistent with this, previous investigators have reported that vitamin D restores chemotaxis and phagocytosis in vitamin D-deficient mice [24] and can facilitate bacterial killing in human cells [25]. Others have also shown that vitamin D induces expression of membrane and cytosolic components of NADPH-oxidase in monocyte-like cell lines [26]. The effects of vitamin D on respiratory burst activity are likely dependent on specifics of dose and endpoint, and this will be investigated in further studies.

Conclusions
We found that 1,25-vit D 3 decreased LPS-induced expression of macrophage inflammatory protein-1β and VEGF in adult, but not neonatal, neutrophils. Our findings suggest that neonates may be resistant to the modulatory effects of vitamin D in neutrophilic inflammation and that this may be due to decreased expression of VDR and 1α-hydroxylase. Decreased responsiveness to vitamin D in neonates may compound the effects of vitamin D deficiency, which is now recognized as an important public health problem. Investigations of vitamin D status have revealed a high incidence of deficiency in both mothers and infants [26], and the American Academy of Pediatrics recently issued recommendations for increased dietary supplementation with vitamin D in neonates [27]. Understanding the immunemodulatory actions of vitamin D in neonates at the cellular level may strengthen efforts to adequately address this public health challenge, using strategies such as therapeutic levels of supplementation or treatment with synthetic VDR agonists. Abbreviation 1α-hydroxylase: 25-hydroxyvitamin D 3 -1α-hydroxylase COX-2: Cyclooxygenase-2 5-LOX: 5-lipoxygenase LPS: Bacterial lipopolysaccharide MIP-1β: Macrophage inflammatory protein-1β PMA: Phorbol 12-myristate 13-acetate VEGF: Vascular endothelial growth factor VDR: Vitamin D receptor 1,25-vit D 3 : 1,25-dihydroxycholecalciferol.

Financial Support
Supported by National Institutes of Health grants HD058019, ES005022, and a research grant from the Foundation of the University of Medicine and Dentistry of New Jersey.