Vitamin D-binding protein directs monocyte responses to 25-hydroxy- and 1,25-dihydroxyvitamin D

Abstract

Background: Serum 25-hydroxyvitamin D (25OHD) is a key factor in determining monocyte induction of the antimicrobial protein cathelicidin, which requires intracrine conversion of 25OHD to 1,25-dihydroxyvitamin D [1,25(OH)(2)D]. Both vitamin D metabolites circulate bound to vitamin D-binding protein (DBP), but the effect of this on induction of monocyte cathelicidin remains unclear.

Methods: Human monocytes were cultured in medium containing 1) serum from DBP knockout (DBP(-/-)) or DBP(+/-) mice, 2) serum-free defined supplement reconstituted with DBP or albumin (control), and 3) human serum with different DBP [group-specific component [Gc]] genotypes with varying affinities for vitamin D metabolites. In each case, response to added 1,25(OH)(2)D(3) or 25OHD(3) was determined by measuring expression of mRNA for cathelicidin and 24-hydroxylase. Monocyte internalization of DBP was assessed by fluorescent tagging followed by microscopic and flow cytometric analysis of tagged DBP.

Results: Monocytes cultured in DBP(-/-) serum showed more potent induction of cathelicidin by 25OHD(3) or 1,25(OH)(2)D(3) when compared with DBP(+/-) serum. Likewise, DBP added to serum-free medium attenuated 25OHD(3)/1,25(OH)(2)D(3) responses. Fluorescently tagged DBP showed low-level uptake by monocytes, but this did not appear to involve a megalin-mediated mechanism. Human serum containing low-affinity Gc2-1S or Gc2-2, respectively, supported 2.75-fold (P = 0.003) and 2.43-fold (P = 0.016) higher induction of cathelicidin by 25OHD relative to cells cultured with high affinity Gc1F-1F.

Conclusion: These data indicate that DBP plays a pivotal role in regulating the bioavailablity of 25OHD to monocytes. Vitamin D-dependent antimicrobial responses are therefore likely to be strongly influenced by DBP polymorphisms.

Figure 1

DBP knockout serum increases monocyte responses to vitamin D metabolites. Human monocytes cultured in medium supplemented with 5% serum from DBP^+/− and DBP^−/− mice were treated with 25OHD₃ (2–200 nm), 1,25(OH)₂D₃ (0.02–2 nm), or vehicle control (C, 0.2% ethanol) for 6 h. RNA from the resulting cells were then analyzed by RT-PCR for cathelicidin (panel A) and 24-hydroxylase (CYP24A1) (panel B). Data are shown as mean (n = 3) changes in RT-PCR ΔΔCt values relative to vehicle-treated cells. *, Statistically different from DBP^+/− serum cells at P < 0.001.

Figure 2

DBP add-back decreases monocyte responses to vitamin D metabolites. Human monocytes were cultured in SF medium supplemented with DBP (0.1–2 μm) or 10 μm BSA control (0 μm DBP) with all treatment groups receiving 10 μm total protein (DBP and BSA). Cells were treated with 25OHD₃ (2–200 nm), 1,25(OH)₂D₃ (0.02–2 nm), or vehicle (C, 0.2% ethanol) for 6 h. RNA from the resulting cells was then analyzed by RT-PCR for cathelicidin (panel A) and 24-hydroxylase (CYP24A1) (panel B). Data are shown as mean (n = 3) changes in RT-PCR ΔΔCt values relative to vehicle-treated cells. *, Statistically different from cells in medium containing no DBP at P < 0.001.

Figure 3

DBP is internalized by monocytes via a non-megalin-mediated mechanism. A, Binding of [³H]25OHD₃ to unlabeled DBP and fluorescently tagged DBP (Alexa-DBP). Nonspecifically bound (NSB) [³H]25OHD₃ was determined in the presence of a molar excess of nontritiated 25OHD₃. Total binding (TB) data are shown as picomoles 25OHD₃ bound per microgram DBP. B, Fluorescence microscopy of megalin-positive BN16 cells and monocytes showing that DBP is internalized by human monocytes. Longer exposure time was used to demonstrate DBP uptake by monocytes. C, Flow cytometric analysis of DBP uptake by monocytes at different stages of differentiation. Aliquots (0.4 μm) of Alexa-DBP were incubated with d-1 or -7 cultures of human monocytes. Flow cytometry was then carried out to identify cells with coexpression of CD14 and Alexa-DBP; dual-label data are shown as mean fluorescence intensity (MFI). D and E, Effect of the megalin inhibitor RAP on BN16 cells (D) and monocytes (E) (d 7). Cells were pretreated with 0.4 μm RAP, a megalin antagonist and then incubated with Alexa-DBP (0.1 μm). Data show flow cytometric data for Alexa-DBP uptake in untreated (control) cells and cells treated with Alexa-DBP in the absence or presence of RAP. Data in C–E show representative flow cytometry plots for each cell treatment incorporating 40,000 cells per plot. Flow cytometry assays were repeated at least twice with similar results. *, Statistically different from unlabeled DBP at P < 0.05. FITC, Fluorescein isothiocyanate.

Figure 4

Dose-responsive induction of cathelicidin by 25OHD₃ in monocytes cultured in medium supplemented with serum from donors with different DBP genotypes. Human monocytes cultured in medium supplemented with 5% serum from sex-matched (male) and age-matched (30–39 yr) donors were treated with 25OHD₃ (0–150 nm) for 6 h. RNA from the resulting cells was then analyzed by RT-PCR for cathelicidin mRNA expression. Data are shown as mean increase in cathelicidin mRNA (ΔΔCt value) relative to vehicle-treated (0.2% ethanol) controls for donor serum with a Gc2-1S genotype (n = 3) vs. a Gc1F-1F genotype (n = 3). *, Statistically different from Gc1F-1F at P < 0.05; **, P < 0.01.

Figure 5

Evolution of low-affinity forms of DBP enhances monocyte responses to vitamin D. Schematic representation of the impact of DBP genotype (Gc type) on monocyte responses to 25OHD₃, the major circulating form of vitamin D is shown. The left part of the panel shows primary DBP peptide sequence variations corresponding to the three major GC forms. The right part of the panel shows proposed variations in sensitivity to the immunomodulatory effects of 25OHD₃ for low- or high-affinity Gc forms.