Regulation of the human cathelicidin antimicrobial peptide gene by 1α,25-dihydroxyvitamin D3 in primary immune cells

Abstract

Production of the human cathelicidin antimicrobial peptide gene (hCAP18/LL-37), is regulated by 1α,25-dihydroxyvitamin D3 (1,25D3) and is critical in the killing of pathogens by innate immune cells. In addition, secreted LL-37 binds extracellular receptors and modulates the recruitment and activity of both innate and adaptive immune cells. Evidence suggests that during infections activated immune cells locally produce increased levels of 1,25D3 thus increasing production of hCAP18/LL-37. The relative expression levels of hCAP18/LL-37 among different immune cell types are not well characterized. The aim of this study was to determine the relative levels of hCAP18/LL-37 in human peripheral blood immune cells and determine to what extent 1,25D3 increased its expression in peripheral blood-derived cells. We show for the first time, a hierarchy of expression of hCAP18 in freshly isolated cells with low levels in lymphocytes, intermediate levels in monocytes and the highest levels found in neutrophils. In peripheral blood-derived cells, the highest levels of hCAP18 following treatment with 1,25D3 were in macrophages, while comparatively lower levels were found in GM-CSF-derived dendritic cells and osteoclasts. We also tested whether treatment with parathyroid hormone in combination with 1,25D3 would enhance hCAP18 induction as has been reported in skin cells, but we did not find enhancement in any immune cells tested. Our results indicate that hCAP18 is expressed at different levels according to cell type and lineage. Furthermore, potent induction of hCAP18 by 1,25D3 in macrophages and dendritic cells may modulate functions of both innate and adaptive immune cells at sites of infection.

Keywords: Dendritic cells; LL-37; Macrophage; Osteoclasts; Parathyroid hormone; hCAP18.

Fig. 1. Expression of hCAP18 in freshly isolated human PBMC and neutrophils

(A) Freshly isolated human PBMCs were stained for lineage markers and hCAP18 and analyzed by two-color flow cytometry. The left column of the FACS density plots shows staining with a control rabbit IgG and lineage markers, while the right column shows the corresponding hCAP18 expression with the same lineage marker. (B) Human neutrophils stained for hCAP18 compared to a control rabbit IgG and unstained cells in a FACS histogram. In both A and B, the mean fluorescence intensity (MFI) for hCAP18 is noted in the upper right of the FACS plots. The plots are from one donor, representative of seven donors tested. (C) Scatter plot of hCAP18 log MFI for seven donors grouped by cell lineage. In each cell group, the central line represents the mean MFI, while the error bars represent the 95% confidence interval as determined by ANOVA. Asterisks denote that all cell lineage groups were statistically different from one another by ANOVA with Tukey’s Multiple comparison test (p < .05).

Fig. 2. Induction of hCAP18 expression by 1,25D3 treatment in monocytes, macrophages and dendritic cells

(A) Monocytes were cultured for 24 h in control media or media with 10 nM 1,25D3 and then stained for both CD14and hCAP18 or control rabbit IgG. The MFI for hCAP18 is noted in the upper right corner of the FACS density plots. (B) Macrophages and dendritic cells were treated with vehicle or with 10 nM 1,25D3 for 48 h and then stained for both CD14 and hCAP18 expression and shown as density plots. The total MFI for hCAP18 is noted in the upper right corner of the density plots. The plots are representative of 3 donors tested. (C) Comparison of hCAP18 MFI between macrophages and dendritic cells by scatter plot from 5 experiments with macrophages and 3 with dendritic cells treated as in part B. In each group, the central line represents the mean, while the error bars depict the standard error, and the asterisk denotes that induction was statistically significant in macrophages as compared to control and to the dendritic cell 1,25D3 treatment group (p < .05).

Fig. 3. Expression of CAMP, CD14 and CYP24A1 mRNA in response to treatment with 1,25D3

(A) Macrophages were treated with vehicle (ethanol) or 1,25D3 for 24 h. Total RNA was harvested and cDNAs generated for use in qRT-PCR. The data were normalized to β-actin and expressed as fold change versus the control treatment. (B) Dendritic cells and macrophages were treated with vehicle or 10 nM 1,25D3 for 48 h. CAMP mRNA levels for different cell types were determined by qRT-PCR and normalized to ribosomal 18S RNA using copy number standards. Data shown are from a single experiment and representative of four experimental replicates. (C) CD14 mRNA expression for samples described in panel B. CD14 levels were normalized to β-actin and are expressed as fold-change versus the vehicle control. Asterisks denote significance between 1,25D3 and controls, while two asterisks denote significant difference between the 1,25D3 treatment groups (p < .05).

Fig. 4. Lack of cooperative induction of CAMP gene expression by 1,25D3 and PTH in osteoclasts and macrophages

(A) Osteoclasts were treated 48 h with combinations of 10 nM 1,25D3 and 10 nM parathyroid hormone (PTH) and then stained for hCAP18 and CD14. FACS density plots show hCAP18 expression on the y-axis and CD14 expression on the x-axis, with the MFI for hCAP18 noted in the upper right hand corner. (B) qRT-PCR of CAMP mRNA expression from osteoclasts and macrophages treated with 1,25D3 and PTH normalized to ribosomal 18S RNA using copy number standards, with the asterisks denoting significance from controls by ANOVA (p < .05). (C) Quantitative PCR measuring CALCR mRNA expression in osteoclasts treated as in panel A. The data are expressed as fold-change normalized to ribosomal 18S RNA relative to the control vehicle treatment, and the asterisk denotes significance by ANOVA versus other treatments (p < .05). (D) Macrophages were treated with combinations of 1,25D3 and PTH for 48 h and then stained for CD14 and hCAP18 expression. FACS density plots for each treatment have the MFI for hCAP18 noted in the upper right corner. Macrophages were also treated with a lower dose of 10⁻¹¹ M PTH in combination with 1,25D3 as in part D (Supplemental Fig. 1). GM-CSF derived dendritic cells were also tested for responses to PTH and 1,25D3 (Supplemental Fig. 2).