Vitamin D supplementation does not change expresion of human cathelicidin in neutrophils of young healthy subjects

MASTAGLIA SR; MAROTTE C; BRYK G; SOMOZA J; BRITO G; SEIJO M; FORMARI MC; DIEZ RA ; OLIVERI B

San Diegp

Congreso; Anual Meeting ASBMR; 2011

ASBMR

VITAMIN D SUPLEMENTATION DOES NOT CHANGE EXPRESION OF HUMAN CATHELICIDIN IN NEUTROPHILS OF YOUNG HEALTHY SUBJECTS. SR Mastaglia1,3, C Marotte1,3, G Bryk1, J Somoza1,3, G Brito1, M Seijo1, MC Fornari4, RA Diez2, B Oliveri1,3. 1Sección Osteopatías Médicas, Hospital de clínicas, Universidad de Buenos Aires. Argentina 2 2º Cátedra de Farmacología, Facultad de Medicina, Universidad de Buenos Aires, Argentina. 3 National Council for Scientific and Technologic Research (CONICET) 4 Laboratorio de Análisis Clínicos Fornari Bioalpha SA, Buenos Aires, Argentina. The antimicrobial peptide cathelicidin (hCAP18) is stored in several cell types of the system immune, mainly in the granules of neutrophils (PMN). 1,25 (OH)2D is a direct inducer of expression of the gene encoding hCAP18. The aim of the present study was to evaluate the expression of leukocytes’ cathelicidin in young healthy volunteers supplemented with vitamin D2 and D3. Thirty-three young healthy volunteers (7M/ 26F) with an age of (mean ± SD) 33.4 ± 6 years and BMI of 22.6 ± 2 Kg/m2 were included in this randomized placebo controlled trial for 11 weeks (Sep-Dec 2010).  Subjects were allocated to the following groups: Vitamin D2 (n=11); Vitamin D3 (n=11) and placebo (n=11). At the start of the study a single dose (100,000 IU/day (d) of vitamin D2 or D3 according to the group) was administered. Thereafter, from d 7th to 21st of the study, volunteers received 4,800 IU/d of the corresponding vitamin D.  No vitamin D was administrated from 22 to 77 d. No infection or allergic symptoms were reported during the study. At baseline and d: 7, 21, and 63 blood samples were obtained to measure serum Ca, P, BAP and 25OHD (RIA-DIASORIN). At each sampling point, the expression of hCAP18 in peripheral blood PMN was assessed by flow cytometry with a FACSort® (Becton-Dickinson, San Jose, CA, USA) and the CellQuest® software (Becton-Dickinson), using the mouse monoclonal antibody OSX12 (Abcam, UK), specific for hCAP18, and a secondary incubation with FITC anti-murine IgG (Abcam). A protocol of permeation used for neutrophil myeloperoxidase was used, and myeloperoxidase labeling was included as control. Region corresponding to PMN was defined by side and forward scattering; PMN expressing a higher level of hCAP18 were identified in the fluorescence histogram of the neutrophils´ region and defined as M1. Most (>95%) PMN expressed intra-granular cathelicidin, either at the basal sample or after vitamin D or placebo treatment. Only 1% to 2% depicted higher expression (Figure1). No significant differences were observed among groups in the expression hCAP18 in PMN, either as level of expression (mean fluorescence) or as percentage of cells over-expressing hCAP18, in spite of the significant increment of 25OHD levels in both supplemented groups (d7:100%;d21:90% and d63:60% compared to basal levels). Under these experimental conditions, PMN´s cathelicidin is not modified by vitamin D supplementation, independently of calciferol used. Figure 1: Comparison of hCAP18 expression in PMN-M1 at baseline and at D21 of the study.