Vitamin D Binding Protein and Complement Factor C5a Influence Neutrophil Chemotaxis Following HSCT

Abstract

Background Vitamin D binding protein (VDBP) is an abundant protein in human serum, related to albumin and alpha-fetoprotein. The VDBP gene is polymorphic, with three major alleles (Gc1F, Gc1S and Gc2) and four frequent genotypes. These genotype differences effect the glycosylation state of the molecule. VDBP has many diverse functions in addition to transporting vitamin D, and only about 4% of VDBP is bound to vitamin D at any one time. Acute inflammation leading to graft-vs-host disease (GVHD) may involve chemotaxis of leukocytes into damaged tissue mediated by chemotactic factors. Complement activation peptide C5a is a potent chemoattractant and together with VDBP has been shown to markedly enhance the motility of leukocytes. In previous work, we have shown that VDBP levels are significantly lower in those with acute GVHD (p=0.026), and 1-year NRM (p=0.03). Here we have reproduced these data analyzing serum from Day 100 post-HSCT with a new 110 patient cohort. Acute GVHD was reduced in those with VDBP levels above the median at Day 100 (0.03 v 0.18, p=0.037). 1-year NRM cumulative incidence (0.02 vs 0.2, p=0.002) was similarly reduced in those with VDBP levels above the median at Day 100, in agreement with our previous findings. In an effort to identify mechanisms whereby VDBP improves survival, we hypothesized that higher levels of VDBP will have a beneficial effect on neutrophil chemotaxis following HSCT. Previous VDBP animal model studies show chemotaxis of neutrophils to lung injuries can exacerbate pulmonary inflammation, but too little chemotaxis and the animals can succumb to infection. Here we observe a similar paradoxical effect with VDBP and chemotaxis. Methods VDBP was measured in serum at Day 100 in 110 transplant recipients using a polyclonal VDBP ELISA. Genotypes were determined by Taqman allele discrimination. Neutrophils from whole blood were negatively enriched from a normal adult donor and incubated in 10% patient serum in HBSS for 1 hr. For modified Transwell assay, put 4 × 105 cells in a 3-um-pore, 12-mm-diameter Transwell and place on top of 20 nM C5a in HBSS in a fibrinogen coated 12-well plate. Allow neutrophils to migrate for 2 hrs then count cells by hemocytometer. Results VDBP levels above the median at Day 100 were associated with significantly lower cumulative incidence of both NRM and GVHD 1-year post transplant (Fig 1 & 2). High levels of VDBP in patients with GcS1/GcS1 genotype decelerate chemotaxis towards a C5a bait. However, VDBP of a similar level but different genotype gives contrasting results; high VDBP levels with a GcS1/Gc2 genotype increase chemotaxis (Fig 3). Conclusions Our data demonstrate that VDBP-containing serum from children post-BMT modifies chemotaxis, potentially in a genotype dependent manner. We are extending our studies to test additional samples and genotypes. Moreover, we are exploring the effect of VDBP genotype on other transplant outcomes.