Macrophage inflammatory protein-3α is upregulated during differentiation of intestinal macrophages

Abstract

Background: Intestinal macrophages (MAC) are a central component in the defense of the intestinal mucosa against luminal microbes. In normal mucoca monocytes (MO) differentiate to immunological tolerant intestinal MAC with a typical phenotype. We could show that in the lamina propria display of markers such as CD14, CD16, CDtlb or T-cell costimulatory molecules such as CD80 or CD86 on MAC is low. Recently we could evaluate CD33 as a positive marker for intestinal MAC and isolate MAC by anti-CD33 magnetic beads. Methods: CD33 + cells (MAC) were isolated from normal intestinal mucosa with the help of immunomagnetic beads armed with CD33 antibody, mRNA was isolated by polyT magnetic beads. A subtractive screening of the mRNA populations (RDA) was performed subtracting mRNA from normal MAC from those of in vitro differentiated MAC. Macrophaoe inflammatory protein 3 alpha (MIP-3a) mRNA expression was determined by RT PCR, protein expression was detarmined by flow cytometry and immunohistochemiatry. Results: 3 of 70 clones obtained by the subtractive mRNA screening and subsequent sequencing showed >99% homology to mRNA of MIP-3a. MIP-3~z is a chemokine found in dendritic cells, fibroblasts and other cells and is responsible for the selective recruitment of dendritic cells and subsets of T cells. For confirmation of the RDA data MIP-3a expression was demonstrated in cryostat sections of normal intestinal mucoea by immunohistocbemiat~ in the lamina propria in a pattern similar to CD68 (MAC marker). FACS analysis of purified intestinal MAC clearly indicated expression of MIP-3a in these cells in contrast to blood MO and in vitro differentiated MAC which lacked a MIP-3=x signal. Furthermore, RT-PCR amplification of a 250 bp hagment of MIP-3~z was only successful in cDNA from purified mucoeal MAC but not from blood MO or in vitro differentiated MAC. In an in vitro differentiation model for intestinal MAC recently established by our group MIP-3(x protein expression was absent on day 1 of co-culture but easily detectable by immunohtatochemiatry on day 7 further confirming the induction of MIP-3a in intestinal MAC during differentiation. Conclusion: MIP-3a mRNA is upregulated during differentiation of blood mononcytes to macrophages in the intestinal mucosa correlating with enhanced expression of MIP-3a protein. Thus the secretion of this chemokine by macrophages could play a central role in the recruitment of immune cells into the intestinal mucoea.