Isolation and phenotypic characterization of colonic macrophages

Abstract

Macrophages play an important role in the intestinal mucosal immune system. However, they are a poorly defined cell population. We therefore determined their phenotype in normal colonic mucosa. Macrophages were isolated from colonic biopsies and surgical specimens by collagenase digestion. Colonic macrophages were positively sorted by anti-CD33 magnetic beads. Flow cytometric triple fluorescence analysis was applied to study CD14, CD16, CD33, CD44, CD11b, CD11c, CD64, HLA-DR, CD80, CD86 and CD3/CD19 expression. CD33 was evaluated as a positive marker for intestinal macrophages. CD33+ cells isolated from normal colonic mucosa showed co-expression of the established intracellular macrophage marker CD68 in FACS analysis. CD33+ cells were capable of phagocytosis. Isolation of this cell population by magnetic anti-CD33 beads and culture resulted in a 4.2-40-fold increase in IL-1beta and 4.5-44-fold increase in tumour necrosis factor-alpha (TNF-alpha) secretion compared with unsorted lamina propria mononuclear cells (LPMC). Of the CD33+ cells, 90.9 +/- 6.9% (mean +/- s.d.) were CD44+. However, macrophages from colonic mucosa showed only a low expression of CD14 (10.5 +/- 3.8%), CD16 (10.1 +/- 3.9%), HLA-DR (27.3 +/- 9.2%), CD11b (17.4 +/- 6.8%), CD11c (17.8 +/- 10.4%). Furthermore, expression of CD80 (9.2 +/- 4.2%) and CD86 (15.1 +/- 7.3%) was low, suggesting a low ability of normal intestinal macrophages to activate T cells and T cell-mediated immune responses. We conclude that CD33 is useful for the isolation and flow cytometric characterization of colonic macrophages. These cells exhibit a single phenotype in normal mucosa (CD33++, CD44++, CD14-, CD16-, CD11b-, CD11c-, HLA-DRlow, CD80-, CD86-) lacking lipopolysaccharide (LPS) receptor and costimulatory molecules.

Lack of CD47 on nonhematopoietic cells induces split macrophage tolerance to CD47nullcells

“…Residential intestinal macrophages display inflammatory anergy characterized by downregulated expression of innate response and growth factor receptors and reduced production of inflammatory cytokines but remain highly phagocytic (14)(15)(16). This unique property of intestinal macrophages is essential to their undiminished capacity to protect the host from foreign pathogens while being less inflammatory in character.…”

Generalised reduction of putative endothelial progenitors and CXCR4-positive peripheral blood cells in type 2 diabetes

“…Laboratory and diagnostic assays were from fasted blood samples; smoking refers to previous or current smokers ***p<0.001 adjusted in forward and side scatter dot plot to exclude cellular debris. Monocytic and lymphocytic populations were identified by size and light-scattering properties [34]. CD14 and CD45 staining was used to further verify the presence of monocytic and lymphocytic cell populations, respectively.…”

Systematic expression profiling of innate immune genes defines a complex pattern of immunosenescence in peripheral and intestinal leukocytes

“…The distribution of subpopulations in the LPMNC preparations was found to be similar to that in earlier flow cytometry studies. [11][12][13] Contamination of epithelial cells was seen as less than 0.5% by both morphology and cytokeratin antibody (Dako) staining. 14 …”