Isolation and Characterization of Intestinal Epithelial Cells from Normal and SIV-Infected Rhesus Macaques

Impairment of intestinal epithelial barriers contributes to the progression of HIV/SIV infection and leads to generalized HIV-induced immune-cell activation during chronic infection. Rhesus macaques are the major animal model for studying HIV pathogenesis. However, detailed characterization of isolated rhesus epithelial cells (ECs) from intestinal tissues is not well defined. It is also not well documented whether isolated ECs had any other cell contaminants from intestinal tissues during the time of processing that might hamper interpretation of EC preparations or cultures. In this study, we identify and characterize ECs based on flow cytometry and immunohistochemistry methods using various enzymatic and mechanical isolation techniques to enrich ECs from intestinal tissues. This study shows that normal healthy ECs differentially express HLA-DR, CD23, CD27, CD90, CD95 and IL-10R markers. Early apoptosis and upregulation of ICAM-1 and HLA-DR in intestinal ECs are thought to be the key features in SIV mediated enteropathy. The data suggest that intestinal ECs might be playing an important role in mucosal immune responses by regulating the expression of different important regulatory and adhesion molecules and their function

Role of CD45 Signaling Pathway in Galactoxylomannan-Induced T Cell Damage

Previously, we reported that Galactoxylomannan (GalXM) activates the extrinsic and intrinsic apoptotic pathways through an interaction with the glycoreceptors on T cells. In this study we establish the role of the glycoreceptor CD45 in GalXM-induced T cell apoptosis, using CD45+/+ and CD45−/− cell lines, derived from BW5147 murine T cell lymphoma. Our results show that whereas CD45 expression is not required for GalXM association by the cells, it is essential for apoptosis induction. In CD45+/+ cells, CD45 triggering by GalXM reduces the activation of Lck, ZAP70 and Erk1/2. Conversely, in CD45−/− cells, Lck was hyperphosphorylated and did not show any modulation after GalXM stimulation. On the whole, our findings provide evidence that the negative regulation of Lck activation occurs via CD45 engagement. This appears to be related to the capacity of GalXM to antagonize T cell activation and induce T cell death. Overall this mechanism may be responsible for the immune paralysis that follows GalXM administration and could explain the powerful immunosuppression that accompanies cryptococcosis

Regulation of CD45 engagement by the B-cell receptor CD22.

The B-cell receptor CD22 binds sialic acid linked alpha-2-6 to terminal galactose residues on N-linked oligosaccharides associated with several cell-surface glycoproteins. The first of these sialoglycoproteins to be identified was the receptor-linked phosphotyrosine phosphatase CD45, which is required for antigen/CD3-induced T-cell activation. In the present work, we examine the effect of interaction between the extracellular domain of CD45 and CD22 on T-cell activation. Using soluble CD22-immunoglobulin fusion proteins and T cells expressing wild-type and chimeric CD45 forms, we show that engagement of CD45 by soluble CD22 can modulate early T-cell signals in antigen receptor/CD3-mediated stimulation. We also show that addition of sialic acid by beta-galactoside alpha-2,6-sialyltransferase to the CD22 molecule abrogates interactions between CD22 and its ligands. Together, these observations provide direct evidence for a functional role of the interaction between the extracellular domain of CD45 and a natural ligand and suggest another regulatory mechanism for CD22-mediated ligand engagement