Figure 2.

Intracellular LPS receptor expression by primary IECs. (A) RT-PCR analysis for TLR4 and MD-2 expression in intestinal epithelium from fetal, 1-, and 6-d-old newborn as well as adult intestinal tissue obtained by laser microdissection. Detection of CD45 expression was used to demonstrate the absence of myeloid cells in the dissected tissue. Macrophage-like RAW 264.7 cells were used as positive control for CD45 expression. β₂-microglobulin (β₂m) expression was used as a housekeeping control. The hematoxylin-stained tissue section illustrates the microdissection procedure. Bar, 500 μm. (B) FACS analysis of isolated primary IECs from fetal (day −1) and adult (day 28) murine intestine for the expression of the LPS receptor complex TLR4/MD-2. RAW 264.7 cells were used as positive control. Dotted line, isotype control. (C) FACS analysis using double immunostaining of primary IECs for isotype antibodies (left) or TLR4/MD-2 and E-cadherin (right) to confirm epithelial TLR4 expression. Primary IECs were isolated from 6-d-old mice. (D) Staining of murine peritoneal macrophages as well as primary IECs isolated from adult (day 28) and fetal (day −1) intestinal tissue for TLR4/MD-2 with or without permeabilization of the cellular membrane to illustrate surface TLR expression. Dotted line, isotype control. (E) LPS internalization of primary IECs. Cells were incubated in the presence of 100 ng/ml S. typhimurium LPS and immunostained using two monoclonal antibodies directed against the LPS O-antigen. Counterstaining with FITC-phalloidin and DAPI. Bar, 20 μm.