Toll-like Receptor 4 Resides in the Golgi Apparatus and Colocalizes with Internalized Lipopolysaccharide in Intestinal Epithelial Cells

Abstract

Toll-like receptor (TLR) 4 is mainly found on cells of the myelopoietic lineage. It recognizes lipopolysaccharide (LPS) and mediates cellular activation and production of proinflammatory cytokines. Less is known about the distribution and role of TLR4 in epithelial cells that are continuously exposed to microbes and microbial products. Here we show that the murine small intestinal epithelial cell line m-ICcl2 is highly responsive to LPS and expresses both CD14 and TLR4. Transcription and surface membrane staining for CD14 were up-regulated upon LPS exposure. Surprisingly, TLR4 immunostaining revealed a strictly cytoplasmic paranuclear distribution. This paranuclear compartment could be identified as the Golgi apparatus. LPS added to the supernatant was internalized by m-ICcl2 cells and colocalized with TLR4. Continuous exposure to LPS led to a tolerant phenotype but did not alter TLR4 expression nor cellular distribution. Thus, intestinal epithelial cells might be able to provide the initial proinflammatory signal to attract professional immune cells to the side of infection. The cytoplasmic location of TLR4, which is identical to the final location of internalized LPS, further indicates an important role of cellular internalization and cytoplasmic traffic in the process of innate immune recognition.

Ligand-induced differential cross-regulation of Toll-like receptors 2, 4 and 5 in intestinal epithelial cells

“…The addition of LPS (1-10 ng/ml) to mIC cl2 cells also caused a dose-dependent NF-B activation ( Fig. 2A), in agreement with earlier observations (Hornef et al, 2002(Hornef et al, , 2003. To ensure the relevance of the results obtained with the NF-B luciferase reporter system, we analyzed the effect of TLR activation on the expression of the chemokines KC (CXCL1) and MIP-2 (CXCL2).…”

“…KC and MIP-2 are mouse orthologs of human IL-8 (Bozic et al, 1994), which is one of the mainly secreted chemokines by epithelial cells upon inflammation (Rimoldi et al, 2005). Similar to stimulation of TLR4 (Hornef et al, 2002), activation of TLR2 (Fig. 2B) and TLR5 (Fig.…”

“…The mouse intestinal epithelial cell line mIC cl2 (Bens et al, 1996) has been shown to express functional TLR4 (Hornef et al, 2002(Hornef et al, , 2003. Real-time RT-PCR demonstrated that mIC cl2 cells also express TLR2 and TLR5 (Table 1).…”

“…The mouse intestinal epithelial cell line mIC cl2 (Bens et al, 1996;Hornef et al, 2002), stably transfected with an NF-B luciferase reporter construct (Hornef et al, 2003), was cultured in DMEM/Ham's F-12 (1:l, v/v), 60 nM sodium selenite, 5 g/ml bovine transferrin, 2 mM glutamine, 50 nM dexamethasone, 1 nM triiodothyronine, 5 g/ml bovine insulin, 20 mM d-glucose, 10 nM mouse epidermal growth factor, 2% FCS, and 20 mM N-2-hydroxyethylpiperazine-N -2-ethanesulfonic acid (HEPES) (pH 7.4). Propagation of the mIC cl2 cell line in IMDM with 10% FCS for several passages to exclude possible effects of the additives present in the complex medium, yielded no noticeable differences in TLR function.…”

“…A novel epithelial (mIC cl2 ) cell line derived from intestinal mouse crypts recently has been shown to respond to LPS (Bens et al, 1996;Hornef et al, 2002Hornef et al, , 2003. mIC cl2 Cells form a tight monolayer separated by tightjunctions with a distinct brush-border (apical) and basolateral membrane compartment and have retained specific epithelial features such as dome formation, membrane-associated enzyme activities and expression of pIgRs (Bens et al, 1996).…”