Enteric mucosal surfaces withstand constant exposure to a great burden of bacteria and bacterial products, a prerequisite for the intimate relationship between the host and its beneficial normal microflora. Low or absent intestinal expression of molecules involved in LPS recognition intriguingly explains local endotoxin hyporesponsiveness (10
). However, here we describe a highly LPS-responsive phenotype of the murine small intestinal cell line m-ICcl2
. Significant levels of TLR4 and MD2 expression were found, and even minute amounts of LPS led to rapid and pronounced cell activation. In fact, the maximal stimulatory LPS concentration and the kinetic of MIP-2 secretion were found to be similar in m-ICcl2
cells and macrophage-like RAW 264.7 cells. However, the response of m-ICcl2
cells was limited to the production of the chemokine MIP-2. No secretion of the proinflammatory cytokines TNF-α, or IL-6 was detected. Interestingly, m-ICcl2
cells expressed the LPS coreceptor CD14. Although the basal level of CD14 expression was low, m-ICcl2
cells maintained full LPS susceptibility under serum-free conditions. Furthermore, LPS exposure induced a dose-dependent increase leading to a significant intensity of CD14-specific surface staining. Hence, m-ICcl2
cells seem to be well equipped to respond to LPS in order to initiate a proinflammatory response and attract professional immune cells.
How can intact recognition of LPS be present in the gastrointestinal tract without evoking proinflammatory stimulation? Fetal enterocytes were shown to express TLR4 and ex vivo–derived intestinal organ cultures from fetal tissue demonstrated significantly higher LPS susceptibility as compared with infant tissue (28
). Therefore, down-regulation of endotoxin recognition only seems to arise with microbial colonization of the intestine after birth. It is noteworthy that few if any resident Gram-negative microflora were found in the small intestine (with the exception of the terminal ileum) in contrast to the colon (30
). Interestingly, a recent thorough investigation of the phenotypic markers of m-ICcl2
cells identified a number of important properties and differentiated functions as found in intestinal epithelial crypt cells. m-ICcl2
cells show a high nucleus to cytoplasma ratio, rudimentary brush border, and regular tight junctions that delineate the apical membrane domain. They express glycoconjugates, polymeric immunoglobulin receptor, the cystic fibrosis transmembrane conductance regulator, and the multidrug resistance-associated protein MRP-1, which is characteristic of intestinal crypt epithelium (14
). Although little is known about the exact environmental conditions in the intestinal crypts in vivo, recent work indicates that the local conditions might differ significantly from the situation in the intestinal lumen. High local concentrations of α-defensins along with a permanent secretory flow might create a largely bacteria-free environment in the enteric crypts of healthy individuals (31
). The LPS-neutralizing capacity of antimicrobial peptides could further diminish the local soluble LPS concentration (32
). These exceptional conditions in enteric crypts would allow the presence of an intact endotoxin recognition system. Indeed, crypts that are isolated from murine small intestinal tissue showed a strong TLR4 expression. This is in accordance with the finding that human TLR4 expression in fetal small intestine was predominantly found in crypt enterocytes (28
). The fact that prominent Gram-negative enteric pathogens specifically target the small intestine might even demand the preservation of intact LPS recognition in this part of the gastrointestinal tract. Tissue destruction by pathogenic microorganisms would expose crypt cells to LPS and consequently initiate an inflammatory response.
Surprisingly, immunostaining of m-ICcl2
cells revealed a dense cytoplasmic perinuclear localization of TLR4. This cytoplasmic compartment could be identified as the Golgi apparatus using three independent markers. Pharmacological disruption of the Golgi structure significantly altered the TLR4 staining morphology, confirming the identified location. No secondary cellular location or cycling between the cell surface and the Golgi complex could be demonstrated as it was reported for TGN38 or the convertase furin (23
). Thus, TLR4 of m-ICcl2
cells seems to be permanently located in the Golgi apparatus. Interestingly, the Golgi apparatus has previously been associated with LPS-mediated cell activation (34
). In contrast to m-ICcl2
cells, surface staining of TLR4 was detected on nonpermeabilized murine peritoneal macrophages, using a monoclonal anti-TLR4 antibody and FACS®
). Moreover, TLR2 was demonstrated on the surface of cells using transient transfection and a flag-based detection system (36
). Consequently, internalization of zymosan particles was accompanied by the concentration of TLR2 to phagosomes. Since peritoneal macrophages in the healthy individual reside in a microbe-free environment, the localization of TLR4 on the plasma membrane exposed to the surrounding environment might ensure the highest LPS sensitivity. In contrast, the intracellular location of TLR4 in epithelial cells would add an additional regulatory barrier to prevent uncontrolled stimulation. Similarly, an intracellular system of LPS recognition has recently been suggested in intestinal epithelial cells (37
). However, further analysis of cellular TLR distribution in intestinal tissue in vivo is clearly needed to validate the importance of this finding.
Close physical contact between LPS and TLR4 was clearly demonstrated to be necessary to mediate cell activation (20
). However, aside from the prominent paranuclear location, membrane staining of TLR4 was not detected in m-ICcl2
cells. Chase treatment, protein synthesis inhibition, and cell activation with LPS also did not reveal any detectable surface membrane staining. Moreover, all efforts to block TLR4-mediated signaling using an antibody directed against the TLR4–MD-2 complex failed, although this strategy was successful in macrophages using the same antibody (16
). These results indicate that TLR4 is not on the plasma membrane of m-ICcl2
cells during LPS-induced cell activation, but rather in an intracellular compartment. The recent finding that oligomers of MD-2 associate with TLR4 in the endoplasmic reticulum–cis Golgi network strengthens the hypothesis that the complete LPS recognition complex might be present in the Golgi compartment (38
). Considering the highly LPS-responsive phenotype, this exclusively intracellular location of the LPS recognition complex strongly suggests that internalization and transport of endotoxin is needed to facilitate close proximity between ligand and receptor at the Golgi complex.
Indeed, uptake of fluorophore-conjugated LPS and cytoplasmic transport could be observed in m-ICcl2
cells. Most importantly, endocytosed cytoplasmic LPS colocalized to the TLR4-positive cellular compartment. Our data therefore give an explanation for the recently described transport of LPS to the Golgi compartment (34
). Internalization of LPS in the cytoplasm of several cell types has been demonstrated in a number of studies (34
). Colocalization strategies could identify several intracellular compartments. LPS was found in early endosomes, lysosomes, sarcomeres, and the Golgi apparatus (34
). Moreover, it seems that more than one way of LPS uptake exists and the kind of internalization determines both the consequences and the fate of the LPS (39
). It was suggested that large aggregates of LPS are internalized along with CD14 and deacylated via the lysosomal pathway, whereas monomeric LPS is transported to the Golgi apparatus and initiates cell activation (34
). In addition, the structure of LPS and the functional integrity of TLR4 is at least indirectly implicated in this internalization process (45
). The importance of LPS uptake for the process of cell activation still remains controversial (40
). However, an increasing body of evidence suggests that LPS internalization is an obligatory event directly linked to recognition and cell activation in several cell types (38
). We are currently evaluating the possible routes involved in LPS internalization and their importance for subsequent cell activation in our cell model.
After the initial signal that directs professional immune cells to the site of infection, the release of proinflammatory mediators needs to be controlled to avoid ongoing phagocyte infiltration and tissue damage. A negative regulator of endotoxin-mediated cell activation is the serine protease inhibitor SLPI, which inhibits LPS transfer to CD14, internalization, and prostaglandin synthesis (50
). Indeed, intestinal m-ICcl2
cells showed a marked up-regulation of SLPI upon LPS stimulation. Another mechanism to limit the proinflammatory response might be the induction of tolerance. Epithelial m-ICcl2
cells abrogated MIP-2 secretion in response to LPS after endotoxin pretreatment. Reduction of LPS responsiveness was achieved by very low amounts of LPS and could not be overcome even with high concentrations of LPS. This reduced LPS susceptibility was recently found to coincide with the temporary down-regulation of TLR4 after endotoxin exposure, which suggests a regulatory role of TLR4 expression (25
). However, in m-ICcl2
cells neither the transcription of TLR4 or MD-2, nor the total signal intensity or cytoplasmic distribution of TLR4 staining, was significantly altered after LPS exposure. The preservation of susceptibility to TNF-α, but not to IL-1β, in tolerant cells suggested that the cause of LPS hyporesponsiveness is located downstream of TLR4. These findings are in accordance with recent reports describing induction of cross-tolerance by ligands using different members of the TLR family, and overexpression studies on tolerant Chinese hamster ovary cells (52
In conclusion, we propose a model of intact LPS recognition in small intestinal crypts to ensure immediate host-defense activation upon microbial infection. Murine crypt intestinal m-ICcl2 cells seem to be well equipped to recognize LPS and show a highly sensitive phenotype. Continuous LPS stimulation is inhibited by a sensitive and tight state of tolerance, induced by inhibition of the cytoplasmic TLR4 signaling pathway. TLR4 does not seem to be present on the cell surface membrane, but instead resides at high concentration in a cytoplasmic compartment, the Golgi apparatus. LPS internalization and colocalization with TLR4 indicates that LPS uptake represents an obligatory step in the process of endotoxin recognition. Thus, we present a novel model of the process of TLR4-mediated LPS recognition in intestinal epithelial cells. The results from this study provide, for the first time, an explanation to the functional importance of LPS internalization and transport to the Golgi complex. Membrane dynamics and cell transport might therefore represent a novel level of regulation of innate immune recognition, cell activation, and host defense.