Toll-like receptor 2-mediated expression of beta-defensin-2 in human corneal epithelial cells

We previously showed that human corneal epithelial cells (HCECs) express Toll-like receptors (TLRs), which recognize gram-positive bacteria and respond to Staphylococcus aureus infection by the expression and secretion of proinflammatory cytokines and beta-defensin-2 (hBD2). In this study, we further elucidated the underlying mechanisms regulating hBD-2 expression and its role in innate defense in HCECs in response to S. aureus challenge. Exposure of HUCL cells, a telomerase-immortalized HCEC line, to S. aureus, its exoproducts (1:10 dilution), or synthetic lipopeptide Pam3Cys (10 microg/ml) resulted in the up-regulation of hBD-2, but not hBD1 and hBD3. Similar to HUCL cells, primary HCECs responded to S. aureus-exoproducts and Pam3Cys challenge by expressing hBD2 mRNA and secreting hBD2 into the culture media. Furthermore, these stimuli induced the expression of TLR2 at both mRNA and protein levels. Consistently with its role as a major pattern-recognizing receptor, TLR2 was located at the cell surface by cell surface biotinylation. The treatment of HUCL cells with TLR2 neutralizing antibody resulted in a significant decrease in Pam3Cys-induced hBD2 production as well as IL-6, IL-8, and TNF-alpha secretion. The Pam3Cys-induced hBD2 expression was completely blocked by NF-kappaB inhibitors and partially inhibited by p38 MAP kinase and the JNK inhibitors. Conditioned media derived from HCECs challenged with S. aureus-exoproducts or Pam3Cys exhibited antibacterial activity against S. aureus, Pseudomonas aeruginosa and Escherichia coli. These findings suggest that S. aureus induces hBD2 production through TLR2-mediated pathways in HCECs and that pathogen-challenged, TLR-activated HCECs possess antimicrobial activity. Thus, the epithelium might play a role in innate defense against bacterial infection by directly killing bacteria in the cornea.     

Staphylococcus aureus protein A induced inflammatory response in human corneal epithelial cells

In the present study, we examined the role of Staphylococcus aureus protein A (SpA) in inducing inflammatory response in human corneal epithelial cells (HCECs). Exposure of HCECs to SpA induces rapid NF-kappaB activation and secretion of proinflammatory cytokine/chemokines (TNF-alpha and IL-8) in both concentration and time-dependent manner. Challenge of HCECs with live SpA(-/-) mutant S. aureus strains resulted in significantly reduced production of the cytokines when compared to the wild-type S. aureus strain. SpA also elicited the activation of MAP Kinases P38, ERK, but not JNK, in HCECs. SpA-induced production of proinflammatory cytokine were completely blocked by the NF-kappaB and p38 inhibitors and partially inhibited by the Jnk inhibitor. Pretreatment with anti-TLR2 neutralizing antibody had no effect on SpA-induced inflammatory response in HCECs, suggesting that this response is independent of TLR2 signaling. Moreover, unlike TLR2 ligands, SpA failed to induce the expression of antimicrobial peptides (hBD2 and LL-37) in HCECs. These studies indicate that SpA is a S. aureus virulence factor that stimulates HCEC inflammatory response through a pathway distinct from TLR2 in HCECs.     

Induction of tolerance to lipopolysaccharide and mycobacterial components in Chinese hamster ovary/CD14 cells is not affected by overexpression of Toll-like receptors 2 or 4

Down-regulation of cell surface expression of Toll-like receptor (TLR) 4 following LPS stimulation has been suggested to underlie endotoxin tolerance. In this study, we examined whether overexpression of TLR2 or TLR4 would affect the ability of cells to become tolerant to LPS or the mycobacterial components, arabinose-capped lipoarabinomannan (LAM) and soluble tuberculosis factor (STF). To this end, Chinese hamster ovary/CD14 cells stably transfected with a NF-kappaB-dependent reporter construct, endothelial leukocyte adhesion molecule CD25 (the 3E10 clone), were engineered to overexpress either human TLR2 or TLR4. Transfected TLRs exhibited proper signaling functions, as evidenced by increased LPS responsiveness of 3E10/TLR4 cells and acquisition of sensitivity to TLR2-specific ligands upon transfection of TLR2 into TLR2-negative 3E10 cells. Pretreatment of cells with LPS, LAM, or STF did not modulate TLR2 or TLR4 cell surface expression. Following LPS exposure, 3E10, 3E10/TLR2, and 3E10/TLR4 cells exhibited comparable decreases in LPS-mediated NF-kappaB activation and mitogen-activated protein (MAP) kinase phosphorylation. Likewise, LPS pretreatment profoundly inhibited LPS-induced NF-kappaB translocation in Chinese hamster ovary cells that concomitantly overexpressed human TLR4 and myeloid differentiation protein-2 (MD-2), but failed to modulate TLR4 or MD-2 cell surface expression. Pretreatment of 3E10/TLR2 cells with LAM or STF decreased their NF-kappaB responses induced by subsequent stimulation with these substances or LPS. Conversely, prior exposure of 3E10/TLR2 cells to LPS led to hyporesponsiveness to LPS, LAM, and STF, indicating that LPS and mycobacterial products induce cross-tolerance. Thus, tolerance to LPS and mycobacterial components cannot be attributed solely to a decrease in TLR/MD-2 expression levels, suggesting inhibition of expression or function of other signaling intermediates.     

Regions of the mouse CD14 molecule required for toll-like receptor 2- and 4-mediated activation of NF-kappa B

Regions of mouse CD14 required for Toll-like receptor 2 (TLR2)- and TLR4-mediated activation of NF-kappaB were studied in transiently transfected 293 cells. Wild-type CD14 enhanced lipopolysaccharide (LPS)-induced NF-kappaB-dependent reporter activity in cells expressing TLR4/MD-2, and deletion of amino acid regions 35-44, 144-153, 235-243, and 270-275 impaired the TLR4-mediated activation. Unlike human CD14, mouse CD14 truncated at amino acid 151 lost the activity. Deletion of amino acids 35-44 or 235-243 also abrogated TLR2-mediated activation of NF-kappaB, whereas mutants lacking 144-153 and 270-275 retained the activity. Deletion and alanine substitution experiments revealed that amino acids 151-153 and 273-275 were required for the TLR4-mediated activation. Both deletion mutants lacking amino acids 35-44 and 235-243 and alanine substitution mutants in regions 151-153 and 273-275 were expressed on the cell surface and retained the ability to associate with TLR4. A cross-linking study with photoreactive LPS showed that the labeling intensities to CD14 mutants/TLR4/MD-2 were paralleled by the ability of CD14 mutants to increase TLR4-mediated activation. These results indicate that different regions of mouse CD14 are required for TLR4- and TLR2-mediated activation of NF-kappaB and suggest that amino acids 35-44, 151-153, 235-243, and 273-275 of mouse CD14 play an important role in LPS binding and its transfer to TLR4/MD-2.     

Cutting edge: direct interaction of TLR4 with NAD(P)H oxidase 4 isozyme is essential for lipopolysaccharide-induced production of reactive oxygen species and activation of NF-kappa B

LPS, the primary constituent of the outer membrane of Gram-negative bacteria, is recognized by TLR4. Binding of TLR4 to LPS triggers various cell signaling pathways including NF-kappaB activation and reactive oxygen species (ROS) production. In this study, we present the data that LPS-induced ROS generation and NF-kappaB activation are mediated by a direct interaction of TLR4 with (NAD(P)H oxidase 4 (Nox) 4), a protein related to gp91phox (Nox2) of phagocytic cells, in HEK293T cells. Yeast two hybrid and GST pull-down assays indicated that the COOH-terminal region of Nox4 interacted with the cytoplasmic tail of TLR4. Knockdown of Nox4 by transfection of small interference RNA specific to the Nox4 isozyme in HEK293T cells expressing TLR4 along with MD2 and CD14 resulted in inhibition of LPS-induced ROS generation and NF-kappaB activation. Taken together, these results indicate that direct interaction of TLR4 with Nox4 is involved in LPS-mediated ROS generation and NF-kappaB activation.     

Lactoferrin activates macrophages via TLR4-dependent and -independent signaling pathways

Lactoferrin (LF) is a component of innate immunity and is known to interact with accessory molecules involved in the TLR4 pathway, including CD14 and LPS binding protein, suggesting that LF may activate components of the TLR4 pathway. In the present study, we have asked whether bovine LF (bLF)-induced macrophage activation is TLR4-dependent. Both bLF and LPS stimulated IL-6 production and CD40 expression in RAW 264.7 macrophages and in BALB/cJ peritoneal exudate macrophages. However, in macrophages from congenic TLR4(-/-) C.C3-Tlr4(lps-d) mice, CD40 was not expressed while IL-6 secretion was increased relative to wild-type cells. The signaling components NF-kappaB, p38, ERK and JNK were activated in RAW 264.7 cells and BALB/cJ macrophages after bLF or LPS stimulation, demonstrating that the TLR4-dependent bLF activation pathway utilizes signaling components common to LPS activation. In TLR4 deficient macrophages, bLF-induced activation of NF-kappaB, p38, ERK and JNK whereas LPS-induced cell signaling was absent. We conclude from these studies that bLF induces limited and defined macrophage activation and cell signaling events via TLR4-dependent and -independent mechanisms. bLF-induced CD40 expression was TLR4-dependent whereas bLF-induced IL-6 secretion was TLR4-independent, indicating potentially separate pathways for bLF mediated macrophage activation events in innate immunity.     

MyD88 functions as a negative regulator of TLR3/TRIF-induced corneal inflammation by inhibiting activation of c-Jun N-terminal kinase

The adaptor molecule MyD88 is necessary for responses to all Toll-like receptors except TLR3 and a subset of TLR4 signaling events, which are mediated by the adaptor molecule TRIF. To determine the role of TRIF in host inflammatory responses, corneal epithelium of C57BL/6, TLR3(-/-), TRIF(-/-), and MyD88(-/-) mice was abraded and stimulated with the synthetic TLR3 ligand poly(I:C). We found that poly(I:C) induced a pronounced cellular infiltration into the corneal stroma, which was TLR3- and TRIF-dependent. Unexpectedly, the inflammatory response was exacerbated in MyD88(-/-) mice, with enhanced neutrophil and F4/80(+) cell infiltration into the corneal stroma and elevated corneal haze, which is an indicator of loss of corneal transparency. To determine whether MyD88-dependent inhibition of TLR3/TRIF responses is a general phenomenon, we examined cytokine production by MyD88(-/-) bone marrow-derived macrophages; however, no significant difference was observed between MyD88(+/+) or MyD88(-/-) macrophages. In contrast, human corneal epithelial cells (HCECs) transfected with MyD88 small interfering RNA had significantly increased (2.5-fold) CCL5/RANTES production compared with control HCECs, demonstrating a negative regulatory role for MyD88 in TLR3/TRIF responses in these cells. Finally, knockdown of MyD88 in HCECs resulted in increased phosphorylation of c-Jun N-terminal kinase (JNK), but not p38, IRF-3, or NF-kappaB. Consistent with this finding, the JNK inhibitor SP600125, but not p38 inhibitor SB203580, ablated this response. Taken together, these findings demonstrate a novel JNK-dependent inhibitory role for MyD88 in the TLR3/TRIF activation pathway.     

Bacterial lipopolysaccharide and IFN-gamma induce Toll-like receptor 2 and Toll-like receptor 4 expression in human endothelial cells: role of NF-kappa B activation

Toll-like receptor (TLR) 4 has been identified as the primary receptor for enteric LPS, whereas TLR2 has been implicated as the receptor for Gram-positive and fungal cell wall components and for bacterial, mycobacterial, and spirochetal lipoproteins. Vascular endothelial cell (EC) activation or injury by microbial cell wall components such as LPS is of critical importance in the development of sepsis and septic shock. We have previously shown that EC express predominantly TLR4, and have very little TLR2. These cells respond vigorously to LPS via TLR4, but are unresponsive to lipoproteins and other TLR2 ligands. Here we show that LPS, TNF-alpha, or IFN-gamma induce TLR2 expression in both human dermal microvessel EC and HUVEC. Furthermore, LPS and IFN-gamma act synergistically to induce TLR2 expression in EC, and LPS-induced TLR2 expression is NF-kappaB dependent. LPS and IFN-gamma also up-regulate TLR4 mRNA expression in EC. These data indicate that TLR2 and TLR4 expression in ECs is regulated by inflammatory molecules such as LPS, TNF-alpha, or IFN-gamma. TLR2 and TLR4 molecules may render EC responsive to TLR2 ligands and may help to explain the synergy between LPS and lipoproteins, and between LPS and IFN-gamma, in inducing shock associated with Gram-negative sepsis.     

Bacterial lipopolysaccharide activates nuclear factor-kappaB through interleukin-1 signaling mediators in cultured human dermal endothelial cells and mononuclear phagocytes

Bacterial lipopolysaccharide (LPS)-mediated immune responses, including activation of monocytes, macrophages, and endothelial cells, play an important role in the pathogenesis of Gram-negative bacteria-induced sepsis syndrome. Activation of NF-kappaB is thought to be required for cytokine release from LPS-responsive cells, a critical step for endotoxic effects. Here we investigated the role and involvement of interleukin-1 (IL-1) and tumor necrosis factor (TNF-alpha) signal transducer molecules in LPS signaling in human dermal microvessel endothelial cells (HDMEC) and THP-1 monocytic cells. LPS stimulation of HDMEC and THP-1 cells initiated an IL-1 receptor-like NF-kappaB signaling cascade. In transient cotransfection experiments, dominant negative mutants of the IL-1 signaling pathway, including MyD88, IRAK, IRAK2, and TRAF6 inhibited both IL-1- and LPS-induced NF-kappaB-luciferase activity. LPS-induced NF-kappaB activation was not inhibited by a dominant negative mutant of TRAF2 that is involved in TNF signaling. LPS-induced activation of NF-kappaB-responsive reporter gene was not inhibited by IL-1 receptor antagonist. TLR2 and TLR4 were expressed on the cell surface of HDMEC and THP-1 cells. These findings suggest that a signal transduction molecule in the LPS receptor complex may belong to the IL-1 receptor/toll-like receptor (TLR) super family, and the LPS signaling cascade uses an analogous molecular framework for signaling as IL-1 in mononuclear phagocytes and endothelial cells.     

Lipopolysaccharide increases alveolar type II cell number in fetal mouse lungs through Toll-like receptor 4 and NF-kappaB

Chorioamnionitis is a major cause of preterm delivery. Infants exposed to inflammation in utero and then born preterm may have improved lung function in the immediate postnatal period. We developed a mouse model of chorioamnionitis to study the inflammatory signaling mechanisms that might influence fetal lung maturation. With this in vivo model, we found that Escherichia coli lipopolysaccharide (LPS) increased the number of alveolar type II cells in the fetal mouse lung. LPS also increased type II cell number in cultured fetal lung explants, suggesting that LPS could directly signal the fetal lung in the absence of maternal influences. Using immunostaining, we localized cells within the fetal mouse lung expressing the LPS receptor molecule Toll-like receptor 4 (TLR4). Similar to the signaling pathways in inflammatory cells, LPS activated NF-kappaB in fetal lung explants. Activation of the TLR4/NF-kappaB pathway appeared to be required, as LPS did not increase the number of type II cells in C.C3H-Tlr4(Lps-d) mice, a congenic strain containing a loss of function mutation in tlr4. In addition, the sesquiterpene lactone parthenolide inhibited NF-kappaB activation following LPS exposure and blocked the LPS-induced increase in type II cells. On the basis of these data from our mouse model of chorioamnionitis, it appears that LPS specifically activated the TLR4/NF-kappaB pathway, leading to increased type II cell maturation. These data implicate an important signaling mechanism in chorioamnionitis and suggest the TLR4/NF-kappaB pathway can influence lung development.     

CD14 Mediates Toll-like Receptor 4 (TLR4) Endocytosis and Spleen Tyrosine Kinase (Syk) and Interferon Regulatory Transcription Factor 3 (IRF3) Activation in Epithelial Cells and Impairs Neutrophil Infiltration and Pseudomonas aeruginosa Killing in Vivo

In the current study, we examined the role of CD14 in regulating LPS activation of corneal epithelial cells and Pseudomonas aeruginosa corneal infection. Our findings demonstrate that LPS induces Toll-like receptor 4 (TLR4) internalization in corneal epithelial cells and that blocking with anti-CD14 selectively inhibits TLR4 endocytosis, spleen tyrosine kinase (Syk) and IRF3 phosphorylation, and production of CCL5/RANTES and IFN-β, but not IL-8. Using a murine model of P. aeruginosa corneal infection, we show that although infected CD14^−/− corneas produce less CCL5, they exhibit significantly increased CXC chemokine production, neutrophil recruitment to the corneal stroma, and bacterial clearance than C57BL/6 mice. We conclude that CD14 has a critical role in mediating TLR4 signaling through IRF3 in resident corneal epithelial cells and macrophages and thereby modulates TLR4 cell surface activation of the MyD88/NF-κB/AP-1 pathway and production of CXC chemokines and neutrophil infiltration to infected tissues.     

Decreased expression of Toll-like receptor-4 and MD-2 correlates with intestinal epithelial cell protection against dysregulated proinflammatory gene expression in response to bacterial lipopolysaccharide

The lumenal surface of the colonic epithelium is continually exposed to Gram-negative commensal bacteria and LPS. Recognition of LPS by Toll-like receptor (TLR)-4 results in proinflammatory gene expression in diverse cell types. Normally, however, commensal bacteria and their components do not elicit an inflammatory response from intestinal epithelial cells (IEC). The aim of this study is to understand the molecular mechanisms by which IEC limit chronic activation in the presence of LPS. Three IEC lines (Caco-2, T84, HT-29) were tested for their ability to activate an NF-kappaB reporter gene in response to purified, protein-free LPS. No IEC line responded to LPS, whereas human dermal microvessel endothelial cells (HMEC) did respond to LPS. IEC responded vigorously to IL-1beta in this assay, demonstrating that the IL-1 receptor signaling pathway shared by TLRs was intact. To determine the reason for LPS hyporesponsiveness in IEC, we examined the expression of TLR4 and MD-2, a critical coreceptor for TLR4 signaling. IEC expressed low levels of TLR4 compared with HMEC and none expressed MD-2. To determine whether the low level of TLR4 expression or absent MD-2 was responsible for the LPS signaling defect in IEC, the TLR4 or MD-2 gene was transiently expressed in IEC lines. Transient transfection of either gene individually was not sufficient to restore LPS signaling, but cotransfection of TLR4 and MD-2 in IEC led to synergistic activation of NF-kappaB and IL-8 reporter genes in response to LPS. We conclude that IEC limit dysregulated LPS signaling by down-regulating expression of MD-2 and TLR4. The remainder of the intracellular LPS signaling pathway is functionally intact.     

Synergy and cross-tolerance between toll-like receptor (TLR) 2- and TLR4-mediated signaling pathways

A family of Toll-like receptor (TLR) mediates the cellular response to bacterial cell wall components; murine TLR2 and TLR4 recognize mycoplasmal lipopeptides (macrophage-activating lipopeptides, 2 kDa (MALP-2)) and LPS, respectively. Costimulation of mouse peritoneal macrophages with MALP-2 and LPS results in a marked increase in TNF-alpha production, showing the synergy between TLR2- and TLR4-mediated signaling pathways. Macrophages pretreated with LPS show hyporesponsiveness to the second LPS stimulation, termed LPS tolerance. The LPS tolerance has recently been shown to be primarily due to the down-regulation of surface expression of the TLR4-MD2 complex. When macrophages were treated with MALP-2, the cells showed hyporesponsiveness to the second MALP-2 stimulation, like LPS tolerance. Furthermore, macrophages pretreated with MALP-2 showed reduced production of TNF-alpha in response to LPS. LPS-induced activation of both NF-kappaB and c-Jun NH(2)-terminal kinase was severely impaired in MALP-2-pretreated cells. However, MALP-2-pretreated macrophages did not show any reduction in surface expression of the TLR4-MD2 complex. These findings indicate that LPS-induced LPS tolerance mainly occurs through the down-regulation of surface expression of the TLR4-MD2 complex; in contrast, MALP-2-induced LPS tolerance is due to modulation of the downstream cytoplasmic signaling pathways.     

N-linked glycosylations at Asn(26) and Asn(114) of human MD-2 are required for toll-like receptor 4-mediated activation of NF-kappaB by lipopolysaccharide.

MD-2 is physically associated with Toll-like receptor 4 (TLR4) and is required for TLR4-mediated LPS signaling. Western blotting analysis revealed the presence of three forms of human (h)MD-2 with different electrophoretic mobilities. After N-glycosidase treatment of the cellular extract prepared from cells expressing hMD-2, only a single form with the fastest mobility was detected. Mutation of either one of two potential glycosylation sites (Asn(26) and Asn(114)) of MD-2 resulted in the disappearance of the slowest mobility form, and only the fastest form was detected in hMD-2 carrying mutations at both Asn(26) and Asn(114). Although these mutants were expressed on the cell surface and maintained its ability to associate with human TLR4, these mutations or tunicamycin treatment substantially impaired the ability of MD-2 to complement TLR4-mediated activation of NF-kappaB by LPS. LPS binding to cells expressing CD14, TLR4, and MD-2 was unaffected by these mutations. These observations demonstrate that hMD-2 undergoes N-linked glycosylation at Asn(26) and Asn(114), and that these glycosylations are crucial for TLR4-mediated signal transduction of LPS.     

Induction of in vitro reprogramming by Toll-like receptor (TLR)2 and TLR4 agonists in murine macrophages: effects of TLR "homotolerance" versus "heterotolerance" on NF-kappa B signaling pathway components

In this study, tolerance induction by preexposure of murine macrophages to Toll-like receptor (TLR)2 and TLR4 agonists was revisited, focusing on the major signaling components associated with NF-kappaB activation. Pretreatment of macrophages with a pure TLR4 agonist (protein-free Escherichia coli (Ec) LPS) or with TLR2 agonists (Porphyromonas gingivalis LPS or synthetic lipoprotein Pam3Cys) led to suppression of TNF-alpha secretion, IL-1R-associated kinase-1, and IkappaB kinase (IKK) kinase activities, c-jun N-terminal kinase, and extracellular signal-regulated kinase phosphorylation, and to suppression of NF-kappaB DNA binding and transactivation upon challenge with the same agonist (TLR4 or TLR2 "homotolerance," respectively). Despite inhibited NF-kappaB DNA binding, increased levels of nuclear NF-kappaB were detected in agonist-pretreated macrophages. For all the intermediate signaling elements, heterotolerance was weaker than TLR4 or TLR2 homotolerance with the exception of IKK kinase activity. IKK kinase activity was unperturbed in heterotolerance. TNF-alpha secretion was also suppressed in P. gingivalis LPS-pretreated, Ec LPS-challenged cells, but not vice versa, while Pam3Cys and Ec LPS did not induce a state of cross-tolerance at the level of TNF-alpha. Experiments designed to elucidate novel mechanisms of NF-kappaB inhibition in tolerized cells revealed the potential contribution of IkappaBepsilon and IkappaBxi inhibitory proteins and the necessity of TLR4 engagement for induction of tolerance to Toll receptor-IL-1R domain-containing adapter protein/MyD88-adapter-like-dependent gene expression. Collectively, these data demonstrate that induction of homotolerance affects a broader spectrum of signaling components than in heterotolerance, with selective modulation of specific elements within the NF-kappaB signaling pathway.     

Dysregulation of LPS-induced Toll-like receptor 4-MyD88 complex formation and IL-1 receptor-associated kinase 1 activation in endotoxin-tolerant cells

Prior exposure to LPS induces a transient state of cell refractoriness to subsequent LPS restimulation, known as endotoxin tolerance. Induction of LPS tolerance has been reported to correlate with decreased cell surface expression of the LPS receptor complex, Toll-like receptor 4 (TLR4)/MD-2. However, other results have underscored the existence of mechanisms of LPS tolerance that operate downstream of TLR4/MD-2. In the present study we sought to delineate further the molecular basis of LPS tolerance by examining the TLR4 signaling pathway in endotoxin-tolerant cells. Pretreatment of human monocytes with LPS decreased LPS-mediated NF-kappaB activation, p38 mitogen-activated protein kinase phosphorylation, and TNF-alpha gene expression, documenting the induction of endotoxin tolerance. FACS and Western blot analyses of LPS-tolerant monocytes showed increased TLR2 expression, whereas TLR4 expression levels were not affected. Comparable levels of mRNA and protein for myeloid differentiation factor 88 (MyD88), IL-1R-associated kinase 1 (IRAK-1), and TNFR-associated factor-6 were found in normal and LPS-tolerant monocytes, while MD-2 mRNA expression was slightly increased in LPS-tolerant cells. LPS induced the association of MyD88 with TLR4 and increased IRAK-1 activity in medium-pretreated cells. In LPS-tolerant monocytes, however, MyD88 failed to be recruited to TLR4, and IRAK-1 was not activated in response to LPS stimulation. Moreover, endotoxin-tolerant CHO cells that overexpress human TLR4 and MD-2 also showed decreased IRAK-1 kinase activity in response to LPS despite the failure of LPS to inhibit cell surface expression of transfected TLR4 and MD-2 proteins. Thus, decreased TLR4-MyD88 complex formation with subsequent impairment of IRAK-1 activity may underlie the LPS-tolerant phenotype.