Role of innate immunity and altered intestinal motility in LPS- and MnCl2-induced intestinal intussusception in mice.

Intestinal intussusception (ISS) commonly causes intestinal obstruction in children. One mechanism that has been proposed to cause ISS is inflammation-induced alteration of intestinal motility. We investigated whether innate inflammatory factors or altered motility is required for induction of ISS by LPS. We compared rates of ISS among BALB/c and C57BL/6 mice, mice lacking lymphocytes or depleted of phagocytes, or mice with defects in the Toll-like receptor 4 (TLR4) signaling pathway following administration of LPS or the Ca(2+) analog MnCl2. At 6 or 2 h after administration of LPS or MnCl2, respectively, mice underwent image analysis to assess intestinal contraction rate or laparotomy to identify ISS. LPS-induced ISS (LPS-ISS) was observed in BALB/c mice, but not in C57BL/6 mice or any BALB/c mice with disruptions of TLR4 signaling. LPS-induced serum TNF-α, IL-6, and nitric oxide (NO) and intestinal NO levels were similar in BALB/c and C57BL/6 mice. The rate of LPS-ISS was significantly reduced in phagocyte-depleted, but not lymphocyte-deficient, mice. Intestinal contraction rates were reduced in LPS-ISS-susceptible BALB/c mice, but not in LPS-ISS-resistant C57BL/6 or TLR4 mutant mice, suggesting a role for reduced intestinal contraction rate in LPS-ISS susceptibility. This was tested with MnCl2, a Ca(2+) antagonist that reduced intestinal contraction rates and induced ISS, irrespective of mouse strain. Therefore, LPS-ISS is initiated by innate immune signaling that requires TLR4 and phagocytes but may be independent of TNF-α, IL-6, and NO levels. Furthermore, alteration of intestinal motility, specifically, reduced intestinal contraction rate, is a key factor in the development of ISS.

Differential expression of CD14-dependent and independent pathways for chemokine induction regulates neutrophil trafficking in infection.

Previous studies have shown that CD14(-/-) mice are resistant to peritoneal infection with some clinical isolates of Escherichia coli and that this resistance is accompanied by an enhanced ability to clear the bacteria; in contrast, normal mice expressing CD14 fail to clear the bacteria, causing severe sepsis and death. The enhanced clearance in CD14(-/-) mice is dependent on early neutrophil recruitment to the local foci of infection in the PC. The studies described show that neutrophil recruitment in CD14(-/-) mice occurs as a result of the local induction of the CXCL1 and CXCL2 chemokines, KC and MIP-2. Although local induction of these chemokines also occurs in normal mice, their effects on neutrophil recruitment to the PC appear to be counterbalanced by very high levels of these chemokines in the blood of normal, but not CD14(-/-), mice. Neutrophil recruitment to the PC is also inhibited in normal mice in response to LPS, which also induces high chemokine levels in the blood of normal, but not CD14(-/-), mice. However, MPLA, a monophosphorylated derivative of LPS, is able to induce early neutrophil recruitment in normal mice; this is because MPLA, unlike LPS or E. coli, induces MIP-2 and KC in the PC but not in the blood of normal mice. The pretreatment of normal mice with MPLA is able to protect them from a lethal E. coli infection. Thus, stimulation of a local CD14-independent chemokine induction pathway without triggering a systemic CD14-dependent chemokine pathway can protect against severe E. coli infections.

Analysis of Cd14 as a genetic modifier of experimental inflammatory bowel disease (IBD) in mice.

BACKGROUND AND AIM: By combining QTL and gene expression analyses, we have previously identified Cd14 as a potential candidate gene contributing to the differential IBD susceptibility of C3H/HeJBir (C3/J)-Il10(-/-) mice [carrying IBD-resistance alleles at this QTL (Cdcs6)] and C57BL/6J (B6)-Il10(-/-) mice, corroborating studies that showed an association of a CD14-promoter polymorphism with Crohn's disease and ulcerative colitis. The aim of the present study was to analyze the molecular mechanisms leading to differential intestinal expression of Cd14 and its contribution to IBD development. METHODS: Intestinal CD14 expression was assessed by FACS, immunohistochemistry, and ELISA on supernatants of primary epithelial cell and tissue cultures. RAW264.7 cells were stimulated with LPS and PGN in the presence or absence of CD14. Cd14 alleles were sequenced and promoters cloned for luciferase assays in transfected RAW264.7 cells. The severity of typhlocolitis between Cd14(-/-) and wild-type mice was compared in 2 distinct mouse models of IBD (acute DSS and Il10(-/-) ). RESULTS: In the gut, CD14 was detected mainly in its soluble form (sCD14), with higher expression in C3/J-Il10(-/-) mice. Polymorphisms in C3/J mice caused higher activity of the Cd14 promoter (luciferase assays). Intestinal sCD14 concentrations influenced the LPS and PGN responses of RAW264.7 cells. In vivo, genetic deletion of Cd14 aggravated colitis in both mouse models of IBD. CONCLUSIONS: Our study shows that Cd14-promoter polymorphisms affect CD14 expression and confirms the protective effect of CD14 against experimental IBD, potentially mediated by TLR2- and TLR4-dependent effects on intestinal barrier function. These findings support the concept that human CD14-promoter polymorphisms contribute to disease development.

Pivotal role of the alpha(2A)-adrenoceptor in producing inflammation and organ injury in a rat model of sepsis.

BACKGROUND: Norepinephrine (NE) modulates the responsiveness of macrophages to proinflammatory stimuli through the activation of adrenergic receptors (ARs). Being part of the stress response, early increases of NE in sepsis sustain adverse systemic inflammatory responses. The intestine is an important source of NE release in the early stage of cecal ligation and puncture (CLP)-induced sepsis in rats, which then stimulates TNF-alpha production in Kupffer cells (KCs) through the activation of the alpha(2)-AR. It is important to know which of the three alpha(2)-AR subtypes (i.e., alpha(2A), alpha(2B) or alpha(2C)) is responsible for the upregulation of TNF-alpha production. The aim of this study was to determine the contribution of alpha(2A)-AR in this process. METHODOLOGY/PRINCIPAL FINDINGS: Adult male rats underwent CLP and KCs were isolated 2 h later. Gene expression of alpha(2A)-AR was determined. In additional experiments, cultured KCs were incubated with NE with or without BRL-44408 maleate, a specific alpha(2A)-AR antagonist, and intraportal infusion of NE for 2 h with or without BRL-44408 maleate was carried out in normal animals. Finally, the impact of alpha(2A)-AR activation by NE was investigated under inflammatory conditions (i.e., endotoxemia and CLP). Gene expression of the alpha(2A)-AR subtype was significantly upregulated after CLP. NE increased the release of TNF-alpha in cultured KCs, which was specifically inhibited by the alpha(2A)-AR antagonist BRL-44408. Equally, intraportal NE infusion increased TNF-alpha gene expression in KCs and plasma TNF-alpha which was also abrogated by co-administration of BRL-44408. NE also potentiated LPS-induced TNF-alpha release via the alpha(2A)-AR in vitro and in vivo. This potentiation of TNF-alpha release by NE was mediated through the alpha(2A)-AR coupled Galphai protein and the activation of the p38 MAP kinase. Treatment of septic animals with BRL-44408 suppressed TNF-alpha, prevented multiple organ injury and significantly improved survival from 45% to 75%. CONCLUSIONS/SIGNIFICANCE: Our novel finding is that hyperresponsiveness to alpha(2)-AR stimulation observed in sepsis is primarily due to an increase in alpha(2A)-AR expression in KCs. This appears to be in part responsible for the increased proinflammatory response and ensuing organ injury in sepsis. These findings provide important feasibility information for further developing the alpha(2A)-AR antagonist as a new therapy for sepsis.

Milk fat globule epidermal growth factor-factor VIII is down-regulated in sepsis via the lipopolysaccharide-CD14 pathway.

Phagocytosis prevents the release of potentially harmful or immunogenic materials from dying cells. Milk fat globule epidermal growth factor (EGF)-factor VIII (MFG-E8) mediates the clearance of apoptotic cells. We have previously shown that the administration of MFG-E8-rich exosomes from immature dendritic cells promotes the phagocytosis of apoptotic cells and improves survival in sepsis. Because endotoxin is elevated in polymicrobial sepsis, we hypothesized that down-regulation of MFG-E8 is mediated via the LPS-CD14 pathway, eventually leading to the accruement of apoptotic cells. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) in CD14-deficient (CD14(-/-)), TLR4-mutated and wild-type (WT) mice. In addition, endotoxemia was elicited by i.p. injection of LPS. LPS was also neutralized by pretreating CLP-induced WT mice with polymyxin B. Splenic MFG-E8 expression, phagocytic activity, and apoptosis were assessed 5 and 20 h after CLP or 5 h after LPS administration. In septic WT mice, MFG-E8 mRNA and protein levels were suppressed by 49 and 33%, respectively. Endotoxemia reduced MFG-E8 mRNA expression in a dose dependent manner and the down-regulation of MFG-E8 mRNA expression in CLP-induced sepsis was attenuated by polymyxin B. This CLP-induced suppression was not observed in both CD14(-/-) and TLR4-mutated mice. CLP significantly decreased phagocytic activity of peritoneal macrophages in WT (by 30%), but not in CD14(-/-) mice. CLP also induced significant apoptosis in the spleen of WT (by 61%), but less in CD14(-/-) mice. Thus, MFG-E8 production is down-regulated in sepsis by LPS-CD14 dependent fashion, leading to a reduction of phagocytosis of apoptotic cells.

Injection of lipopolysaccharide induces the migration of splenic neutrophils to the T cell area of the white pulp: role of CD14 and CXC chemokines.

There is increasing evidence that neutrophils are involved in the regulation of adaptive immunity. We therefore tested whether these cells may colocalize with T lymphocytes in lymphoid organs. Our results demonstrate that administration of the microbial product LPS induces the migration of neutrophils in the spleen from the red pulp and the marginal zone to the area of the white pulp where T cells reside. This movement is CD14-dependent, whereas the recruitment of neutrophils in the peritoneal cavity is increased in the absence of CD14. Our data further suggest the involvement of the chemokine MIP-2 and keratinocyte-derived chemokine and their receptor CXCR2. We conclude that neutrophils may interact with naïve T cells upon infection/inflammation and that the migration of neutrophils in the lymphoid organs and in the periphery is regulated differently by a signal transduced by CD14.

Expression of Legionella pneumophila paralogous lipid A biosynthesis genes under different growth conditions.

Legionella pneumophila is an opportunistic pathogen that in the environment colonizes biofilms and replicates within amoebae. The bacteria employ the intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV secretion system to grow intracellularly in a specific vacuole. Using Acanthamoeba castellanii as a host cell, we have previously identified lcsC (Legionella cytotoxic suppressor), a paralogue of the lipid A disaccharide synthase lpxB, as a cytotoxic factor of L. pneumophila. A bioinformatic analysis of the genome revealed that L. pneumophila is unique in harbouring two paralogues of lpxB and two and three paralogues of the lipid A biosynthesis acyltransferases lpxA and lpxD, respectively. LcsC (lpxB1) forms a transcriptional unit with gnnA, encoding a putative UDP-GlcNAc oxidase in the biosynthetic pathway leading to 3-aminoglucosamine analogues of lipid A. LpxB2 clusters with lpxD2, lpxA2 and lpxL paralogues, encoding secondary acyltransferases. LcsC/lpxB1 and lpxB2 were found to partially complement the growth defect of an Escherichia coli lpxB conditional mutant strain, indicating that both corresponding enzymes possess lipid A disaccharide synthase activity. The two L. pneumophila lpxB paralogues are not functionally equivalent, since expression of lcsC/lpxB1 but not lpxB2 in an L. pneumophila icmG mutant is cytotoxic for A. castellanii, and LPS purified from the two strains triggers CD14-dependent tumour necrosis factor (TNF)alpha production by macrophages with a different potency. The lpxB and lpxA paralogues are expressed under various growth conditions, including broth, biofilms and in A. castellanii. While the flagellar gene flaA is mainly expressed in late stationary phase, the lpxB and lpxA paralogues are preferentially expressed in the exponential and early stationary phases. Upon exposure to hypotonic stress and nutrient deprivation, lpxA1, and to a lesser extent lcsC/lpxB1, is upregulated. The differential regulation of lpxB or lpxA paralogues in response to changing environmental conditions might allow L. pneumophila to adapt its lipid A structure.

Role of CD14 in responses to clinical isolates of Escherichia coli: effects of K1 capsule expression.

Severe bacterial infections leading to sepsis or septic shock can be induced by bacteria that utilize different factors to drive pathogenicity and/or virulence, leading to disease in the host. One major factor expressed by all clinical isolates of gram-negative bacteria is lipopolysaccharide (LPS); a second factor expressed by some Escherichia coli strains is a K1 polysaccharide capsule. To determine the role of the CD14 LPS receptor in the pathogenic effects of naturally occurring E. coli, the responses of CD14-/- and CD14+/+ mice to three different isolates of E. coli obtained from sepsis patients were compared; two isolates express both smooth LPS and the K1 antigen, while the third isolate expresses only LPS and is negative for K1. An additional K1-positive isolate obtained from a newborn with meningitis and a K1-negative isogenic mutant of this strain were also used for these studies. CD14-/- mice were resistant to the lethal effects of the K1-negative isolates. This resistance was accompanied by significantly lower levels of systemic tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) in these mice than in CD14+/+ mice, enhanced clearance of the bacteria, and significantly fewer additional gross symptoms. In contrast, CD14-/- mice were as sensitive as CD14+/+ mice to the lethal effects of the K1-positive isolates, even though they had significantly lower levels of TNF-alpha and IL-6 than CD14+/+ mice. These studies show that different bacterial isolates can use distinctly different mechanisms to cause disease and suggest that new, nonantibiotic therapeutics need to be directed against multiple targets.

In silico and in vivo approach to elucidate the inflammatory complexity of CD14-deficient mice.

The inflammatory phenotype of genetically modified mice is complex, and the role of Gram-negative lipopolysaccharide (LPS) in acute inflammation induced by surgical cannulation trauma, alone or in combination with hemorrhage and resuscitation ("hemorrhagic shock"), is both complex and controversial. We sought to determine if a mathematical model of acute inflammation could elucidate both the phenotype of CD14-deficient (CD14(-/-)) mice--following LPS, cannulation, or hemorrhagic shock--and the role of LPS in trauma/hemorrhage-induced inflammation. A mathematical model of inflammation initially calibrated in wild-type (C57Bl/6) mice subjected to LPS, cannulation, and hemorrhagic shock was recalibrated in CD14(-/-) mice subjected to the same insults, yielding an ensemble of models that suggested specific differences at the cellular and molecular levels (for example, 43-fold lower activation of leukocytes by LPS). The CD14(-/-)-specific model ensemble could account for complex changes in inflammatory analytes in these mice following LPS treatment. Model prediction of similar organ damage in CD14(-/-) and wild-type mice subjected to cannulation alone or with hemorrhagic shock was verified in vivo (similar ALT levels). These studies suggest that LPS-CD14 responses do not cause inflammation in surgical trauma/hemorrhagic shock and demonstrate a novel use of combined in silico and in vivo methods to elucidate the complex inflammatory phenotype of genetically modified animals.

Influence of CD14 on ligand interactions between lipopolysaccharide and its receptor complex.

The interaction of LPS (endotoxin) with the CD14-TLR4 receptor complex modulates the host innate immune response. Several studies using partial structures of LPS have suggested that TLR4 determines the ligand specificity of this complex, and that CD14 indiscriminately serves to deliver the ligand to TLR4. This conclusion has been made despite observations that the response of TLR4(+/+),CD14(-/-) macrophages to LPS is very weak. To determine whether CD14 itself plays a role in specific ligand recognition, the influences of various partial structures of LPS on induction of the proinflammatory cytokine, TNF, by CD14(+/+) and CD14(-/-) macrophages were compared. These studies show that the ligand specificities of CD14(+/+) and CD14(-/-) macrophages are very different. When CD14 is present, the receptor complex shows exquisite specificity for smooth LPS, the major form expressed by Gram-negative bacteria; however, as increasing amounts of carbohydrate are removed from smooth LPS, the sensitivity of CD14(+/+) macrophages decreases as much as 500-fold. In contrast, CD14(-/-) macrophages are unable to distinguish between smooth LPS and its various partial structures. Furthermore, CD14(-/-) macrophages are 150,000-fold less sensitive than CD14(+/+) macrophages to smooth LPS. A similar ability to distinguish the differing LPS structures of various bacteria such as Bacteroides fragilis and Salmonella abortus are observed for CD14(+/+), but not CD14(-/-), macrophages. Thus, CD14(+/+), but not CD14(-/-), macrophages are highly sensitive to stimulation by natural forms of LPS and show the ability to distinguish between various LPS ligands, consistent with CD14 being a highly specific receptor.

Signaling through CD14 attenuates the inflammatory response to Borrelia burgdorferi, the agent of Lyme disease.

Lyme disease is a chronic inflammatory disorder caused by the spirochetal bacterium, Borrelia burgdorferi. In vitro evidence suggests that binding of spirochetal lipoproteins to CD14, a pattern recognition receptor expressed on monocytes/macrophages and polymorphonuclear cells, is a critical requirement for cellular activation and the subsequent release of proinflammatory cytokines that most likely contribute to symptomatology and clinical manifestations. To test the validity of this notion, we assessed the impact of CD14 deficiency on Lyme disease in C3H/HeN mice. Contrary to an anticipated diminution in pathology, CD14(-/-) mice exhibited more severe and persistent inflammation than did CD14(+/+) mice. This disparity reflects altered gene regulation within immune cells that may engender the higher bacterial burden and serum cytokine levels observed in CD14(-/-) mice. Comparing their in vitro stimulatory activity, live spirochetes, but not lysed organisms, were a potent CD14-independent stimulus of cytokine production, triggering an exaggerated response by CD14(-/-) macrophages. Collectively, our in vivo and in vitro findings support the provocative notion that: 1) pattern recognition by CD14 is entirely dispensable for elaboration of an inflammatory response to B. burgdorferi, and 2) CD14-independent signaling pathways are inherently more destructive than CD14-dependent pathways. Continued study of CD14-independent signaling pathways may provide mechanistic insight into the inflammatory processes that underlie development of chronic inflammation.

Persistence of apoptotic cells without autoimmune disease or inflammation in CD14-/- mice.

Interaction of macrophages with apoptotic cells involves multiple steps including recognition, tethering, phagocytosis, and anti-inflammatory macrophage responses. Defective apoptotic cell clearance is associated with pathogenesis of autoimmune disease. CD14 is a surface receptor that functions in vitro in the removal of apoptotic cells by human and murine macrophages, but its mechanism of action has not been defined. Here, we demonstrate that CD14 functions as a macrophage tethering receptor for apoptotic cells. Significantly, CD14(-/-) macrophages in vivo are defective in clearing apoptotic cells in multiple tissues, suggesting a broad role for CD14 in the clearance process. However, the resultant persistence of apoptotic cells does not lead to inflammation or increased autoantibody production, most likely because, as we show, CD14(-/-) macrophages retain the ability to generate anti-inflammatory signals in response to apoptotic cells. We conclude that CD14 plays a broad tethering role in apoptotic cell clearance in vivo and that apoptotic cells can persist in the absence of proinflammatory consequences.

Bacterial endotoxin stimulates macrophages to release HMGB1 partly through CD14- and TNF-dependent mechanisms.

Bacterial endotoxin [lipopolysaccharide (LPS)] stimulates macrophages to sequentially release early [tumor necrosis factor (TNF)] and late [high mobility group box 1 (HMGB1)] proinflammatory cytokines. The requirement of CD14 and mitogen-activated protein kinases [MAPK; e.g., p38 and extracellular signal-regulated kinase (ERK)1/2] for endotoxin-induced TNF production has been demonstrated previously, but little is known about their involvement in endotoxin-mediated HMGB1 release. Here, we demonstrated that genetic disruption of CD14 expression abrogated LPS-induced TNF production but only partially attenuated LPS-induced HMGB1 release in cultures of primary murine peritoneal macrophages. Pharmacological suppression of p38 or ERK1/2 MAPK with specific inhibitors (SB203580, SB202190, U0126, or PD98059) significantly attenuated LPS-induced TNF production but failed to inhibit LPS-induced HMGB1 release. Consistently, an endogenous, immunosuppressive molecule, spermine, failed to inhibit LPS-induced activation of p38 MAPK and yet, still significantly attenuated LPS-mediated HMGB1 release. Direct suppression of TNF activity with neutralizing antibodies or genetic disruption of TNF expression partially attenuated HMGB1 release from macrophages induced by LPS at lower concentrations (e.g., 10 ng/ml). Taken together, these data suggest that LPS stimulates macrophages to release HMGB1 partly through CD14- and TNF-dependent mechanisms.

CD14 mediates the innate immune responses to arthritopathogenic peptidoglycan-polysaccharide complexes of Gram-positive bacterial cell walls.

Bacterial infections play an important role in the multifactorial etiology of rheumatoid arthritis. The arthropathic properties of Gram-positive bacteria have been associated with peptidoglycan-polysaccharide complexes (PG-PS), which are major structural components of bacterial cell walls. There is little agreement as to the identity of cellular receptors that mediate innate immune responses to PG-PS. A glycosylphosphatidylinositol-linked cell surface protein, CD14, the lipopolysaccharide receptor, has been proposed as a PG-PS receptor, but contradictory data have been reported. Here, we examined the inflammatory and pathogenic responses to PG-PS in CD14 knockout mice in order to examine the role for CD14 in PG-PS-induced signaling. We found that PG-PS-induced responses in vitro, including transient increase in intracellular calcium, activation of nuclear factor-kappaB, and secretion of the cytokines tumor necrosis factor-alpha and interleukin-6, were all strongly inhibited in CD14 knockout macrophages. In vivo, the incidence and severity of PG-PS induced acute polyarthritis were significantly reduced in CD14 knockout mice as compared with their wild-type counterparts. Consistent with these findings, CD14 knockout mice had significantly inhibited inflammatory cell infiltration and synovial hyperplasia, and reduced expression of inflammatory cytokines in PG-PS arthritic joints. These results support an essential role for CD14 in the innate immune responses to PG-PS and indicate an important role for CD14 in PG-PS induced arthropathy.

CD14-deficient mice are protected against lipopolysaccharide-induced cardiac inflammation and left ventricular dysfunction.

BACKGROUND: The molecular mechanisms responsible for sepsis-induced myocardial dysfunction remain undefined. CD14 mediates the inflammatory response to lipopolysaccharide (LPS) in various organs including the heart. In this study we investigated the role of CD14 in LPS-induced myocardial dysfunction in vivo. METHODS AND RESULTS: Wild-type and CD14-deficient (CD14-D) mice were challenged with Escherichia coli LPS. Myocardial tumor necrosis factor, interleukin-1beta (IL-1beta), and NOS2 induction was measured before and 6 hours after LPS challenge. Echocardiographic parameters of left ventricular function were measured before and 6 hours after LPS administration. LPS challenge induced a significant increase in myocardial tumor necrosis factor and IL-1beta mRNA and protein expression in wild-type mice. In contrast, mRNA and protein levels for TNF and IL-1beta were significantly blunted in CD14-D mice. An increase in NOS2 protein was noted within 6 hours of LPS provocation only in the hearts of wild-type mice. This was associated with an increase in ventricular cGMP levels. Activation of nuclear factor-kappaB was observed within 30 minutes of LPS in the hearts of wild-type mice but not in CD14-D mice. In wild-type mice, LPS significantly decreased left ventricular fractional shortening, velocity of circumferential shortening, and dP/dt(max). LPS-treated CD14-D mice maintained normal cardiac function. CONCLUSIONS: These results suggest that CD14 is important in mediating the proinflammatory response induced by LPS in the heart and that CD14 is necessary for the development of left ventricular dysfunction during LPS-induced shock in vivo.

Lipopolysaccharide-induced leukocyte-endothelial cell interactions: a role for CD14 versus toll-like receptor 4 within microvessels.

The objective of this study was to systematically assess leukocyte-endothelial cell interactions in vivo in response to LPS in CD14-deficient (CD14(-/-)) and Toll-like receptor 4-deficient (TLR4(d); C3H/HeJ) mice. Local injection of LPS (0.05 micro g/kg) into muscle at a concentration that did not cause systemic effects produced a significant reduction in the speed with which leukocytes roll and a substantial increase in leukocyte adhesion and emigration 4 h postinjection. There was no response to LPS in the muscle microvasculature of CD14(-/-) mice or TLR4(d) animals. Systemic LPS induced leukopenia and significant sequestration of neutrophils in lungs in wild-type mice but not in CD14(-/-) or TLR4(d) mice. P-selectin expression was examined in numerous mouse organs using a dual radiolabeling mAb technique. The results revealed a 20- to 50-fold increase in P-selectin expression in response to LPS in all wild-type tissues examined but no response in any TLR4(d) tissues. Surprisingly, there was consistently a partial, significant increase in P-selectin expression in numerous microvasculatures including skin and pancreas, but no increase in P-selectin was detected in lung, muscle, and other organs in CD14(-/-) mice in response to LPS. Next, the skin and muscle microcirculation were visualized using intravital microscopy after systemic LPS treatment, and the results confirmed a CD14-independent mechanism of leukocyte sequestration in skin but not muscle. In summary, our results suggest that the LPS-induced leukocyte sequestration to some tissues is entirely dependent on both CD14 and TLR4 but there are CD14-independent, TLR4-dependent endothelial cell responses in some microvascular beds.

Activation of human and mouse Kupffer cells by lipopolysaccharide is mediated by CD14.

Upregulation of CD14 in Kupffer cells has been implicated in the pathogenesis of several forms of liver injury, including alcoholic liver disease. However, it remains unclear whether CD14 mediates lipopolysaccharide (LPS) signaling in this specialized liver macrophage population. In this series of experiments, we determined the role of CD14 in LPS activation of Kupffer cells by using several complementary approaches. First, we isolated Kupffer cells from human livers and studied the effects of anti-CD14 antibodies on LPS activation of these cells. Kupffer cells were incubated with increasing concentrations of LPS in the presence and absence of recombinant human LPS binding protein (LBP). With increasing concentrations of LPS, human Kupffer cell tumor necrosis factor-alpha (TNF-alpha) production (a marker for Kupffer cell activation) increased in a dose-dependent manner in the presence and absence of LBP. In the presence of anti-human CD14 antibodies, the production of TNF-alpha was significantly diminished. Second, we compared LPS activation of Kupffer cells isolated from wild-type and CD14 knockout mice. Kupffer cells from CD14 knockout mice produced significantly less TNF-alpha in response to the same amount of LPS. Together, these data strongly support a critical role for CD14 in Kupffer cell responses to LPS.