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Infection with Plasmodium vivax results in strong activation of monocytes, which are important components of both the systemic inflammatory response and parasite control. The overall goal of this study was to define the role of monocytes during P. vivax malaria. Here, we demonstrate that P. vivax-infected patients display significant increase in circulating monocytes, which were defined as CD14(+)CD16- (classical), CD14(+)CD16(+) (inflammatory), and CD14loCD16(+) (patrolling) cells. While the classical and inflammatory monocytes were found to be the primary source of pro-inflammatory cytokines, the CD16(+) cells, in particular the CD14(+)CD16(+) monocytes, expressed the highest levels of activation markers, which included chemokine receptors and adhesion molecules. Morphologically, CD14(+) were distinguished from CD14lo monocytes by displaying larger and more active mitochondria. CD14(+)CD16(+) monocytes were more efficient in phagocytizing P. vivax-infected reticulocytes, which induced them to produce high levels of intracellular TNF-α and reactive oxygen species. Importantly, antibodies specific for ICAM-1, PECAM-1 or LFA-1 efficiently blocked the phagocytosis of infected reticulocytes by monocytes. Hence, our results provide key information on the mechanism by which CD14(+)CD16(+) cells control parasite burden, supporting the hypothesis that they play a role in resistance to P. vivax infection.

Innate immunity is the first line defense against invading pathogens. During Gram-negative bacterial infection, the Toll-like receptor 4 and MD-2 complex recognize lipopolysaccharide present in the bacterial cell wall. This recognition can be enhanced 100-1000-fold by CD14. However, the beneficial role provided by CD14 becomes detrimental in the context of sepsis and septic shock. An understanding of how CD14 functions will therefore benefit treatments targeted at both immune suppression and immune enhancement. In the present study, we use site-directed mutagenesis to address the role of disulfide bonds and N-linked glycosylation on CD14. A differential impact is observed for the five disulfide bonds on CD14 folding, with the first two (Cys(6)-Cys(17) and Cys(15)-Cys(32)) being indispensable, the third and fourth (Cys(168)-Cys(198) and Cys(222)-Cys(253)) being important, and the last (Cys(287)-Cys(333)) being dispensable. A functional role is observed for the first disulfide bond because the C6A substitution severely reduces the ability of CD14 to confer lipopolysaccharide responsiveness to U373 cells. Two of the four predicted glycosylation sites, asparagines 132 and 263, are actually involved in N-linked glycosylation, resulting in heterogeneity in CD14 molecular weight. Furthermore, glycosylation at Asn(132) plays a role in CD14 trafficking and upstream and/or downstream ligand interactions. When mapped onto the crystal structure of mouse CD14, the first two disulfide bonds and Asn(132) are in close proximity to the initial beta strands of the leucine rich repeat domain. Thus, disulfide bonds and N-linked glycosylation in the initial beta sheets of the inner concave surface of CD14 are crucial for structure and function.

Members of the Toll-like receptor (TLR) family have been shown to be important in the activation of cells by a variety of microbial ligands. TLRs are thought to mediate the 'recognition event' that follows an encounter between a mammalian cell and a microbial agent. In the case of the response to bacterial lipopolysaccharide (LPS), it is clear that the ability of these cell surface proteins to initiate the events necessary for activation of cells to produce cytokines is dependent upon 'accessory proteins' such as the pattern recognition protein CD14 and the lipopolysaccharide binding protein (LBP). While the role of these proteins in the LPS-specific response is defined, their role in other TLR responses has not been defined, but it is important in understanding these events and, potentially, in designing new therapeutic strategies. Here we report on the role of these proteins in the response to yeast zymosan. The requirements for this response (which unlike the response to LPS is a response to a particulate antigen) and the role of other serum proteins are defined.

Amphotericin B is the most effective drug for treating many life-threatening fungal infections. Amphotericin B administration is limited by infusion-related toxicity, including fever and chills, an effect postulated to result from proinflammatory cytokine production by innate immune cells. Because amphotericin B is a microbial product, we hypothesized that it stimulates immune cells via Toll-like receptors (TLRs) and CD14. We show here that amphotericin B induces signal transduction and inflammatory cytokine release from cells expressing TLR2 and CD14. Primary murine macrophages and human cell lines expressing TLR2, CD14, and the adapter protein MyD88 responded to amphotericin B with NF-kappaB-dependent reporter activity and cytokine release, whereas cells deficient in any of these failed to respond. Cells mutated in TLR4 were less responsive to amphotericin B stimulation than cells expressing normal TLR4. These data demonstrate that TLR2 and CD14 are required for amphotericin B-dependent inflammatory stimulation of innate immune cells and that TLR4 may also provide stimulation of these cells. Our results provide a putative molecular basis for inflammatory responses elicited by amphotericin B and suggest strategies to eliminate the acute toxicity of this drug.

Human cytomegalovirus (CMV) is a ubiquitous opportunistic pathogen that causes significant morbidity and mortality in immunocompromised people. An understanding of how CMV induces and circumvents host immunity is of critical importance in efforts to design effective therapeutics. It was recently discovered that mere cell contact by CMV particles leads to profound modulation of cellular gene expression, including induction of inflammatory cytokines and interferon-stimulated genes characteristic of innate immune detection. These findings suggest that a membrane receptor recognizes a CMV envelope protein(s), leading to innate immune activation. Here, we show that the pattern recognition receptors Toll-like receptor 2 (TLR2) and CD14 recognize CMV virions and trigger inflammatory cytokine production. Induction of inflammatory cytokines is mediated via TLR2-dependent activation of NF-kappa B. Since many of the pathological processes associated with CMV disease are facilitated or directly mediated by inflammatory cytokines, identification of the host membrane detection machinery may ultimately lead to improved therapeutics.

Bacterial lipopolysaccharides (LPSs) are recognized in mammals by a receptor complex composed of CD14, Toll-like receptor 4 (TLR4), and MD-2. The mechanism of TLR4 function remains to be elucidated. We constructed chimeric TLR molecules C-terminally fused to fluorescent proteins and stably expressed these chimeric constructs in cells. Confocal microscopy revealed TLR4 to be expressed on the plasma membrane and the Golgi apparatus. Time-lapse confocal imaging showed rapid recycling of TLR4/CD14/MD-2 complexes between the Golgi and the plasma membrane. Membrane TLR4 engagement by antibody was sufficient to induce signaling and pharmacological disruption of the Golgi did not affect cellular responses to LPS. Thus, LPS signaling commences after LPS recognition by surface-expressed TLR4 independent of LPS trafficking to the Golgi.

Mammalian responses to LPS require the expression of Toll-like receptor 4 (TLR4), CD14, and MD-2. We expressed fluorescent TLR4 in cell lines and found that TLR4 densely localized to the surface and the Golgi. Similar distributions were observed in human monocytes. Confocal imaging revealed rapid recycling of TLR4-CD14-MD-2 complexes between the Golgi and the plasma membrane. Fluorescent LPS followed these trafficking pathways in CD14-positive cells. The TLR4- adapter protein, MyD88, translocated to the cell surface upon LPS exposure, and cross-linking of surface TLR4 with antibody induced signaling. Golgi-associated TLR4 expression was disrupted by brefeldin A, yet LPS signaling was preserved. We conclude that LPS signaling may be initiated by surface aggregation of TLR4 and is not dependent upon LPS trafficking to the Golgi.

Group B streptococcus (GBS) imposes a major health threat to newborn infants. Little is known about the molecular basis of GBS-induced sepsis. Both heat-inactivated whole GBS bacteria and a heat-labile soluble factor released by GBS during growth (GBS-F) induce nuclear translocation of NF-kappaB, the secretion of TNF-alpha, and the formation of NO in mouse macrophages. Macrophages from mice with a targeted disruption of MyD88 failed to secrete TNF-alpha in response to both heat-inactivated whole bacteria and GBS-F, suggesting that Toll-like receptors (TLRs) are involved in different aspects of GBS recognition. Immune cell activation by whole bacteria differed profoundly from that by secreted GBS-F. Whole GBS activated macrophages independently of TLR2 and TLR6, whereas a response to the secreted GBS-F was not observed in macrophages from TLR2-deficient animals. In addition to TLR2, TLR6 and CD14 expression were essential for GBS-F responses, whereas TLR1 and TLR4 or MD-2 did not appear to be involved. Heat lability distinguished GBS-F from peptidoglycan and lipoproteins. GBS mutants deficient in capsular polysaccharide or beta-hemolysin had GBS-F activity comparable to that of wild-type streptococci. We suggest that CD14 and TLR2 and TLR6 function as coreceptors for secreted microbial products derived from GBS and that cell wall components of GBS are recognized by TLRs distinct from TLR1, 2, 4, or 6.

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.

Lipopolysaccharide (LPS) and related bacterial products can be recognized by host inflammatory cells in a particulate, bacterium-bound form, as well as in various soluble, released forms. In the present study we have compared the mechanisms used by LPS, detoxified LPS (DLPS), and mannuronic acid polymers (M-polymers), in solution or covalently linked to particles, in stimulating monocytes to tumor necrosis factor (TNF) production. The addition of recombinant LPS binding protein (LBP) and/or soluble CD14 (sCD14) enhanced the production of TNF from monocytes stimulated with soluble LPS, DLPS, or M-polymer, but did not affect the response to M-polymer or DLPS attached to particles. Treatment of monocytes with antibody to CD14, CD18, or CD11b showed that CD14, but not CR3 (CD11b/CD18), mediated monocyte TNF production in response to the soluble antigens. In contrast, anti-CD14, anti-CD11b and anti-CD18 monoclonal antibodies all inhibited the response to the particulate stimuli. On the other hand, B975, a synthetic analog of Rhodobacter capsulatus lipid A, completely abrogated the monocyte TNF response induced by LPS but did not affect the TNF induction by DLPS or M-polymer, either in soluble or particulate forms. These data demonstrate that the engagement of immune receptors by bacterial products such as LPS, DLPS, and M-polymer is dependent upon the presentation form of their constituent carbohydrates, and that factors such as aggregation state, acylation, carbohydrate chain length, and solid versus liquid phase of bacterial ligands influence the mechanisms used by cells in mediating proinflammatory responses.

The innate immune system contributes to the earliest phase of the host defense against foreign organisms and has both soluble and cellular pattern recognition receptors for microbial products. Two important members of this receptor group, CD14 and the Toll-like receptor (TLR) pattern recognition receptors, are essential for the innate immune response to components of Gram-negative and Gram-positive bacteria, mycobacteria, spirochetes and yeast. We now find that these receptors function in an antiviral response as well. The innate immune response to the fusion protein of an important respiratory pathogen of humans, respiratory syncytial virus (RSV), was mediated by TLR4 and CD14. RSV persisted longer in the lungs of infected TLR4-deficient mice compared to normal mice. Thus, a common receptor activation pathway can initiate innate immune responses to both bacterial and viral pathogens.

Gram-negative bacteria and the LPS constituent of their outer membranes stimulate the release of inflammatory mediators believed to be responsible for the clinical manifestations of septic shock. The GPI-linked membrane protein, CD14, initiates the signaling cascade responsible for the induction of this inflammatory response by LPS. In this paper, we report the generation and characterization of CD14-null mice in which the entire coding region of CD14 was deleted. As expected, LPS failed to elicit TNF-alpha and IL-6 production in macrophages taken from these animals, and this loss in responsiveness is associated with impaired activation of both the NF-kappaB and the c-Jun N-terminal mitogen-activated protein kinase pathways. The binding and uptake of heat-killed Escherichia coli, measured by FACS analysis, did not differ between CD14-null and wild-type macrophages. However, in contrast to the findings with LPS, whole E. coli stimulated similar levels of TNF-alpha release from CD14-null and wild-type macrophages at a dose of 10 bioparticles per cell. This effect was dose dependent, and at lower bacterial concentrations CD14-deficient macrophages produced significantly less TNF-alpha than wild type. Approximately half of this CD14-independent response appeared to be mediated by CD11b/CD18, as demonstrated by receptor blockade using neutrophil inhibitory factor. An inhibitor of phagocytosis, cytochalasin B, abrogated the induction of TNF-alpha in CD14-deficient macrophages by E. coli. These data indicate that CD14 is essential for macrophage responses to free LPS, whereas other receptors, including CD11b/CD18, can compensate for the loss of CD14 in response to whole bacteria.

Lipopolysaccharide (LPS) is the main inducer of shock and death in Gram-negative sepsis. Recent evidence suggests that LPS-induced signal transduction begins with CD14-mediated activation of 1 or more Toll-like receptors (TLRs). The lipid A analogues lipid IVa and Rhodobacter sphaeroides lipid A (RSLA) exhibit an uncommon species-specific pharmacology. Both compounds inhibit the effects of LPS in human cells but display LPS-mimetic activity in hamster cells. We transfected human TLR4 or human TLR2 into hamster fibroblasts to determine if either of these LPS signal transducers is responsible for the species-specific pharmacology. RSLA and lipid IVa strongly induced NF-kappaB activity and IL-6 release in Chinese hamster ovary fibroblasts expressing CD14 (CHO/CD14), but these compounds antagonized LPS antagonists in CHO/CD14 fibroblasts that overexpressed human TLR4. No such antagonism occurred in cells overexpressing human TLR2. We cloned TLR4 from hamster macrophages and found that human THP-1 cells expressing the hamster TLR4 responded to lipid IVa as an LPS mimetic, as if they were hamster in origin. Hence, cells heterologously overexpressing TLR4 from different species acquired a pharmacological phenotype with respect to recognition of lipid A substructures that corresponded to the species from which the TLR4 transgene originated. These data suggest that TLR4 is the central lipid A-recognition protein in the LPS receptor complex.

Mammalian Toll-like receptor (TLR) proteins are new members of the IL-1 receptor family that participate in activation of cells by bacteria and bacterial products. Several recent reports indicate that TLR proteins mediate cellular activation by bacterial LPS via a signaling pathway that is largely shared by the type I IL-1 receptor. We previously showed that Chinese hamster ovary (CHO) fibroblasts engineered to express CD14 (CHO/CD14) were responsive to LPS, but not to a distinct CD14 ligand, mycobacterial lipoarabinomannan (LAM). These CHO/CD14 cells were subsequently found to possess a frame-shift mutation within the TLR2 gene which resulted in their inability to express functional TLR2 protein. Thus, we hypothesized that TLR2, but not TLR4, was necessary for LAM signaling. In this paper we show that CHO/CD14 cells engineered to express functional TLR2 protein acquired the ability to be activated by LAM. Similarly, overexpression of TLR2 in murine macrophages conferred enhanced LAM responsiveness. Together, our data demonstrate that the distinct CD14 ligands LAM and LPS utilize different TLR proteins to initiate intracellular signals. These findings suggest a novel receptor signaling paradigm in which the binding of distinct ligands is mediated by a common receptor chain, but cellular activation is initiated via distinct signal-transducing chains that confer ligand specificity. This paradigm contrasts with many cytokine receptor complexes in which receptor specificity is conferred by a unique ligand-binding chain but cellular activation is initiated via shared signal-transducing chains.

CD14-transfected Chinese hamster ovary K1 fibroblasts (CHO/CD14) respond to lipopolysaccharide (LPS) by metabolizing arachidonic acid and with translocation of NF-kappaB to the nucleus. Although previous experiments failed to identify the production of tumor necrosis factor-alpha and interleukin (IL)-1beta by CHO/CD14 cells, LPS did induce the expression of IL-6 mRNA and the subsequent release of the IL-6 protein. To identify additional LPS-inducible genes, a cDNA library derived from LPS-stimulated CHO/CD14 cells was screened by subtractive hybridization. Fourteen genes were found to be expressed differentially, and two were analyzed in detail: hop (Hsp70/Hsp90-organizing protein), which is the hamster homologue of the stress-inducible yeast gene, STI1, and clone H411, which encodes a novel LPS-inducible growth factor. In response to LPS, the expression of Hop mRNA was also increased in both the murine macrophage cell line, RAW 264.7, as well as in primary hamster macrophages. This suggested that the up-regulation of Hop expression is part of the macrophage stress response to LPS. Clone H411 encodes a protein in the epidermal growth factor-like repeat protein family. Overexpression of H411 cDNA in the RAW 264.7 macrophage cell line promoted an increased growth rate, suggesting that expression of H411 is part of the proliferative cell response to LPS. Both Hop and H411 represent novel gene products not previously recognized as part of the complex biological response to endotoxin.

The activation of phagocytes by lipopolysaccharide (LPS) has been implicated in the pathogenesis of Gram-negative sepsis. Although the interaction between CD14 and LPS is a key event in the signaling cascade, the molecular mechanism by which cellular activation occurs remains obscure. We hypothesized that the main function of CD14 was to bind LPS and transfer it to a second receptor, which then initiates the subsequent signal for cellular activation. Thus, surface binding of LPS to the cell membrane would be the critical step that CD14 carries out. To test this hypothesis, we examined the activity of two other proteins known to bind LPS, lipopolysaccharide-binding protein and bactericidal/permeability-increasing protein. We found that when these normally soluble proteins were expressed in Chinese hamster ovary-K1 fibroblasts as glycosylphosphatidylinositol-anchored proteins, both could substitute for CD14 in initiating LPS signaling. Pharmacological studies with synthetic lipid A analogues demonstrated that these surface expressed LPS-binding proteins had characteristics that were qualitatively identical to membrane CD14. These data support the hypothesis that a receptor distinct from CD14 functions as the actual signal transducer and suggest that surface binding of LPS to the cell membrane is the crucial first step for initiating downstream signaling events.

The activation of phagocytes by the lipid A moiety of LPS has been implicated in the pathogenesis of Gram-negative sepsis. While two LPS receptors, CD14 and CD11/CD18, have been associated with cell signaling, details of the LPS signal transduction cascade remain obscure. CD14, which exists as a GPI-anchored and a soluble protein, lacks cytoplasmic-signaling domains, suggesting that an ancillary molecule is required to activate cells. The CD11/CD18 integrins are transmembrane proteins. Like CD14, they are capable of mediating LPS-induced cellular activation when expressed on the surface of hamster fibroblasts Chinese hamster ovary (CHO)-K1. The observation that a cytoplasmic deletion mutant is still capable of activating transfected CHO-K1 argues that CD11/CD18 also utilizes an associated signal transducer. We sought to identify further similarities between the signaling systems utilized by CD14 and CD11/CD18. LPS-binding protein, which transfers LPS to CD14, enhanced both LPS-induced cellular activation and binding of Gram-negative bacteria in CD11/CD18-transfected CHO-K1, thus implying that LPS-binding protein can also transfer LPS to CD11/CD18. When synthetic lipid A analogues were analyzed for their ability to function as LPS agonists, or antagonists, in the CHO transfectants, we found the effects were identical regardless of which LPS receptor was expressed. This supports the hypothesis that a receptor distinct from CD14 and CD11/CD18 is responsible for discriminating between the lipid A of LPS and the LPS antagonists. We propose that this receptor, which is the target of the LPS antagonists, functions as the true signal transducer in LPS-induced cellular activation for both CD14 and CD11/CD18.

Helicobacter pylori gastritis is characterized by leukocyte infiltration of the gastric mucosa. The aims of this study were to determine whether H. pylori-derived factors stimulate chemokine release from human monocytes and to ascertain whether H. pylori lipopolysaccharide (LPS) may be responsible for this effect. Human peripheral blood monocytes were exposed to an H. pylori water extract (HPE) or to purified H. pylori LPS. Levels of the chemokines interleukin-8 (IL-8), epithelial neutrophil-activating peptide 78 (ENA-78), and monocyte chemotactic protein 1 (MCP-1) were measured by enzyme-linked immunosorbent assay. The contribution of H. pylori LPS to monocyte activation was determined by using the LPS antagonist Rhodobacter sphaeroides lipid A (RSLA) and a blocking monoclonal antibody to CD14 (60bca). HPE increased monocyte secretion of IL-8, ENA-78, and MCP-1. Heat treatment of HPE did not reduce its ability to activate monocytes. Purified H. pylori LPS also stimulated monocyte chemokine production but was 1,000-fold less potent than Salmonella minnesota lipid A. RSLA blocked H. pylori LPS-induced monocyte IL-8 release in a dose-dependent fashion (maximal inhibition 82%, P < 0.001). RSLA also inhibited HPE-induced IL-8 release (by 93%, P < 0.001). The anti-CD14 monoclonal antibody 60bca substantially inhibited IL-8 release from HPE-stimulated monocytes (by 88%, P < 0.01), whereas the nonblocking anti-CD14 monoclonal antibody did not. These experiments with potent and specific LPS inhibitors indicate that the main monocyte-stimulating factor in HPE is LPS. H. pylori LPS, acting through CD14, stimulates human monocytes to release the neutrophil-activating chemokines IL-8 and ENA-78 and the monocyte-activating chemokine MCP-1. Despite its low relative potency, H. pylori LPS may play an important role in the pathogenesis of H. pylori gastritis.

Macrophage activation by gram-negative lipopolysaccharide (LPS) has been extensively studied in an attempt to define the mechanisms that underlie innate immunity against bacterial pathogens. Dysregulation of these same mechanisms contributes to the pathophysiological consequences of bacterial sepsis. The biological actions of LPS are mediated, at least in part, by both LPS-binding proteins and LPS receptors. Several LPS receptors (CD14, the macrophage scavenger receptor, and the beta2 integrins), as well as the serum LPS-binding protein LBP, have been cloned and studied in detail. In addition, insights gained through the use of LPS antagonists have led to a better understanding of a molecule believed to function in conjunction with LPS receptors to transduce signals from the membrane to the cytosol. More recently, the use of knockout mice has greatly expanded our knowledge of the biology of LPS receptors and binding proteins. This review will summarize various phenotypes of mice that lack genes encoding CD14, the scavenger receptor, and LBP. These knockout mice have revealed several unexpected features of LPS action in vivo. Together, these animal models may provide a means to develop and evaluate novel therapeutic approaches to the control of endotoxin shock.

This study was undertaken to evaluate the role of CD14 and complement receptors type 3 (CR3) and 4 (CR4) in mediating TNF release and NF-kappaB activation induced by LPS and cell wall preparations from group B streptococci type III (GBS). LPS and GBS caused TNF secretion from human monocytes in a CD14-dependent manner, and soluble CD14, LPS binding protein, or their combination potentiated both LPS- and GBS-induced activities. Blocking of either CD14 or CD18, the common beta-subunit of CR3 and CR4, decreased GBS-induced TNF release, while LPS-mediated TNF production was inhibited by anti-CD14 mAb only. Chinese hamster ovary cell transfectants (CHO) that express human CD14 (CHO/CD14) responded to both LPS and GBS with NF-kappaB translocation, which was inhibited by anti-CD14 mAb and enhanced by LPS binding protein. While LPS showed fast kinetics of NF-kappaB activation in CHO/CD14 cells, a slower NF-kappaB response was induced by GBS. LPS also activated NF-kappaB in CHO cells transfected with either human CR3 or CR4 cDNA, although responses were delayed and weaker than those of CHO/CD14 cells. In contrast to LPS, GBS failed to induce NF-kappaB in CHO/CR3 or CHO/CR4 cells. Both C3H/OuJ (Lps[n]) and C3H/HeJ (Lps[d]) mouse peritoneal macrophages responded to GBS with TNF production and NF-kappaB translocation, whereas LPS was active only in C3H/OuJ macrophages. Thus, LPS and GBS differentially involve CD14 and CR3 or CR4 for signaling NF-kappaB activation in CHO cells and TNF release in human monocytes, and engage a different set of receptors and/or intracellular signaling pathways in mouse macrophages.

Alveolar macrophages, which play a central role in lung defense, produce cytokines that help orchestrate local inflammatory responses. In sepsis and other pathological conditions, bacterial lipopolysaccharide endotoxin can induce alveolar macrophages (AM) to release proinflammatory cytokines, including tumor necrosis factor-alpha, interleukin-1, and interleukin-6. Studying the mechanisms that control alveolar macrophage cytokine production may lead to better therapies for conditions involving inflammatory lung injury. We and others have noted significant differences between alveolar macrophages and peritoneal macrophages, but large numbers of human or murine alveolar macrophages are rarely available for detailed mechanistic studies. We have obtained three murine alveolar macrophage cell lines (AMJ2C8, AMJ2C11, and AMJ2C20) and have begun to characterize their cytokine responses to proinflammatory stimuli. We measured the effects of endotoxin, interferon gamma, and the combination of the two on production of tumor necrosis factor, interleukin-1 beta, and interleukin-6 in each line. We also studied the expression of the endotoxin receptor CD14 by these cells, and investigated the effect of serum on their endotoxin responsiveness. We show here that all three of the cell lines responded in a manner comparable to that of primary murine alveolar macrophages. Observed variations between these lines may reflect the documented heterogeneity seen in populations of primary alveolar macrophages. These cell lines should expand the repertoire of tools available to investigators studying regulation of murine alveolar macrophage responses.

Phagocytic leukocytes respond to a variety of bacterial products including Gram-negative bacterial LPS and mycobacterial lipoarabinomannan (LAM). Anti-CD14 mAbs have been shown to block LPS and LAM activation of myeloid cells, suggesting that CD14 is required for cellular recognition of both ligands. Activation of undifferentiated promonomyelocytic THP-1 cells with LAM or LPS under serum-free conditions was enhanced in the presence of recombinant soluble CD14 (rsCD14). LPS binding protein (LBP), which is present in normal serum, further enhanced the sensitivity of undifferentiated THP-1 cells to both ligands even in the absence of rsCD14. Although CD14-transfected Chinese hamster ovary and human HT1080 fibrosarcoma cell lines can be activated by LPS, neither cell line was activated by LAM. Furthermore, U373 astrocytoma cells, which respond to LPS using sCD14 and LBP, failed to be activated by LAM in the presence of rsCD14 and rLBP. We then tested the effects of lipid IVA and Rhodobacter sphaeroides lipid A, compounds that function as endotoxin inhibitors in human cells by interacting with a molecule thought to be a CD14-dependent LPS signal transducer. Both lipid IVA and R. sphaeroides lipid A inhibited the effects of LPS and LAM in THP-1 cells. Thus, the LPS and LAM receptors share CD14, LBP, and a putative endotoxin antagonist-inhibitable signal transducing component. However, the LAM signaling system appears to require an additional receptor component whose expression is restricted to cells of hemopoietic origin.

Binding of the lipid A portion of bacterial lipopolysaccharide (LPS) to leukocyte CD14 activates phagocytes and initiates the septic shock syndrome. Two lipid A analogs, lipid IVA and Rhodobacter sphaeroides lipid A (RSLA), have been described as LPS-receptor antagonists when tested with human phagocytes. In contrast, lipid IVA activated murine phagocytes, whereas RSLA was an LPS antagonist. Thus, these compounds displayed a species-specific pharmacology. To determine whether the species specificity of these LPS antagonists occurred as a result of interactions with CD14, the effects of lipid IVA and RSLA were examined by using human, mouse, and hamster cell lines transfected with murine or human CD14 cDNA expression vectors. These transfectants displayed sensitivities to lipid IVA and RSLA that reflected the sensitivities of macrophages of similar genotype (species) and were independent of the source of CD14 cDNA. For example, hamster macrophages and hamster fibroblasts transfected with either mouse or human-derived CD14 cDNA responded to lipid IVA and RSLA as LPS mimetics. Similarly, lipid IVA and RSLA acted as LPS antagonists in human phagocytes and human fibrosarcoma cells transfected with either mouse or human-derived CD14 cDNA. Therefore, the target of these LPS antagonists, which is encoded in the genomes of these cells, is distinct from CD14. Although the expression of CD14 is required for macrophage-like sensitivity to LPS, CD14 cannot discriminate between the lipid A moieties of these agents. We hypothesize that the target of the LPS antagonists is a lipid A recognition protein which functions as a signaling receptor that is triggered after interaction with CD14-bound LPS.