MyD88-dependent pathway is essential for the innate immunity to Enterocytozoon bieneusi.

Enterocytozoon bieneusi is clinically the most significant microsporidian parasite associated with persistent diarrhoea, wasting and cholangitis in 30-50% of individuals with HIV/AIDS, as well as in malnutritional children and in the recipients of immunosuppressive therapy. However, the host immune responses to E. bieneusi have not been investigated until recently because of lack of sources of spores, cell culture system and animal models. In this study, we purified spores from heavily infected human or monkey faeces by serial salt-Percoll-sucrose-iodixanol centrifugation, and the purity of spores was confirmed by FACS and scanning electron microscopy. Exposure of dendritic cells to E. bieneusi spores induced the upregulation of the surface markers and production of pro-inflammatory cytokines. The cytokine production was independent of toll-like receptor 4, but MyD88 dependent, because dendritic cells from MyD88 knockout mice failed to secrete these pro-inflammatory cytokines, whereas dendritic cells from C3H/HeJ (a toll-like receptor 4 mutant) were activated by E. bieneusi and secreted these cytokines. Furthermore, MyD88-deficient mice were susceptible to E. bieneusi infection, in contrast to wild-type mice that resisted the infection. Collectively, the data demonstrate innate recognition of E. bieneusi by dendritic cells and the importance of MyD88-dependent signalling in resisting infection in a murine challenge model.

CD11c/CD18, a transmembrane signaling receptor for lipopolysaccharide.

CD11c/CD18 is a member of the leukocyte integrin family, heterodimeric adhesion molecules that interact with a diverse repertoire of ligands, including bacterial lipopolysaccharide (LPS). Their role as signal transducing receptors remains uncertain. We used a heterologous expression system to determine if CD11c/CD18 was capable of initiating signal transduction in response to LPS-binding, as assessed by the induced translocation of nuclear factor-kappa B. We have previously reported that Chinese hamster ovary (CHO)-K1 fibroblasts, normally unresponsive to LPS, acquire serum-dependent macrophage-like responses to LPS when transfected with CD14 (Golenbock, D.T., Y. Liu, F. Millham, M. Freeman, and R. Zoeller. 1993. J. Biol. Chem. 268:22055-22059), a known LPS receptor. In contrast, CHO cells acquired serum-independent responses to Gram-negative bacteria and LPS when transfected with CD11c/CD18 (CHO/CD11c). In comparison to CHO cells transfected with CD14 (CHO/CD14), responses in CHO/CD11c cells were slower, required higher endotoxin concentrations for maximal response, and were not inhibited by the presence of antibodies to CD14. CD11c/CD18 is, thus, the second phagocyte receptor, in addition to CD14, which has been shown to have the capacity to activate cells after binding to LPS. The function of this receptor in normal phagocytes may be limited to the recognition of LPS in infected tissues, where LPS-CD14 interactions are not favored because of the absence of serum proteins.

Molecular genetic analysis of an endotoxin nonresponder mutant cell line: a point mutation in a conserved region of MD-2 abolishes endotoxin-induced signaling.

Somatic cell mutagenesis is a powerful tool for characterizing receptor systems. We reported previously two complementation groups of mutant cell lines derived from CD14-transfected Chinese hamster ovary--K1 fibroblasts defective in responses to bacterial endotoxin. Both classes of mutants expressed a normal gene product for Toll-like receptor (TLR)4, and fully responded to stimulation by tumor necrosis factor (TNF)-alpha or interleukin (IL)-1 beta. We identified the lesion in one of the complementation groups in the gene for MD-2, a putative TLR4 coreceptor. The nonresponder phenotype of this mutant was reversed by transfection with MD-2. Cloning of MD-2 from the nonresponder cell line revealed a point mutation in a highly conserved region resulting in a C95Y amino acid exchange. Both forms of MD-2 colocalized with TLR4 on the cell surface after transfection, but only the wild-type cDNA reverted the lipopolysaccharide (LPS) nonresponder phenotype. Furthermore, soluble MD-2, but not soluble MD-2(C95Y), functioned to enable LPS responses in cells that expressed TLR4. Thus, MD-2 is a required component of the LPS signaling complex and can function as a soluble receptor for cells that do not otherwise express it. We hypothesize that MD-2 conformationally affects the extracellular domain of TLR4, perhaps resulting in a change in affinity for LPS or functioning as a portion of the true ligand for TLR4.

Toll-like receptor 4 imparts ligand-specific recognition of bacterial lipopolysaccharide.

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.

The biology of Toll-like receptors.

In 1997, a human homologue of the Drosophila Toll protein was described, a protein later to be designated Toll-like receptor 4 (TLR4). Since that time, additional human and murine TLR proteins have been identified. Mammalian TLR proteins appear to represent a conserved family of innate immune recognition receptors. These receptors are coupled to a signaling pathway that is conserved in mammals, insects, and plants, resulting in the activation of genes that mediate innate immune defenses. Numerous studies have now identified a wide variety of chemically-diverse bacterial products that serve as putative ligands for TLR proteins. More recent studies have identified the first endogenous protein ligands for TLR proteins. TLR signaling represents a key feature of innate immune response to pathogen invasion.

Chlamydia pneumoniae and chlamydial heat shock protein 60 stimulate proliferation of human vascular smooth muscle cells via toll-like receptor 4 and p44/p42 mitogen-activated protein kinase activation.

An early component of atherogenesis is abnormal vascular smooth muscle cell (VSMC) proliferation. The presence of Chlamydia pneumoniae in many atherosclerotic lesions raises the possibility that this organism plays a causal role in atherogenesis. In this study, C pneumoniae elementary bodies (EBs) rapidly activated p44/p42 mitogen-activated protein kinases (MAPKs) and stimulated proliferation of VSMCs in vitro. Exposure of VSMCs derived from human saphenous vein to C pneumoniae EBs (3x10(7) inclusion forming units/mL) enhanced bromodeoxyuridine (BrdU) incorporation 12+/-3-fold. UV- and heat-inactivated C pneumoniae EBs also stimulated VSMC proliferation, indicating a role of direct stimulation by chlamydial antigens. However, the mitogenic activity of C pneumoniae was heat-labile, thus excluding a role of lipopolysaccharide. Chlamydial hsp60 (25 microg/mL) replicated the effect of C pneumoniae, stimulating BrdU incorporation 7+/-3-fold. Exposure to C pneumoniae or chlamydial hsp60 rapidly activated p44/p42 MAPK, within 5 to 10 minutes of exposure. In addition, PD98059 and U0126, which are two distinct inhibitors of upstream MAPK kinase 1/2 (MEK1/2), abolished the mitogenic effect of C pneumoniae and chlamydial hsp60. Toll-like receptors (TLRs) act as sensors for microbial antigens and can signal via the p44/p42 MAPK pathway. Human VSMCs were shown to express TLR4 mRNA and protein, and a TLR4 antagonist abolished chlamydial hsp60-induced VSMC proliferation and attenuated C pneumoniae-induced MAPK activation and VSMC proliferation. Together these results indicate that C pneumoniae and chlamydial hsp60 are potent inducers of human VSMC proliferation and that these effects are mediated, at least in part, by rapid TLR4-mediated activation of p44/p42 MAPK.

LPS-binding proteins and receptors.

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.

Differential roles of TLR2 and TLR4 in the host response to Gram-negative bacteria: lessons from a lipopolysaccharide-deficient mutant of Neisseria meningitidis.

The inflammatory response to bacterial infections plays an important role in the detection and elimination of invading micro-organisms. Various components of the bacterial cell wall are capable of activating this pro-inflammatory response. In the case of Gram-negative bacteria, lipopolysaccharide (LPS) is the dominant trigger, although other bacterial factors are also capable of activating this systemic inflammatory response. Recently, Toll-like receptors (TLRs) have been implicated in host responses to bacterial pathogens. Specifically, TLR4 mediates LPS responses while TLR2 plays a broader role in the recognition of a variety of bacteria and bacterial antigens. The experiments in this study were designed to examine the role of Gram-negative cell wall components, other than LPS, and their cellular receptors in the host response to infection using an LPS-deficient mutant of Neisseria meningitidis. Although less potent than the parental strain, we found the LPS-deficient mutant to be a capable inducer of the inflammatory response in a variety of cell types. Moreover, cellular activation by this mutant required expression of CD14 and TLR2.

Characterization of low-density lipoprotein uptake by murine macrophages exposed to Chlamydia pneumoniae.

Exposure to Chlamydia pneumoniae is correlated with atherosclerosis in a variety of clinical and epidemiological studies, but how the organism may initiate and promote the disease is poorly understood. One pathogenic mechanism could involve modulation of macrophage function by C. pneumoniae. We recently demonstrated that C. pneumoniae induces macrophages to accumulate excess cholesterol and develop into foam cells, the hallmark of early atherosclerotic lesions. To determine if C. pneumoniae-induced foam cell formation involved increased uptake of low-density lipoprotein (LDL), the current study examined macrophage association of a fluorescent carbocyanine (DiI)-labeled LDL following infection. C. pneumoniae enhanced the association of DiI-LDL with macrophages in a dose-dependent manner with respect to both C. pneumoniae and DiI-LDL. Interestingly, increased association was inhibited by native LDL and occurred in the absence of oxidation byproducts and in the presence of antioxidants. However, enhanced DiI-LDL association occurred without the participation of the classical Apo B/E native LDL receptor, since C. pneumoniae increased DiI-LDL association and induced foam cell formation in macrophages isolated from LDL-receptor-deficient mice. Surprisingly, DiI-LDL association was inhibited not only by unlabeled native LDL but also by high-density lipoprotein, very low density lipoprotein, and oxidized LDL. These data indicate that exposure of macrophages to C. pneumoniae increases the uptake of LDL and foam cell formation by an LDL-receptor-independent mechanism.

Pyomyositis and hepatic abscess in association with Aeromonas hydrophila sepsis.

This report describes a patient with chronic liver disease in whom Aeromonas hydrophila sepsis developed following ingestion of fresh lake water. A hepatic abscess and bilateral calf pyomyositis, an extremely rare and previously fatal complication of A. hydrophila septicemia, subsequently developed. The patient recovered following bilateral fasciotomies and systemic antibiotic therapy with gentamicin and trimethoprim/sulfamethoxazole. No previous survivors of A. hydrophila pyomyositis secondary to metastatic infection have been described. A discussion of these rare sequelae of A. hydrophila septicemia and their effective management is included.

Absence of infection with human immunodeficiency virus in Peruvian prostitutes.

We serologically tested 140 female prostitutes (mean age, 30 years) from the port city of Callao, Peru, for evidence of infection with human immunodeficiency virus (HIV), Chlamydia trachomatis, Treponema pallidum, herpes simplex viruses (HSV) I and II, and hepatitis B virus. The women had worked as prostitutes for an average of 5 years; one-fourth serviced foreign visitors exclusively, mainly sailors. Only 4 women used condoms, and only 1 woman gave a history of parenteral narcotic abuse, although 53% were regularly exposed to unsterile needles outside the medical setting for injections of vitamins, antibiotics, or steroids; another 29% are thought to probably use unsterile needles. None of the 140 prostitutes screened was seropositive for HIV, despite a very high prevalence of antibody to T. pallidum (24%), C. trachomatis (97%), HSV I and II (100%), and hepatitis B (51%); 5% were HbsAg positive. These data indicate that HIV has not yet been introduced into female prostitutes in the Peruvian port city. We believe that widespread use of unsterile needles in developing countries, such as Peru, represents a serious health threat and will amplify the spread of HIV, once introduced.

Structure and function of Toll-like receptor proteins.

Beginning in 1997 with the identification of the first human homologue of the Drosophila protein Toll, a family of related molecules have been identified in both humans and other mammals. These Toll-like receptor (TLR) proteins appear to represent a conserved family of innate immune recognition receptors. TLR proteins share extended homology with receptors for the cytokines interleukin 1 (IL-1) and interleukin 18 (IL-18). These receptors are coupled to a signaling pathway that is conserved in mammals, insects, and plants, resulting in cellular activation, thereby stimulating innate immune defenses. A variety of bacterial and fungal products have been identified that serve as TLR ligands, and more recent studies have identified the first endogenous protein ligands for TLR proteins. While TLR signaling is likely to be a key feature of innate immune responses, these proteins may also regulate homeostasis via interaction with endogenous protein ligands.

Characterization of proinflammatory cytokine production and CD14 expression by murine alveolar macrophage cell lines.

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.

Lipopolysaccharide antagonists.

Bacterial lipopolysaccharide (LPS) is a potent and pleiotropic stimulus of immune cells. LPS has important clinical relevance because it has a direct role in the pathogenesis of Gram-negative bacterial infection. The lipid A moiety of LPS is responsible for the toxic effects of LPS. The identification of structural analogs and precursors of lipid A, which are apparently competitive antagonists of the biological actions of LPS, is strong evidence that the actions of LPS are mediated by a specific LPS receptor or family of receptors. Identification and analysis of these LPS receptors with LPS antagonists should help to define the pathways of cellular activation by LPS and lead to the development of novel anti-LPS strategies in the therapy of bacterial diseases.

Neither CD14 nor serum is absolutely necessary for activation of mononuclear phagocytes by bacterial lipopolysaccharide.

The stimulation of mononuclear phagocytes by lipopolysaccharide (LPS) is facilitated by the binding of complexes of LPS and LPS-binding protein to CD14. Although it is clear that CD14 is involved in LPS-induced signaling, other investigators have hypothesized the existence of additional signaling pathways in macrophages. We sought to determine whether CD14-independent pathways of monocyte activation might exist. Washed human mononuclear cells responded with reduced sensitivity to LPS in the absence of serum. Anti-CD14 monoclonal antibody (MAb) inhibited the response to LPS in serum-free conditions, but this was easily reversed at higher concentrations of LPS. We established a human monocytic cell line, designated SFM (derived from THP-1), in serum-free medium to examine LPS responses under defined conditions. Differentiation of SFM cells with 1,25-dihydroxycholecalciferol promoted the expression of abundant cell surface CD14. Differentiated SFM cells responded to LPS despite the complete absence of serum proteins for > 20 generations of growth. LPS stimulation of differentiated SFM cells was inhibited by anti-CD14 MAbs only when serum was present. In contrast to anti-CD14 MAb, the LPS antagonists lipid IVa and Rhodobacter sphaeroides lipid A inhibited monocyte activation under serum-free conditions, implying that these compounds compete with LPS at a site distinct from CD14. Undifferentiated SFM cells (expressing minimal CD14) still responded to LPS in serum-free conditions, and anti-CD14 MAb had little inhibitory effect. The addition of purified LPS-binding protein or human serum promoted a CD14-dependent pathway of monocyte activation by LPS in these cells. We conclude that monocytes do not absolutely require serum proteins to be stimulated by LPS and that CD14-independent LPS signaling pathways exist which are inhibitable by lipid IVa and R. sphaeroides lipid A.

Lipid A binding sites in membranes of macrophage tumor cells.

Lipopolysaccharide affects a variety of eukaryotic cells and mammalian organisms. These actions are involved in the pathogenesis of Gram-negative septicemia. Many of the actions of lipopolysaccharide are believed to be caused by its active moiety, lipid A. Our laboratory has previously identified a bioactive lipid A precursor, termed lipid IVA (Raetz, C. R. H., Purcell, S., Meyer, M. V., Qureshi, N., and Takayama, K. (1985) J. Biol. Chem. 260, 16080-16888), which can be labeled with 32P of high specific activity and purified. In this work we have used the labeled probe, 4'-32P-lipid IVA, to develop a novel assay for the specific binding of lipid IVA to whole cells. We have also demonstrated its use in a ligand blotting assay of immobilized cellular proteins. Using the whole cell assay, we show that 4'-32P-lipid IVA specifically binds to RAW 264.7 macrophage-like cultured cells. The binding is saturable, is inhibited with excess unlabeled lipid IVA, and is proteinase K-sensitive. It displays cellular and pharmacological specificity. Using the ligand blotting assay, we show that several RAW 264.7 cell proteins can bind 4'-32P-lipid IVA. The two principal binding proteins have Mr values of 31 and 95 kDa, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fractionation studies indicate that the 31-kDa protein is enriched in the nuclear fraction and may be a histone, whereas the 95-kDa protein is enriched in the membrane fraction. The binding assays that we have developed should lead to a clearer understanding of lipid A/animal cell interactions.

Outside-in signaling by lipopolysaccharide through a tailless integrin.

Ligand binding to integrins activates intracellular signaling pathways that coordinate and regulate a variety of cellular responses. There is evidence to suggest that the cytoplasmic tails play a key role in several of these signaling events. We sought to determine whether the beta2 integrin complement receptor type 3 (CR3; CD11b/CD18), a receptor for LPS, could initiate an intracellular signal in the absence of its cytoplasmic domains. Expression of full length CR3 in a Chinese hamster ovary-K1 fibroblast line enabled serum-independent translocation of nuclear factor-kappaB in response to binding LPS. Unexpectedly, a cell line expressing a mutated form of CR3 deficient in the cytoplasmic domains was also competent for transmitting a signal in response to LPS. In contrast, phagocytosis of whole Gram-negative bacteria and iC3b-coated erythrocytes took place only with a full length receptor. Thus, while full length CR3 is necessary for productive phagocytic signals, LPS activation does not require the cytoplasmic domains. CR3 may function to activate cells by presenting LPS to a downstream signal transducer.

Membrane expression of soluble endotoxin-binding proteins permits lipopolysaccharide signaling in Chinese hamster ovary fibroblasts independently of CD14.

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.