RESUMEN
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.
Asunto(s)
Toxinas Bacterianas/metabolismo , Endotoxinas/metabolismo , Integrina alfaXbeta2/fisiología , Lipopolisacáridos/metabolismo , Activación de Linfocitos , Receptores Inmunológicos/metabolismo , Transducción de Señal , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/genética , Antígenos de Diferenciación Mielomonocítica/metabolismo , Toxinas Bacterianas/farmacología , Células CHO , Cricetinae , Endotoxinas/farmacología , Integrina alfaXbeta2/genética , Integrina alfaXbeta2/metabolismo , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Receptores de Lipopolisacáridos , Lipopolisacáridos/farmacología , FN-kappa B/metabolismo , Receptores Inmunológicos/genética , Proteínas Recombinantes de Fusión/metabolismo , Salmonella/metabolismoRESUMEN
Gram-negative bacterial lipopolysaccharide (LPS) stimulates phagocytic leukocytes by interacting with the cell surface protein CD14. Cellular responses to LPS are markedly potentiated by the LPS-binding protein (LBP), a lipid-transfer protein that binds LPS aggregates and transfers LPS monomers to CD14. LBP also transfers LPS to lipoproteins, thereby promoting the neutralization of LPS. LBP present in normal plasma has been shown to enhance the LPS responsiveness of cells in vitro. The role of LBP in promoting LPS responsiveness in vivo was tested in LBP-deficient mice produced by gene targeting in embryonic stem cells. Whole blood from LBP-deficient animals was 1,000-fold less responsive to LPS as assessed by the release of tumor necrosis factor (TNF)-alpha. Blood from gene-targeted mice was devoid of immunoreactive LBP, essentially incapable of transferring LPS to CD14 in vitro, and failed to support cellular responses to LPS. These activities were restored by the addition of exogenous recombinant murine LBP to the plasma. Despite these striking in vitro findings, no significant differences in TNF-alpha levels were observed in plasma from wild-type and LBP-deficient mice injected with LPS. These data suggest the presence of an LBP-independent mechanism for responding to LPS. These LBP knockout mice may provide a tool for discovering the nature of the presumed second mechanism for transferring LPS to responsive cells.
Asunto(s)
Proteínas de Fase Aguda/genética , Proteínas Portadoras/fisiología , Eliminación de Gen , Lipopolisacáridos/farmacología , Glicoproteínas de Membrana , Animales , Proteínas Portadoras/genética , Quimera , Técnicas In Vitro , Riñón/química , Riñón/efectos de los fármacos , Receptores de Lipopolisacáridos/metabolismo , Hígado/química , Hígado/efectos de los fármacos , Ratones , Ratones Noqueados , Ratones Mutantes , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Drosophila , Receptores de Lipopolisacáridos/metabolismo , Lipopolisacáridos/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Células CHO , Cricetinae , Glucolípidos/metabolismo , Humanos , Ligandos , Lípido A/análogos & derivados , Lípido A/metabolismo , Receptores de Lipopolisacáridos/genética , Lipopolisacáridos/antagonistas & inhibidores , Macrófagos/citología , Macrófagos/metabolismo , Glicoproteínas de Membrana/genética , Imitación Molecular , Receptores de Superficie Celular/genética , Proteínas Recombinantes/metabolismo , Rhodobacter sphaeroides , Transducción de Señal , Especificidad de la Especie , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-LikeRESUMEN
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.
Asunto(s)
Proteínas de Drosophila , Lipopolisacáridos/inmunología , Glicoproteínas de Membrana/inmunología , Neisseria meningitidis/inmunología , Receptores de Superficie Celular/inmunología , Animales , Células CHO , Pared Celular/inmunología , Cricetinae , Humanos , Técnicas In Vitro , Mediadores de Inflamación/inmunología , Leucocitos Mononucleares/inmunología , Receptores de Lipopolisacáridos/genética , Glicoproteínas de Membrana/genética , Mutación , Neisseria meningitidis/genética , Receptores de Superficie Celular/genética , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-Like , TransfecciónRESUMEN
The ability of a host to sense invasion by a pathogenic organism, and to respond appropriately to control infection, is paramount to survival. To that end, an array of receptors and binding proteins has evolved as part of the innate immune system to detect Gram-negative bacteria. This article reviews the role of CD14, other LPS binding proteins, and the Toll family of receptors in the innate recognition of bacterial lipopolysaccharide.
Asunto(s)
Proteínas de Drosophila , Receptores de Lipopolisacáridos/fisiología , Lipopolisacáridos/toxicidad , Animales , Antígenos CD11/fisiología , Antígenos CD18/fisiología , Endotoxinas/antagonistas & inhibidores , Humanos , Lípido A/farmacología , Receptores de Lipopolisacáridos/metabolismo , Receptores de Lipopolisacáridos/farmacología , Lipopolisacáridos/antagonistas & inhibidores , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Noqueados/genética , Receptores de Superficie Celular/fisiología , Choque Séptico/etiología , Transducción de Señal , Receptores Toll-LikeRESUMEN
Neisseria gonorrhoeae is a common bacterial sexually transmitted infection. Like all Gram-negative bacteria, the outer membrane of the gonococcus is rich in endotoxin, a known ligand for Toll-like receptor (TLR)4. However, the role of endotoxin and that of its cognate receptor TLR4 in the mucosal response to acute gonococcal infection in the genital tract of women is unclear. To test this, we examined the course of infection after vaginal inoculation of N. gonorrhoeae in mice carrying the Lps(d) mutation in Tlr4, which renders them unresponsive to endotoxin. Although there was no difference in the duration of colonization, Lps(d) mice had a significantly higher peak bacterial burden which coincided with a massive polymorphonuclear cell influx and concomitant upregulation of a subset of inflammatory cytokine and chemokine markers. Notably, infected Lps(d) mice showed a decrease in interleukin-17, suggesting that Th17 responses are more dependent on TLR4 signaling in vivo. Defective polymorphonuclear cell-mediated and complement-independent serum killing of gonococci in Lps(d) mice was also observed and may account for the increased bacterial burden. This is the first in vivo evidence that TLR4-regulated factors modulate early inflammatory responses to gonococcal infection in the female reproductive tract and control bacterial replication.
Asunto(s)
Gonorrea/inmunología , Neisseria gonorrhoeae/fisiología , Células Th17/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Movimiento Celular/genética , Movimiento Celular/inmunología , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Citotoxicidad Inmunológica/genética , Citotoxicidad Inmunológica/inmunología , Modelos Animales de Enfermedad , Femenino , Gonorrea/genética , Humanos , Mediadores de Inflamación/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Mutantes , Mutación/genética , Neisseria gonorrhoeae/patogenicidad , Transducción de Señal/genética , Transducción de Señal/inmunología , Células Th17/inmunología , Células Th17/patología , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunología , Regulación hacia ArribaRESUMEN
The recent isolation of a lipopolysaccharide (LPS)-deficient mutant of Neisseria meningitidis has allowed us to explore the roles of other gram-negative cell wall components in the host response to infection. The experiments in this study were designed to examine the ability of this mutant strain to activate cells. Although it was clearly less potent than the parental strain, we found the LPS-deficient mutant to be a capable inducer of the inflammatory response in monocytic cells, inducing a response similar to that seen with Staphylococcus aureus. Cellular activation by the LPS mutant was related to expression of CD14, a high-affinity receptor for LPS and other microbial products, as well as Toll-like receptor 2, a member of the Toll family of receptors recently implicated in host responses to gram-positive bacteria. In contrast to the parental strain, the synthetic LPS antagonist E5564 did not inhibit the LPS-deficient mutant. We conclude that even in the absence of LPS, the gram-negative cell wall remains a potent inflammatory stimulant, utilizing signaling pathways independent of those involved in LPS signaling.
Asunto(s)
Proteínas Bacterianas/fisiología , Proteínas de Drosophila , Lípido A/análogos & derivados , Lipopolisacáridos/toxicidad , Glicoproteínas de Membrana/fisiología , Proteínas de la Membrana/fisiología , Neisseria meningitidis/inmunología , Receptores de Superficie Celular/fisiología , Animales , Células CHO , Pared Celular/fisiología , Cricetinae , Humanos , Lípido A/farmacología , Receptores de Lipopolisacáridos/fisiología , Mutación , Receptor Toll-Like 2 , Receptores Toll-Like , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
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.
Asunto(s)
Antígenos CD18/metabolismo , Lipopolisacáridos/farmacología , Transducción de Señal/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Antígenos CD18/genética , Células CHO , Cricetinae , Humanos , Lipopolisacáridos/metabolismo , Datos de Secuencia Molecular , MutaciónRESUMEN
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.
Asunto(s)
Receptores de Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Proteínas de la Membrana/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Péptidos Catiónicos Antimicrobianos , Secuencia de Bases , Actividad Bactericida de la Sangre , Proteínas Sanguíneas/metabolismo , Antígenos CD11/metabolismo , Células CHO , Cricetinae , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Bacterias Gramnegativas/metabolismo , Humanos , Datos de Secuencia MolecularRESUMEN
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.
Asunto(s)
Endotoxinas/metabolismo , Receptores de Lipopolisacáridos/fisiología , Lipopolisacáridos/inmunología , Mycobacterium/inmunología , Receptores Inmunológicos/fisiología , Transducción de Señal/inmunología , Animales , Astrocitoma , Células CHO , Cricetinae , Fibrosarcoma , Humanos , Interleucina-1/biosíntesis , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , ARN Mensajero/biosíntesis , Células Tumorales CultivadasRESUMEN
Exaggerated responses by phagocytes to bacterial endotoxin [lipopolysaccharide (LPS)] may result in the sepsis syndrome. While a number of LPS-binding proteins have been identified on immune cells, only CD14 has been definitively shown to be involved in signal transduction in response to LPS. The beta2 leukocyte integrins are a family of transmembrane receptors whose expression is restricted to leukocytes. Among their many functions, the beta2 integrins are phagocytic receptors that bind a variety of bacterial products, including LPS. We hypothesize that this binding results in signal transduction. Chinese hamster ovary (CHO) fibroblast cell lines expressing the CD11a/CD18 or CD11b/CD18 antigen were engineered by gene transfection. The cell lines were stimulated with LPS. LPS-induced nuclear translocation of nuclear factor kappa B (NF-kappaB) was analyzed by electrophoretic mobility shift assay. Heterologous expression of CD11a/CD18 and CD11b/CD18 in otherwise LPS-nonresponsive fibroblasts imparted the ability to respond to LPS. Responses to LPS were observed at levels of LPS of 100 ng/ml, as were responses to whole Gram-negative bacteria. The CD11/CD18 leukocyte integrins mediate cellular responses to the LPS component of Gram-negative bacteria. CD11/CD18-mediated responses of cells to LPS are likely to affect the phagocytosis, intracellular trafficking, and killing of invading bacteria as well as to help mediate cytokine responses during endotoxemia. The development of novel therapies to prevent the end-organ damage frequently observed during sepsis will require an understanding of these complex cellular events.
Asunto(s)
Antígenos CD11/fisiología , Antígenos CD18/fisiología , Receptores Inmunológicos/fisiología , Transducción de Señal , Animales , Antígenos CD11/genética , Antígenos CD18/genética , Células CHO , Cricetinae , Expresión Génica , Cinética , Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , FN-kappa B/metabolismo , Proteínas Recombinantes , TransfecciónRESUMEN
Gram-negative bacterial LPS is a potent activator of inflammatory responses. The binding of LPS to CD14 initiates signal transduction; however, the molecular processes immediately following this event remain unclear. We engineered an LPS-inducible fibroblast reporter cell line to facilitate the use of molecular genetic techniques to study the LPS signaling pathway. A plasmid containing the human Tac Ag cDNA under transcriptional control of the human E selectin promoter was cotransfected into Chinese hamster ovary (CHO)-K1 cells together with a CD14 expression plasmid. A cell line was obtained, 3E10, which upregulated expression of Tac following stimulation with LPS. Pools of mutagenized cells were exposed to LPS and then labeled with anti-Tac mAb. Cells that failed to up-regulate Tac expression were enriched by flow cytometry. Thirty clonal mutant cell lines were identified that continued to express CD14 and bind LPS, but failed to express Tac or translocate nuclear factor-kappaB (NF-kappaB) following LPS exposure. TNF-alpha-treated mutant cells continued to express Tac and translocate NF-kappaB. An analysis of LPS-induced NF-kappaB activity in heterokaryons derived from polyethylene glycol-fused cell lines indicated that recessive mutations in genes encoding components of the LPS signaling pathway accounted for the signaling defects. To date, two complementation groups have been identified from 11 cell lines analyzed. These data demonstrate that the TNF-alpha signaling pathway diverges from the LPS pathway early in the signal-transduction cascade despite similarities in LPS- and TNF-alpha-induced responses. Identification of the genes affected in these mutant reporter cells should identify heretofore-elusive components of the LPS signaling cascade.
Asunto(s)
Proteínas de Fase Aguda , Endotoxinas/genética , Genes Reporteros/inmunología , Lipopolisacáridos/inmunología , Glicoproteínas de Membrana , Mutagénesis/inmunología , Transducción de Señal/genética , Factor de Necrosis Tumoral alfa/genética , Animales , Transporte Biológico/inmunología , Northern Blotting , Células CHO , Proteínas Portadoras/fisiología , Técnicas de Cultivo de Célula/métodos , Fusión Celular/genética , Fusión Celular/inmunología , Línea Celular , Separación Celular , Cricetinae , Endotoxinas/deficiencia , Citometría de Flujo , Prueba de Complementación Genética , Humanos , Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , FN-kappa B/metabolismo , Fenotipo , Receptores de Interleucina-2/biosíntesis , Receptores de Interleucina-2/efectos de los fármacos , Receptores de Interleucina-2/genética , Transducción de Señal/inmunología , Transfección/inmunología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Invasive infection with Gram-positive and Gram-negative bacteria often results in septic shock and death. The basis for the earliest steps in innate immune response to Gram-positive bacterial infection is poorly understood. The LPS component of the Gram-negative bacterial cell wall appears to activate cells via CD14 and Toll-like receptor (TLR) 2 and TLR4. We hypothesized that Gram-positive bacteria might also be recognized by TLRs. Heterologous expression of human TLR2, but not TLR4, in fibroblasts conferred responsiveness to Staphylococcus aureus and Streptococcus pneumoniae as evidenced by inducible translocation of NF-kappaB. CD14 coexpression synergistically enhanced TLR2-mediated activation. To determine which components of Gram-positive cell walls activate Toll proteins, we tested a soluble preparation of peptidoglycan prepared from S. aureus. Soluble peptidoglycan substituted for whole organisms. These data suggest that the similarity of clinical response to invasive infection by Gram-positive and Gram-negative bacteria is due to bacterial recognition via similar TLRs.
Asunto(s)
Proteínas de Drosophila , Bacterias Grampositivas/inmunología , Glicoproteínas de Membrana/inmunología , Receptores de Superficie Celular/inmunología , Animales , Proteínas Bacterianas , Células CHO , Pared Celular/inmunología , Pared Celular/metabolismo , Cricetinae , Drosophila melanogaster/inmunología , Bacterias Grampositivas/patogenicidad , Infecciones por Bacterias Grampositivas/inmunología , Humanos , Inmunidad Innata , Lipopolisacáridos/inmunología , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , FN-kappa B/genética , Peptidoglicano/inmunología , Peptidoglicano/aislamiento & purificación , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/genética , Proteínas Recombinantes de Fusión/biosíntesis , Staphylococcus aureus/inmunología , Streptococcus pneumoniae/inmunología , Estreptolisinas/metabolismo , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-Like , Transfección/inmunologíaRESUMEN
Previously, we demonstrated that monoclonal antibodies (MAb) directed against any of the three defined complement receptors (CR) for the third component of complement (CR1, CR3, and CR4) profoundly inhibited the binding of serum-opsonized Cryptococcus neoformans to monocyte-derived macrophages. These studies suggested either that a synergistic interaction between multiple CR was required for optimal binding of C. neoformans or that the MAb were exerting nonspecific effects (such as receptor coassociation). In the present studies, we took a novel approach to dissecting out the contributions of individual receptors to binding of a microbial pathogen. Chinese hamster ovary (CHO) cells stably transfected with human CR1, CR3, or CR4 were challenged with serum-opsonized C. neoformans. We found that CHO cells transfected with any of the three receptors bound C. neoformans, with the avidity of binding to CR3 being the greatest followed in decreasing order by CR1 and CR4. Following binding of C. neoformans to transfected CHO cells, most organisms remained surface attached only, although for each receptor a significant percentage (18.5 to 27.3%) of C. neoformans was internalized. Both C. neoformans and sheep erythrocytes that were selectively opsonized with the fragments of the third component of complement, C3b and iC3b, were bound preferentially by CHO cells transfected with CR1 and CR3, respectively. These data establish CR1, CR3, and CR4 as receptors independently capable of binding C. neoformans opsonized with fragments of C3. Moreover, our study demonstrates the usefulness of transfected cell lines as a powerful tool for identifying the contribution of individual receptors to the binding of a microbial pathogen.
Asunto(s)
Cryptococcus neoformans/inmunología , Receptores de Complemento/fisiología , Animales , Anticuerpos Monoclonales/inmunología , Células CHO , Cricetinae , Humanos , Ratones , Fagocitosis , Receptores de Complemento/genética , TransfecciónRESUMEN
Chlamydia trachomatis is a major etiologic agent of sexually transmitted diseases. Although C. trachomatis is a gram-negative pathogen, chlamydial infections are not generally thought of as endotoxin-mediated diseases. A molecular characterization of the acute immune response to chlamydia, especially with regard to the role of its lipopolysaccharide (LPS), remains to be undertaken. We extracted 15 mg of LPS from 5 x 10(12) C. trachomatis elementary bodies (EB) for analysis of structure and biological activity. When methylated lipid A was subjected to high-pressure liquid chromatography followed by mass spectrometry, the majority of the lipid A was found to be pentaacyl. The endotoxin activities of whole C. trachomatis EB and purified LPS were characterized in comparison with whole Salmonella minnesota R595 and with S. minnesota R595 LPS and lipooligosaccharide from Neisseria gonorrhoeae. Both C. trachomatis LPS and whole EB induced the release of tumor necrosis factor alpha from whole blood ex vivo, and C. trachomatis LPS was capable of inducing the translocation of nuclear factor kappa B in a Chinese hamster ovary fibroblast cell line transfected with the LPS receptor CD14. In both assays, however, C. trachomatis was approximately 100-fold less potent than S. minnesota and N. gonorrhoeae. The observation that C. trachomatis is a weak inducer of the inflammatory cytokine response correlates with the clinical observation that, unlike N. gonorrhoeae infection, genital tract infection with C. trachomatis is often asymptomatic. The ability of specific LPS antagonists to completely inhibit the tumor necrosis factor alpha-inducing activity of whole C. trachomatis EB suggests that the inflammatory cytokine response to chlamydia infection may be mediated primarily through LPS. This implies that the role of other surface protein antigens, at least in terms of eliciting the proinflammatory cytokine response, is likely to be minor.
Asunto(s)
Antígenos CD/fisiología , Antígenos de Diferenciación Mielomonocítica/fisiología , Infecciones por Chlamydia/inmunología , Chlamydia trachomatis/inmunología , Endotoxinas/inmunología , Lipopolisacáridos/inmunología , Chlamydia trachomatis/química , Humanos , Técnicas In Vitro , Lípido A/química , Lípido A/inmunología , Receptores de Lipopolisacáridos , Lipopolisacáridos/química , Activación de Macrófagos , Macrófagos/inmunología , FN-kappa B/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
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.
Asunto(s)
Receptores de Lipopolisacáridos/inmunología , Lipopolisacáridos/inmunología , Antígeno de Macrófago-1/inmunología , FN-kappa B/inmunología , Streptococcus agalactiae/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Células CHO , Pared Celular/inmunología , Cricetinae , Humanos , Lipopolisacáridos/farmacología , Ratones , Transducción de Señal/inmunologíaRESUMEN
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.
Asunto(s)
Escherichia coli/inmunología , Eliminación de Gen , Receptores de Lipopolisacáridos/genética , Lipopolisacáridos/inmunología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/microbiología , Animales , Adhesión Bacteriana/genética , Adhesión Bacteriana/inmunología , Proteínas de la Membrana Bacteriana Externa/fisiología , Antígenos CD18/fisiología , Línea Celular , Citocinas/biosíntesis , Escherichia coli/fisiología , Femenino , Receptores de Lipopolisacáridos/sangre , Activación de Macrófagos/genética , Antígeno de Macrófago-1/fisiología , Macrófagos Peritoneales/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutagénesis Insercional , Fagocitosis/inmunología , Transducción de Señal/inmunologíaRESUMEN
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.
Asunto(s)
Proteínas de Fase Aguda , Alginatos/farmacología , Antígenos CD18/fisiología , Receptores de Lipopolisacáridos/fisiología , Lipopolisacáridos/farmacología , Glicoproteínas de Membrana , Animales , Células CHO , Proteínas Portadoras/fisiología , Cricetinae , Ácido Glucurónico , Ácidos Hexurónicos , Antígeno de Macrófago-1/fisiología , Monocitos/efectos de los fármacos , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
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.
Asunto(s)
Proteínas de Fase Aguda , Antígenos CD11/fisiología , Antígenos CD18/fisiología , Lípido A/fisiología , Receptores de Lipopolisacáridos/fisiología , Glicoproteínas de Membrana , Transducción de Señal/inmunología , Animales , Antígenos CD11/genética , Células CHO , Proteínas Portadoras/fisiología , Línea Celular , Cricetinae , Bacterias Gramnegativas/inmunología , Humanos , Lípido A/análogos & derivados , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/química , Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Activación de Macrófagos/efectos de los fármacos , Ratones , Mycobacterium/inmunología , Peptidoglicano/farmacología , Transfección/efectos de los fármacos , Transfección/inmunología , Células Tumorales CultivadasRESUMEN
Toll-like receptors (TLRs) 2 and 4 are signal transducers for lipopolysaccharide, the major proinflammatory constituent in the outer membrane of Gram-negative bacteria. We observed that membrane lipoproteins/lipopeptides from Borrelia burgdorferi, Treponema pallidum, and Mycoplasma fermentans activated cells heterologously expressing TLR2 but not those expressing TLR1 or TLR4. These TLR2-expressing cells were also stimulated by living motile B. burgdorferi, suggesting that TLR2 recognition of lipoproteins is relevant to natural Borrelia infection. Importantly, a TLR2 antibody inhibited bacterial lipoprotein/lipopeptide-induced tumor necrosis factor release from human peripheral blood mononuclear cells, and TLR2-null Chinese hamster macrophages were insensitive to lipoprotein/lipopeptide challenge. The data suggest a role for the native protein in cellular activation by these ligands. In addition, TLR2-dependent responses were seen using whole Mycobacterium avium and Staphylococcus aureus, demonstrating that this receptor can function as a signal transducer for a wide spectrum of bacterial products. We conclude that diverse pathogens activate cells through TLR2 and propose that this molecule is a central pattern recognition receptor in host immune responses to microbial invasion.