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1.
J Endotoxin Res ; 7(6): 447-50, 2001.
Artículo en Inglés | MEDLINE | ID: mdl-11753215

RESUMEN

In mice, defense against an intraperitoneal Salmonella infection depends on a vigorous innate immune response. Mutations which lead to an inadequate early response to the pathogen thus identify genes involved in innate immunity. The best studied host resistance factor, NRAMP-1, is an endosomal membrane protein whose loss leads to an inability of the animals to hold the infection in check. However, innate defense against Salmonella is not restricted to mechanisms which directly attack the pathogen within macrophages. Here we have examined the contribution of the LBP, CD14 and TLR4 gene products to innate defense against Salmonella. To this end, we have generated mice which carry a wild-type allele of NRAMP-1, but which are deficient for the LBP, CD14 or TLR4 genes. Loss of any of these genes leads to a susceptibility to Salmonella as dramatic as that seen in animals lacking functional NRAMP-1 protein. This indicates that LBP, CD14 and TLR4 are all critical elements required in the proper induction of this innate defense system.


Asunto(s)
Proteínas Portadoras/fisiología , Proteínas de Drosophila , Receptores de Lipopolisacáridos/fisiología , Glicoproteínas de Membrana/fisiología , Peritoneo/microbiología , Receptores de Superficie Celular/fisiología , Salmonelosis Animal/inmunología , Proteínas de Fase Aguda/fisiología , Alelos , Animales , Disparidad de Par Base , Secuencia de Bases , Proteínas Portadoras/genética , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Receptores de Lipopolisacáridos/genética , Receptores de Lipopolisacáridos/inmunología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Datos de Secuencia Molecular , Mutación , Peritoneo/patología , Peritonitis/genética , Peritonitis/inmunología , Peritonitis/patología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Salmonelosis Animal/genética , Salmonelosis Animal/patología , Salmonella typhimurium/patogenicidad , Receptor Toll-Like 4 , Receptores Toll-Like
2.
J Immunol Methods ; 204(1): 99-102, 1997 May 12.
Artículo en Inglés | MEDLINE | ID: mdl-9202714

RESUMEN

Chinese hamster ovary (CHO) cells transfected with various genes are widely used as adherent cell monolayers to produce recombinant proteins. In this report we present a new culture technique for CHO cells transfected with the vector pPOL-DHFR-CD14 using a minifermenter (miniPERM, Heraeus) for the production of recombinant human endotoxin receptor CD14 (rCD14). The transfectants were cultured for 12-17 days under serum-free conditions and formed spheroids. From this system we harvested supernatants containing up to 3.1 mg/ml recombinant CD14 (rCD14). This represents a 200-fold increase of rCD14 yield compared to conventional adherent CHO cell culture.


Asunto(s)
Células CHO , Clonación Molecular/métodos , Animales , Células CHO/metabolismo , Técnicas de Cultivo de Célula , Cricetinae , Humanos , Receptores de Lipopolisacáridos/biosíntesis , Receptores de Lipopolisacáridos/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Transfección
3.
Arch Virol ; 150(2): 247-59, 2005 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-15480855

RESUMEN

Hepatitis B surface antigen, when produced in yeast (rHBsAg), is capable of binding to cells that express the lipopolysaccharide coreceptor CD14. This interaction is enhanced by a serum protein, the lipopolysaccharide binding protein (LBP). Here we report that most of the rHBsAg particles that attached to monocytes at 0 degrees C, were not endocytosed but were released back into the serum-containing binding buffer at 37 degrees C. Additionally, serum-dependent binding at 37 degrees C was weak when compared to the serum-dependent attachment at 0 degrees C. Pre-incubation at 37 degrees C of cells together with serum did not abolish binding of freshly added rHBsAg at 0 degrees C. However, pre-incubation of rHBsAg with serum at 37 degrees C reduced attachment to cells following incubation at 0 degrees C. Soluble CD14 and LBP, two serum proteins which can act as phospholipid transfer molecules, were shown not to be responsible for the inhibitory effect. Pre-incubation at 37 degrees C of rHBsAg in serum-free hepatoma cell line-conditioned media resulted in a pronounced reduction in subsequent binding to cells at 0 degrees C. These observations suggest that the temperature-dependent inhibitory effect is caused by serum factors that are probably secreted by hepatocytes.


Asunto(s)
Antígenos de Superficie de la Hepatitis B/inmunología , Monocitos/inmunología , Suero/inmunología , Temperatura , Proteínas de Fase Aguda , Animales , Células CHO , Proteínas Portadoras , Línea Celular Tumoral , Cricetinae , Medios de Cultivo Condicionados , Antígenos de Superficie de la Hepatitis B/biosíntesis , Antígenos de Superficie de la Hepatitis B/genética , Humanos , Receptores de Lipopolisacáridos , Glicoproteínas de Membrana , Ratones , Unión Proteica , Proteínas Recombinantes/biosíntesis , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factores de Tiempo
4.
Biomed Biochim Acta ; 49(12): 1237-41, 1990.
Artículo en Inglés | MEDLINE | ID: mdl-2097995

RESUMEN

Complement component 3 (C3) phenotype and allele frequencies were defined in 312 patients with type-1 diabetes (insulin-dependent diabetes mellitus), 256 patients with type-2 diabetes (non-insulin-dependent diabetes mellitus), 114 apparently non-diabetic first-degree relatives of type-1 diabetics, in 10 families (29 members) with a familial history of type-1 or type-2 diabetes, in 181 patients with coronary heart disease and 255 subjects with arterial hypertension. 512 blood donors served as controls. All persons investigated were Europeans. There is no evidence that genes linked to C3 influence susceptibility to type-1 and type-2 diabetes and to their late complications as well as to atherosclerosis and essential hypertension. The distribution of apolipoprotein E phenotypes in patients and controls was likewise not significantly different. The combined evaluation of data from linked genes (C3 and apo E) could not improve the results. Deductions of C3 as a genetic disease marker have to be interpreted with caution.


Asunto(s)
Complemento C3/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 2/genética , Alelos , Apolipoproteínas E/genética , Arteriosclerosis/etiología , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 2/complicaciones , Humanos , Hipertensión/etiología , Fenotipo
5.
Exp Clin Endocrinol ; 92(3): 287-96, 1988 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-3253105

RESUMEN

Complement factor 3 (C3) phenotype and allele frequencies were defined in 312 patients with Type 1 diabetes (IDDM), 256 patients with Type 2 diabetes mellitus (NIDDM), 114 apparently healthy first-degree relatives of Type 1 diabetics, in 10 families (29 members) with a familial history of Type 1 or Type 2 diabetes, and 512 controls (blood donors). All persons investigated were Europeans. There is no evidence to suggest that genes linked to C3 influence susceptibility to Type 1 and Type 2 diabetes and to their late complications. C3 levels in blood plasma were found to be slightly elevated in both types of diabetes. But the C3 concentrations varied considerably within the groups. C3 split products were demonstrable in a high percentage in the blood plasma of freshly manifested Type 1 diabetic persons as well as in Type 1 diabetics with a duration of the disease of 1 to 3 years. C3 proteolysis could also be found in plasma of Type 2 diabetics (26%).


Asunto(s)
Complemento C3/genética , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 2/inmunología , Adolescente , Adulto , Anciano , Alelos , Niño , Complemento C3/análisis , Electroforesis en Gel de Agar , Femenino , Frecuencia de los Genes , Humanos , Masculino , Linaje , Fenotipo , Factores de Tiempo
6.
Eur J Immunol ; 25(5): 1436-41, 1995 May.
Artículo en Inglés | MEDLINE | ID: mdl-7539760

RESUMEN

Tissue macrophages and their precursors-the blood monocytes-respond rapidly to a bacterial infection with the release of inflammatory mediators. These mediators are involved in the recruitment of phagocytic cells, principally neutrophils, from the blood to the site of infection. To initiate this process macrophages and monocytes must be able to detect the presence of bacteria in a reliable, but nevertheless nonspecific, fashion. It is thought that this is achieved by means of receptors on the cell surface which recognize structures common to many different bacteria. One candidate for such a "pattern recognition element" is the cell surface glycoprotein CD14. CD14 has been shown to bind components of the Gram-positive cell wall and it also binds soluble lipopolysaccharide released from Gram-negative bacteria. In both cases the interaction with CD14 leads to an activation of the cell. Here we show that human peripheral blood monocytes can, in addition, bind intact Gram-negative bacteria in the presence of serum and this process involves CD14. When CD14 expression is induced on the myelomonocytic cell line U937 by treatment with vitamin D3 the cells concomittently acquire the capacity to bind bacteria. Furthermore, a non-monocytic cell line which does not bind bacteria acquires the capacity to do so when transfected with either the human or mouse CD14 gene. This binding can be inhibited by blocking the CD14 receptor with anti-CD14 antibody or by blocking the ligand on the bacteria with soluble CD14. Finally we demonstrate binding of sCD14 to Escherichia coli. We conclude that in the presence of serum both membrane-bound and soluble forms of CD14 can bind to Gram-negative bacteria. This suggests that CD14 may play a role in the detection and elimination of intact bacteria in vivo.


Asunto(s)
Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Bacterias Gramnegativas/metabolismo , Glicoproteínas de Membrana/metabolismo , Animales , Antígenos CD/química , Antígenos de Diferenciación Mielomonocítica/química , Secuencia de Bases , Células CHO , Pared Celular/metabolismo , Colecalciferol , Cricetinae , Escherichia coli/metabolismo , Humanos , Receptores de Lipopolisacáridos , Lipopolisacáridos/metabolismo , Datos de Secuencia Molecular , Monocitos/efectos de los fármacos , Monocitos/inmunología , Proteínas Recombinantes/metabolismo , Solubilidad , Transfección , Células Tumorales Cultivadas
7.
Infect Immun ; 67(6): 2964-8, 1999 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-10338506

RESUMEN

The CD14 molecule expressed on monocytes and macrophages is a high-affinity receptor for bacterial lipopolysaccharide (LPS) and hence an important component of the innate immune system. LPS binding protein (LBP) is required to facilitate the binding of LPS to CD14 in vitro and is necessary for the induction of an inflammatory response to LPS in vivo. Here we show that CD14 and LBP can also bind to lipoteichoic acid from the gram-positive bacterium Bacillus subtilis. Although CD14 does not interact with intact B. subtilis organisms, a brief exposure of the bacteria to serum converts them into a form which can bind to CD14 in an LBP-dependent reaction. When serum-pretreated B. subtilis organisms are incubated with the myelomonocytic cell line U937, which expresses CD14, the bacteria are rapidly phagocytosed. The phagocytosis is strictly dependent both on LBP and on CD14. These in vitro results suggest that LBP plays a role in the innate response not only to gram-negative but also to gram-positive infections.


Asunto(s)
Proteínas de Fase Aguda , Bacillus subtilis/inmunología , Proteínas Portadoras/inmunología , Receptores de Lipopolisacáridos/inmunología , Lipopolisacáridos/inmunología , Glicoproteínas de Membrana , Animales , Células CHO , Pared Celular , Cricetinae , Humanos , Fagocitosis , Ácidos Teicoicos/inmunología , Células U937
8.
Eur J Clin Invest ; 28(3): 205-13, 1998 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-9568466

RESUMEN

BACKGROUND: About 50% of septic shock cases are attributed to Gram-negative bacteria or their cell wall compound lipopolysaccharide (LPS, endotoxin). An attractive therapeutic strategy could target the binding of LPS to its cellular receptors. In vitro the soluble form of the endotoxin receptor CD14 (sCD14) competitively prevents binding of LPS to membrane-bound CD14 and inhibits LPS-stimulated macrophage responses. METHODS: We tested the in vivo endotoxin-neutralizing capacity of human recombinant sCD14 using a mouse model of shock induced by 8 micrograms g-1 of LPS from Salmonella abortus equi. RESULTS: In this model, treatment with sCD14 reduced mortality if administered before or simultaneously with LPS. However, application of sCD14 had no effect on the secretion of early proinflammatory cytokines and did not protect the animals against the development of apparent shock symptoms and liver injury. sCD14 also failed to prevent LPS-inducible (7.5 ng g-1) liver injury in galactosamine-sensitized mice. CONCLUSION: In line with these findings, sCD14 did not block LPS-induced activation of Kupffer cells in vitro, which might explain why the compound only partially protected in vivo.


Asunto(s)
Receptores de Lipopolisacáridos/farmacología , Lipopolisacáridos/toxicidad , Choque Séptico/prevención & control , Animales , Células CHO , Cricetinae , Modelos Animales de Enfermedad , Femenino , Humanos , Técnicas In Vitro , Interferón gamma/sangre , Interleucina-6/sangre , Macrófagos del Hígado/efectos de los fármacos , Macrófagos del Hígado/inmunología , Lipopolisacáridos/administración & dosificación , Lipopolisacáridos/antagonistas & inhibidores , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes/farmacología , Choque Séptico/etiología , Solubilidad , Factor de Necrosis Tumoral alfa/metabolismo
9.
Eur J Immunol ; 23(9): 2053-8, 1993 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-7690321

RESUMEN

The CD14+/CD16+ cells account for about 10% of all blood monocytes. They are characterized by a low level expression of the CD14 molecule and a high level expression of the CD16 (Fc gamma R III) molecule. Polymerase chain reaction analysis of mRNA prevalence in CD14+/CD16+ cells (compared to the regular CD14++ blood monocytes) demonstrates low levels of CD14 transcripts and high levels of CD16 transcripts, suggestive of a transcriptional control for both of these proteins. Analysis of additional cell surface molecules in three-color immunofluorescence reveals that CD14+/CD16+ cells express the Fc gamma R II in all, and Fc gamma R I and ICAM-1 in some donors. Furthermore, class II antigens are expressed at fourfold higher levels, while both, CD11b and CD33 cell surface proteins, are decreased by a factor of two. Transcript levels were reduced in CD14+/CD16+ cells for all three cell surface molecules. Since these phenotypic markers of the CD14+/CD16+ blood monocytes are reminiscent of tissue macrophages, we performed a comparative analysis with alveolar macrophages (AM). These cells are similar to the CD14+/CD16+ monocytes in that they show low levels of CD14 and strong expression of CD16. Furthermore, similar to the CD14+/CD16+ cells, the AM also exhibit higher levels of class II and lower levels of CD11b and CD33 when compared to the regular CD14++ blood monocytes. In vitro induction of maturation of blood monocytes by 5 day culture of peripheral blood mononuclear cells in 10% human serum will result in decreased CD14 and increased CD16 cell surface expression on the monocyte derived macrophages. At the same time, these cells acquire increased levels of class II and decreased levels of CD11b and CD33. Taken together, these data show that CD14+/CD16+ monocytes, while still in circulation, have acquired features in common with mature tissue macrophages.


Asunto(s)
Antígenos CD/análisis , Antígenos de Diferenciación Mielomonocítica/análisis , Macrófagos/inmunología , Monocitos/inmunología , Receptores de IgG/análisis , Secuencia de Bases , Células Cultivadas , Humanos , Receptores de Lipopolisacáridos , Datos de Secuencia Molecular , Lectina 3 Similar a Ig de Unión al Ácido Siálico
10.
Eur J Biochem ; 243(1-2): 100-9, 1997 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-9030727

RESUMEN

As a key receptor for lipopolysaccharide (LPS) on the surface of monocytes and macrophages, the CD14 molecule is primarily involved in non-specific host defense mechanisms against gram-negative bacteria. To delineate the structural basis of LPS binding, 23 mutants in the N-terminal 152 amino acids of human CD14 were generated and stably transfected into CHO cells. In each mutant, a block of five amino acids was substituted by alanine. Reactivity of the mutants with anti-CD14 mAbs, and their ability to interact with LPS and Escherichia coli were tested. 4 of 21 expressed CD14 mutants, ([Ala9-Ala13]CD14, [Ala39-Ala41, Ala43, Ala44]CD14, [Ala51-Ala55]CD14 and [Ala57, Ala59, Ala61-Ala63]CD14), are not recognized by anti-CD14 mAbs that interfere with the binding of LPS to human monocytes. However, only [Ala39-Ala41, Ala43, Ala44]CD14 is unable to react with fluorescein-isothiocyanate-labeled LPS or with FITC-labeled E. coli (055:B5). In addition, [Ala39-Ala4l, Ala43, Ala44]CD14 does not mediate LPS (E. coli 055:B5; 10 ng/ml)-induced translocation of nuclear factor kappaB in CHO-cell transfectants. The results indicate that the region between amino acids 39 and 44 forms an essential part of the LPS-binding site of human CD14.


Asunto(s)
Receptores de Lipopolisacáridos/química , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Sitios de Unión , Células CHO , Cricetinae , Mapeo Epitopo , Escherichia coli/metabolismo , Humanos , Receptores de Lipopolisacáridos/metabolismo , Lipopolisacáridos/metabolismo , Datos de Secuencia Molecular , FN-kappa B/metabolismo , Relación Estructura-Actividad
11.
Infect Immun ; 63(3): 1020-6, 1995 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-7532623

RESUMEN

During infection or inflammation, cells of the blood vessel wall, such as endothelial cells (EC) and smooth muscle cells (SMC), contribute to the regulation of the immune response by production of cytokines or expression of adhesion molecules. Little is known about the mechanism(s) involved in the stimulation of vascular cells by endotoxin (lipopolysaccharide [LPS]). As reported previously, LPS antagonists reduce LPS-induced cytokine production or adhesion in vitro specifically, suggesting a specific LPS recognition mechanism. We thus investigated the role of CD14 for stimulation of vascular SMC by LPS. Complement-fixing antibodies directed against CD14 (LeuM3, RoMo I, or Mo2) lysed monocytes but failed to mediate lysis of EC or SMC, indicating the lack of endogenous membrane CD14 in vascular cells. In addition, we did not detect expression of CD14 protein on EC and SMC in cell sorting analysis or cell immunoassay experiments. These observations are in line with our finding that a CD14 probe did not hybridize with mRNA or EC or SMC in Northern (RNA) blot experiments, although it hybridized well with monocyte-derived mRNA. We obtained the same results with the much more sensitive reverse transcription-PCR. Since the vascular SMC did not express endogenous CD14, we investigated the role of human serum-derived soluble CD14 (sCD14) for activation of SMC by LPS. In medium containing human serum, anti-CD14 antibodies inhibited activation of SMC by LPS. In contrast, the same antibodies did not inhibit activation of cells cultured in medium containing fetal calf serum. SMC cultured in sCD14-depleted medium responded 1,000-fold less to LPS than cells cultured in presence of sCD14. Reconstitution of sCD14-depleted serum or supplementation of serum-free medium with recombinant CD14 restored the capacity of the cells to respond to LPS. These results show that specific activation of vascular SMC by LPS does not involve binding to endogenous membrane CD14, but that the activation of vascular SMC by LPS is mediated to a great extent by serum-derived sCD14.


Asunto(s)
Antígenos CD/inmunología , Antígenos de Diferenciación Mielomonocítica/inmunología , Endotelio Vascular/inmunología , Músculo Liso Vascular/inmunología , Antígenos CD/biosíntesis , Antígenos CD/genética , Antígenos de Diferenciación Mielomonocítica/biosíntesis , Antígenos de Diferenciación Mielomonocítica/genética , Secuencia de Bases , Northern Blotting , Pruebas de Fijación del Complemento , Endotelio Vascular/efectos de los fármacos , Humanos , Interleucina-6/biosíntesis , Receptores de Lipopolisacáridos , Lipopolisacáridos/farmacología , Datos de Secuencia Molecular , Músculo Liso Vascular/efectos de los fármacos , ARN Mensajero/análisis , Solubilidad , Venas/citología
12.
Eur J Immunol ; 25(2): 604-10, 1995 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-7533093

RESUMEN

The receptor for lipopolysaccharide LPS (CD14) exists in a membrane-associated (mCD14) and a soluble form (sCD14). Previous studies indicate that monocytes produce sCD14 by limited proteolysis of the membrane-bound receptor. In this study we demonstrate that human monocytes also produce sCD14 by a protease-independent mechanism. To investigate the molecular nature of this second pathway we studied sCD14 formation in the monocytic cell line Mono Mac 6 (MM6) and in CD14 transfectants. Both MM6 and the CD14 transfectants constitutively produce sCD14 by a protease-independent mechanism. Structural analysis of sCD14 produced by the CD14 transfectants reconfirmed the presence of the COOH terminus predicted from the cDNA. Since glycosylphosphatidylinositol anchor attachment is associated with the removal of a hydrophobic C-terminal signal peptide, our finding demonstrates that the transfectants secrete sCD14 which escaped this posttranslational modification. Identical results obtained for sCD14 derived from peritoneal dialysis fluid of a patient with kidney dysfunction show the in vivo relevance of this pathway for sCD14 production.


Asunto(s)
Antígenos CD/biosíntesis , Antígenos de Diferenciación Mielomonocítica/biosíntesis , Monocitos/metabolismo , Receptores Inmunológicos/biosíntesis , Secuencia de Aminoácidos , Antígenos CD/química , Antígenos CD/genética , Antígenos de Diferenciación Mielomonocítica/química , Antígenos de Diferenciación Mielomonocítica/genética , Humanos , Receptores de Lipopolisacáridos , Datos de Secuencia Molecular , Acetato de Tetradecanoilforbol/farmacología , Transfección , Células Tumorales Cultivadas
13.
Infect Immun ; 66(9): 4151-7, 1998 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-9712761

RESUMEN

Legionella pneumophila, a gram-negative bacterium causing Legionnaires' disease and Pontiac fever, was shown to be highly reactive in in vitro gelation of Limulus lysate but not able to induce fever and the local Shwartzman reaction in rabbits and mice. We analyzed the capacity of purified L. pneumophila lipopolysaccharide (LPS-Lp) to induce activation of the human monocytic cell line Mono Mac 6, as revealed by secretion of proinflammatory cytokines and desensitization to subsequent LPS stimulation. We showed that despite normal reactivity of LPS-Lp in the Limulus amoebocyte lysate assay, induction of cytokine secretion in Mono Mac 6 cells and desensitization to an endotoxin challenge required LPS-Lp concentrations 1,000 times higher than for LPS of Salmonella enterica serovar Minnesota. Therefore, we examined the interaction of LPS-Lp with the LPS receptor CD14. We demonstrated that LPS-Lp did not bind to membrane-bound CD14 expressed on transfected CHO cells, nor did it react with soluble CD14. Our results suggest that the low endotoxic potential of LPS-Lp is due to a failure of interaction with the LPS receptor CD14.


Asunto(s)
Legionella pneumophila/inmunología , Receptores de Lipopolisacáridos/inmunología , Lipopolisacáridos/inmunología , Monocitos/inmunología , Animales , Células CHO , Línea Celular , Cricetinae , Citocinas/biosíntesis , Cangrejos Herradura , Humanos , Monocitos/efectos de los fármacos
14.
J Immunol ; 157(9): 4119-25, 1996 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-8892647

RESUMEN

Phagocytosis of bacteria by monocytes and neutrophil granulocytes provides an important first line of defense against bacterial infections. Opsonization of bacteria with complement and phagocytosis by neutrophils is dependent on divalent cations and does not take place in blood that has been anticoagulated with EDTA. Monocytes, however, do carry out phagocytosis even in the presence of EDTA. We show here that this divalent cation-independent phagocytosis pathway requires the presence of the LPS receptor CD14 on the cell surface. This pathway is dependent on the availability of LPS binding protein, can be blocked by anti-CD14 Abs, by an excess of soluble CD14, by excess free LPS, or by an excess of unlabeled Gram-negative bacteria. In contrast, intact Gram-positive bacteria fail to inhibit this process. These experiments define a CD14-dependent phagocytosis pathway for Gram-negative bacteria that operates in monocytes in human whole blood. This pathway may be able to deal with bacterial pathogens that have developed resistance to complement-dependent opsonization and phagocytosis by neutrophils.


Asunto(s)
Proteínas de Fase Aguda , Calcio/fisiología , Bacterias Gramnegativas , Receptores de Lipopolisacáridos/fisiología , Glicoproteínas de Membrana , Monocitos/fisiología , Neutrófilos/fisiología , Fagocitosis/fisiología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Anticoagulantes/farmacología , Proteínas Portadoras/genética , Proteínas Portadoras/farmacología , Ácido Edético/farmacología , Escherichia coli/metabolismo , Heparina/farmacología , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/farmacología , Receptores de Lipopolisacáridos/inmunología , Lipopolisacáridos/metabolismo , Linfoma de Células B Grandes Difuso/patología , Monocitos/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Proteínas Opsoninas/inmunología , Fagocitosis/efectos de los fármacos , Proteínas Recombinantes/farmacología , Células Tumorales Cultivadas
15.
Res Immunol ; 146(6): 339-50, 1995.
Artículo en Inglés | MEDLINE | ID: mdl-8719658

RESUMEN

Monocytes and macrophages express a glycosyl phosphatidylinositol (GPI)-anchored lipopolysaccharide (LPS) receptor on the cell surface which enables them to detect minute amounts of LPS released from Gram-negative bacteria. A soluble form of CD14 is also found free in serum, though its physiological function is unknown. the interaction of LPS with CD14 on the monocyte surface leads to an activation of the cells which is manifested in the sudden release of reactive oxygen species, a process referred to as an oxidative burst. In patients suffering from the condition known as paroxysmal nocturnal haemoglobinuria (PNH), the synthesis of GPI anchors is blocked in haematopoietic cells which are therefore unable to express GPI-linked proteins on their surface. In severe cases, over 90% of monocytes lack membrane-bound CD14, though normal levels of the soluble form of the receptor-sCD14-are found in the serum. Despite this lack of membrane-bound CD14, monocytes from PNH patients can respond to low concentrations of LPS. Here we show that the LPS-induced oxidative burst of these PNH monocytes requires a component present in serum. The serum-dependent activation can be inhibited by monoclonal antibodies to CD14, can be removed from the serum by passage over a matrix to which an anti-CD14 antibody has been bound, and the depleted serum can be reconstituted by the addition of either purified natural or purified recombinant soluble CD14. We conclude that an LPS-dependent oxidative burst in PNH monocytes can be mediated by soluble CD14.


Asunto(s)
Proteínas de Fase Aguda , Receptores de Lipopolisacáridos/metabolismo , Glicoproteínas de Membrana , Monocitos/inmunología , Monocitos/metabolismo , Estallido Respiratorio , Animales , Secuencia de Bases , Células CHO , Proteínas Portadoras/metabolismo , Membrana Celular/inmunología , Cricetinae , Cartilla de ADN/genética , Hemoglobinuria Paroxística/inmunología , Hemoglobinuria Paroxística/metabolismo , Humanos , Técnicas In Vitro , Receptores de Lipopolisacáridos/genética , Lipopolisacáridos/farmacología , Datos de Secuencia Molecular , Monocitos/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Solubilidad
16.
Eur J Biochem ; 236(2): 457-64, 1996 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-8612616

RESUMEN

The myeloid differentiation antigen CD14 acts as the major receptor for bacterial lipopolysaccharide (LPS). A soluble form of the protein (sCD14) is present in human serum which functions as a soluble LPS receptor. We have compared the isoform patterns of soluble CD14 derived from human serum and of the recombinant proteins produced by CHO cells transfected with either the wild-type CD14 gene or with a cDNA coding for a truncated protein which lacks the C-terminal 21 amino acids [sCD14-(1-335)-peptide]. Using SDS/PAGE, two dominant isoforms (53 and 50 kDa) and two minor forms (46 and 43 kDa) can be detected in serum as well as in the supernatants of both transfectants. sCD14 is a glycoprotein which carries N- and O-linked carbohydrates. The different isoforms of sCD14-(1-335)-peptide are due to differences in the content of N-linked sugars. However after the removal of N- and O-linked carbohydrates from serum- and CHO-derived wild-type proteins, two isoforms are still present. These results indicate that N-linked glycosylation contributes to but does not fully explain the different forms of soluble CD14. We further examined whether the mutation of individual N-linked glycosylation sites influences the expression of membrane-bound and soluble CD14 forms and the ability of the membrane-bound molecule to bind LPS. As with the wild-type proteins, the different isoforms of the soluble mutants are partially due to differences in N-linked glycosylation. A truncated mutant which lacks the two N-terminal glycosylation sites {[Asp18, Asp132]CD14-(1-335)peptide} does not give rise to multiple forms on SDS gels. Like CD14-(1-335)-peptide, this mutant is not expressed on the cell surface suggesting that a smaller isoform present in the wild-type preparations results from proteolytic cleavage of the membrane-bound molecule. N-linked carbohydrates do not seem to be important for the binding of LPS to membrane-bound CD14.


Asunto(s)
Receptores de Lipopolisacáridos/química , Animales , Secuencia de Bases , Células CHO , Compartimento Celular , Membrana Celular/metabolismo , Cricetinae , Cartilla de ADN/química , Glicosilación , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes , Solubilidad
17.
Prog Clin Biol Res ; 397: 301-13, 1998.
Artículo en Inglés | MEDLINE | ID: mdl-9575571

RESUMEN

The CD14 molecule is a key receptor on myeloid lineage cells involved in the recognition of lipopolysaccharide (LPS) and Gram-negative bacteria. The application of its soluble form, sCD14, has been shown to protect mice from lethality in LPS-induced shock. Therefore the protein or its derivatives may be considered as a possible therapeutic alternative for the treatment of patients suffering from Gram-negative septic shock. In this study we performed an alanine scan of amino acids 1 to 152 of human CD14. Twenty-three substitution mutants were generated and stably transfected into CHO-cells. In each mutant five amino acids were substituted by alanine. We analyzed (a) whether mutant proteins expressed on the surface of transfectants were recognized by a panel of anti-CD14 monoclonal antibodies (mAb's), (b) the ability of mCD14-mutants to bind LPS and E. coli in a serum- or LBP-dependent manner, and (c) the capacity of soluble mutants to mediate the LPS-induced IL 6 release of U 373 astrocytoma cells. Twenty-one CD14-mutants were expressed on the surface of transfectants and 18 were present as soluble forms in the culture supernatants. We demonstrated that only CD14(39-41,43-44)A completely lacked the ability to bind LPS and E. coli. In addition, a combined mutant CD14(9-13/57,59,61-63)A had very limited capacity to interact with LPS indicating that the LPS-binding site of human CD14 is a conformational epitope. Analysis of LPS-induced activation of CD14-negative U 373 cells revealed that the regions 9-13 and 91-101 are most important for sCD14-mediated signalling.


Asunto(s)
Proteínas de Fase Aguda , Receptores de Lipopolisacáridos/fisiología , Glicoproteínas de Membrana , Animales , Anticuerpos Monoclonales/metabolismo , Células CHO , Proteínas Portadoras/metabolismo , Cricetinae , Humanos , Receptores de Lipopolisacáridos/química , Lipopolisacáridos/metabolismo , Ratones , FN-kappa B/metabolismo , Transducción de Señal , Relación Estructura-Actividad , Tetrahidrofolato Deshidrogenasa/metabolismo
18.
J Immunol ; 163(11): 6035-44, 1999 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-10570291

RESUMEN

The soluble form of the endotoxin receptor CD14 is required for the LPS-induced activation of cells lacking membrane-bound CD14. It has been shown that a deletion mutant of human CD14 consisting of the N-terminal 152 amino acids has the capacity to mediate the stimulation of different cell types by LPS. To identify the structural domains of the molecule related to this functional property, we screened a set of alanine substitution mutants using CD14-negative U373 astrocytoma cells. We show that 3 of 18 soluble mutants of human CD14 failed to mediate the LPS-induced IL-6 production in U373 cells. These mutants were located in two regions of the molecule (aa 9-13 and 91-101) that are not essential for LPS binding. In addition, the mutants had a reduced capacity to mediate LPS-stimulated IL-6 production in human vascular endothelial and SMC. In contrast, the potential of sCD14(91-94,96)A, and sCD14(97-101)A to signal LPS-induced activation of human PBMC was not significantly reduced. These results show that the regions 9-13 and 91-101 are involved in the sCD14-dependent stimulation of cells by LPS but that the mechanisms by which different cell types are activated may not be identical.


Asunto(s)
Interleucina-6/metabolismo , Receptores de Lipopolisacáridos/metabolismo , Lipopolisacáridos/metabolismo , Alanina/genética , Astrocitos/citología , Astrocitos/metabolismo , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Receptores de Lipopolisacáridos/genética , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Mutagénesis , Unión Proteica , Estallido Respiratorio , Eliminación de Secuencia , Transducción de Señal , Solubilidad
19.
Infect Immun ; 65(3): 858-64, 1997 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-9038288

RESUMEN

Previously, we were able to show that soluble peptidoglycan (sPG)-induced monokine production in human peripheral monocytes is inhibited by anti-CD14 monoclonal antibodies and by lipid A partial structures. This suggested but did not prove that monocytic surface protein CD14 is involved in the activation of human monocytes not only by cell wall components of gram-negative bacteria such as lipopolysaccharide (LPS) but also by cell wall components of gram-positive bacteria such as sPG. In the present study, we provide experimental evidence that CD14 indeed constitutes a binding site for sPG recognition and activation of human monocytes. The results show that fluorescein isothiocyanate-sPG (FITC-sPG) binds to human monocytes in a saturable, dose-dependent, and specific manner. For maximal binding, 2 to 3 microg of FITC-sPG per ml was sufficient, and this binding is completed within 90 min; about 40% of the binding is completed within the first 3 min. The FITC-sPG binding is considered specific because unlabeled sPG and also muramyldipeptide (MDP), the minimal bioactive structure of sPG, inhibit the binding of sPG to monocytes in a dose-dependent manner. This specific binding was also inhibited by an anti-CD14 monoclonal antibody, LPS, and lipid A partial structure compound 406. Direct evidence for an interaction of sPG with CD14 is provided by experiments involving native polyacrylamide gel electrophoresis that showed a shift of the electrophoretic mobility of CD14 by LPS as well as by sPG. These results allow the conclusion that sPG binds directly to CD14, that MDP represents the active substructure of sPG, and that CD14 may be a lectin-like receptor which plays a key role in cellular stimulation by bioactive components of not only gram-negative but also gram-positive bacteria.


Asunto(s)
Acetilmuramil-Alanil-Isoglutamina/metabolismo , Receptores de Lipopolisacáridos/metabolismo , Monocitos/metabolismo , Peptidoglicano/metabolismo , Animales , Sitios de Unión , Fluoresceína-5-Isotiocianato , Humanos , Interleucina-6/biosíntesis , Lipopolisacáridos/farmacología , Ratones , Polimixina B/farmacología
20.
Eur J Immunol ; 29(1): 265-76, 1999 01.
Artículo en Inglés | MEDLINE | ID: mdl-9933108

RESUMEN

T cell activation is controlled by the coordination of stimulatory and negative regulatory signals which are not completely defined. In this study we tested for a possible direct effect of CD14 on the regulation of T cell activation and function. We show that soluble CD14 (sCD14) induces inhibition of antigen-mediated peripheral blood mononuclear cells (PBMC) proliferation and anti-CD3-mediated proliferation of CD4+CD8+, CD4+CD8+ and CD4+CD8+ Tcell clones. This effect is not due to cell death, but results from a marked inhibition of IL-2 production. Proliferation of T cell clones due to exogenous IL-2 is not affected by sCD14. We also found that sCD14 inhibits production of another Th1-like cytokine, IFN-gamma and a Th2-like cytokine, IL-4. Importantly, sCD14 induces a progressive accumulation of the inhibitory protein IkappaB-alpha. We show that sCD14 binds to activated T cells. Following cell activation, biotinylated sCD14 stains CD3+ PBMC, as well as human T cell clones with varying intensity. The binding is saturable, can be inhibited by excess of unlabeled sCD14 and, following binding, sCD14 is internalized. Collectively, these findings reveal a previously unrecognized function of sCD14, namely its capacity to negatively regulate T lymphocyte activation and function by interacting directly with activated T cells.


Asunto(s)
Proteínas I-kappa B , Receptores de Lipopolisacáridos/metabolismo , Receptores de Lipopolisacáridos/farmacología , Activación de Linfocitos , Linfocitos T/inmunología , Transporte Biológico Activo , Biotina , División Celular , Supervivencia Celular , Células Clonales , Proteínas de Unión al ADN/metabolismo , Humanos , Interferón gamma/biosíntesis , Interleucina-2/biosíntesis , Interleucina-2/farmacología , Interleucina-4/biosíntesis , Células Jurkat , Cinética , Inhibidor NF-kappaB alfa , Receptores de Interleucina-2/metabolismo , Solubilidad , Linfocitos T/citología , Linfocitos T/metabolismo
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