RESUMEN
MHCII, Tlr4, and Nramp1 genes are each independently important in pulmonary immunity. To determine the effect of these genes on host resistance, mice carrying various combinations of functional alleles for these three genes were experimentally challenged with the opportunistic bacterium, Pasteurella pneumotropica. MHCII-/-, Tlr4d/d, and Nramp1s/s mice were significantly more susceptible to experimental infections by P. pneumotropica after intranasal challenge compared to mice carrying functional alleles at only one of those genes. P. pneumotropica were cultured from the lungs of challenged mice, and the severity of the pneumonia strongly correlated with the number of isolated bacteria. Mice with the genotype MHCII-/- Tlr4n/n genotype were less susceptible to pneumonia than MHCII+/+, Tlr4d/d mice. It is interesting that the Nramp1 gene contribution to host resistance was apparent only in the absence of functional MHCII or Tlr4 genes. These data suggest that MHCII, Tlr4, and Nramp1 genes are important to pulmonary bacterial resistance.
Asunto(s)
Proteínas Portadoras/genética , Proteínas de Transporte de Catión , Proteínas de Drosophila , Genes MHC Clase II/inmunología , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/genética , Infecciones por Pasteurella/genética , Infecciones por Pasteurella/inmunología , Neumonía Bacteriana/genética , Neumonía Bacteriana/inmunología , Receptores de Superficie Celular/genética , Alelos , Animales , Proteínas Portadoras/inmunología , Cruzamientos Genéticos , Femenino , Genes MHC Clase II/genética , Predisposición Genética a la Enfermedad/genética , Inmunidad Innata/genética , Inmunidad Innata/inmunología , Pulmón/microbiología , Pulmón/patología , Masculino , Glicoproteínas de Membrana/inmunología , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Pasteurella , Receptores de Superficie Celular/inmunología , Receptor Toll-Like 4 , Receptores Toll-LikeRESUMEN
The mechanism of how superantigens function to activate cells has been linked to their ability to bind and cross-link the major histocompatibility complex class II (MHCII) molecule. Cells that lack the MHCII molecule also respond to superantigens, however, with much less efficiency. Therefore, the purpose of this study was to confirm that staphylococcal enterotoxin A (SEA) could bind the MHCI molecule and to test the hypothesis that cross-linking SEA bound to MHCII-deficient macrophages would induce a more robust cytokine response than without cross-linking. We used a capture enzyme-linked immunosorbent assay and an immunprecipitation assay to directly demonstrate that MHCI molecules bind SEA. Directly cross-linking MHCI using monoclonal antibodies or cross-linking bound SEA with an anti-SEA antibody or biotinylated SEA with avidin increased TNF-alpha and IL-6 secretion by MHCII(-/-) macrophages. The induction of a vigorous macrophage cytokine response by SEA/anti-SEA cross-linking of MHCI offers a mechanism to explain how MHCI could play an important role in superantigen-mediated pathogenesis.
Asunto(s)
Enterotoxinas/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Macrófagos Peritoneales/inmunología , Staphylococcus aureus/inmunología , Superantígenos/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Células Cultivadas , Reactivos de Enlaces Cruzados , Antígenos de Histocompatibilidad Clase I/genética , Interleucina-6/biosíntesis , Macrófagos Peritoneales/citología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
The standard method for quantitating bone marrow precursor cells has been to count the number of colony-forming units that form in semisolid (0.3%) agar. Recently we adapted this assay for use in hardware, the Fluid Processing Apparatus, that is flown in standard payload lockers of the space shuttle. When mouse or rat macrophage colony-forming units were measured with this hardware in ground-based assays, we found significantly more colony growth than that seen in standard plate assays. The improved growth correlates with increased agar thickness but also appears to be due to properties inherent to the Fluid Processing Apparatus. This paper describes an improved method for determining bone marrow macrophage precursor numbers in semisolid agar.
Asunto(s)
Células de la Médula Ósea/citología , Ensayo de Unidades Formadoras de Colonias/métodos , Células Madre Hematopoyéticas/citología , Macrófagos/citología , Animales , Recuento de Células , Diferenciación Celular , División Celular , Ensayo de Unidades Formadoras de Colonias/instrumentación , Ratones , Ratones Endogámicos C3H , Ratas , Ratas Sprague-Dawley , Vuelo EspacialRESUMEN
Sprague-Dawley rats were subjected to two 8-day spaceflights on the space shuttle. Rats housed in the National Aeronautics and Space Administration's animal enclosure were injected (iv or sc) with pegylated interleukin-2 (PEG-IL-2) or a placebo. We tested the hypothesis that PEG-IL-2 would ameliorate some of the effects of spaceflight. We measured body and organ weights; blood cell differentials; plasma corticosterone; colony-forming units (macrophage and granulocyte macrophage); lymphocyte mitogenic, superantigenic, and interferon-gamma responses; bone marrow cell and peritoneal macrophage cytokine secretion; and bone strength and mass. Few immunological parameters were affected by spaceflight. However, some spaceflight effects were observed in each flight. Specifically, peritoneal macrophage spontaneous secretion of tumor necrosis factor-alpha occurred in the first but not in the second flight. A significant monocytopenia and lymphocytopenia were detected in the second but not in the first flight. The second mission produced bone changes more consistent with past spaceflight investigations. PEG-IL-2 did not appear to be beneficial; however, this was mostly due to the lack of spaceflight effects. These studies reflect the difficulty in reproducing experimental models by using current space shuttle conditions.
Asunto(s)
Inmunidad/fisiología , Interleucina-2/análogos & derivados , Vuelo Espacial , Animales , Peso Corporal/efectos de los fármacos , Peso Corporal/fisiología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/fisiología , Ensayo de Unidades Formadoras de Colonias , Corticosterona/biosíntesis , Citocinas/biosíntesis , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Inmunidad/efectos de los fármacos , Interleucina-2/farmacología , Recuento de Leucocitos , Ganglios Linfáticos/citología , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/fisiología , Masculino , Tamaño de los Órganos/efectos de los fármacos , Tamaño de los Órganos/fisiología , Polietilenglicoles , Ratas , Ratas Sprague-Dawley , Bazo/citología , Bazo/efectos de los fármacos , Bazo/fisiologíaRESUMEN
Certain strains of Staphylococcus aureus express one or both of two related, but immunologically distinct, exfoliative toxins (ETA and ETB). These toxins induce the symptoms associated with staphylococcal scalded skin syndrome. Both ETs have been shown to stimulate T cell proliferation. Recently, it was reported that ETA is a superantigen that stimulates T cells bearing human Vbeta2 or several murine Vbetas. However, other investigators have proposed that the superantigenicity reported for ETA resulted from contaminants in commercial preparations. This present study addresses those conflicting reports by assessing the biological and immunologic activities of highly purified rETs. ETA and ETB required APCs to induce selective polyclonal expansion of several human Vbetas (huVbetas), although, neither toxin expanded huVbeta2. ETB induced expansion of murine T cells bearing Vbetas 7 and 8, those that have the highest homology to the huVbetas expanded by ETA and ETB. Although flow cytometry of ETB-stimulated T cells matched PCR results, stimulation by ETA reduced percentages of T cells positive for several huVbetas that had been shown to have increased levels of mRNA transcripts. ETA and ETB induced contrasting reactions in vivo. In rabbits, ETB was moderately pyrogenic and enhanced susceptibility to lethal shock, while ETA lacked both activities. Predictions based on comparisons with other superantigens suggest molecular regions potentially involved in receptor binding in the ETA crystal structure and a modeled ETB three-dimensional structure. These results show that ETs are superantigens with unique properties that could account for the discrepancies reported.
Asunto(s)
Exfoliatinas/inmunología , Superantígenos/inmunología , Animales , Células Cultivadas , Células Clonales , Epítopos de Linfocito T/inmunología , Exfoliatinas/química , Exfoliatinas/toxicidad , Regulación de la Expresión Génica/inmunología , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T/inmunología , Humanos , Inmunofenotipificación , Inyecciones Intravenosas , Activación de Linfocitos , Recuento de Linfocitos , Ratones , Ratones Endogámicos C3H , Modelos Moleculares , Conejos , Superantígenos/química , Superantígenos/toxicidad , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
We tested the hypothesis that insulin-like growth factor-1 (IGF-1) would ameliorate space flight-induced effects on the immune system. Twelve male, Sprague-Dawley rats, surgically implanted with mini osmotic pumps, were subjected to space flight for 10 days on STS-77. Six rats received 10 mg/kg/day of IGF-1 and 6 rats received saline. Flight animals had a lymphocytopenia and granulocytosis which were reversed by IGF-1. Flight animals had significantly higher corticosterone levels than ground controls but IGF-1 did not impact this stress hormone. Therefore, the reversed granulocytosis did not correlate with serum corticosterone. Space flight and IGF-1 also combined to induce a monocytopenia that was not evident in ground control animals treated with IGF-1 or in animals subjected to space flight but given physiological saline. There was a significant increase in spleen weights in vivarium animals treated with IGF-1, however, this change did not occur in flight animals. We observed reduced agonist-induced lymph node cell proliferation by cells from flight animals compared to ground controls. The reduced proliferation was not augmented by IGF-1 treatment. There was enhanced secretion of TNF, IL-6 and NO by flight-animal peritoneal macrophages compared to vivarium controls, however, O2(-) secretion was not affected. These data suggest that IGF-1 can ameliorate some of the effects of space flight but that space flight can also impact the normal response to IGF-1. Grant Numbers: NAGW-1197, NAGW-2328.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/farmacología , Linfocitos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Vuelo Espacial , Ingravidez , Animales , Corticosterona/metabolismo , Citocinas/metabolismo , Factor I del Crecimiento Similar a la Insulina/inmunología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/fisiología , Linfocitos/inmunología , Linfocitos/fisiología , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/inmunología , Macrófagos/fisiología , Masculino , Óxido Nítrico/metabolismo , Tamaño de los Órganos , Ratas , Ratas Sprague-Dawley , Bazo/citología , Bazo/fisiología , Timo/citología , Timo/fisiologíaRESUMEN
Previous experiments have shown that skeletal unloading resulting from exposure to microgravity induces osteopenia in rats. In maturing rats, this is primarily a function of reduced formation, rather than increased resorption. Insulin-like growth factor-I (IGF-I) stimulates bone formation by increasing collagen synthesis by osteoblasts. The ability of IGF-I to prevent osteopenia otherwise caused by spaceflight was investigated in 12 rats flown for 10 days aboard the Space Shuttle, STS-77. The effect IGF-I had on cortical bone metabolism was generally anabolic. For example, humerus periosteal bone formation increased a significant 37.6% for the spaceflight animals treated with IGF-I, whereas the ground controls increased 24.7%. This increase in humeral bone formation at the periosteum is a result of an increased percent mineralizing perimeter (%Min.Pm), rather than mineral apposition rate (MAR), for both spaceflight and ground control rats. However, IGF-I did inhibit humerus endocortical bone formation in both the spaceflight and ground control rats (38.1% and 39.2%, respectively) by limiting MAR. This effect was verified in a separate ground-based study. Similar histomorphometric results for spaceflight and ground control rats suggest that IGF-I effects occur during normal weight bearing and during spaceflight. Microhardness measurements of the newly formed bone indicate that the quality of the bone formed during IGF-I treatment or spaceflight was not adversely altered. Spaceflight did not consistently change the structural (force-deflection) properties of the femur or humerus when tested in three-point bending. IGF-I significantly increased femoral maximum and fracture strength.
Asunto(s)
Resorción Ósea/prevención & control , Húmero/fisiología , Factor I del Crecimiento Similar a la Insulina/farmacología , Vuelo Espacial , Tibia/fisiología , Animales , Fenómenos Biomecánicos , Peso Corporal/efectos de los fármacos , Peso Corporal/fisiología , Densidad Ósea/efectos de los fármacos , Densidad Ósea/fisiología , Resorción Ósea/fisiopatología , Húmero/efectos de los fármacos , Húmero/patología , Masculino , Ratas , Ratas Sprague-Dawley , Organismos Libres de Patógenos Específicos , Tibia/efectos de los fármacos , Tibia/patología , Soporte de Peso , IngravidezRESUMEN
Staphylococcal enterotoxins A and C1 (SEA or SEC1) bound to major histocompatibility-I (MHCI) molecules with high affinity (binding constants ranging from 1.1 microM to 79 nM). SEA and SEC1 directly bound MHCI molecules that had been captured by monoclonal antibodies specific for H-2Kk, H-2Dk, or both. In addition, MHCI-specific antibodies inhibited the binding of SEC1 to LM929 cells and SEA competitively inhibited SEC1 binding; indicating that the superantigens bound to MHCI on the cell surface. The affinity and number of superantigen binding sites differed depending on whether MHCI was expressed in the membrane of LM929 cells or whether it was captured. These data support the hypothesis that MHCI molecules can serve as superantigen receptors.
Asunto(s)
Antígenos Bacterianos/metabolismo , Enterotoxinas/metabolismo , Antígenos H-2/metabolismo , Superantígenos/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/inmunología , Línea Celular , Enterotoxinas/inmunología , Antígenos H-2/inmunología , Antígeno de Histocompatibilidad H-2D , Ratones , Unión ProteicaRESUMEN
Two bone-marrow-derived macrophage cell lines, C2D and C2Dt, were isolated from major histocompatibility class II negative knock-out mice. The C2D cell line was stabilized by continuous culture in colony-stimulating factor-1 and the C2Dt cell line was transformed with SV40 virus large T antigen. These cells exhibited phenotypic properties of macrophages including morphology and expression of Mac 1 and Mac 2 cell surface molecules. These cells also had comparable growth to the bone-marrow-derived macrophage cell line B6MP102. These new cell lines were not spontaneously cytotoxic and were only capable of modest killing of F5b tumor cells when stimulated with LPS and interferon-gamma, but not when stimulated with LPS alone or with staphylococcal exotoxin. C2D and C2Dt cells phagocytosed labeled Staphylococcus aureus similarly to B6MP102 cells but less well than C2D peritoneal macrophages. These cell lines secreted interleukin-6, but not tumor necrosis factor or nitric oxide in response to LPS or staphlococcal enterotoxins A or B C2D(t) cells were tumorigenic in C2D and C57BL/6J mice but C2D cells were not. These data suggest that macrophage cell lines can be established from bone marrow cells of major histocompatibility complex II-negative mice.
Asunto(s)
Antígenos de Histocompatibilidad Clase II/fisiología , Macrófagos/fisiología , Animales , Pruebas de Carcinogenicidad , Línea Celular , Línea Celular Transformada , Citocinas/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fagocitosis , Fenotipo , Superantígenos/metabolismoRESUMEN
We examined the pathogenesis of the facultative intracellular bacterium, Salmonella typhimurium in MHCII-/-, C2D knock-out mice, and wild-type C57BL/6J mice. The MHCII knock-out shortened the kinetics of animal death and reduced the dose of S. typhimurium needed to kill mice. We measured the physiological and cytokine responses of both mouse strains after S. typhimurium injection. Animal weight loss, spleen weights, liver weights, thymus weights, and serum corticosterone concentrations were comparable after injection with several doses of bacteria. The only physiological differences observed between the two strains were observed 3 days after injection of the highest dose of bacteria tested. Serum concentrations of tumor necrosis factor alpha, interleukin-2, and interleukin-6 increased in a dose-dependent fashion irrespective of mouse MHCII expression. Therefore, even in the absence of MHCII, mice are able to mount relatively normal physiological and immunological responses. Consistent with these normal responses, an increased percentage of MHCII-/- mice, primed with a low dose of bacteria 13 days earlier, were able to survive a lethal challenge of Salmonella compared with unprimed controls. Lastly, C2D mice had significantly higher serum interleukin-10 concentrations than C57BL/6J mice 48 h after infection with all doses of S. typhimurium. C2D macrophages also secreted significantly more IL-10 and less NO and O2- after lipopolysaccharide or phorbol ester stimulation in vitro than wild-type macrophages.
Asunto(s)
Antígenos de Histocompatibilidad Clase II/fisiología , Salmonelosis Animal/inmunología , Salmonella typhimurium , Animales , Femenino , Antígenos de Histocompatibilidad Clase II/biosíntesis , Antígenos de Histocompatibilidad Clase II/genética , Interleucina-10/biosíntesis , Interleucina-2/sangre , Interleucina-6/sangre , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico/metabolismo , Tamaño de los Órganos , Salmonelosis Animal/sangre , Salmonelosis Animal/genética , Superóxidos/metabolismo , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismo , Pérdida de PesoRESUMEN
PURPOSE: Keratan sulfate proteoglycans (KSPGs) of the cornea exhibit a characteristic change in glycosylation resulting from stromal inflammation and scarring. To examine potential roles for these molecules in the pathobiology of the cornea, the authors investigated interaction of inflammatory macrophages with KSPGs in vitro. METHODS: Attachment and spreading of mouse peritoneal macrophages were examined on surfaces coated with corneal proteoglycans, intact or with modified glycosylation. Solution-phase interactions were demonstrated using soluble proteoglycans labeled with 125I-Iodine or with fluorescein. The affinity and specificity of these interactions were determined by competitive inhibition with unlabeled proteoglycans. RESULTS: Macrophages did not adhere to intact corneal KSPGs but did attach and spread rapidly on the lumican core protein after the removal of keratan sulfate chains. Arterial lumican, a nonsulfated form of this proteoglycan, also stimulated macrophage attachment. Labeled arterial lumican specifically bound to macrophages with high affinity. Flow cytometry demonstrated a high proportion of macrophages binding lumican. Lumican binding was inhibited by divalent cation-chelators and by polyanions. Inhibition and kinetics of lumican binding were distinct from interaction of macrophages with maleated bovine serum albumin, collagen, laminin, and fibronectin. CONCLUSIONS: The highly sulfated KSPGs of cornea do not promote macrophage adhesion; however, the low-sulfate lumican present in pathologic corneas may act to localize macrophages in regions of inflammation. The lumican receptor differs from macrophage scavenger receptors and from receptors for several other extracellular matrix molecules.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Córnea/metabolismo , Sulfato de Queratano/metabolismo , Macrófagos Peritoneales/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Aniones/farmacología , Unión Competitiva , Bovinos , Adhesión Celular , Quelantes/farmacología , Proteoglicanos Tipo Condroitín Sulfato/antagonistas & inhibidores , Ácido Edético/farmacología , Citometría de Flujo , Fluoresceína , Fluoresceínas , Sulfato de Queratano/antagonistas & inhibidores , Lumican , Macrófagos Peritoneales/fisiología , Ratones , Ratones Endogámicos C3HRESUMEN
The pleiotropic cytokine interleukin-6 (IL-6) is produced in and secreted from anterior pituitary (AP) cells of a number of species. Bacterial endotoxin (END) may enhance the transcription of IL-6 and its secretion from the AP. In the studies presented here, we evaluated pig AP cells for the presence of IL-6 mRNA. In addition, because we had observed previously that END stimulated the secretion of prostaglandin E2 from cultured porcine AP cells, the effects of the inhibition of END-stimulated cyclooxygenase products on IL-6 mRNA abundance and the secretion of IL-6 were evaluated. In the first experiment, RNA was extracted from cultured pig AP cells that had been treated with END for 0.5 or 1 hr and subjected to reverse transcription followed by polymerase chain reaction and hybridization after Southern transfer. Bands of expected amplified product size, corresponding to IL-6, were observed only from cells treated with END, although specific hybridization was observed from both control and END-treated wells. In the next experiment, RNA was extracted from cultured AP cells treated with END or END in the presence of the cyclooxygenase inhibitor indomethacin (IND). Amplification of the expected product could be observed from all cultured cells except those treated with IND. However, hybridization data indicated that IND did not eliminate IL-6 mRNA entirely. Next, we measured IL-6 secretion from cultured AP cells exposed to END or END and IND. Treatment with END stimulated IL-6 secretion (P < 0.001) above controls, whereas IND blocked END stimulation of IL-6 secretion (P < 0.001). Finally, using immunostaining, we confirmed the presence of CD14, an END receptor, in cultured pig AP cells. These studies clearly establish the presence of IL-6 mRNA and secretion of the cytokine from cultured porcine AP cells. In addition, END stimulates the secretion of IL-6, perhaps through cells expressing CD14, and END-stimulated IL-6 secretion appears to be mediated by products of the cyclooxygenase pathway.
Asunto(s)
Endotoxinas/farmacología , Interleucina-6/genética , Interleucina-6/metabolismo , Adenohipófisis/metabolismo , ARN Mensajero/metabolismo , Porcinos , Animales , Células Cultivadas , Inhibidores de la Ciclooxigenasa/farmacología , Indometacina/farmacología , Receptores de Lipopolisacáridos/análisis , Adenohipófisis/inmunología , Reacción en Cadena de la Polimerasa , Prostaglandina-Endoperóxido Sintasas/metabolismoRESUMEN
We screened a panel of monoclonal antibodies against selected macrophage cell surface molecules for their ability to inhibit enterotoxin binding to major histocompatibility complex class II-negative C2D (H-2b) macrophages. Two monoclonal antibodies, HB36 and TIB126, that are specific for the alpha 2 domain of major histocompatibility complex class I, blocked staphylococcal enterotoxins A and B (SEA and SEB, respectively) binding to C2D macrophages in a specific and concentration-dependent manner. Inhibitory activities were haplotype-specific in that SEA and SEB binding to H-2k or H-2d macrophages was not inhibited by either monoclonal antibody. HB36, but not TIB126, inhibited enterotoxin-induced secretion of cytokines by H-2b macrophages. Lastly, passive protection of D-galactosamine-sensitized C2D mice by injection with HB36 antibody prevented SEB-induced death. Therefore, SEA and SEB binding to the alpha 2 domain of the H-2Db molecule induces biological activity and has physiological consequences.
Asunto(s)
Enterotoxinas/inmunología , Antígenos H-2/inmunología , Macrófagos/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/farmacología , Antígenos H-2/química , Antígeno de Histocompatibilidad H-2D , Antígenos de Histocompatibilidad Clase II/genética , Interleucina-6/biosíntesis , Cinética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Unión Proteica , Staphylococcus aureus , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Cluster of differentiation antigen 14 (CD14) functions as a receptor for lipopolysaccharide (LPS) LPS-binding protein (LBP) complexes. Because LPS has varying effects on CD14 expression in vitro, we evaluated CD14 expression in response to LPS with a fully differentiated macrophage phenotype, the alveolar macrophage. By using flow microfluorometric analysis and a radioimmunoassay with an anti-human CD14 monoclonal antibody (My4) that cross-reacts with porcine CD14, we found that macrophages stimulated with LPS for 24 h exhibited a two- to fivefold increase in CD14-like antigen compared with unstimulated cells. At low concentrations of LPS, up-regulation of the CD14-like antigen was dependent on serum; at higher concentrations of LPS, serum was not required. In the absence of serum a 10-fold higher dose of LPS (10 ng/ml) was required to increase CD14-like expression. In addition, LPS-induced CD14-like up-regulation correlated with secretion of tumor necrosis factor-alpha, regardless of serum concentration. Blockade with My4 antibody significantly inhibited LPS-induced tumor necrosis factor-alpha secretion at 1 ng/ml of LPS. However, inhibition decreased as we increased the LPS concentration, suggesting the existence of CD14-independent pathways of macrophage activation in response to LPS. Alternatively, My4 may have a lower affinity for the porcine CD14 antigen than LPS, which may have only partially blocked the LPS-LBP binding site at high concentrations of LPS. Therefore, these data suggest that LPS activation of porcine alveolar macrophages for 24 h increased CD14-like receptor expression. The degree of CD14-like up-regulation was related to LPS concentration, however, activation did not require the presence of serum at high concentrations of LPS.
Asunto(s)
Antígenos CD/fisiología , Antígenos de Diferenciación Mielomonocítica/fisiología , Lipopolisacáridos/farmacología , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/fisiología , Animales , Anticuerpos Monoclonales/farmacología , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Células Cultivadas , Medio de Cultivo Libre de Suero , Cinética , Receptores de Lipopolisacáridos , Macrófagos Alveolares/metabolismo , Ensayo de Unión Radioligante , Estimulación Química , Porcinos , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Antiorthostatically suspended mice had suppressed macrophage development in both unloaded and loaded bones, indicating a systemic effect. Bone marrow cells from those mice secreted less macrophage colony-stimulating factor (M-CSF) and interleukin-6 (IL-6) than did control mice. Because M-CSF and IL-6 are important to bone marrow macrophage maturation, we formulated the hypothesis that suppressed macrophage development occurred as a result of the depressed levels of either M-CSF or IL-6. To test the hypothesis, mice were administered recombinant M-CSF or IL-6 intraperitoneally. We showed that recombinant M-CSF therapy, but not recombinant IL-6 therapy, reversed the suppressive effects of antiorthostatic suspension on macrophage development. These data suggest that bone marrow cells that produce M-CSF are affected by antiorthostatic suspension and may contribute to the inhibited maturation of bone marrow macrophage progenitors.
Asunto(s)
Terapia de Inmunosupresión , Interleucina-6/farmacología , Factor Estimulante de Colonias de Macrófagos/farmacología , Postura , Soporte de Peso , Animales , Células de la Médula Ósea , División Celular , Senescencia Celular , Citocinas/metabolismo , Hematopoyesis/fisiología , Macrófagos/fisiología , Masculino , Ratones , Ratones Endogámicos C3H , Proteínas Recombinantes , Estrés Fisiológico/fisiopatología , Linfocitos T/citologíaRESUMEN
We used weak electric fields to monitor macrophage spreading in microgravity. Using this technique, we demonstrated that bone marrow-derived macrophages responded to microgravity within 8 s. We also showed that microgravity differentially altered two processes associated with bone marrow-derived macrophage development. Spaceflight enhanced cellular proliferation and inhibited differentiation. These data indicate that the space/microgravity environment significantly affects macrophages.
Asunto(s)
Médula Ósea/inmunología , Hematopoyesis , Macrófagos/fisiología , Vuelo Espacial , Ingravidez , Animales , Antígenos de Diferenciación/biosíntesis , Adhesión Celular , Diferenciación Celular , División Celular/efectos de los fármacos , Movimiento Celular , Galectina 3 , Hematopoyesis/efectos de los fármacos , Antígenos de Histocompatibilidad Clase II/biosíntesis , Interleucina-2/farmacología , Interleucina-6/metabolismo , Macrófagos/citología , Masculino , Ratones , Ratones Endogámicos C3H , TemperaturaRESUMEN
The tumor necrosis factor-alpha (TNF)-resistant, SV40-transformed, murine fibroblast cell lines, F5b and F5m, became sensitive to TNF-mediated cytolysis after treatment with a biologically active 18 kDa peptide fragment (SGP) derived from a 66-kDa parental cell surface sialoglycoprotein. Neither TNF nor the SGP alone exhibited cytotoxicity to the two SV40-transformed cell lines. However, Balb/c 3T3 cells, incubated with SGP alone or with SGP and TNF, were not killed. Therefore, SGP can selectively sensitize cells for TNF alpha-mediated cytotoxicity. This selective sensitization may be due to the previously documented ability of the SGP to selectively mediate cell cycle arrest.
Asunto(s)
Sustancias de Crecimiento/farmacología , Sialoglicoproteínas/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Células 3T3 , Animales , Línea Celular Transformada , Supervivencia Celular/efectos de los fármacos , Cicloheximida/farmacología , Ratones , Ratones Endogámicos BALB C , Fragmentos de Péptidos/farmacologíaRESUMEN
Macrophages from C2D transgenic mice deficient in the expression of major histocompatibility complex (MHC) class II proteins were used to identify binding sites for superantigens distinct from the MHC class II molecule. Iodinated staphylococcal enterotoxins A and B (SEA and SEB) and exfoliative toxins A and B (ETA and ETB) bound to C2D macrophages in a concentration-dependent and competitive manner. All four toxins increased F-actin concentration within 30 s of their addition to C2D macrophages, indicating that signal transduction occurred in response to toxin in the absence of class II MHC. Furthermore, ETA, ETB, SEA, and, to a lesser extent, SEB induced C2D macrophages to produce interleukin 6. Several molecular species on C2D macrophages with molecular masses of 140, 97, 61, 52, 43, and 37 kDa bound SEA in immunoprecipitation experiments. These data indicate the presence of novel, functionally active toxin binding sites on murine macrophages distinct from MHC class II molecules.
Asunto(s)
Enterotoxinas/metabolismo , Antígenos de Histocompatibilidad Clase II/fisiología , Activación de Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Superantígenos/metabolismo , Actinas/metabolismo , Animales , Sitios de Unión , Citocinas/metabolismo , Enterotoxinas/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Using antiorthostatic suspension, we characterized hematopoietic changes that may be responsible for the detrimental effect of skeletal unloading on macrophage development. Skeletally unloaded mice had suppressed macrophage development in unloaded and loaded bones, which indicated a systemic effect. Bone marrow cells from unloaded mice secreted less macrophage colony-stimulating factor and interleukin-6 than control mice. Additionally, T-lymphocyte proliferation was reduced after skeletal unloading. We show that polyethylene glycol-interleukin-2 therapy reversed the effects of skeletal unloading on macrophage development and cell proliferation.
Asunto(s)
Células de la Médula Ósea , Inmovilización , Terapia de Inmunosupresión , Interleucina-2/farmacología , Macrófagos/citología , Animales , Peso Corporal/efectos de los fármacos , Peso Corporal/fisiología , Médula Ósea/efectos de los fármacos , Médula Ósea/inmunología , Recuento de Células , Citocinas/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C3H , Tamaño de los Órganos/efectos de los fármacos , Tamaño de los Órganos/fisiología , Proteínas Recombinantes/farmacología , Células Madre/citología , Células Madre/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Linfocitos T/fisiologíaRESUMEN
Both spaceflight and skeletal unloading suppress the haematopoietic differentiation of macrophages (Sonnenfeld et al., Aviat. Space Environ. Med., 61:648-653, 1990; Armstrong et al., J. Appl. Physiol., 75:2734-2739, 1993). The mechanism behind this reduction in haematopoiesis has yet to be elucidated. However, changes in bone marrow extracellular matrix (ECM) may be involved. To further understand the role of ECM products in macrophage differentiation, we have performed experiments evaluating the effects of fibronectin, laminin, collagen type I, and collagen type IV on macrophage development and function. Bone marrow-derived macrophages cultured on four different ECM substrates in liquid culture medium showed less growth than those cultured on plastic. Significant morphological differences were seen on each of the substrates used. Phenotypically and functionally, as measured by class II major histocompatibility molecule (MHCII) expression, MAC-2 expression, and the secretion of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), these macrophages were similar. In contrast, bone marrow-derived macrophages cultured in suspension, using agar, showed no difference in growth when exposed to ECM proteins. However, IL-6 and TNF-alpha secretion was affected by fibronectin, laminin, collagen type I, and collagen type IV in a concentration-dependent manner. We conclude that the ECM products fibronectin, laminin, collagen type I, and collagen type IV have profound effects on macrophage development and function. Additionally, we suggest that an ECM-supplemented agar culture system provides an environment more analogous to in vivo bone marrow than does a traditional liquid culture system.