RESUMEN

Neutrophil accumulation is a hallmark of immune complex-mediated inflammatory disorders. Current models of neutrophil recruitment envision the capture of circulating neutrophils by activated endothelial cells. We now demonstrate that immobilized immune complexes alone support the rapid attachment of neutrophils, under physiologic flow conditions. Initial cell tethering requires the low-affinity Fc gamma receptor IIIB (Fc gamma RIIIB), and the beta(2) integrins are additionally required for the subsequent shear-resistant adhesion. The attachment function of Fc gamma RIIIB may be facilitated by its observed presentation on neutrophil microvilli. In vivo, in a model of acute antiglomerular basement membrane nephritis in which immune complexes are accessible to circulating neutrophils, Fc gamma RIII-deficient mice had a significant reduction in neutrophil recruitment. Thus, the interaction of immune complexes with Fc gamma RIII may mediate early neutrophil recruitment in immune complex-mediated inflammation.

Asunto(s)

Enfermedad por Anticuerpos Antimembrana Basal Glomerular/inmunología , Complejo Antígeno-Anticuerpo/inmunología , Antígenos CD/inmunología , Neutrófilos/inmunología , Receptores de IgG/inmunología , Animales , Anticuerpos/inmunología , Antígenos CD/genética , Autoanticuerpos , Membrana Basal/inmunología , Antígenos CD18/inmunología , Adhesión Celular , Proteínas Ligadas a GPI , Humanos , Células K562 , Glomérulos Renales/inmunología , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Antígeno de Macrófago-1/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microvellosidades/inmunología , Neutrófilos/fisiología , Receptores de IgG/genética

RESUMEN

Receptors for human immunoglobulin (Ig)G and IgA initiate potent cytolysis of antibody (Ab)-coated targets by polymorphonuclear leukocytes (PMNs). Mac-1 (complement receptor type 3, CD11b/CD18) has previously been implicated in receptor cooperation with Fc receptors (FcRs). The role of Mac-1 in FcR-mediated lysis of tumor cells was characterized by studying normal human PMNs, Mac-1-deficient mouse PMNs, and mouse PMNs transgenic for human FcR. All PMNs efficiently phagocytosed Ab-coated particles. However, antibody-dependent cellular cytotoxicity (ADCC) was abrogated in Mac-1(-/-) PMNs and in human PMNs blocked with anti-Mac-1 monoclonal Ab (mAb). Mac-1(-/-) PMNs were unable to spread on Ab-opsonized target cells and other Ab-coated surfaces. Confocal laser scanning and electron microscopy revealed a striking difference in immunologic synapse formation between Mac-1(-/-) and wild-type PMNs. Also, respiratory burst activity could be measured outside membrane-enclosed compartments by using Mac-1(-/-) PMNs bound to Ab-coated tumor cells, in contrast to wild-type PMNs. In summary, these data document an absolute requirement of Mac-1 for FcR-mediated PMN cytotoxicity toward tumor targets. Mac-1(-/-) PMNs exhibit defective spreading on Ab-coated targets, impaired formation of immunologic synapses, and absent tumor cytolysis.

Asunto(s)

Antígenos CD18/fisiología , Citotoxicidad Inmunológica , Antígeno de Macrófago-1/fisiología , Proteínas de Transporte de Membrana , Neutrófilos/fisiología , Receptores Fc/fisiología , Animales , Anticuerpos Monoclonales/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Neoplasias de la Mama/patología , Candida albicans , Carcinoma/patología , Adhesión Celular , Cruzamientos Genéticos , Exocitosis , Femenino , Glucuronidasa/metabolismo , Humanos , Inmunoglobulina A/inmunología , Inmunoglobulina G/inmunología , Lactoferrina/metabolismo , Fusión de Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , NADPH Deshidrogenasa/metabolismo , NADPH Oxidasas , Neutrófilos/inmunología , Neutrófilos/ultraestructura , Proteínas Opsoninas/inmunología , Fagocitosis , Fosfoproteínas/metabolismo , Transporte de Proteínas , Estallido Respiratorio , Células Tumorales Cultivadas

RESUMEN

Neutrophils undergo constitutive death by apoptosis, leading to safe nonphlogistic phagocytosis and clearance by macrophages. Recent work has shown that before secondary necrosis, neutrophils exhibiting classical features of apoptosis can progress to a morphologically defined late apoptotic state. However, whether such neutrophils could be safely cleared was unknown. We now report that human late apoptotic neutrophils could be purified from cultured neutrophil populations undergoing constitutive death and were subsequently ingested by human monocyte-derived macrophages by serum-independent mechanisms that did not trigger the release of IL-8 or TNF-alpha. Such ingestion was specifically inhibited by Abs to thrombospondin-1 and the alpha(v)beta(3) vitronectin receptor. Murine bone marrow-derived macrophage phagocytosis of late and early apoptotic neutrophils occurred by similar mechanisms, proceeding with the same efficiency as that observed for wild-type controls when macrophages from [alpha(m)](-/-) or [beta(2)](-/-) mice were used. We conclude that specific nonphlogistic, beta(2) integrin-independent mechanisms involving thrombospondin-1 and alpha(v)beta(3) allow macrophages to ingest late apoptotic neutrophils without eliciting inflammatory cytokine secretion.

Asunto(s)

Apoptosis/inmunología , Integrinas/fisiología , Macrófagos/inmunología , Neutrófilos/citología , Neutrófilos/inmunología , Fagocitosis/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Antígenos CD/inmunología , Apoptosis/genética , Sangre/inmunología , Células de la Médula Ósea/inmunología , Separación Celular , Células Cultivadas , Humanos , Inmunosupresores/farmacología , Integrina alfaV , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/inmunología , Neutrófilos/metabolismo , Fagocitosis/genética , Receptores de Vitronectina/inmunología , Trombospondina 1/inmunología

RESUMEN

Overproduction of inflammatory mediators by macrophages in response to Gram-negative LPS has been implicated in septic shock. Recent reports indicate that three membrane-associated proteins, CD14, CD11b/CD18, and Toll-like receptor (TLR) 4, may serve as LPS recognition and/or signaling receptors in murine macrophages. Therefore, the relative contribution of these proteins in the induction of cyclooxygenase 2 (COX-2), IL-12 p35, IL-12 p40, TNF-alpha, IFN-inducible protein (IP)-10, and IFN consensus sequence binding protein (ICSBP) genes in response to LPS or the LPS-mimetic, Taxol, was examined using macrophages derived from mice deficient for these membrane-associated proteins. The panel of genes selected reflects diverse macrophage effector functions that contribute to the pathogenesis of septic shock. Induction of the entire panel of genes in response to low concentrations of LPS or Taxol requires the participation of both CD14 and TLR4, whereas high concentrations of LPS or Taxol elicit the expression of a subset of LPS-inducible genes in the absence of CD14. In contrast, for optimal induction of COX-2, IL-12 p35, and IL-12 p40 genes by low concentrations of LPS or by all concentrations of Taxol, CD11b/CD18 was also required. Mitigated induction of COX-2, IL-12 p35, and IL-12 p40 gene expression by CD11b/CD18-deficient macrophages correlated with a marked inhibition of NF-kappa B nuclear translocation and mitogen-activated protein kinase (MAPK) activation in response to Taxol and of NF-kappa B nuclear translocation in response to LPS. These findings suggest that for expression of a full repertoire of LPS-/Taxol-inducible genes, CD14, TLR4, and CD11b/CD18 must be coordinately engaged to deliver optimal signaling to the macrophage.

Asunto(s)

Antígenos CD11/fisiología , Antígenos CD18/fisiología , Proteínas de Drosophila , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Receptores de Lipopolisacáridos/fisiología , Lipopolisacáridos/farmacología , Glicoproteínas de Membrana/fisiología , Paclitaxel/farmacología , Receptores de Superficie Celular/fisiología , Transporte Activo de Núcleo Celular/inmunología , Adyuvantes Inmunológicos/biosíntesis , Adyuvantes Inmunológicos/genética , Adyuvantes Inmunológicos/fisiología , Animales , Antígenos CD11/biosíntesis , Antígenos CD11/genética , Antígenos CD18/biosíntesis , Antígenos CD18/genética , Células Cultivadas , Quimiocina CXCL10 , Quimiocinas CXC/biosíntesis , Quimiocinas CXC/genética , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/farmacología , Factores Reguladores del Interferón , Interferón gamma/metabolismo , Interferón gamma/fisiología , Interleucina-12/antagonistas & inhibidores , Interleucina-12/biosíntesis , Interleucina-12/genética , Isoenzimas/biosíntesis , Isoenzimas/genética , Proteínas Quinasas JNK Activadas por Mitógenos , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Fosforilación , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Prostaglandina-Endoperóxido Sintasas/genética , Proteínas Represoras/biosíntesis , Proteínas Represoras/genética , Receptor Toll-Like 4 , Receptores Toll-Like , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genética , Proteínas Quinasas p38 Activadas por Mitógenos

RESUMEN

P-selectin is a leukocyte adhesion receptor stored in platelets and endothelial cells and is translocated to the surface upon cell activation. Purified P-selectin is oligomeric and has increased avidity for its ligand relative to the monomeric form, but whether P-selectin self-associates in the membrane of intact cells is not known. A chemical cross-linking approach was used to show that P-selectin is present as noncovalent dimers in resting platelets, human umbilical vein endothelial cells, and heterologous RIN5F cells expressing P-selectin. The results of 2-dimensional isoelectric focusing are consistent in showing P-selectin dimers as homodimers, but they are composed of a more basic subset of P-selectin than the monomers. This suggests that the dimers are a biochemically distinct subset of P-selectin. P-selectin dimers form in the endoplasmic reticulum and Golgi compartments of human umbilical vein endothelial cells only after synthesis of the mature P-selectin subunit, and are not preferentially stored in Weibel-Palade bodies as compared with the monomeric form. Platelet activation with thrombin receptor-activating peptide leads to the presence of P-selectin monomers and homodimers on the cell surface as well as P-selectin heterodimers, which are composed of P-selectin and an unidentified protein of approximately 81 kd molecular weight. In summary, these studies demonstrate that P-selectin is homodimeric in situ and that platelet activation leads to the formation of an additional activation-specific heterodimeric species. In addition, the homodimer has unique biochemical characteristics compared with the monomeric form, and dimerization occurs in the endoplasmic reticulum and Golgi compartments of endothelial cells.

Asunto(s)

Plaquetas/fisiología , Endotelio Vascular/fisiología , Selectina-P/química , Selectina-P/metabolismo , Sustitución de Aminoácidos , Línea Celular , Células Cultivadas , Reactivos de Enlaces Cruzados , Dimerización , Retículo Endoplásmico/fisiología , Aparato de Golgi/fisiología , Humanos , Técnicas In Vitro , Leucemia Eritroblástica Aguda , Orgánulos/fisiología , Selectina-P/sangre , Mutación Puntual , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Células Tumorales Cultivadas , Venas Umbilicales

RESUMEN

BACKGROUND: Several lines of evidence suggest that cellular immune mechanisms contribute to glomerulonephritis. METHODS: The roles of alphabeta and gammadelta T cells in the pathogenesis of glomerulonephritis were investigated in a model of nephrotoxic nephritis in mice deficient in either T-cell population [T-cell receptor (TCR)beta and TCRdelta knockout mice]. The model, induced by the injection of rabbit anti-mouse glomerular basement membrane antibody, is characterized by the development of proteinuria and glomerular damage over a 21-day observation period in wild-type mice. RESULTS: Mice deficient in either alphabeta or gammadelta T cells developed minimal proteinuria and glomerular lesions and had a significant reduction in macrophage accumulation compared with wild-type mice. In gammadelta T-cell-deficient mice, circulating levels and glomerular deposition of autologous IgG were comparable to wild-type levels, while alphabeta T-cell-deficient mice had no autologous IgG production. Autologous antibody production was not required for the development of glomerulonephritis since mice that lack IgG and B cells (micro-chain-/-) developed similar proteinuria to that observed in wild-type mice. CONCLUSIONS: These studies suggest a proinflammatory role for both alphabeta and gammadelta T cells in glomerular injury, independent of the humoral response. This is the first demonstration, to our knowledge, that both T-cell subsets contribute to the progression of a disease, and it suggests that complex regulatory interactions between alphabeta and gammadelta T cells play a role in glomerular injury.

Asunto(s)

Glomerulonefritis/inmunología , Glomerulonefritis/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Animales , Anticuerpos , Linfocitos B/inmunología , Membrana Basal/inmunología , Linfocitos T CD8-positivos/inmunología , Proteínas del Sistema Complemento/análisis , Expresión Génica/inmunología , Glomerulonefritis/patología , Inmunidad Celular/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Glomérulos Renales/inmunología , Glomérulos Renales/patología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteinuria/inmunología , Proteinuria/metabolismo , Proteinuria/patología , ARN Mensajero/análisis , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T gamma-delta/genética

RESUMEN

Liver macrophages (Kupffer cells) play a major role in blood clearance of both C3-opsonized immune complexes and therapeutic beta-glucan polysaccharides. Human Kupffer cells express three types of C3-receptors: CR1 (C3b-receptor; CD35), CR3 (iC3b- and beta-glucan-receptor), and CR4 (iC3b-receptor; CD11c/CD18). Studies of isolated macrophages have suggested that CR3 is the major receptor mediating capture of either C3-opsonized erythrocytes (E) or beta-glucans. In this investigation, the organ distribution and function of CR3 in the clearance of IgM-opsonized E and soluble CR3-binding polysaccharides were explored in normal vs. CR3-knockout (CR3-KO) mice. Analysis of intravenously (i.v.) injected 125I-anti-CR3 showed that the major vascular reservoir of CR3 was the liver, followed by spleen and lungs. By contrast, clearance of 125I-anti-CR1 appeared to be mediated predominantly by splenic B lymphocytes, as only subsets of splenic macrophages or Kupffer cells were found to express CR1. Clearance of IgM-opsonized 51Cr-E occurred rapidly to the livers of normal mice but was nearly absent in CR3-KO mice. Soluble 125I-beta-glucan exhibited rapid clearance to the liver in normal mice, whereas clearance in CR3-KO mice was significantly reduced. In conclusion, Kupffer cell CR3 plays a crucial role in the clearance of both IgM-opsonized E and beta-glucans.

Asunto(s)

Eritrocitos/metabolismo , Glucanos/farmacocinética , Inmunoglobulina M/metabolismo , Macrófagos del Hígado/metabolismo , Antígeno de Macrófago-1/farmacología , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Antígenos CD18 , Radioisótopos de Cromo , Eritrocitos/efectos de los fármacos , Femenino , Glucanos/metabolismo , Glucanos/uso terapéutico , Inmunoglobulina M/farmacología , Radioisótopos de Yodo , Cinética , Macrófagos del Hígado/inmunología , Antígeno de Macrófago-1/inmunología , Antígeno de Macrófago-1/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Proteínas Opsoninas/metabolismo , Proteínas Opsoninas/farmacología , Especificidad de Órganos , Receptores de Complemento 3b/inmunología , Zimosan/metabolismo , Zimosan/farmacocinética , Zimosan/uso terapéutico

RESUMEN

The activation of endothelium is important in recruiting neutrophils to sites of inflammation and in modulating their function. We demonstrate that conditioned medium from cultured, activated endothelial cells acts to significantly delay the constitutive apoptosis of neutrophils, resulting in their enhanced survival and increased phagocytic function. The antiapoptotic activity is, in part, attributable to granulocyte/macrophage colony-stimulating factor (GM-CSF) secreted by activated endothelial cells. The in vivo relevance of these findings was investigated in a cytokine-induced model of acute meningitis in mice. Peripheral blood neutrophils (PBNs) from mice with meningitis exhibited a delay in apoptosis compared with untreated mice. Furthermore, neutrophils recovered from the inflamed cerebrospinal fluid (CSF) exhibited enhanced survival compared with neutrophils isolated from the peripheral blood of the same animals. In unchallenged GM-CSF-deficient mice, the apoptosis of circulating PBNs was similar to wild-type animals; however, after cytokine-induced meningitis, the delay in neutrophil apoptosis typically observed in wild-type mice was attenuated. In contrast, the apoptosis of neutrophils recovered from the CSF of mice of both genotypes was comparable. Taken together, these studies suggest that neutrophil apoptosis is regulated during an inflammatory response, in both intravascular and extravascular compartments. GM-CSF released by activated endothelium can act to increase neutrophil survival and function in the peripheral blood, whereas other factor(s) appear to perform this function in the extravascular space.

Asunto(s)

Apoptosis/fisiología , Antígenos CD18/fisiología , Citocinas/fisiología , Citocinas/toxicidad , Endotelio Vascular/fisiopatología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Antígeno de Macrófago-1/fisiología , Meningitis/fisiopatología , Neutrófilos/citología , Neutrófilos/fisiología , Animales , Antígenos CD18/genética , Células Cultivadas , Quimiotaxis de Leucocito , Endotelio Vascular/fisiología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/líquido cefalorraquídeo , Humanos , Interleucina-1/toxicidad , Antígeno de Macrófago-1/genética , Masculino , Meningitis/líquido cefalorraquídeo , Meningitis/patología , Ratones , Ratones Noqueados , Factor de Necrosis Tumoral alfa/toxicidad , Venas Umbilicales

RESUMEN

beta-Glucans were identified 36 years ago as a biologic response modifier that stimulated tumor rejection. In vitro studies have shown that beta-glucans bind to a lectin domain within complement receptor type 3 (CR3; known also as Mac-1, CD11b/CD18, or alphaMbeta2-integrin, that functions as an adhesion molecule and a receptor for factor I-cleaved C3b, i.e., iC3b) resulting in the priming of this iC3b receptor for cytotoxicity of iC3b-opsonized target cells. This investigation explored mechanisms of tumor therapy with soluble beta-glucan in mice. Normal mouse sera were shown to contain low levels of Abs reactive with syngeneic or allogeneic tumor lines that activated complement, depositing C3 onto tumors. Implanted tumors became coated with IgM, IgG, and C3, and the absent C3 deposition on tumors in SCID mice was reconstituted with IgM or IgG isolated from normal sera. Therapy of mice with glucan- or mannan-rich soluble polysaccharides exhibiting high affinity for CR3 caused a 57-90% reduction in tumor weight. In young mice with lower levels of tumor-reactive Abs, the effectiveness of beta-glucan was enhanced by administration of a tumor-specific mAb, and in SCID mice, an absent response to beta-glucan was reconstituted with normal IgM or IgG. The requirement for C3 on tumors and CR3 on leukocytes was highlighted by therapy failures in C3- or CR3-deficient mice. Thus, the tumoricidal function of CR3-binding polysaccharides such as beta-glucan in vivo is defined by natural and elicited Abs that direct iC3b deposition onto neoplastic cells, making them targets for circulating leukocytes bearing polysaccharide-primed CR3. Therapy fails when tumors lack iC3b, but can be restored by tumor-specific Abs that deposit iC3b onto the tumors.

Asunto(s)

Anticuerpos Antineoplásicos/metabolismo , Antígenos CD18/inmunología , Citotoxicidad Inmunológica/inmunología , Glucanos/uso terapéutico , Factores Inmunológicos/uso terapéutico , Antígeno de Macrófago-1/inmunología , Neoplasias Mamarias Experimentales/inmunología , Animales , Anticuerpos Antineoplásicos/biosíntesis , Antígenos de Neoplasias/inmunología , Complemento C3/metabolismo , Complemento C3b/inmunología , Femenino , Glucanos/administración & dosificación , Sueros Inmunes/metabolismo , Inmunidad Celular , Inmunoglobulina G/metabolismo , Inmunoglobulina M/metabolismo , Factores Inmunológicos/administración & dosificación , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/terapia , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones SCID , Células Tumorales Cultivadas , Zimosan/administración & dosificación , Zimosan/uso terapéutico

RESUMEN

Single gene knock-outs in mice have been used to define the biological role of leukocyte adhesion receptors, Fc-gamma receptors and complement in animal models of immune complex glomerulonephritis. These studies have shown important differences in the role of P-selectin in glomerular inflammation and inflammation at other sites, and have given a new appreciation of the dominant role played by Fc-gamma receptors in immune complex-induced glomerular injury.

Asunto(s)

Moléculas de Adhesión Celular/fisiología , Glomerulonefritis/genética , Glomerulonefritis/inmunología , Receptores de IgG/fisiología , Animales , Moléculas de Adhesión Celular/genética , Proteínas del Sistema Complemento/fisiología , Modelos Animales de Enfermedad , Glomerulonefritis/etiología , Enfermedades del Complejo Inmune/etiología , Enfermedades del Complejo Inmune/genética , Enfermedades del Complejo Inmune/inmunología , Leucocitos/inmunología , Ratones , Ratones Noqueados , Receptores de IgG/deficiencia , Receptores de IgG/genética , Selectinas/genética , Selectinas/fisiología

RESUMEN

Even though more immunoglobulin A (IgA) is produced in humans than all other isotypes combined, relatively little is known about receptors that bind the Fc part of IgA. The myeloid IgA receptor, FcalphaRI (CD89), triggers various effector functions in vitro, but its in vivo role remains unclear. Here, a transgenic mouse model is described in which FcalphaRI is expressed under its own regulatory sequences. Receptor expression and regulation by cytokines was comparable to the human situation and hFcalphaRI can trigger phagocytosis and lysis of tumor cells. To analyze the contribution of the FcR gamma chain or the beta2 integrin CR3 (CD11b/CD18) in FcalphaRI biological function, FcalphaRI transgenic mice were crossed with either FcR gamma chain -/- or CR3 -/- mice. In contrast to in vitro data, FcR gamma chain was essential for surface expression of hFcalphaRI in vivo. Functional studies in hFcalphaRI/ gamma-/-mice were, therefore, limited. In vitro studies showed FcR gamma chain to be necessary for phagocytosis. Neither hFcalphaRI expression nor phagocytosis, triggered via hFcalphaRI, were influenced by CR3. Remarkably, the capacity to lyse tumor targets was ablated in hFcalphaRI transgenic/ CR3-/- mice, although binding of neutrophils to tumor cells was intact. This shows a previously unrecognized importance of CR3 for hFcalphaRI-mediated antibody-dependent cellular cytotoxicity (ADCC).

Asunto(s)

Antígenos CD/inmunología , Antígenos CD11/inmunología , Antígenos CD18/inmunología , Receptores Fc/inmunología , Receptores de IgG/inmunología , Animales , Citotoxicidad Celular Dependiente de Anticuerpos , Antígenos CD/genética , Antígenos CD11/genética , Antígenos CD18/genética , Cruzamientos Genéticos , Citocinas/farmacología , Expresión Génica , Humanos , Ratones , Ratones Transgénicos , Fagocitosis , Receptores Fc/genética , Receptores de IgG/genética

RESUMEN

Taxol, a potent antitumor agent that binds beta-tubulin and promotes microtubule assembly, results in mitotic arrest at the G2/M phase of the cell cycle. More recently, Taxol was shown to be a potent LPS mimetic in murine, but not in human macrophages, stimulating signaling pathways and gene expression indistinguishably from LPS. Although structurally unrelated to LPS, Taxol's LPS-mimetic activities are blocked by inactive structural analogues of LPS, indicating that despite the species-restricted effects of Taxol, LPS and Taxol share a common receptor/signaling complex that might be important in LPS-induced human diseases. To identify components of the putatively shared Taxol/LPS receptor, a novel, photoactivatable Taxol analogue was employed to identify unique Taxol-binding proteins in murine macrophage membranes. Seven major Taxol-binding proteins, ranging from approximately 50 to 200 kDa, were detected. Although photoactivatable Taxol analogue failed to bind to CD14, the prominent Taxol-binding protein was identified as CD18, the approximately 96-kDa common component of the beta2 integrin family. This finding was supported by the concomitant failure of macrophage membranes from Mac-1 knockout mice to express immunoreactive CD18 and the major Taxol-binding protein. In addition, Taxol-induced IL-12 p40 mRNA was markedly reduced in Mac-1 knockout macrophages and anti-Mac-1 Ab blocked secretion of IL-12 p70 in Taxol- and LPS-stimulated macrophages. Since CD18 has been described as a participant in LPS-induced binding and signal transduction, these data support the hypothesis that the interaction of murine CD18 with Taxol is involved in its proinflammatory activity.

Asunto(s)

Antígenos CD18/metabolismo , Proteínas Portadoras/metabolismo , Regulación de la Expresión Génica/inmunología , Antígeno de Macrófago-1/fisiología , Macrófagos Peritoneales/metabolismo , Paclitaxel/análogos & derivados , Paclitaxel/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos CD18/aislamiento & purificación , Células CHO , Proteínas Portadoras/aislamiento & purificación , Línea Celular , Membrana Celular/metabolismo , Cricetinae , Humanos , Sueros Inmunes/farmacología , Interleucina-12/antagonistas & inhibidores , Interleucina-12/biosíntesis , Interleucina-12/genética , Interleucina-12/metabolismo , Lipopolisacáridos/farmacología , Antígeno de Macrófago-1/genética , Antígeno de Macrófago-1/inmunología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/inmunología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Paclitaxel/farmacología , Etiquetas de Fotoafinidad/metabolismo , ARN Mensajero/biosíntesis

RESUMEN

The advent of gene targeting has led to the generation of several mouse strains deficient in select leukocyte adhesion receptors. These strains of mice have been very informative about the roles of cell adhesion molecules in leukocyte-endothelium interaction and have produced some surprises: roles for leukocyte adhesion receptors have been demonstrated in development as well as pathologies like obesity, and evidence for functional synergies between adhesion receptors have been provided. We attempt in this review to first outline the technique of gene targeting and give an overview of leukocyte adhesion receptors and mice deficient in these receptors. Second, we discuss models of experimental glomerulonephritis and what we have learned about leukocyte adhesion receptors in the pathogenesis of glomerulonephritis through studies in knockout mice.

Asunto(s)

Moléculas de Adhesión Celular/fisiología , Endotelio Vascular/fisiología , Glomerulonefritis/fisiopatología , Integrinas/fisiología , Leucocitos/fisiología , Animales , Adhesión Celular , Moléculas de Adhesión Celular/genética , Comunicación Celular , Glomerulonefritis/genética , Humanos , Integrinas/genética , Ratones , Ratones Noqueados

RESUMEN

P-selectin is a leukocyte adhesion receptor present in endothelial cells and platelets. We examined the role of P-selectin in the autologous phase of an accelerated model of anti-glomerular basement membrane (GBM) glomerulonephritis using P-selectin-deficient mice and chimeric mice expressing P-selectin only in platelets or endothelial cells. P-selectin-deficient mice exhibited more severe glomerular damage with increased interstitial mononuclear leukocytic infiltrates, and had significantly increased proteinuria and mortality when compared to wild-type mice. P-selectin on the endothelium was predominantly responsible for protection from the exacerbated disease, because chimeric mice with endothelial P-selectin, and not mice with platelet P-selectin, showed glomerular injury similar to that in wild-type animals. Levels of soluble circulating P-selectin were increased in nephritic wild-type mice and in chimeric mice with endothelial P-selectin, but not platelet P-selectin. Levels of soluble P-selectin, which has been shown to be anti-inflammatory in vitro, were inversely associated with the severity of disease. P-selectin was not expressed in the endothelium of the glomerulus or interstitium. Thus, the protective effect in wild-type mice may be accounted for, in part by soluble P-selectin shed by non-renal endothelial cells, although other endothelial P-selectin-dependent mechanisms cannot be ruled out.

Asunto(s)

Enfermedad por Anticuerpos Antimembrana Basal Glomerular/genética , Enfermedad por Anticuerpos Antimembrana Basal Glomerular/inmunología , Selectina-P/genética , Selectina-P/inmunología , Animales , Enfermedad por Anticuerpos Antimembrana Basal Glomerular/patología , Endotelio Vascular/inmunología , Endotelio Vascular/patología , Inmunohistoquímica , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados

RESUMEN

BACKGROUND AND PURPOSE: Macrophage-1 antigen (Mac-1) (CD11b/CD18), a leukocyte beta2 integrin, facilitates neutrophil adhesion, transendothelial migration, phagocytosis, and respiratory burst, all of which may mediate reperfusion-induced injury to ischemic brain tissue in conditions such as stroke. To determine the role of Mac-1 during ischemia and reperfusion in the brain, we analyzed the effect of transient focal cerebral ischemia in mice genetically engineered with a specific deficiency in Mac-1. METHODS: Transient focal ischemia/reperfusion was induced by occluding the left middle cerebral artery for 3 hours followed by a 21-hour reperfusion period in Mac-1-deficient (n=12) and wild-type (n=11) mice. Regional cerebral blood flow was determined with a laser-Doppler flowmeter. Brain sections were stained with 2% 2,3,5-triphenyltetrazolium chloride to determine the infarct volume. Neutrophil accumulation was determined by staining the brain sections with dichloroacetate esterase to identify neutrophils. RESULTS: Compared with the wild-type cohort, Mac-1-deficient mice had a 26% reduction in infarction volume (P<0.05). This was associated with a 50%, but statistically insignificant, reduction in the number of extravasated neutrophils in the infarcted areas of the brains in the mutant mice. There were no differences in regional cerebral blood flow between the 2 groups. CONCLUSIONS: Mac-1 deficiency reduces neutrophil infiltration and cerebral cell death after transient focal cerebral ischemia. This finding may be related to a reduction in neutrophil extravasation in Mac-1-deficient mice.

Asunto(s)

Antígenos CD18/genética , Ataque Isquémico Transitorio/genética , Antígeno de Macrófago-1/genética , Daño por Reperfusión/genética , Animales , Antígenos CD18/metabolismo , Infarto Cerebral/genética , Infarto Cerebral/metabolismo , Trastornos Cerebrovasculares/genética , Quimiotaxis/fisiología , Susceptibilidad a Enfermedades , Molécula 1 de Adhesión Intercelular/metabolismo , Ataque Isquémico Transitorio/metabolismo , Antígeno de Macrófago-1/metabolismo , Ratones , Ratones Noqueados , Neutrófilos/citología , Neutrófilos/metabolismo , Fagocitosis/fisiología , Daño por Reperfusión/metabolismo , Estallido Respiratorio/fisiología

RESUMEN

Mac-1 (CD11b/CD18, CR3), a beta2 integrin expressed on leukocytes, is important in leukocyte migration. We demonstrate that Mac-1 is also expressed on peritoneal mast cells and LPS stimulated bone marrow-derived cultured mast cells, and that Mac-1-deficient mice, which lack this receptor, have significant reductions in the numbers of mast cells resident in the peritoneal cavity, peritoneal wall, and dorsal skin. The reduced numbers of mast cells in Mac-1-deficient mice may have important functional consequences, in that Mac-1-deficient mice exhibit significantly increased mortality after cecal ligation and puncture, a model of acute septic peritonitis in which host resistance has been shown to be dependent on both mast cells and complement. These findings demonstrate that Mac-1 is required for the expression of normal levels of mast cells in the peritoneal cavity, peritoneal wall, and certain areas of the skin, as well as for maintaining adequate mast cell-dependent host defense against bacterial infection.

Asunto(s)

Inmunidad Innata , Síndromes de Inmunodeficiencia/patología , Antígeno de Macrófago-1/fisiología , Mastocitos/patología , Peritonitis/inmunología , Enfermedad Aguda , Animales , Recuento de Células , Síndromes de Inmunodeficiencia/complicaciones , Síndromes de Inmunodeficiencia/inmunología , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Perforación Intestinal/complicaciones , Antígeno de Macrófago-1/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Cavidad Peritoneal/patología , Peritonitis/complicaciones , Peritonitis/patología , Piel/patología

RESUMEN

P-selectin is an adhesion receptor for leukocytes expressed on activated platelets and endothelial cells. The cytoplasmic domain of P-selectin was shown in vitro to contain signals required for both the sorting of this protein into storage granules and its internalization from the plasma membrane. To evaluate in vivo the role of the regulated secretion of P-selectin, we have generated a mouse that expresses P-selectin lacking the cytoplasmic domain (DeltaCT mice). The deletion did not affect the sorting of P-selectin into alpha-granules of platelets but severely compromised the storage of P-selectin in endothelial cells. Unstored P-selectin was proteolytically shed from the plasma membrane, resulting in increased levels of soluble P-selectin in the plasma. The DeltaCT-P-selectin appeared capable of mediating cell adhesion as it supported leukocyte rolling in the mutant mice. However, a secretagogue failed to upregulate leukocyte rolling in the DeltaCT mice, indicating an absence of a releasable storage pool of P-selectin in the endothelium. Furthermore, the neutrophil influx into the inflamed peritoneum was only 30% of the wild-type level 2 h after stimulation. Our results suggest that different sorting mechanisms for P-selectin are used in platelets and endothelial cells and that the storage pool of P-selectin in endothelial cells is functionally important during early stages of inflammation.

Asunto(s)

Hepatitis Animal/metabolismo , Hígado/inmunología , Selectina-P/sangre , Animales , Plaquetas/inmunología , Plaquetas/metabolismo , Plaquetas/ultraestructura , Citoplasma/química , Gránulos Citoplasmáticos/metabolismo , Endotelio/metabolismo , Femenino , Citometría de Flujo , Expresión Génica/efectos de los fármacos , Expresión Génica/inmunología , Células HL-60 , Hepatitis Animal/inmunología , Humanos , Lipopolisacáridos/farmacología , Hígado/química , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Inmunoelectrónica , Mutagénesis/fisiología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Selectina-P/química , Selectina-P/genética , Peritonitis/inmunología , Peritonitis/metabolismo , Estructura Terciaria de Proteína , Solubilidad , Tioglicolatos , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

Obesity is a complex disease, and multiple genes contribute to the trait. The description of five genes (ob, db, tub, Ay, and fat) responsible for distinct syndromes of spontaneous monogenic obesity in mice has advanced our knowledge of the genetics of obesity. However, many other genes involved in the expression of this disease remain to be determined. We report here the identification of an additional class of genes involved in the regulation of adipose tissue mass. These genes encode receptors mediating leukocyte adhesion. Mice deficient in intercellular adhesion molecule-1 became spontaneously obese in old age on normal mouse chow or at a young age when provided with a diet rich in fat. Mice deficient in the counterreceptor for intercellular adhesion molecule-1, the leukocyte integrin alphaMbeta2 (Mac-1), showed a similar obesity phenotype. Since all mice consumed approximately the same amount of food as controls, the leukocyte function appears to be in regulating lipid metabolism and/or energy expenditure. Our results indicate that (i) leukocytes play a role in preventing excess body fat deposition and (ii) defects in leukocyte adhesion receptors can result in obesity.

Asunto(s)

Molécula 1 de Adhesión Intercelular/genética , Obesidad/genética , Receptores de Adhesión de Leucocito/genética , Envejecimiento , Animales , Dieta , Femenino , Ratones , Ratones Noqueados

RESUMEN

P- and E-selectins are adhesion molecules expressed on activated endothelium and platelets at sites of vascular injury and inflammation. The selectins are important for leukocyte recruitment. Because little is known about the role of selectins in wound healing, we studied cutaneous wound repair of full-thickness excisional skin wounds in mice lacking P-selectin, E-selectin, or both of these selectins. The absence of either selectin alone had no notable effect on healing, and the only deficit observed was a delay in early neutrophil extravasation in the P-selectin-deficient mice. Mice deficient in both P- and E-selectins had markedly reduced recruitment of inflammatory cells and impaired closure of the wounds. Wound sections, studied up to 3 days after wounding, showed significant impairment of neutrophil influx. Macrophage numbers were also reduced in the double mutants at 3 and 7 days after wounding as compared with wild-type mice. Additionally, a wider epithelial gap in the wounds of the P- and E-selectin-double-deficient mice 3 days after wounding indicated delayed keratinocyte migration. These results demonstrate an important combined role for P- and E-selectins in processes leading to wound healing.

Asunto(s)

Endotelio Vascular/fisiología , Selectinas/fisiología , Cicatrización de Heridas/fisiología , Animales , Movimiento Celular/genética , Femenino , Tejido de Granulación/patología , Queratinocitos/fisiología , Macrófagos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/fisiología , Selectinas/genética , Piel/patología , Cicatrización de Heridas/genética

RESUMEN

P-selectin is expressed on activated endothelium and platelets where it can bind monocytes, neutrophils, stimulated T cells, and platelets. Because recruitment of these cells is critical for atherosclerotic lesion development, we examined whether P-selectin might play a role in atherosclerosis. We intercrossed P-selectin-deficient mice with mice lacking the low density lipoprotein receptor (LDLR) because these mice readily develop atherosclerotic lesions on diets rich in saturated fat and cholesterol. The atherogenic diet stimulated leukocyte rolling in the mesenteric venules of LDLR-deficient mice, and the increase in adhesiveness of the vessels was P-selectin-dependent. Most likely due to the reduced leukocyte interaction with the vessel wall, P-selectin-deficient mice on diet for 8-20 wk formed significantly smaller fatty streaks in the cusp region of the aortae than did P-selectin-positive mice. This difference was more prominent in males. At 37 wk on diet, the lesions in the LDLR-deficient animals progressed to the fibrous plaque stage and were distributed throughout the entire aorta; their size or distribution was no longer dependent on P-selectin. Our results show that P-selectin-mediated adhesion is an important factor in the development of early atherosclerotic lesions, and that adhesion molecules such as P-selectin are involved in the complex process of atherosclerosis.

Asunto(s)

Arteriosclerosis/genética , Arteriosclerosis/metabolismo , Adhesión Celular/fisiología , Receptores de LDL/genética , Selectinas/genética , Selectinas/fisiología , Animales , Aorta/patología , Arteriosclerosis/etiología , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Colesterol/análisis , Colesterol/sangre , Colesterol en la Dieta/farmacología , Dieta Aterogénica , Grasas de la Dieta/farmacología , Femenino , Leucocitos/efectos de los fármacos , Lipoproteínas/análisis , Lipoproteínas/sangre , Macrófagos , Masculino , Venas Mesentéricas/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Músculo Liso/citología , Factores Sexuales , Triglicéridos/análisis , Triglicéridos/sangre