RESUMEN
Complement subcomponent C1q has been recently implicated in the modulation of autocrine binding of TNF-alpha to murine macrophages for induction of nitric oxide synthase. In the present study, the putative role of C1q in increasing TNF-alpha binding to L929 cells to mediate cytotoxicity was explored. TNF-sensitive L929 cells (L929-S) had higher total endogenous cellular and surface C1q levels and bound correspondingly more phycoerythrin-labelled rTNF-alpha (PE-TNF) than did a TNF-resistant L929 variant (L929-R). Pretreatment of L929-S with soluble C1q increased their sensitivity to TNF-mediated cytotoxicity coincident with increased binding of PE-TNF, but similar treatment of L929-R had no effect. Pretreatment of L929-S with an inhibitor of C1q secretion, 3,4 dehydro-D,L-proline (DHP), resulted in a decrease in their TNF-mediated cytotoxicity, as well as reduced binding of PE-TNF. Subsequent exposure of DHP-treated L929-S with exogenous soluble C1q restored their TNF-mediated cytotoxicity and binding of PE-TNF. These results provide evidence for the modulation of TNF-alpha binding to TNF sensitive tumour targets L929 by either endogenously synthesized or exogenously added C1q to promote TNF-mediated cytotoxicity by mechanisms which remain to be elucidated.
Asunto(s)
Complemento C1q/fisiología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Ratones , Prolina/análogos & derivados , Prolina/farmacología , Receptores del Factor de Necrosis Tumoral/análisis , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
AKR mouse peritoneal macrophages (PM) were previously found to have a defect in their response to lipid A for nitric oxide (NO)-mediated tumor cytotoxicity, which was related to a lower level of C1q synthesis and reconstituted by exogenous IFN-gamma or C1q. We used AKR-PM as a model to further define the role of IFN-alpha beta in modulation of induction of macrophage nitric oxide synthase (NOS) in response to lipid A. Studies have revealed that AKR-PM produced a significantly lower level of IFN-alpha beta than responsive C3H-PM in response to lipid A. AKR-PM failed to increase NOS mRNA synthesis and NO generation when exposed to lipid A, although they had normal levels of TNF-alpha bioactivity and mRNA expression. This partial deficiency of AKR-PM to lipid A stimulation was reconstituted completely by exogenous IFN-alpha beta for both synthesis of NOS mRNA and release of NO. The failure of AKR-PM to produce NOS to lipid A stimulation appears to be related to reduced secretion of IFN-alpha beta and the resultant failure to express TNF-alpha type II receptor (TNF-RII) mRNA, which in turn decreases TNF-alpha binding to its receptor for autocrine induction of NOS. Insufficient synthesis and secretion of endogenous IFN-alpha beta may be the primary reason for AKR-PM refractoriness to induction of NOS in response to lipid A. furthermore, the close correlation between lack of IFN-alpha beta secretion and decreased TNF-RII mRNA synthesis may implicate a critical role for IFN-alpha beta in the upregulation of macrophage TNF-RII receptor expression for autocrine induction of NOS during lipid A stimulation.
Asunto(s)
Interferón-alfa/farmacología , Interferón beta/farmacología , Lípido A/farmacología , Activación de Macrófagos/efectos de los fármacos , Macrófagos Peritoneales/enzimología , Macrófagos Peritoneales/inmunología , Óxido Nítrico Sintasa/deficiencia , Animales , Anticuerpos/farmacología , Secuencia de Bases , Citotoxicidad Inmunológica/efectos de los fármacos , Fibrosarcoma , Interferón-alfa/biosíntesis , Interferón-alfa/inmunología , Interferón beta/biosíntesis , Interferón beta/inmunología , Leucemia L1210 , Macrófagos Peritoneales/metabolismo , Masculino , Ratones , Ratones Endogámicos AKR , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Óxido Nítrico/biosíntesis , Óxido Nítrico/toxicidad , Óxido Nítrico Sintasa/biosíntesis , ARN Mensajero/biosíntesis , Receptores del Factor de Necrosis Tumoral/genética , Células Tumorales CultivadasRESUMEN
The role of endogenously synthesized complement subcomponent C1q on autocrine binding of tumor necrosis factors (TNF-alpha) and on TNF-alpha receptor (TNF-R) mRNA synthesis by mouse macrophages was investigated. Activation of C3H mouse peritoneal macrophages (C3H-PM phi) by Lipid A induced TNF-alpha and nitric oxide (NO) to kill tumor targets. Such activation also increased macrophage-endogenous C1q synthesis and secretion in a dose-dependent fashion. Antibody for C1q markedly inhibited C3H-PM phi NO production in response to Lipid A, but had no effect on TNF-alpha production. C3H-PM phi treated with C1q or Lipid A displayed increased TNF-R mRNA synthesis and in combination with Lipid A and anti-C1q antibody inhibited TNF-R and nitric oxide synthase (NOS) mRNA synthesis compared with Lipid A only, but had no effect on TNF mRNA synthesis. In vitro treatment of C3H-PM phi with C1q also increased TNF-alpha binding to their surfaces. Taken together, the data indicate that endogenously synthesized C1q is operative in promoting TNF-R mRNA synthesis and resultant autocrine binding of TNF-alpha for induction of NOS in the process of NO-mediated tumor cytotoxicity by Lipid A-activated macrophages.
Asunto(s)
Complemento C1q/fisiología , Lípido A/farmacología , Activación de Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Óxido Nítrico/biosíntesis , Receptores del Factor de Necrosis Tumoral/biosíntesis , Factor de Necrosis Tumoral alfa/metabolismo , Adyuvantes Inmunológicos/fisiología , Animales , Secuencia de Bases , Complemento C1q/biosíntesis , Complemento C1q/inmunología , Humanos , Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Óxido Nítrico Sintasa/biosíntesis , Unión Proteica/efectos de los fármacos , Unión Proteica/inmunología , ARN Mensajero/biosíntesis , Receptores del Factor de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The role of complement subcomponent C1q in the modulation of TNF-alpha binding to L929 cells to mediate cytotoxicity and nitric oxide (NO) generation was investigated. Transfection of L929 with murine C1q B-chain antisense plasmid cDNA rendered them markedly less susceptible to TNF-mediated cytotoxicity coincident with a decreased capacity for TNF-alpha binding and expression of cell surface C1q protein. The inhibitory effects of L929 transfection with C1q B-chain antisense were transiently expressed at 24 hr post-transfection with full recovery of cellular functions by 72 hr. Transfected L929 cells were fully reconstituted in their TNF-alpha binding and in their cytotoxic response following exposure to soluble C1q which was bound to their cell surface. Transfection with C1q B-chain antisense also significantly inhibited NO generation by L929 cells in response to stimulation by TNF-alpha, IFN-alpha/beta, and LPS. Taken together, these results indicate that endogenously synthesized C1q is prerequisite for binding of TNF-alpha to L929 cells to mediate cytotoxicity and NO generation.
Asunto(s)
Complemento C1q/fisiología , Citotoxicidad Inmunológica , Óxido Nítrico/biosíntesis , Factor de Necrosis Tumoral alfa/metabolismo , Animales , ADN sin Sentido , ADN Complementario/genética , Células L , Macrófagos/fisiología , Ratones , Transducción de Señal , TransfecciónRESUMEN
Murine macrophages (M phi) are activated either by interferon-gamma (IFN-gamma) or interferon-alpha/beta (IFN-alpha/beta) in combination with bacterial lipopolysaccharide (LPS) to induce synthesis of tumor necrosis factor alpha (TNF-alpha) and nitric oxide synthase (iNOS) mRNA synthesis for generation of tumor cytotoxic nitric oxide (NO). In the present study, the effect of exogenous IFN-gamma on the induction of endogenous mRNA synthesis and secretion of IFN-alpha/beta by murine M phi was investigated. Neutralizing antibodies to IFN-alpha/beta reversed TNF-alpha and NOS mRNA synthesis, as well as nitric oxide (NO)-mediated tumor cytotoxicity. Quantitative reverse transcription polymerase chain reaction (RT-PCR) revealed that treatment of M phi with IFN-gamma induced increases in both IFN-alpha and IFN-beta mRNA synthesis by approximately 2-fold and 10-fold, respectively, which corresponded to a 2-fold increase in secretion of IFN-alpha/beta by ELISA. These data indicate that exogenous IFN-gamma induces endogenous synthesis and secretion of IFN-alpha/beta by M phi, which appears to act in concert with endogenously synthesized TNF-alpha for the autocrine induction of NOS mRNA synthesis.
Asunto(s)
Inductores de Interferón/farmacología , Interferón Tipo I/biosíntesis , Macrófagos/efectos de los fármacos , Óxido Nítrico Sintasa/biosíntesis , Animales , Antineoplásicos/farmacología , Secuencia de Bases , Inducción Enzimática , Ensayo de Inmunoadsorción Enzimática , Interferón Tipo I/metabolismo , Leucemia L1210/tratamiento farmacológico , Leucemia L1210/patología , Lipopolisacáridos/farmacología , Macrófagos/enzimología , Masculino , Ratones , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Transcripción Genética , Células Tumorales CultivadasRESUMEN
Studies were designed to further define the modulatory role of complement subcomponent C1q in macrophage activation by Lipid A to mediate production of TNF and cytotoxic nitric oxide (NO). Pretreatment of macrophages for 24 h with 2.5 mM 3,4,dehydro-D,L-proline (DHP), an inhibitor of C1q secretion, suppressed their response to Lipid A activation for cytotoxicity of P815 tumor targets which correlated with a corresponding decrease in NO production. In contrast, DHP-pretreated macrophages displayed an increase in the release of TNF in response to Lipid A as compared to untreated controls. Time kinetic studies indicated that DHP-pretreated macrophages produced higher sustained levels of TNF activity during 1 to 24 h culture with Lipid A than did untreated control macrophages. This was confirmed by increased TNF mRNA expression in response to Lipid A by DHP-treated cells. DHP-pretreated macrophages had reduced levels of cell surface C1q as determined by cytofluorometric analysis of the binding of FITC-labeled anti-C1q, F(ab')2. Macrophages were also found to have reduced binding capacity for phycoerythrin-labeled rTNF (PE-TNF) by cytofluorometric analysis following DHP treatment. Exposure of DHP-pretreated macrophage to soluble C1q at 4 degrees C restored their reduced binding of PE-TNF. C1q was confirmed to bind to macrophages at 4 degrees C as detected by FITC anti-C1q, F(ab')2 and such C1q binding promoted a corresponding increased binding of PE-TNF. Macrophages which were plated over immobilized C1q were also markedly enhanced in their binding of PE-TNF probe. Our results indicate that the inhibition of macrophage secretion of C1q by DHP pretreatment, was accompanied by an increased TNF mRNA expression and release with a decrease in NO generation following Lipid A activation. Since TNF binding to DHP-treated macrophages was reconstituted by the binding of exogenous C1q to the cells, it appears that C1q may be involved in the modulation of autocrine binding of TNF for subsequent generation of cytotoxic NO.
Asunto(s)
Complemento C1q/antagonistas & inhibidores , Lípido A/farmacología , Macrófagos/metabolismo , Óxido Nítrico/biosíntesis , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Complemento C1q/metabolismo , Citotoxicidad Inmunológica , Citometría de Flujo , Humanos , Activación de Macrófagos , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C3H , Prolina/análogos & derivados , Prolina/farmacología , ARN Mensajero/metabolismo , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Regulation of macrophage Fc receptor (Fc gamma R)-mediated phagocytic function by histidine-rich glycoprotein (HRG) was investigated. Pretreatment of oil-elicited inflammatory mouse peritoneal macrophages with HRG for 1-3 hr increased their Fc gamma R-mediated binding and phagocytosis of IgG-opsonized sheep erythrocyte conjugates (EA). A significant reduction of Fc gamma R-dependent EA binding and phagocytosis occurred after pretreatment of macrophages with HRG for more than 8 hr. These results indicate that HRG is capable of modulating Fc gamma R expression in a biphasic fashion, which directly affects the overall efficiency of phagocytosis. HRG differentially regulated the functions of Fc gamma R subclasses. For example, HRG reduced the efficiency of Fc gamma RII (Fc gamma 2b/gamma 1R)-dependent phagocytosis of erythrocytes conjugated with monoclonal IgG2b or IgG1 by macrophages pretreated with HRG for 24 hr. However, when similar studies were performed using erythrocytes coated with monoclonal IgG2a, HRG was less effective in inhibiting Fc gamma RI (Fc gamma 2aR)-dependent phagocytosis. As an HRG-binding glycosaminoglycan, heparin failed to block the regulatory function of HRG on macrophages. Similarly, interferon-gamma (IFN-gamma) was not capable of blocking the functional activity of HRG. These studies suggest that HRG regulates macrophage function via a novel pathway different from that of heparin or IFN-gamma.
Asunto(s)
Macrófagos/inmunología , Fagocitosis/inmunología , Proteínas/inmunología , Receptores de IgG/análisis , Animales , Heparina/inmunología , Inmunoglobulina G/inmunología , Interferón gamma/inmunología , Masculino , Ratones , Ratones Endogámicos C3H , Proteínas/aislamiento & purificaciónRESUMEN
The modulation of complement functional efficiency by serum histidine-rich glycoprotein (HRG) was investigated. Addition of exogenous HRG to prewarmed diluted serum, followed immediately by sensitized sheep erythrocytes (EA), resulted in enhanced hemolysis. However, when HRG was incubated with diluted serum for 10 minutes at 37 degrees C, inhibition of hemolysis occurred. The biphasic modulation of complement function was also obtained with the complement alternative pathway when HRG was added to diluted serum for hemolysis of rabbit erythrocytes. Partial reduction of complement functional activity was shown when serum was absorbed by an HRG-Sepharose 6MB column. Western blot analysis showed that complement C8, C9, factor D, and S-protein in diluted serum were bound by nylon membrane-immobilized HRG. However, by immunoprecipitation of relatively undiluted serum with anti-HRG IgG beads, HRG was found to coprecipitate with S-protein and plasminogen, which suggested that HRG may complex with these proteins in serum. In functional tests, HRG inhibited C8 hemolytic activity, probably by preventing C8 binding to EAC1-7 cells. HRG also enhanced polymerization of purified C9 as well as the generation of a 45-Kd C9 fragment. Such an effect was even more pronounced in the presence of divalent cations with the reaction mixtures of C9 and HRG. Partial dimerization of C9 was shown when exogenous HRG was added to normal serum. In contrast, polymerization of serum C9 was inhibited by exogenous HRG during poly I:C activation of serum or incubation under low ionic strength conditions. HRG was further shown to inhibit factor D-mediated cleavage of factor B when bound by cobra venom factor. The molecular basis by which HRG regulates serum complement function is not clear. Hypothetically, the tandem repetitions of a consensus histidine-rich penta-peptide sequence in HRG may provide a highly charged area that interacts with complement components.
Asunto(s)
Proteínas del Sistema Complemento/inmunología , Glicoproteínas/farmacología , Proteínas/farmacología , Animales , Complemento C8/antagonistas & inhibidores , Complemento C8/inmunología , Complemento C8/metabolismo , Complemento C9/química , Complemento C9/metabolismo , Factor B del Complemento/metabolismo , Factor D del Complemento/antagonistas & inhibidores , Factor D del Complemento/metabolismo , Vía Alternativa del Complemento/efectos de los fármacos , Venenos Elapídicos/metabolismo , Eritrocitos/inmunología , Glicoproteínas/metabolismo , Hemólisis , Calor , Humanos , Sustancias Macromoleculares , Proteínas/metabolismo , Conejos , Ovinos , Factores de Tiempo , VitronectinaRESUMEN
The staphylococcal exotoxins toxic shock syndrome toxin 1 (TSST-1) and enterotoxin B were tested for their ability to stimulate murine peritoneal macrophages (PM) for tumoricidal activity. Both toxins were found to stimulate oil-elicited, gamma interferon-primed PM monolayers to kill nonadherent P815 tumor targets. The mechanism of killing of toxin-stimulated tumoricidal activity involved the production of nitric oxide, as nitrite could be demonstrated in culture fluids, and NG-monomethyl-L-arginine, an inhibitor of nitric oxide production, abrogated toxin-stimulated tumoricidal activity. TSST-1 stimulated the secretion of tumor necrosis factor by PM monolayers in the presence and absence of gamma interferon. The mechanism of toxin-stimulated tumoricidal activity was also determined to be independent of the production of reactive oxygen intermediates in that TSST-1 failed to stimulate H2O2 production by PM. These results demonstrate that the staphylococcal exotoxins are capable of stimulating macrophage production of nitric oxide for tumor cytotoxicity and suggest that the nitric oxide thus produced may subsequently play a role in the pathogenesis of the diseases caused by these toxins.
Asunto(s)
Toxinas Bacterianas , Enterotoxinas/farmacología , Macrófagos/inmunología , Óxido Nítrico/metabolismo , Staphylococcus aureus , Superantígenos , Animales , Arginina/análogos & derivados , Arginina/farmacología , Citotoxicidad Inmunológica , Peróxido de Hidrógeno/metabolismo , Interferón gamma/farmacología , Activación de Linfocitos , Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C3H , Óxido Nítrico/antagonistas & inhibidores , Células Tumorales Cultivadas/efectos de los fármacos , Factor de Necrosis Tumoral alfa/biosíntesis , omega-N-MetilargininaRESUMEN
Murine resident peritoneal macrophages (PM) were refractory to activation for antibody-dependent cellular cytotoxicity (ADCC) of SRBC targets as compared with either oil or thioglycollate-elicited inflammatory macrophages. Western blot analysis of macrophage cellular lysates indicated a direct correlation between the endogenous C1q levels and their innate response to activation for ADCC. Inflammatory PM had 7- to 14-fold higher C1q levels (ca. 23 to 45 ng C1q/100 micrograms protein) than resident PM (ca. 3 ng C1q/100 micrograms protein) as determined by densitometric scanning of blots. Purified exogenous mouse or human C1q were found to reconstitute the response of resident PM for ADCC mediated by C-activating mouse IgG2a or IgG2b mAb, but not by non-C-activating IgG1. Thioglycollate-elicited PM with highest endogenous C1q levels were unaffected by exogenous C1q, whereas oil-elicited PM with intermediate C1q levels were slightly augmented in their ADCC response by exogenous C1q. Augmentation of the resident PM response for ADCC activation was accomplished by either coincubation of effector macrophages with physiologic concentrations of C1q (0.5 to 4.0 micrograms/ml), IgG, and SRBC targets or by IgG and C1q preopsonized targets. FcR-dependent phagocytosis by resident PM was similarly reconstituted by exogenous C1q. The results indicate that resident macrophages with low potential for C1q biosynthesis and secretion were reconstituted by exogenous C1q in their FcR-dependent phagocytosis and ADCC, whereas inflammatory macrophages with sufficient endogenous C1q levels were largely unaffected. Thus C1q appears to have a pivotal mechanistic role in the initiation of macrophage activation for FcR-dependent effector functions.
Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos , Complemento C1q/farmacología , Macrófagos/inmunología , Fagocitosis , Receptores Fc/fisiología , Adyuvantes Inmunológicos/farmacología , Animales , Western Blotting , Fraccionamiento Celular , Activación de Complemento , Complemento C1q/aislamiento & purificación , Complemento C1q/fisiología , Humanos , Inmunoglobulina G/fisiología , Isotipos de Inmunoglobulinas/fisiología , Sustancias Macromoleculares , Activación de Macrófagos , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C3H , Cavidad PeritonealRESUMEN
Mouse resident peritoneal macrophages (PM) were reconstituted in their response to activation for antibody-dependent cellular cytotoxicity (ADCC) for sheep erythrocyte targets (SRBC) by subhemolytic dilutions of homologous or autologous sera. ADCC-responsive inflammatory PM were largely unaffected in their activation by exogenous serum. Augmentation of resident PM for ADCC by homologous serum was correlated with the complement-activating potential of the mouse monoclonal anti-SRBC IgG isotype in that serum augmented IgG gamma 2a greater than IgG gamma 2b much greater than IgG gamma 1. The active component of mouse serum was heat-labile at 56 degrees C for 30 min and was present in both C5-deficient AKR and C5-sufficient homologous C3H mouse sera. Western blot analysis of the cell lysates for Clq confirmed that oil-elicited and thioglycollate-elicited inflammatory PM had greater levels of endogenous Clq than did resident PM which correlated with their innate responsiveness for ADCC activation. Depletion of Clq from serum by immunoprecipitation with IgG antibody to Clq or by ion exchange chromatography removed the active reconstituting activity for ADCC. Purified mouse Clq (0.4 microgram) partially replenished the ADCC augmenting activity of Clq-depleted AKR mouse serum. SRBC targets preopsonized with IgG gamma 2a and purified mouse Clq (0.075-5.0 microgram/ml) fully reconstituted the ADCC response of resident PM similar to homologous serum indicating that the major active component of serum was Clq. Thus resident PM with low endogenous levels of Clq were reconstituted for ADCC by the addition of exogenous Clq, whereas inflammatory PM with sufficiently high endogenous levels of Clq were not further enhanced by exogenous Clq. Our findings indicate that Clq may provide an essential second signal in concert with Fc receptor binding of IgG to initiate ADCC activation of macrophages.
Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos , Enzimas Activadoras de Complemento/fisiología , Complemento C1/fisiología , Macrófagos/inmunología , Animales , Fenómenos Fisiológicos Sanguíneos , Enzimas Activadoras de Complemento/análisis , Complemento C1/análisis , Complemento C1q , Inmunoglobulina G/fisiología , Activación de Macrófagos , Masculino , Ratones , Ratones Endogámicos C3H , Cavidad Peritoneal/citologíaRESUMEN
Resident and oil-elicited inflammatory peritoneal macrophages (PM phi) from competent C3HeB/FeJ and genetically deficient C3H/HeJ mice were characterized for their Fc receptor (FcR)-dependent binding, phagocytic and ADCC functions during in vitro differentiation under the influence of mouse recombinant interferon-gamma (rIFN-gamma), interferon-alpha/beta (IFN-alpha/beta) fetal bovine serum (FBS), or in serum-free medium. Freshly cultured resident PM phi from C3HeB/FeJ mice had low levels of FcR-mediated phagocytosis in response to mouse monoclonal IgG gamma 2a, IgG gamma 2b or IgG gamma 1, opsonized sheep erythrocytes as compared to oil-elicited inflammatory PM phi from the same strain. Resident PM phi were uniformly upregulated in their FcR-dependent phagocytosis after 24-48 h in vitro culture with FBS to levels approximating that of freshly cultured inflammatory PM phi which were also further upregulated after 24 h in vitro culture with FBS. Both resident and inflammatory PM phi were upregulated largely by an autostimulatory process in that they increased their FcR-mediated phagocytosis in serum-free RPMI-1640 medium without the addition of rIFN-gamma or IFN-alpha/beta, although FBS further augmented FcR upregulation. A synergistic effect of FBS and rIFN-gamma was required for total reconstitution of FcR-mediated phagocytosis of FcR-incompetent C3H/HeJ inflammatory PM phi in that FBS or rIFN-gamma alone only partially reconstituted FcR function, whereas in combination full reconstitution occurred. Thus, macrophages from competent C3HeB/FeJ mice were upregulated in their FcR-mediated functions largely by an autostimulatory process, presumably dependent on endogenous of IFN-beta, whereas, genetically-deficient C3H/HeJ macrophages required exogenous rIFN-gamma in combination with fetal bovine serum for synergistic reconstitution of FcR functions. The uniform upregulation of FcR-dependent effector functions in vitro appears to provide an efficient system for enhanced immune function during differentiation which may be applicable to in vivo situations.
Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Interferones/farmacología , Macrófagos/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Receptores Fc/fisiología , Animales , Bovinos , Células Cultivadas , Sangre Fetal/fisiología , Inflamación , Macrófagos/clasificación , Masculino , Ratones , Ratones Endogámicos C3HRESUMEN
Mouse peritoneal macrophages possess distinct Fc receptors (FcR) for binding the various murine IgG isotypes. FcRI binds monomeric IgG gamma 2a, but not monomeric IgG gamma 2b or IgG gamma 1 with high affinity at 4 degrees C and is sensitive to trypsin degradation. We have assessed the functional consequences of the cytophilic binding at 4 degrees C of monomeric IgG gamma 2a to FcRI of mouse peritoneal macrophages using newly developed photometric microassays for quantification of binding, phagocytosis, and antibody dependent cellular cytotoxicity (ADCC) of post-opsonized sheep red blood cell (SRBC) targets. Dose-dependent binding specificity of monomeric IgG gamma 2a, but not IgG gamma 2b or IgG gamma 1 to FcRI of oil-elicited mouse peritoneal macrophages at 4 degrees C for 2 h was confirmed to display typical saturation kinetics both by the photometric assay and by a cellular enzyme-linked immunosorbent assay (CELISA). Binding of monomeric IgG gamma 2a to macrophage FcRI promoted highly efficient phagocytosis of opsonized SRBC in that most cells that were bound were also rapidly internalized by the phagocytic process during a 1 h incubation at 37 degrees C. Upregulation of FcRI-dependent binding and phagocytosis occurred during 24-48 h in vitro culture of macrophages as shown both by the photometric assays and CELISA. Trypsin treatment of macrophages abrogated FcRI-dependent binding and phagocytosis by monomeric IgG gamma 2a, but had little effect on FcRII-dependent functions. Cytophilic binding of monomeric IgG gamma 2a to FcRI failed to trigger ADCC activation. Thus functional characterization of macrophage FcRI-dependent effector functions confirmed the fidelity of binding specificity of monomeric IgG gamma 2a to a trypsin degradable receptor which mediates highly efficient phagocytosis but fails to initiate the signal for ADCC activation. It appears that passively bound immune monomeric IgG gamma 2a could provide an efficient mechanism by macrophages in vivo for FcRI-dependent immune clearance of soluble or particulate cellular antigens without elicitation of potentially harmful cytolytic factors associated with ADCC activation.
Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos , Antígenos de Diferenciación/análisis , Citofotometría , Inmunoglobulina G/fisiología , Macrófagos/inmunología , Fagocitosis , Receptores Fc/análisis , Animales , Antígenos de Diferenciación/metabolismo , Citofotometría/métodos , Inmunoglobulina G/metabolismo , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C3H , Cavidad Peritoneal/citología , Fagocitosis/efectos de los fármacos , Receptores Fc/metabolismo , Receptores de IgG , TripsinaRESUMEN
The relationship between Fc receptor (FcR) function and activation of murine macrophage populations for non-specific tumor cytotoxicity was studied. Oil-elicited inflammatory peritoneal macrophages (PM phi) from C3HeB/FeJ mice had higher FcR function upon harvest than resident PM phi from the same strain or elicited PM phi from genetically deficient C3H/HeJ mice. C3HeB/FeJ inflammatory PM phi were uniformly responsive to activation by MAF and the complement activators: LPS, Poly I:C, cobra venom factor (CVF) and zymosan for tumoricidal activity. Resident cells from the same strain and C3H/HeJ-elicited PM phi were uniformly unresponsive to the same activators. In vitro culture of C3HeB/FeJ resident PM phi with fetal bovine serum for 24-48 h produced unregulation of FcR function which coincided with a conversion from an unresponsive to a responsive state for tumoricidal activity. Reconstitution of the FcR function of C3H/HeJ-elicited PM phi during 24-48 h culture with lymphokine or Poly I:C also coincided with the restoration of responsiveness to activation by LPS, CVF, and zymosan for tumor cytotoxicity. Thus, the consistent temporal relationship between upregulated FcR function and the capacity of macrophages to respond to activation for non-specific tumoricidal activity may be more than coincidental. Preincubation of responsive C3HeB/FeJ-elicited PM phi with insoluble immune complex or heat-aggregated IgG was shown to blockade FcR-mediated phagocytosis and to abrogate LPS-mediated tumoricidal activity. Interestingly, FcR blockade by IgG-opsonized sheep erythrocyte conjugates selectively inhibited activation by MAF, LPS, and Poly I:C, but had no inhibitory effect on activation by CVF or zymosan. Similar blockade of C3b receptors produced an identical pattern of selective inhibition of activation. This selective inhibition of non-specific tumoricidal activity by FcR/C3bR blockade suggests the existence of two pathways for antibody-independent activation of macrophages.
Asunto(s)
Citotoxicidad Inmunológica , Macrófagos/inmunología , Fagocitosis , Receptores Fc/inmunología , Animales , Inmunocompetencia , Activación de Linfocitos , Linfocinas/farmacología , Factores Activadores de Macrófagos , Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C3H , Neoplasias Experimentales/inmunología , Cavidad Peritoneal/citología , Poli I-C/farmacologíaRESUMEN
An improved microassay for quantitation of murine macrophage-mediated antibody-dependent cellular cytotoxicity (ADCC) has been developed. The method is based on the turbidimetric measurement of sheep erythrocyte or nucleated (L1210) target cell suspensions at 630 nm with an automatic microtiter plate densitometer. The novel method was applied to demonstrate dose-related increases in murine macrophage mediated ADCC with varying antibody concentration, effector:target ratio, and incubation time. Advantages of the turbidimetric method were shown over the 51Cr-labeled target cell method by direct comparisons in that the new method was 2-4 times more sensitive and allowed repeated readings of the same plate after various incubation time intervals. The method provides further advantages of (1) elimination of the need for expensive and hazardous radioactive materials, (2) relative ease and rapidity in which experiments may be performed and quantitated, (3) sensitivity and reproducibility, and (4) versatility of the assay for measuring cytotoxicity of either erythrocyte or nucleated target cells.
Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos , Técnicas Inmunológicas , Macrófagos/inmunología , Animales , Radioisótopos de Cromo , Eritrocitos/inmunología , Leucemia L1210/inmunología , Masculino , Ratones , Nefelometría y Turbidimetría , OvinosRESUMEN
Saline extracts of logarithmic-phase Pasteurella haemolytica, serotype 1, were separated by chromatofocusing. The resulting fractions were analyzed by immunodiffusion and an enzyme-linked immunosorbant assay, and six antigen groups (AG's) were identified. AG 1 did not bind to the column, AG's 2, 3 and 4 were eluted with a decreasing pH gradient, and AG's 5 and 6 were eluted with an increasing NaC1 gradient. Fractions containing each AG were pooled and further purified by gel filtration. The AG's were subsequently characterized as to protein, carbohydrate and 2-keto-3-deoxyoctanate (KDO) content. AG's 1, 5, and 6 had higher carbohydrate contents than AG's 2, 3 and 4. Only AG 5 contained detectable levels of KDO. The AG's were also analyzed by SDS-PAGE and immunoblotting. Each AG produced a characteristic pattern of proteins and antigens, although two antigenic proteins were common to all AG's. AG 1 contained the greatest number of antigenic proteins. Immunization of mice with each AG in Freund's incomplete adjuvant resulted in a strong antibody response to the homologous AG for four of the six AG's. Limited protection against a P. haemolytica challenge was observed in mice that were immunized with AG 2 or 4.
Asunto(s)
Antígenos Bacterianos/aislamiento & purificación , Pasteurella/inmunología , Animales , Anticuerpos Antibacterianos/biosíntesis , Antígenos Bacterianos/administración & dosificación , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/aislamiento & purificación , Bovinos , Enfermedades de los Bovinos/inmunología , Enfermedades de los Bovinos/prevención & control , Femenino , Inmunización , Ratones , Infecciones por Pasteurella/inmunología , Infecciones por Pasteurella/prevención & control , Infecciones por Pasteurella/veterinaria , Pasteurelosis Neumónica/inmunología , Pasteurelosis Neumónica/prevención & controlRESUMEN
A procedure was developed for separating antigens associated with a saline extract of Pasteurella haemolytica serotype 1. Seven antigens were identified by immunoelectrophoresis to be associated with the extract. The extract was subjected to preparative isoelectrofocusing in a pH range of 3-10. The majority of extracted proteins were found to have pI's of 4-6, whereas the carbohydrate antigen(s) were distributed over a pI range of 3.0-8.0. The fractions that were of interest were pooled and refocused in a narrower pH range to improve resolution of the protein antigens. Specific antigens from defined pH ranges were pooled to form 6 antigen groups. These antigen groups were examined further by immunoelectrophoresis, analytical isoelectrofocusing, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The molecular weights of the proteins found in the capsular extracts ranged from 33 k to greater than 80 k. Injection of mice with capsular extract or antigen Groups 1-6 in Freund's incomplete adjuvant resulted in a serum antibody response to the various antigens as detected by an enzyme-linked immunosorbent assay. Significant protection (P less than 0.05) against challenge with virulent P. haemolytica was seen in mice injected with antigen Groups 2 and 4. Six calves were immunized with saline extract. These calves had greater resistance to experimental pneumonic pasteurellosis than did 6 non-vaccinated calves. A serum antibody response to the crude extract and to each antigen group was detected in vaccinated calves by an enzyme-linked immunosorbant assay.
Asunto(s)
Antígenos Bacterianos/aislamiento & purificación , Proteínas Bacterianas/aislamiento & purificación , Enfermedades de los Bovinos/inmunología , Pasteurella/inmunología , Animales , Anticuerpos Antibacterianos/análisis , Anticuerpos Antibacterianos/biosíntesis , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Bovinos , Ensayo de Inmunoadsorción Enzimática , Femenino , Concentración de Iones de Hidrógeno , Inmunización Secundaria , Focalización Isoeléctrica , Masculino , Ratones , Peso Molecular , Infecciones por Pasteurella/inmunología , Infecciones por Pasteurella/microbiología , Infecciones por Pasteurella/veterinaria , Serotipificación , Cloruro de Sodio , VacunaciónRESUMEN
A photometric microassay has been developed to quantitate macrophage Fc and C3b receptor mediated binding and phagocytosis by measuring the absorbance of macrophage associated erythrocytes at 405 nm on an automated densitometer. The method compares favorably in sensitivity and kinetics to the 51Cr-labeled erythrocyte assay. Saturation and linear dose response kinetics were demonstrable for both total index and phagocytic index of either Fc receptor or C3b receptor. The assay allowed detection of significant differences in Fc receptor function with varying macrophage densities and between Fc receptor competent (C3HeB/FeJ) macrophages and Fc receptor deficient (C3H/HeJ) macrophages. A valid binding index was derived at 37 degrees C by computing the difference between the total and phagocytic indices, which compared favorably with binding studies at 4 degrees C. This new procedure provides a simple, rapid and reproducible microassay for the quantitation of Fc/C3b receptor dependent binding and phagocytosis which offers distinct advantages over the laborious rosette assay and the 51Cr-labeled erythrocyte assay.
Asunto(s)
Macrófagos/fisiología , Receptores de Complemento/fisiología , Receptores Fc/fisiología , Animales , Radioisótopos de Cromo , Eritrocitos/inmunología , Masculino , Ratones , Ratones Endogámicos C3H , Fagocitosis , Fotometría , Receptores de Complemento 3bRESUMEN
Using 6- to 8-month-old beef calves, 3 experiments were conducted to compare the effect of vaccination with live or killed Pasteurella haemolytica on resistance to a transthoracic challenge exposure with the organism and to correlate serum antibody response with resistance. In each experiment, calves were vaccinated twice at 1-week intervals and were challenge exposed 21 days after the first inoculation. Lung lesions were evaluated by a system, such that higher scores indicated the more severe lesions. In each experiment, calves immunized with live P haemolytica had lower lesion scores than calves vaccinated with saline solution or bacterin. In 2 of the experiments, the differences were significant (P less than 0.05). In all experiments, calves vaccinated parenterally with a commercial P haemolytica/P multocida bacterin or with a formalin-killed P haemolytica bacterin had lesion scores that were not significantly different (P greater than 0.05) than for control calves vaccinated with saline solution. Live and killed bacterial preparations induced a significant serum antibody response to P haemolytica as measured by a quantitative fluorometric immunoassay. The antibody response to vaccination was not affected by preexisting titers to P haemolytica. Serum antibody titers were not consistently as high for calves vaccinated with bacterins as for calves vaccinated with live organisms. Although high antibody titers correlated with low lesion scores when calves vaccinated with saline solution or live organisms were analyzed collectively, there was not a significant correlation between the 2 variables when calves, vaccinated with saline solution or with bacterin, were analyzed collectively. These data indicate that, although bacterins may induce a detectable serum antibody response, they do not induce protection against transthoracic challenge exposure to P haemolytica.
Asunto(s)
Anticuerpos Antibacterianos/biosíntesis , Vacunas Bacterianas/inmunología , Enfermedades de los Bovinos/inmunología , Infecciones por Pasteurella/inmunología , Pasteurella/inmunología , Pasteurelosis Neumónica/inmunología , Animales , Bovinos , Enfermedades de los Bovinos/patología , Femenino , Fluorometría , Inmunoensayo , Pulmón/patología , Masculino , Pasteurelosis Neumónica/patología , Vacunación/veterinaria , Vacunas Atenuadas/inmunologíaRESUMEN
Five experiments were conducted that compared aerosol immunization of calves with live Pasteurella haemolytica from logarithmic (6 hour) or stationary (20 to 22 hour) phase cultures. Calves were challenge exposed by transthoracic injection with P haemolytica. In 4 experiments, calves inoculated with 6-hour cultures had slightly lower mean lesion scores (indicating greater resistance to challenge exposure) than those inoculated with 20- to 22-hour cultures. High antibody titers, as detected by a quantitative fluorometric immunoassay or the indirect hemagglutination test, correlated directly with lung resistance (based on lesion scores) regardless of the age of the culture used as the immunogen.