RESUMO
Although nonclassical (class I-b) gene products represent the majority of murine major histocompatibility complex (MHC) genes, the role of these relatively nonpolymorphic molecules remains uncertain. Recently, one such protein, H-2M3 (formerly designated Hmt), was shown to bind and specifically present N-formylated peptides to cytotoxic T lymphocytes. Because N-formylation is characteristic of prokaryotic proteins, this MHC molecule may be especially adapted for a role in the mammalian defense against bacterial attack. The current studies demonstrate that an MHC molecule, indistinguishable from H-2M3, presents antigens derived from the intracellular pathogen Listeria monocytogenes to Listeria-specific CD8+ cells.
Assuntos
Antígenos de Bactérias/imunologia , Antígenos H-2/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Listeria monocytogenes/imunologia , Listeriose/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Formiatos/imunologia , Formiatos/metabolismo , Antígenos H-2/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/imunologia , Peptídeos/metabolismo , Linfócitos T Citotóxicos/imunologiaRESUMO
The effects of IgG in different configurations on the Fc receptor function of human monocytes were studied. Receptor function was assessed by quantitating immune adherence and/or ingestion of human erythrocytes coated with IgG anti-D antibody. Monomeric IgGl in solution inhibited the Fc receptor function of monocytes, but this function was restored completely after washing. In contrast, monomeric IgG that was adsorbed nonspecifically to a plastic surface inhibited the Fc receptor function of monocytes even after washing away unbound IgGl. This loss of function could be blocked by sodium azide and was reversed when the IgG adsorbed to plastic was degraded by trypsin, suggesting that loss of function was the reversible consequence of localized binding of most of the monocyte's receptors at the point of contact with immobilized IgGl. Fluid-phase aggregates of IgGl also reduced the Fc receptor function of monocytes as a consequence of direct binding to the monocyte surface. High concentrations of purified aggregates rapidly reduced Fc receptor function but function was reversed by trypsin even after incubation for 18 h. Lower concentrations of aggregates reduced Fc receptor function more slowly, but after 18 h of incubation, lost function was not restored by trypsin treatment. Because the transition from reversible to irreversible loss was blocked by sodium azide, an energy-dependent process of ingestion, shedding or denaturation of receptors is responsible for this irreversible loss of Fc receptor function. Rabbit IgG anti-human IgG bound to IgG adsorbed to the surface of monocytes also mediated a loss of Fc receptor function as a result of the binding of Fc receptors to the Fc portion of the rabbit IgG molecule, a process analogous to the binding of aggregated IgG. After irreversible depletion of Fc receptor function by anti-IgG, partial recovery of function was detectable within 12-24 h of incubation in vitro, and this recovery was blocked by cycloheximide, suggesting that new receptor synthesis was required for restoration of function.
Assuntos
Fragmentos Fc das Imunoglobulinas/imunologia , Monócitos/imunologia , Anticorpos Anti-Idiotípicos/imunologia , Azidas/farmacologia , Humanos , Imunoglobulina G/imunologia , FagocitoseRESUMO
Fc-receptor-mediated clearance and nonspecific phagocytic clearance were assessed after the infusion of monomeric human IgG, heat-aggregated human IgG, and a monoclonal anti-mouse macrophage FcII receptor antibody (2.4G2) into normal mice. Each agent blocked Fc-receptor function in vivo, but 2.4G2 was much more potent per microgram than the other agents. Monomeric IgG in blocking doses did not affect other aspects of immune function. In contrast, aggregated IgG, and to a lesser extent, 2.4G2 reduced serum complement levels. In addition, these agents also caused moderate reductions in nonspecific phagocytic function. Monoclonal anti-mouse macrophage C3bi receptor antibody (Mac-1), another monoclonal antibody which binds to macrophage CR3 receptors without interfering with Fc-receptor function, also reduced serum complement and inhibited nonspecific phagocytic function. Complement depletion alone (produced by infusion of cobra venom factor) could not account for the observed changes in Fc receptor or nonspecific phagocytic function. We conclude that both monomeric IgG and anti-Fc-receptor antibodies can markedly inhibit Fc-receptor function in vivo; however, the pattern of physiologic changes produced by these agents differs.
Assuntos
Anticorpos Monoclonais , Sistema Imunitário/efeitos dos fármacos , Receptores Fc/imunologia , gama-Globulinas/farmacologia , Animais , Proteínas do Sistema Complemento/metabolismo , Temperatura Alta , Humanos , Imunoglobulina G/farmacologia , Injeções Intravenosas , Masculino , Matemática , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Receptores de Complemento/imunologia , Receptores de Complemento/metabolismo , Receptores de Complemento 3b , Receptores de IgGRESUMO
Covalently cross-linked dimers and oligomers composed of 2-4 subunits of monoclonal human IgG1 were prepared by incubation of purified monomeric IgG1 with glutaraldehyde followed by gelfiltration chromatography. Monomers, dimers, and oligomers then were labeled with (125)I and used to compare the binding properties of IgG Fc receptors on human peripheral blood monocytes and polymorphonuclear leukocytes (PMN). Binding of IgG1 to monocytes at 37 degrees C and of IgG1 polymers to PMN at 4 degrees C could be readily measured and were found to be reversible and saturable. Scatchard plots of binding were linear in each instance. Monocytes bound a mean of 20,200+/-6,800 molecules/cell of IgG1 monomer at saturation and comparable amounts of dimer or oligomer. The mean association constant (Ka) for binding of IgG1 monomer to monocytes was 8.6 x 10(8)M(-1) and the Ka for binding of dimer and oligomer were three-to fivefold greater.In contrast, PMN bound a mean of 460,000+/-130,000 molecules of IgG1 dimer at saturation and comparable amounts of oligomer. The Ka of binding in both cases was 100-1,000-fold lower than the Ka for binding of the same preparations to monocytes. Binding of labeled IgG1 to both cell types was more potently inhibited by unlabeled IgG1 and IgG3 than by IgG4 or IgG2. Binding of labeled polymers of IgG1 to monocytes was 10-100-fold more easily inhibited by monomeric IgG1 than was binding to PMN. Thus, there are significant quantitative and qualitative differences between the binding properties of Fc receptors present on monocytes and PMN.
Assuntos
Imunoglobulina G/metabolismo , Monócitos/metabolismo , Neutrófilos/metabolismo , Receptores Fc/metabolismo , Anticorpos Monoclonais , Humanos , Cinética , Polímeros , Relação Estrutura-AtividadeRESUMO
Monocyte-mediated lysis in vitro of human red cells coated with measured amounts of immunoglobulin G (IgG) or complement were studied. 1,000-1,500 molecules of IgG anti-D are necessary to effect measurable lysis, and lysis increases linearly with increasing levels of antibody sensitization. 100 microgram/ml of IgG1 abolished lysis even at maximal levels of anti-D sensitization (15,000 molecules/cell). Two isoimmune IgG anti-A or anti-B antisera were 5 to 10-fold less efficient in promoting phagocytosis or lysis per molecule of IgG bound; however, because of the greater antigen density of A or B, more than 100,000 molecules IgG/cell could be bound, producing equivalent lysis to anti-D-coated cells. Although inhibition by IgG1 was similar at equivalent levels of sensitization with anti-A, anti-B, or anti-D at high levels of coating with anti-A or anti-B (not attainable with anti-D), lysis was not inhibited by IgG1. Cells coated with human complement components alone were not lysed by monocytes; however, complement coating augmented IgG-mediated lysis and reduced the quantity of anti-D necessary to produce lysis to less than 1,000 molecules/cell. After thorough degradation of C3b by serum to C3d, complement augmentation persisted.
Assuntos
Complemento C3/metabolismo , Eritrócitos/imunologia , Hemólise , Imunoglobulina G , Isoanticorpos , Monócitos/imunologia , Sistema ABO de Grupos Sanguíneos , Citotoxicidade Celular Dependente de Anticorpos , Relação Dose-Resposta Imunológica , Humanos , Alótipos de Imunoglobulina , Fagocitose , Sistema do Grupo Sanguíneo Rh-HrRESUMO
Allospecific CD8(+) T lymphocytes are an important component of the cellular response in allograft rejection. These cells recognize and engage MHC class I antigens, leading to allospecific cytolytic responses and graft rejection. In mouse kidney allografts that survive to 3 wk after transplantation, we noted that the majority of CD8(+) cells do not express surface alpha/beta T cell receptor alpha/beta(TCR), gamma/deltaTCR, or CD3. However, these CD8(+)TCR- cells did express surface markers characteristic of T cells, including Thy1.2, CD2, and CD5. In addition, the CD8(+)TCR- cells expressed mRNA for TCR Vbeta gene families, and nearly half stained positive for cytoplasmic Vbeta8 protein, suggesting that they are T cells that have downregulated alpha/betaTCR protein expression from their cell surfaces. When these surface TCR- cells were isolated from kidney allografts by flow cytometry and cultured in the presence of either allogeneic or syngeneic stimulators, nearly 100% of cells reacquired normal levels of alpha/betaTCR expression with disproportionate usage of Vbeta8 chains. After recovery of their surface TCR expression, the CD8(+)TCR- population demonstrated strong alloreactivity in culture. These results suggest that the substantial number of CD8(+)TCR- cells found in long-term surviving mouse kidney allografts are alpha/beta-T cells that have downregulated their cell surface expression of TCR. While in other systems this phenotype may identify cells that have engaged antigen, our results indicate that loss of TCR expression by CD8(+) kidney graft-infiltrating cells may not depend on antigen engagement and that elements in the microenvironment of the kidney graft play a key role in this process. Factors that modulate expression of TCR by graft-infiltrating lymphocytes may have an important role in regulating rejection responses.
Assuntos
Linfócitos T CD8-Positivos/química , Transplante de Rim/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Animais , Citocinas/genética , Regulação para Baixo , Rejeição de Enxerto , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , RNA Mensageiro/análise , Transplante HomólogoRESUMO
To identify the role of donor class I alloantigens in regulating the CD8+ T cell response to a kidney allograft, we analyzed and compared the CD8+ infiltrate in kidney transplants from MHC class I-deficient (class I-) mouse donors and class I+ controls. One week after transplantation, there was a prominent CD8+ infiltrate in control allografts, whereas CD8+ T cells were virtually absent in grafts from class I- donors. In class I+ allografts, infiltrating CD8+ cells utilized a wide range of T cell receptor (TCR) Vbeta families and their Vbeta usage was similar to that of the systemic CD8+ population. However, there was a modest but significant overrepresentation of cells bearing Vbeta8 in the graft compared with the spleen due to an expansion of CD8+ Vbeta8.3+ cells. This could be detected as early as 1 week and became more pronounced by 3 weeks after transplantation. In 3-week allografts, only 52% of CD8+ cells expressed alphabetaTCR. Among T cells isolated from class I+ grafts, the CD8+ Vbeta8+ cells demonstrated allospecific responses that were numerically larger than responses of the CD8+ Vbeta8- population. In contrast to the early (1 week) time point, significant numbers of CD8+ cells could be isolated from class I- grafts by 3 weeks after transplantation and their Vbeta repertoire resembled that seen in controls. While increasing numbers of CD8+ Vbeta8+ were present in the class I- grafts at 3 weeks, this increase was not statistically significant. Thus, expression of class I alloantigens on a kidney graft plays an important role in regulating the rate of accumulation of CD8+ T cells in rejecting kidney grafts. However, the TCR Vbeta repertoire of the CD8+ T cell infiltrate is largely determined by factors that are independent of normal class I expression on the graft.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Rejeição de Enxerto/imunologia , Transplante de Rim/imunologia , Animais , Citometria de Fluxo , Interferon gama/biossíntese , Transplante de Rim/patologia , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Baço/imunologiaRESUMO
The effect of murine IgG hybridoma antibodies directed against leukocyte antigens on the Fc receptor function of human cells was studied. For this purpose, the specific binding of 125I-labeled monomeric human IgG1 to a macrophage-like cell-line (U-937) was quantitated before and after incubation in the presence of murine monoclonal hybridoma antibodies. Four monoclonal hybridoma antibodies (A1G3, 23D6, 4F2, and 3A 10), each of which binds to different antigens on the surface of U-937 cells, rapidly and potently inhibited the specific binding of labeled IgG1 to these cells. Inasmuch as inhibition was mediated only by IgG antibodies with an intact Fc fragment and antibody activity against surface antigens found on U-937, inhibition appears to have resulted from the formation of a three-component complex composed of antibody bound by its Fab portion to antigen and by its Fc fragment to a Fc receptor. Equilibrium binding studies performed on treated cells confirmed that reduced Fc receptor-mediated binding was due to a reduction in the number of available receptors. Binding studies employing double isotope labeling methods demonstrated that about 0.5 to 1.0 Fc receptor was blocked for each molecule of intact antibody bound to a U-937 cell. Using several techniques, it was shown that most of the monoclonal antibody bound to cells and the Fc receptors blocked by antibody remained on the cell surface despite incubation at 37 degrees C for 3 hr. Thus, the loss of receptor function observed in these experiments was almost exclusively due to reversible receptor blockade rather than receptor internalization or degradation. The antibodies identified in these studies also markedly inhibited Fc receptors on one other human cell line (HL-60) as well as those on normal human peripheral blood monocytes.
Assuntos
Anticorpos Monoclonais/fisiologia , Antígenos de Superfície/imunologia , Leucócitos/metabolismo , Receptores Imunológicos/análise , Animais , Sítios de Ligação de Anticorpos , Ligação Competitiva , Plaquetas/metabolismo , Linhagem Celular , Humanos , Hibridomas/imunologia , Camundongos , Monócitos/metabolismo , Neutrófilos/metabolismo , Receptores de IgGRESUMO
When peripheral blood lymphocytes and human red cells coated with IgG were incubated in vitro in culture medium, antibody-dependent lymphocyte-mediated lysis was observed. This lysis was markedly inhibited by the addition of purified monoclonal IgG1 (1000 microgram/ml) to the culture medium. In contrast, lysis by lymphocytes of sensitized red cells in the presence of undiluted human serum was equal to or greater than lysis in medium alone, even in the presence of IgG1 at 1000 microgram/ml, despite the high concentration of IgG in human serum (6000--19,000 microgram/ml). Serum heated to 56 degrees C for 30 min also restored lysis in the presence of IgG1. When serum was separated into three fractions by passage through a Sephadex G-200 column, the third fraction, which contained proteins with a molecular weight of less than 100,000 d (but neither of the other two fractions nor purified human albumin), restored lymphocyte-mediated lysis in the presence of IgG1.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Eritrócitos/imunologia , Imunoglobulina G , Linfócitos/imunologia , Sangue , Células Clonais/imunologia , Meios de Cultura , HumanosRESUMO
To examine the physiologic mechanisms responsible for enhanced antibacterial activity during infection with Listeria monocytogenes (LM), we developed an in vitro assay for quantifying leukocyte anti-listerial activity (LAA) in spleen and bone marrow. When LAA was serially measured in C57B1/6 (B6) mice infected i.v. with LM, two distinct phases of response were observed. Splenic LAA increased four- to fivefold during the first 2 days after i.v. infusion of LM (from 2.4 +/- 1.8 U/spleen before infection to 11.8 +/- 2.4, p less than 0.01), dropped significantly on days 3 to 4, and increased again to similar levels from days 5 to 7. A fall in bone marrow activity from the 3.5 +/- 1.5 to 1.6 +/- 0.7 U/mouse (two femurs) coincided with the initial rise in splenocyte activity, and was followed by a gradual return to base line. Bacterial containment in vivo correlated well with splenic LAA in vitro. Carbonyl iron pretreatment of cells from both normal and LM-infected animals ablated LAA, suggesting the effectors were phagocytic. LAA in normal spleens was unaffected by 400 rad; LAA of normal marrow as well as splenocyte and marrow cell suspensions obtained 2 days after LM infection was markedly reduced by this dose of irradiation. Quantitative studies of spleen composition revealed a 10-fold increase in polymorphonuclear neutrophils between day 0 and day 2 followed by a marked decrease on day 3; this pattern closely resembled the changes in LAA observed during the same period. In contrast, splenic macrophage number did not increase from base line until after day 3. To look for evidence of changes in the efficiency of bacterial killing by phagocytes during infection, we calculated LAA/splenic phagocyte. The efficiency of killing increased threefold over base line within 1 day after LM infusion but we detected no additional increases later in infection. Because cytokines may have mediated some or all of the changes observed, we measured the capacity of splenocytes obtained at various times after infection to produce IL-2, TNF, and IFN-gamma in vitro. TNF activity increased in parallel with the first and second LAA peaks, whereas increases in IL-2 and IFN-gamma activity were associated only with the second.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Movimento Celular , Listeriose/patologia , Linfocinas/biossíntese , Fagócitos/microbiologia , Animais , Antibiose , Técnicas Bacteriológicas , Medula Óssea/microbiologia , Contagem de Leucócitos , Listeria/crescimento & desenvolvimento , Listeriose/metabolismo , Listeriose/microbiologia , Macrófagos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos , Fagócitos/fisiologia , Baço/microbiologiaRESUMO
Infection with Listeria monocytogenes stimulates T cell proliferation and T cell-derived lymphokine production. The release of lymphokines, in turn, "activates" macrophages, enhancing their bactericidal capacity. Because prior studies suggest that I-A+ accessory cells play a critical role in this pathway, we assessed the effects of an anti-I-A antibody on the murine host resistance to listerial infection. To this end, we infused Listeria into control C57BL/6 mice (I-Ab haplotype) and mice of the same strain which had been pretreated 18 hr earlier with D3137 (a monoclonal IgG2a anti-I-Ab,d antibody). Preliminary studies demonstrated that this antibody can markedly inhibit antigen-induced proliferation of Listeria-dependent T cells in vitro and (at a dose of 1 mg/animal) can markedly reduce I-A expression on splenocytes in vivo. Even though D3137 pretreatment prevented the splenomegaly normally observed after Listeria infusion into mice, it protected animals infused with otherwise lethal concentrations of Listeria. Because antibody-treated animals had sevenfold fewer organisms in their spleens 18 hr after infection and 1000-fold fewer organisms than control animals 3 days after infection, improved survival resulted from an antibody-induced increase in the bactericidal capacity of the MPS. Protection was not noted when C1.18.4 (an IgG2a myeloma protein without known antibody activity) was infused into C57BL/6 mice or when D3137 was infused in B10.BR (I-Ak) mice. D3137 also protected (B10 X B10.BR)F1 mice (which are hybrids bearing I-Ab and I-Ak), suggesting that complete blockade of antigen presentation is not a prerequisite for its protective action. Further studies into the mechanism for these effects may provide new insights into the pathophysiology of MPS activation in response to immunologic challenge.
Assuntos
Antígenos de Histocompatibilidade Classe II/imunologia , Isoanticorpos/imunologia , Listeriose/imunologia , Linfócitos T/imunologia , Animais , Imunidade Inata , Imunização Passiva , Listeriose/prevenção & controle , Linfocinas/metabolismo , Ativação de Macrófagos , Masculino , Camundongos , Camundongos Endogâmicos/imunologia , Baço/patologia , Linfócitos T/metabolismoRESUMO
To examine the regulation of cytokine synthesis during murine listeriosis, we have monitored IFN-gamma, TNF-alpha, and IL-1 beta mRNA levels in the spleens of C57B1/6 mice after the i.v. infusion of virulent and nonvirulent preparations of Listeria monocytogenes (LM). Messenger RNA coding for TNF, IL-1, or IFN did not become detectable until approximately 12 to 15 h after the infusion of virulent LM. Levels of each cytokine mRNA then increased synchronously reaching peak or near peak levels around 24 h after infection. Levels gradually decreased over the next 4 to 5 days. Unlike virulent LM, neither heat-killed LM, nor nonvirulent LM variants lacking listeriolysin O, stimulated monokine or IFN mRNA accumulation even when administered in very large doses. To gain perspective concerning the response to LM, we examined the early pattern of cytokine mRNA accumulation induced by Salmonella typhimurium (ST), an intracellular pathogen expressing LPS. We noted at least three significant differences between the cytokine responses to LM and ST: 1) monokine mRNA levels increased much more rapidly (within 1 h) after ST infection; 2) unlike LM, ST retained the capacity to stimulate cytokine mRNA production when injected as heat-killed bacteria; 3) in contrast to LM, ST could not trigger the early IFN production characteristic of LM infection. Our data suggest that monokine and IFN production early in listeriosis are critically linked with the process of bacterial invasion of host cells. The timing and pattern of cytokine mRNA accumulation in this setting is qualitatively different from that induced by LPS. The pathway described in these studies may also play a role in the host cytokine response to other intracellular pathogens as well.
Assuntos
Citocinas/biossíntese , Listeriose/imunologia , Baço/metabolismo , Actinas/biossíntese , Animais , Northern Blotting , Expressão Gênica , Interferon gama/biossíntese , Interleucina-1/biossíntese , Listeria monocytogenes/patogenicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Salmonella/imunologia , Salmonella typhimurium , Fatores de Tempo , Fator de Necrose Tumoral alfa/biossíntese , Vacinas Atenuadas/farmacologia , Vacinas de Produtos Inativados/farmacologiaRESUMO
Immune CD8 cells, which play an essential role in the adoptive transfer of antilisterial immunity, can specifically lyse Listeria-bearing macrophages in vitro in an MHC-unrestricted manner. In contrast, the adoptive transfer of immunity by unseparated immune lymphocytes has been reported to be MHC-restricted. To address the restriction properties of CD8 effectors in vivo, we assessed their efficacy in protecting syngeneic and allogeneic recipients. Protection was determined by comparing the number of viable splenic Listeria in naive mice and in recipients of 60 million CD8-enriched, L3T4-depleted, Listeria-immune spleen cells, 2 days after the infusion of 10,000 Listeria. Donor cells from B6 (H-2b) mice transferred about 4 logs of protection in syngeneic recipients and more than 2 logs in allogeneic B10.A (H-2a) or B10.BR (H-2k) mice. Immune B10.A CD8 cells transferred equivalent protection to B6 mice. Protection was almost completely abrogated by the lysis or lethal irradiation of CD8 cells before transfer in vivo. On the other hand, the depletion of macrophages or NK cells did not impair adoptive transfer. By comparison, nonimmune CD8 cells from normal mice or from mice stimulated with an irrelevant Ag in vivo did not transfer substantial immunity to allogeneic recipients. We have noted previously that protective CD8 cells inhibit phagocyte accumulation in the spleen of Listeria-infected syngeneic recipients. In the present studies, we observed similar changes in adoptively immunized allogeneic mice. Reduced phagocyte accumulation may reflect Listeria-dependent lysis of infected phagocytes by immune CD8 cells. In support of this, we showed that Listeria-immune donor cells rapidly acquired the capacity to mediate Listeria-dependent, MHC-unrestricted lysis of macrophages after incubation with small amounts of IL-2 in vitro. In sum, our data establish that Listeria-immune CD8 cells can function in vivo in MHC incompatible hosts, and indirectly support the hypothesis that the destruction of infected phagocytes may be important in T cell-mediated immunity against Listeria and perhaps other intracellular pathogens.
Assuntos
Antígenos de Diferenciação de Linfócitos T , Antígenos H-2/genética , Imunização Passiva , Listeriose/imunologia , Transfusão de Linfócitos , Baço/imunologia , Animais , Antígenos CD8 , Movimento Celular , Separação Celular , Citotoxicidade Imunológica , Epitopos/fisiologia , Antígenos H-2/fisiologia , Imunidade Inata , Células Matadoras Naturais/imunologia , Listeria/imunologia , Listeriose/genética , Listeriose/patologia , Linfócitos/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/patologia , FenótipoRESUMO
IFN-gamma clearly plays an important role in the murine host response against Listeria monocytogenes, but the time course of its production and its precise role in immunity remain controversial. To address these issues, we sequentially monitored IFN production and bacterial accumulation in vivo in C57B1/6 mice during primary listeriosis. IFN-gamma mRNA levels (measured by Northern blot analysis of freshly isolated splenic RNA) and serum IFN (measured by ELISA) were both maximal on day 1 of infection, decreasing steadily after day 2 to barely detectable levels by days 4 to 6. Significantly, there was no direct relationship between IFN levels and listericidal activity in vivo. Between days 1 and 3, the period of maximal IFN production, host bacterial load (assessed by quantitating live L. monocytogenes/spleen) increased approximately 10- to 50-fold. On the other hand, during the immune phase of infection (between days 5 and 7), a period when both IFN mRNA and protein were barely detectable, the host bacterial load decreased 1,000- to 10,000-fold. The paucity of IFN production in vivo during the immune phase was unexpected in light of previous reports demonstrating abundant in vitro lymphokine release by splenocytes isolated during the same time period. By direct comparisons of IFN production in vivo and in vitro, however, we could show that the late (days 6-7) peak of IFN release observed in Ag-stimulated cultures was an in vitro artifact. By contrast the pattern of spontaneous IFN release (obtained when freshly isolated cells were incubated in the absence of Ag) conformed more closely to that observed in vivo. Because listerial Ag stimulated vigorous lymphokine release in vitro, we sought to determine whether an analogous effect could be observed in vivo. In fact, even the infusion of very large doses of live bacteria (5-20,000,000/mouse) did not stimulate endogenous IFN-gamma production in mice infected for 6 to 7 days. These studies suggest three major conclusions: 1) IFN production in vivo occurs primarily during the early phase of listeriosis; 2) the dramatic decrease in bacterial numbers observed late in infection cannot be directly attributed to increased IFN production by LM-immune T cells; 3) although Ag-driven cultures of freshly isolated cells can provide useful information about the potential lymphokine-producing capabilities of Ag-specific T cells, these results have limited relevance in understanding patterns of T cell lymphokine production in vivo.
Assuntos
Interferon gama/biossíntese , Listeriose/imunologia , RNA Mensageiro/biossíntese , Animais , Interferon gama/genética , Listeriose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia , Fatores de TempoRESUMO
IL-12, a recently described cytokine, is an important mediator in the early production of IFN-gamma during infection. To evaluate the timing of IL-12 production, and its relationship to TNF-alpha, and IFN-gamma production during primary murine listeriosis, we measured cytokine mRNA and protein levels in C57B1/6 mice infected intravenously with Listeria monocytogenes (LM). IL-12 is a disulfide-linked heterodimer containing two chains (designated P35 and P40); however, bioactive cytokine production has been more closely linked with P40 expression. Consequently, we monitored mRNA and protein levels of P40 in the spleen as a marker for IL-12 production in vivo. Splenic P40 mRNA levels (assayed using RNase protection methods) were low in uninfected animals, but increased markedly beginning 15 to 18 hr after LM infection. In sublethally infected animals, P40 mRNA levels remained elevated for 5 days, returning to baseline with the resolution of infection. P40 protein (assayed using an antibody capture ELISA) could be detected in the spleens of LM-infected animals beginning around 18 hr postinfection confirming linkage between P40 mRNA accumulation and the generation of a protein product. In comparing P40 and IFN-gamma mRNA levels in vivo, we found in each case that substantial increases in mRNA accumulation did not appear until 15-18 hr postinfection. In comparable studies using BALB/c animals, cytokine production began slightly earlier (between 12 and 15 hr) but once again P40 and IFN-gamma mRNA levels increased in a coordinated manner. P40 mRNA (like IFN-gamma and TNF-alpha mRNA) only accumulated in animals infused with live, virulent bacteria. Although we could detect no obvious lag between the time of onset of IL-12 and IFN-gamma accumulation in vivo, infusions of anti-IL-12 antibodies markedly reduced IFN-gamma expression implying that IL-12 production precedes and directs IFN-gamma production. TNF-alpha production, on the other hand, was not diminished by anti-IL-12 treatment. Our studies demonstrate that IL-12 generation is an essential step in normal IFN-gamma production during listeriosis, and suggest that IL-12, once produced, may begin enhancing IFN-gamma production in vivo in less than 3 hr.
Assuntos
Interferon gama/biossíntese , Interleucina-12/biossíntese , Listeriose/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Animais , Anticorpos Monoclonais/imunologia , Antígenos/metabolismo , Ensaio de Imunoadsorção Enzimática , Interferon gama/genética , Interleucina-12/fisiologia , Interleucina-9/genética , Interleucina-9/imunologia , Fígado/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , RNA Mensageiro/biossíntese , Baço/imunologia , Fatores de Tempo , Fator de Necrose Tumoral alfa/genética , VirulênciaRESUMO
The goal of these experiments was to assess the relationship between the binding and processing of IgG by Fc-receptor-bearing cells. Cells of the U937 human macrophage-like cell line were incubated with 125I-labeled monomers, dimers, oligomers (composed of 2-4 IgG1 subunits), and HP (heavy polymers composed of 5 or more subunits per polymer) of monoclonal human IgG1 in vitro. Binding was assessed by spinning cells through a layer of phthalate oils. Internalization of IgG1 was assessed by quantitating residual binding to cells after surface-bound IgG was removed by a brief treatment with a solution containing 0.25 M acetic acid and 0.5 M sodium chloride. Catabolism was assessed by measuring the release of radioactive fragments of IgG1, which were not precipitated by 10% trichloroacetic acid. Unstimulated U937 bound about 10,000 molecules per cell of IgG1 monomer, with an equilibrium binding constant (Ka) of 5 X 10(8) M-1. After stimulation with a conditioned medium in vitro, binding per cell was increased 3-7--fold, and the Ka was decreased 2-4--fold. Both unstimulated and stimulated cells internalized and catabolized labeled IgG1 HP, but stimulated cells internalized and digested much more IgG1 HP per cell than unstimulated cells. Both monomers and dimers of IgG1 were internalized and degraded very slowly by stimulated cells, even though both preparations readily bound to cells. In contrast, oligomers and (to an even greater extent) IgG1 HP were internalized and degraded much more rapidly. Internalization of IgG1 HP was markedly inhibited by incubation at 4 degrees C, but not by incubation with a variety of metabolic inhibitors. Catabolism was inhibited by chloroquine and monensin (inhibitors of lysosomal acidification) and by cytochalasin (an inhibitor of microfilament polymerization). Binding to the surface of cells was not markedly inhibited by any agent tested. The capacity of cells to bind labeled IgG1 was markedly reduced by prior incubation in the presence of unlabeled IgG1. This reduction was in part due to the steric blockade of receptors caused by the avid, but reversible, binding of IgG1. In addition, IgG1 oligomers or HP (but not IgG1 monomers or dimers) also caused an irreversible reduction in the number of Fc receptors by a process analogous to receptor down-regulation, as observed in other receptor--ligand systems.
Assuntos
Anticorpos Monoclonais/imunologia , Acetatos/farmacologia , Ácido Acético , Linhagem Celular , Humanos , Imunoglobulina G/imunologia , Receptores Fc/metabolismo , Cloreto de Sódio/farmacologiaRESUMO
The ability of lymphocytes to lyse human red cells coated with anti-D antibody was assessed by measuring 51 Cr release from labeled red cells incubated with peripheral blood leukocyte suspensions from 12 normal donors. Mixed mononuclear cell suspensions (containing monocytes and lymphocytes) from all donors produced lysis of sensitized red cells. Treatment with carbonyl iron reduced monocyte concentration to less than 1.2% in all donors, as measured by morphologic criteria, esterase staining and ingestion of latex particles. Lysis of red cells following monocyte depletion was markedly reduced in 8 of the 12 donors. Despite depletion of monocytes, unchanged or increased lysis was noticed with the leukocytes of the remaining 4 donors. This lysis was due to lymphocytes, not to residual monocytes. If target red cells were treated with papain or trypsin prior to sensitization, marked lysis occurred with lymphocytes of all donors, including those which did not lyse unmodified red cells. Direct cytolysis of sensitized red cells during contact with small lymphocytes was recorded using microcinematography, which confirmed the role of lymphocytes in mediating lysis. Lymphocyte-mediated lysis of red cells increased with mounting levels of antibody sensitization regardless to prior treatment with papain. Papain increased antibody coating per red cell, yet lysis per molecule of antibody bound was also increased. Lysis was inhibited by IgG1 and IgG3 in the fluid phase but not by IgG2 or IgG4. At an equivalent level of antibody sensitization lysis was augmented by concurrent coating of the red cells with C3b, C3d and/or C4b, though these components could not produce lysis in the absence of antibody coating.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Eritrócitos/imunologia , Hemólise , Linfócitos/imunologia , Sítios de Ligação de Anticorpos , Eritrócitos/efeitos dos fármacos , Humanos , Imunoglobulina G/imunologia , Monócitos/imunologia , Papaína/farmacologia , Tripsina/farmacologiaRESUMO
MRL-lpr/lpr mice develop an autoimmune disease similar to systemic lupus erythematosus. To determine whether mice of this strain develop defects in mononuclear phagocyte system (MPS) function similar to those observed in patients, the pattern of sequestration of labeled immune complexes was compared 90 min after infusion into MRL-lpr/lpr and into normal B6D2 mice. The amount of complexes persisting in the blood was increased, and the amount sequestered in the liver was significantly reduced in MRL-lpr/lpr mice in comparison to normal B6D2 controls. This defect was most evident in MRL-lpr/lpr mice of the ages of 25-26 weeks; mice of this age also demonstrated the greatest elevation of anti-DNA antibody levels. The role of the MRL strain background and of the lpr gene in determining this defect was investigated by analysis of MRL-+/-/+/- and of other lpr congenic strains (B6-lpr/lpr, AKR-lpr/lpr, and C3H-lpr/lpr). Both MRL-+/-/+/- and congenic lpr animals showed similar defects, although to a lesser degree than MRL-lpr/lpr mice. In contrast, MRL-lpr/lpr mice demonstrated normal clearance of heat-damaged red blood cells and heat-aggregated albumin. Thus MRL-lpr/lpr mice display a selective defect in MPS Fc receptor function and may provide a valuable model for elucidating the etiology and importance of MPS dysfunction in immune complex deposition disease.
Assuntos
Doenças Autoimunes/imunologia , Camundongos Mutantes/imunologia , Fagócitos/imunologia , Animais , Anticorpos/imunologia , Anticorpos Antinucleares/análise , Complexo Antígeno-Anticorpo/imunologia , DNA/imunologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Eritrócitos/imunologia , Temperatura Alta , Masculino , Camundongos , Albumina Sérica/imunologiaRESUMO
To study the assembly of DNA-anti-DNA complexes in vivo, we have measured the clearance from blood and organ localization of a murine IgG2a monoclonal anti-DNA antibody, called 6/0, following the infusion of DNA intravenously or intraperitoneally. Intraperitoneal DNA caused a profound acceleration of 6/0 anti-DNA clearance that was dose dependent and demonstrable after the infusion of as little as 1.9 microgram per gram of body weight of single-stranded DNA. The antibody was cleared primarily in the liver without increased deposition in the kidney. Intraperitoneal infusions of DNA also accelerated the clearance of 6/0 in autoimmune MRL-lpr/lpr mice. In contrast, intravenous DNA given in comparable doses caused only a slight increase in 6/0 antibody clearance; this accelerated clearance was seen only at low antigen doses and only during the first 10 min following DNA infusion. Using double-radiolabeling techniques, 6/0 and Cl.18, an IgG2ak myeloma protein without anti-DNA activity, were found to disappear from blood at a comparable rate in both B6D2 mice and MRL-lpr/lpr mice. These results suggest that the DNA-anti-DNA immune complexes can form in vivo but that this process is profoundly affected by the manner in which DNA enters the circulation. In addition, the results suggest that DNA-dependent clearance is not a major pathway for anti-DNA metabolism in normal or at least one strain of autoimmune mice.
Assuntos
Anticorpos Monoclonais , Complexo Antígeno-Anticorpo , Doenças Autoimunes/metabolismo , DNA de Cadeia Simples/imunologia , Animais , DNA de Cadeia Simples/administração & dosagem , Modelos Animais de Doenças , Infusões Parenterais , Radioisótopos do Iodo , Cinética , Masculino , Taxa de Depuração Metabólica , Camundongos , Camundongos Endogâmicos , Especificidade da EspécieRESUMO
Though ingested Ag are readily degraded into peptides within endocytic vesicles, APC usually cannot present these fragments to CD8 cells. Despite this generalization, some exceptions have been noted. For example, murine macrophage targets readily process heat-killed Listeria monocytogenes (HKLM) into a form recognizable by immune CD8 CTL. Using an assay of Listeria-specific, CD8-mediated cytotoxicity to quantitate Ag presentation by C57Bl/6 macrophage targets, we have examined some of the cellular requirements for this form of Ag processing. To assess whether the physical form of the Ag is an important determinant of processing, we compared the ability of macrophages to present intact HKLM, fractionated L. monocytogenes (LM) membranes, and octyl-beta-d-thioglucopyranoside-solubilized extracts of LM membranes. Macrophages presented each Ag form in a similar manner indicating that processing is not critically dependent on the presence of intact bacteria or even on the introduction of Ag in a particulate form. To gain insight into the metabolic requirements for Ag processing, we examined the effects of several inhibitors. As might be expected, listerial Ag presentation was blocked by brefeldin, a known inhibitor of the endogenous pathway of Ag processing. LM Ag presentation, however, was also blocked by inhibitors of endosomal acidification (chloroquine, ammonium chloride, and monensin) and by the acid protease inhibitor pepstatin A, suggesting that endocytic processing may play an essential role in CD8 recognition of this Ag. To formally establish that this pattern of exogenous Ag processing requires the presence of a class I MHC product, we demonstrated that beta-2 microglobulin-deficient macrophages, which lack class I MHC product expression, cannot present HKLM to CD8 cells. However, we could not block Ag presentation by incubating macrophages with monoclonal anti-H-2K or H-2D antibodies, suggesting that LM Ag presentation may be mediated by some other class I MHC product. Additional characterization of this pathway of Ag presentation is warranted in view of its possible role in initiating CD8-mediated immunity against microbial Ag.