RESUMO
We have developed a system to induce oncornavirus-specific secondary cytotoxic response in vitro. When Moloney strain of murine sarcoma virus-immune spleen cells were cultivated with purified infectious Moloney murine leukemia virus (M-MuLV) or with supernates of tissue culture cells containing infectious virus, a virus-specific secondary cytotoxic response directed against type-specific determinant(s) of M-MuLV was generated in vitro, as determined by a 4-h 51Cr-release assay. The effector cells were susceptible to the treatment with anti-Thyl.2 plus complement, but were unrelated to natural killer cells (NK), because they could not lyse some target cells specific for M-MuLV in both the induction phase and the interaction between effector cells and target cells. Furthermore, a product of the env gene of M-MuLV, perhaps gp70, appeared to be responsible for this response, because viruses with recombinations in the env gene between ecotropic M-MuLV and a xenotropic virus failed to induce a response. When infectious M-MuLV was exposed to UV-light at different doses, the ability of UV-treated M-MuLV to induce a secondary cytotoxic response decreased in parallel with infectivity, indicating that infectivity was necessary for the induction of this response.
Assuntos
Citotoxicidade Imunológica , Linfócitos T/imunologia , Infecções Tumorais por Vírus/imunologia , Animais , Reações Antígeno-Anticorpo , Antígenos de Superfície , Memória Imunológica , Isoantígenos , Camundongos , Camundongos Endogâmicos C57BL , Vírus da Leucemia Murina de Moloney/imunologia , Retroviridae/imunologiaRESUMO
Spleens from Moloney sarcoma virus (MSV) tumor-bearing C57BL/6N mice contained four times the normal number of mononuclear cells and displayed a markedly elevated "spontaneous" (mitogen-independent) DNA synthesis on a per cell basis. The number of macrophages were increased three-fold while there was a slight reduction in the percentage of T lymphocytes. The phytohemagglutinin (PHA) response on a per cell basis of spleens from tumor-bearing mice was decreased about 90% when compared with normal control mice. The primary in vitro immune response to sheep red blood cells was also suppressed to levels of less than 10% of normals. The PHA response could be restored by purification of MSV spleen cells by rayon adherence columns and by removal of phagocytic cells by an iron/magnet technique. The activity of suppressor cells in MSV spleens was demonstrated in mixtures with syngeneic normal spleen cells where a marked impairment of the PHA response was observed. Spleen cells from tumor-free nude mice and normal spleen cells treated by anti-theta serum plus guinea pig complement (C'), both totally unreactive to PHA, had no such effect. The inhibitor cell in MSV spleens was shown to be insensitive to inactivation by anti-theta plus C', but could be removed by the adherence columns and the iron/magnet technique. These data suggest that this suppressor cell is a cell of the monocyte/macrophage series. Suggestive evidence was also presented that the suppressor cells belong to a proliferating population in MSV spleens. Similar suppressor cells have been previously demonstrated in spleens of mice during a variety of immune responses. Our data show, that a tumor, although stimulating the immune system, nevertheless may be suppressive on certain immune functions through the activation of suppressor cells.
Assuntos
Terapia de Imunossupressão , Linfócitos/imunologia , Vírus da Leucemia Murina de Moloney/imunologia , Sarcoma Experimental/imunologia , Baço/imunologia , Animais , Formação de Anticorpos , Antígenos , Soro Antilinfocitário/farmacologia , Bromodesoxiuridina/farmacologia , Adesão Celular , Células Cultivadas , Radioisótopos de Cromo , Testes Imunológicos de Citotoxicidade , Eritrócitos/imunologia , Técnica de Placa Hemolítica , Ferro , Lectinas/farmacologia , Magnetismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Vírus Rauscher/imunologia , Baço/citologia , Timo/anormalidadesRESUMO
Spleen cells from C57BL/6 mice bearing primary tumors induced by the Moloney strain of murine sarcoma virus (MuSV) strongly inhibited the uptake of tritiated thymidine (3H-TDR) by RBL-5 lymphoma cells in a 48-hour growth-inhibition assay (GIA). This activity was first detected 7 days after MuSV was injected; it peaked at 14 days, and was usually no longer detectable after 18-21 days. It could be detected at effector cell/target cell ratios between 20:1 and 5:1, at which normal spleen cells had a growth-promoting effect. The effector cells in the GIA were not T cells, and various depletion experiments suggested that they were macrophages. Macrophages of a purity of over 95% were obtained in the glass-adherent fraction of thioglycollate-induced peritoneal exudate cells (PEC). PEC were growth inhibitory when obtained from either normal or MuSV tumor-bearing mice. However, at effector cell/target ratios of 2.5:1, only PEC from MuSV tumor-bearing mice had an effect; PEC from normal mice were inactive. Activity of spleen cells in the GIA appeared distinct from T-cell-dependent specific cytotoxicity, which was not affected by removal of macrophages. Activity in the GIA was nonspecific, and target cells which do not cross-react with RBL-5 cells were equally inhibited. Furthermore, spleen cells from mice bearing primary tumors induced by 3-methylcholanthrene were also fully active against RBL-5 cells. Supernatants from spleen cell cultures obtained from mice 14 days post injection with MuSV also inhibited the incorporation of 3H-TDR by RBL-5 cells in vitro. However, this effect seemed to be an artifact, since the tumor cells proliferated equally well in the presence or absence of the supernatants. In contrast, the direct effect of spleen cells from MuSV tumor-bearing mice was reflected both by an inhibition of cell proliferation and by inhibition of 3H-TDR incorporation.
Assuntos
Linfoma/imunologia , Macrófagos/imunologia , Animais , Líquido Ascítico/citologia , Líquido Ascítico/imunologia , Reações Cruzadas , Testes Imunológicos de Citotoxicidade , Linfoma/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Vírus da Leucemia Murina de Moloney , Neoplasias Experimentais/imunologia , Vírus do Sarcoma Murino , Baço/imunologia , Linfócitos T/imunologiaRESUMO
Both systemic and in situ natural killer (NK) activity was assessed in young and old C57BL/6N and CBA/N mice bearing primary murine sarcoma virus-induced tumors. Splenic NK activity was depressed in tumor-bearing mice relative to normal age-matched controls. In situ NK activity was detected in young mice, and the level paralleled that found in the spleens of the same mice. In older mice, in situ NK activity was not detected. Furthermore, the depressed levels of activity in the spleens of tumor-bearing mice seemed to reflect systemic effects rather than migration of NK cells from the spleen to the tumor site.
Assuntos
Citotoxicidade Imunológica , Células Matadoras Naturais/imunologia , Sarcoma Experimental/imunologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Vírus do Sarcoma Murino , Baço/imunologiaRESUMO
C57BL/6N mice immunized with regressor murine sarcoma virus (MuSV) were studied at different times after inoculation for their cellular immune responses as measured by migration inhibition assays. We used both the direct capillary and indirect agarose dropiet methods and, as the source of antigens, viable tumor cells and 3 M KCl-solubilized extracts. Migration inhibitory factor (MIF) production coulctivity becoming undetectable after 21 days. However, the level of activity and the kinetics of production of this lymphokine were strongly influenced by the source of the antigen and the form in which it was presented to the immune spleen cells (ISC). Using RBL-5 ct 9-10 days, a decrease to low levels at 14 days, and a second peak of activity between 17 and 21 days. However, uith MBL-2 cells or with 3 M KCl-solubilized antigen from fresh RBL-5 ascites cells, MIF production was observed as early as 9-10 days after tumor induction, peaked at 14 days, and decreased substantially by 21 days. T-cells appeared to be required for migration inhibition reactivity, since ISC depleted of T-lymphocytes by treatment with antibody plus complement were unable to produce MIF after antigen stimulation. The results obtained from specificity studies on the response of ISC in migration inhibition to 11 different tumor lines agreed with the results previously obtained from cytotoxicity studies. With the use of RBL-5 cells as the antigen, there appeared to be an inverse relationship between the development of specific cytotoxic effector cells in 51Cr-release assay and the development of specific effector cells needed for MIF production. However, after removal of adherent cells, the level of cytotoxicity observed correlated with MIF release by immune lymphocytes.
Assuntos
Inibição de Migração Celular , Imunidade Celular , Macrófagos/imunologia , Sarcoma Experimental/imunologia , Animais , Antígenos de Neoplasias , Soro Antilinfocitário/farmacologia , Adesão Celular , Técnicas In Vitro , Cinética , Fatores Inibidores da Migração de Macrófagos/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Vírus do Sarcoma Murino , Baço/imunologia , Linfócitos T/imunologia , Infecções Tumorais por Vírus/imunologiaRESUMO
Direct comparison of the effector cells mediating natural killer (NK) activity against mouse tumor cells and antibody-dependent cell-mediated cytotoxicity (ADCC) against mouse tumor target cells coated with alloantisera indicated that NK cells and K-cells (effector cells mediating ADCC) may belong to the same subpopulation of lymphocytes, but they have a different mechanism of killing. Effector cells mediating NK activity and ADCC were nonadherent, nonphagocytic Fc receptor-bearing cells that sediment at 3.5-4.5 mm/hour. Treatment with anti-Thy 1.2 serum in the absence of complement resulted in an increase of NK activity, whereas this treatment caused a substantial loss in ADCC. Both NK activity and ADCC were equally sensitive to the in vivo or in vitro effects of X-irridiation. In vivo inoculations of high doses of hydrocortisone resulted in a reduction of NK activity, but ADCC was not affected. NK cells were trypsin-sensitive, with a profound decrease in the cytolytic activity being observed in a 4-hour 51Cr release assay. The activity, however, could be recovered after overnight incubation at 37 degrees C. Trypsin treatment did not inhibit ADCC as measured by the 18-hour assay.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Imunidade Inata , Células Matadoras Naturais/imunologia , Neoplasias Experimentais/imunologia , Animais , Soro Antilinfocitário/farmacologia , Hidrocortisona/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/efeitos da radiação , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos CBA , Receptores Fc , Linfócitos T/imunologia , Tripsina/farmacologiaRESUMO
Three isotopic release microtoxicity assays--[125I]5-iodo-2'-deoxyuridine release assay (IRA), 51Cr release assay (CRA), and [3H]proline release assay (PRA)--have been utilized to measure cell-mediated immunity to (C58NT)D, a Gross virus-induced lymphoma in rats. These studies were designed so that all three assays were done under physical conditions as comparable as possible between the assays. A considerable difference was noted in the ability of one or another target cell to function well in each assay. The tissue culture line of (C58NT)D proved an excellent target cell in the long-term assays, whereas the ascites line was inadequate in these same long-term assays. The monolayer Gross virus-induced tumor cell line ERTh/G was resistant to lysis in the short-term CRA but functioned well in the long-term assays. The autologous and thymus cell controls utilized in these studies were reasonably neutral baseline controls for the evaluation of both normal and immune activity. Although all three assays were capable of measuring both natural and immune activity in this systemthe PRA appeared more sensitive at 24 hours and the IRA at 48 hours, whereas the CRA activity with these target cells was only useful in the short-term assays.
Assuntos
Vírus AKR da Leucemia Murina/imunologia , Testes Imunológicos de Citotoxicidade/métodos , Imunidade Celular , Linfoma/imunologia , Animais , Soro Antilinfocitário , Linhagem Celular , Cromo/metabolismo , Feminino , Idoxuridina/metabolismo , Técnicas In Vitro , Linfoma/metabolismo , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/metabolismo , Prolina/metabolismo , Ratos , Ratos Endogâmicos WF , Baço/imunologia , Linfócitos T/imunologia , Timo/imunologia , Fatores de TempoRESUMO
Spleen cells from mice bearing primary tumours induced by Moloney strain of murine sarcoma virus (M-MuSV) strongly inhibited the in vitro generation of specific secondary cell-mediated cytotoxic response of spleen cells from M-MusV regressor mice. These suppressor cells were resistant to treatment with anti-theta serum and complement or to X-irradiation. It appeared that suppressor cells may have had a role in limiting the host's immune response against tumor growth.
Assuntos
Testes Imunológicos de Citotoxicidade , Imunidade Celular , Sarcoma Experimental/imunologia , Baço/imunologia , Animais , Antígenos de Neoplasias , Camundongos , Camundongos Endogâmicos C57BL , Vírus da Leucemia Murina de MoloneyRESUMO
Utilizing controlled cryopreservation techniques, we were able to standardize the 51Cr release cytotoxicity assay and thereby ensured reliable comparisons between results obtained on different days. Optimal conditions for freezing of both effector and target cells were quite similar. Dimethyl sulfoxide (DMSO) at a concentration of 7.5-10.0% was employed as the cryoprotective agent and cells were frozen at the rate of -1 degrees C/minute. The handling procedures for the cells before and after freezing were important. Factors affecting recovery of functional reactivity were related to toxicity of DMSO for the cells, the osmotic stress placed upon the cells as the DMSO was being removed after thawing, the handling temperature of the freshly thawed cells, and the susceptibility of cells to mechanical damage immediately after thawing. The recovery of lymphocytes after freezing was about 70%; the recovery of cytotoxicity was around 85%. Syngeneic cytotoxic reactivity induced by inoculation with the Moloney strain of murine sarcoma virus was cryopreserved, as were allogeneic cytotoxicity and natural cytotoxic reactivity. Multiple tests employing effector cells from the same frozen pool gave reproducible results; the standard error of the mean percent cytotoxicity was less than 1.5%. Cryopreserved target cells gave decreased day-to-day variability in susceptibility to lysis, since the same population of cells could be employed in each assay. These results demonstrated conclusively that we can now have a constant source of effector cells and target cells, which can be used from assay to assay as an internal standard.
Assuntos
Testes Imunológicos de Citotoxicidade/métodos , Animais , Antígenos de Neoplasias , Antígenos Virais , Soro Antilinfocitário , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Radioisótopos de Cromo , Crioprotetores , Meios de Cultura , Dimetil Sulfóxido/farmacologia , Congelamento , Imunidade Celular , Linfócitos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos , Vírus da Leucemia Murina de Moloney/imunologia , Neoplasias Experimentais/imunologia , Sarcoma Experimental/imunologia , Temperatura , Fatores de TempoRESUMO
The cytotoxicity inhibition assay is an extremely useful tool for examination of the specificity of cell-mediated cytotoxicity. With this demonstration of cryopreservation of the attacker, target, and inhibitor cells, a powerful new tool is available for the standardization of this assay to better examine the specificity of cytotoxicity assays. Not only can cryopreservation be used to standardize the assay within a single laboratory, but also reagents may now be exchanged between laboratories to better understand the differences in cell-mediated cytotoxicity as observed by different investigators.
Assuntos
Testes Imunológicos de Citotoxicidade/métodos , Imunidade Celular , Neoplasias Experimentais/imunologia , Animais , Congelamento , Técnicas In Vitro , Camundongos , Preservação Biológica , RatosRESUMO
Requirement for macrophages in in vitro augmentation, by interferon (IFN), polyinosinic-polycytidylic acid (poly I:C), or Corynebacterium parvum, of rat, mouse, and human natural killer (NK) activities was examined. Several differences were seen among the species. Mouse NK activity demonstrated some lability at 37 degrees C and a strict macrophage requirement for in vitro production of IFN, and augmentation of NK activity was demonstrated by either poly I:C or C. parvum. In contrast, human peripheral blood leukocytes (PBL) depleted of monocytes by adherence on nylon wool demonstrated NK activity, which was not labile but rather increased substantially upon overnight culture at 37 degrees C alone or with poly I:C or C. parvum. Monocyte-depleted human PBL also produced IFN in these cultures. The pattern of reactivity seen with rat spleen cell cultures was different from either that of mouse and human cells. This pattern of reactivity had no lability at 37 degrees C and had a macrophage requirement for IFN production and NK cell augmentation upon culture alone or with poly I:C but not with C. parvum. These results indicated some major differences among species in the regulation of NK activity in vitro and the requirement for macrophages for the in vitro production of IFN. A better understanding of these differences will be helpful in choosing appropriate models for in vitro and in vivo studies of NK cell activity.
Assuntos
Interferons/imunologia , Células Matadoras Naturais/imunologia , Macrófagos/imunologia , Animais , Linhagem Celular , Separação Celular , Feminino , Humanos , Interferons/análise , Células Matadoras Naturais/efeitos dos fármacos , Linfoma , Camundongos , Monócitos/imunologia , Poli I-C/farmacologia , Propionibacterium acnes/imunologia , Ratos , Especificidade da Espécie , Baço/citologia , TemperaturaRESUMO
The in situ inflammatory response was studied in 2 different tumors, the T1699 mammary adenocarcinoma in inbred DBA/2 mice and a sarcoma induced in inbred C57BL/6 mice by murine sarcoma virus. The presence of Fc receptors (FcR's) on infiltrating host cells was determined by rosette formation with antibody-coated sheep red blood cells (EA). Differences in FcR-binding activity among various cells were detected with the use of EA prepared with a range of antibody concentrations. Cells bearing FcR represented one-third or more of the inflammatory cell population in both tumors. The FcR-positive cells were heterogeneous in regard to both cell type and FcR activity. Monocytes and macrophages had greater FcR activity than did lymphocytes. Different populations of FcR-positive cells could be isolated by their EA-binding characteristics.
Assuntos
Imunidade Celular , Fragmentos Fc das Imunoglobulinas , Neoplasias Experimentais/imunologia , Animais , Sítios de Ligação , Separação Celular , Feminino , Neoplasias Mamárias Experimentais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Formação de Roseta , Sarcoma Experimental/etiologia , Sarcoma Experimental/imunologia , Infecções Tumorais por Vírus/imunologiaRESUMO
The cell-mediated immune response of C57BL/6 mice to murine sarcoma virus (MSV) was examined by the [125I]-iododeoxyuridine release cytotoxicity assay using MSV-induced sarcoma tissue culture cell lines as target cells. Cellular cytotoxicity was detected as early as 3 days after virus inoculation. Most mice assayed between 12 and 17 days after MSV inoculation gave positive results with maximum levels of activity present on Days 13 and 14. Reactivity was frequently detected for up to 100 days after MSV inoculation, although at low levels (5 to 10%). Additional experiments comparing the kinetics of the cellular response as measured by different in vitro cytotoxicity assays were performed. The results showed a good direct correlation between the [125I]iododeoxyuridine release assay and a 51Cr release assay. A similar pattern of reactivity was also observed when the cellular response was measured by a visual microcytotoxicity assay, although reactivity dropped off more rapidly and became undetectable in most instances by 20 days after injection of MSV. Studies on effector cell type revealed that cytotoxicity in all three assays was T-cell dependent, being eliminated by treatment with anti-theta plus complement. Macrophages did not appear to play a role, since treatment with carbonyl iron and magnet had no effect.
Assuntos
Anticorpos Antivirais/análise , Gammaretrovirus/imunologia , Imunidade Celular , Vírus do Sarcoma Murino/imunologia , Animais , Radioisótopos de Cromo , Proteínas do Sistema Complemento , Testes Imunológicos de Citotoxicidade/métodos , Idoxuridina , Soros Imunes , Radioisótopos do Iodo , Ferro/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia , Fatores de TempoRESUMO
Cell-mediated immune reactions appear to play an important role in resistance against growth of leukemia cells in mice. Possible mechanisms for in vivo protection in two tumor systems are discussed. These tumor models, which are a Friend leukemia virus-induced transplantable tumor, FBL-3, and primary murine sarcoma virus (MSV) -induced tumors, are strongly antigenic; under some conditions, tumors regress completely. In mice with regressing FBL-3 tumors, cell-mediated cytotoxicity was measured by release of [125I]iododeoxyuridine. The response was biphasic, with an initial peak at 10 days and a 2nd peak after 30 days. A boost in reactivity could be elicited by later challenge with tumor cells. All of the reactivity was dependent on T-cells, being eliminated by treatment with anti-theta plus complement. The specificity of the reactions was not completely defined, but it was consistent with Friend type-specific antigen plus broader, common antigens. In mice with regressing MSV tumors, strong cell-mediated cytotoxicity, measured mainly by release of 51Cr, was seen against RBL-5, a Rauscher virus-induced leukemia. A single peak of response occurred at about 14 days after virus inoculation. Upon later challenge with RBL-5 cells, a vigorous and rapid secondary response was elicited, mainly in the region of tumor challenge. This cytotoxic reactivity and in vivo resistance to leukemia.lso was completely dependent on T-cells. In addition, macrophage-mediated inhibition of leukemia cell growth in vitro was seen in this system at the time of peak tumor development. The 51Cr release cytotoxicity was specific and directed primarily against an antigen, MEV-SA1, associated with mouse endogenous C-type viruses. The macrophage-induced growth inhibition appeared to be nonspecific. In both the FBL-3 and MSV tumor systems, protection against tumor growth could be adoptively transferred by immune lymphoid cells. In addition to induction of cell-mediated immunity by tumor cell or virus inoculation, cell-mediated cytotoxic reactivity was found to occur naturally in most young mice. This natural killer activity was quite distinct from the experimentally elicited reactions, being mediated by N-cells, a subpopulation of lymphoid cells with no clearly identifiable cell surface markers. The natural cytotoxicity was also directed against antigenic specificities different from those recognized by the MSV-immune cells. The central issue in all of these studies has been to determine the relationships between the in vitro-detected cell-mediated reactivity and in vivo resistance to leukemia.
Assuntos
Antígenos de Neoplasias , Imunidade Celular , Vírus da Leucemia Murina/imunologia , Neoplasias Experimentais/imunologia , Animais , Especificidade de Anticorpos , Testes Imunológicos de Citotoxicidade , Epitopos , Vírus da Leucemia Murina de Friend/imunologia , Imunização Passiva , Leucemia Experimental/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Vírus Rauscher/imunologia , Vírus do Sarcoma Murino/imunologia , Linfócitos T/imunologiaRESUMO
All peritoneal macrophage (pM phi) populations studied exhibited some binding of the anti-asGM1 serum as assessed by flow cytometry. The levels of reactivity varied quantitatively among populations, depending on the combination of eliciting and activating agents employed prior to the harvest of pM phi. Resident pM phi contained a very small percentage (4%) of cells that were strongly asGM1+. Any treatment of these cells that induced them to become stimulated or activated increased the percentage of highly asGM1+ cells. Treatments that enhanced anti-asGM1 binding including eliciting pM phi with proteose peptone (16% asGM1+) or Brewer's thioglycollate medium (66% asGM1+), treatment with the activating biological response modifiers (BRMs) MVE-2 (12% asGM1+) and P acnes (18% asGM1+), or treatment with both peptone + MVE-2 (37% asGM1+) or peptone + poly IC/LC (33%). Increased expression of anti-asGM1 was accompanied by some increase in the reactivity of the various pM phi populations to treatment with anti-asGM1 serum. This conclusion was based on the reduced viabilities of cells treated with both an eliciting agent and an activating agent prior to in vitro treatment with anti-asGM1 + C, as well as by reductions in cytolytic activity of pM phi elicited with peptone and activated by MVE-2, following anti-asGM1 treatment in vitro or administration in vivo. Conversely, the cytolytic activity of resident pM phi activated in vivo by MVE-2 or heat-killed P acnes, agents that induced relatively small increases in the percentage of asGM1+ cells, was resistant to the effects of in vivo and/or in vitro treatment with doses of anti-asGM1 serum that inhibit NK activity. These results indicate that stimulation of pM phi by eliciting or activating agents can increase the level of expression of asGM1. This increased expression of asGM1 may be a useful marker for some aspects of macrophage heterogeneity, but increased expression is not necessarily directly related to expression of tumoricidal activity. In fact, the results of this study demonstrate that anti-asGM1 serum can be used for specific depletion of NK activity in vivo in normal mice and in mice treated with at least some BRMs. However, the results also demonstrate that the use of eliciting agents, particularly thioglycollate, or eliciting agents in conjunction with activating agents can cause pM phi to become reactive with anti-asGM1 serum.
Assuntos
Gangliosídeo G(M1) , Glicoesfingolipídeos/imunologia , Macrófagos/imunologia , Neoplasias Experimentais/imunologia , Animais , Antígenos de Superfície/análise , Citotoxicidade Imunológica , Imunofluorescência , Células Matadoras Naturais/imunologia , Ativação de Macrófagos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
A short term microcytotoxicity assay is described for studying the activation of M0 by lymphokines. Proteose-peptone-induced macrophages were purified by adherence in flat-bottomed microtiter plates and then activated by MAF-containing supernatants for 18 h. 51Cr-labeled tumor target cells were added for an additional 18 h, and then the supernatants were harvested and the % isotope release quantitated. When endotoxin-free medium and FBS were used, we found that small amounts of LPS were absolutely required for macrophage activation in this assay. The advantages of this technique included (a) good reproducibility, (b) the requirement for small number of M0, and (c) the potential of standardizing the assay and thereby testing a large number of samples. Moreover, this assay may have particular value for investigations of M0 activation by exogenous stimuli, since M0 that were not pretreated with activating agents did not exhibit cytotoxicity.
Assuntos
Testes Imunológicos de Citotoxicidade/métodos , Endotoxinas/farmacologia , Linfocinas/imunologia , Macrófagos/imunologia , Animais , Escherichia coli , Feminino , Camundongos , Camundongos Endogâmicos C3H/imunologia , Camundongos Endogâmicos C57BL/imunologiaRESUMO
This report describes the utilization of 111 indium-oxine chelate ([111In]Ox) for studies of macrophage-mediated cytotoxicity. [111In]Ox efficiently labeled both non-adherent and adherent tumor targets with no decrease in cell viability. Spontaneous release of intracellularly incorporated [111In]Ox was very slow (0.25-0.50%/h) from most targets, making isotope-release assays of at least 48 h feasible. In addition, released [111In]Ox was not reutilized. In contrast to its low spontaneous release from intact cells, incorporated [111In]Ox was rapidly released from tumor targets after interaction with activated macrophages. Levels of [111In]Ox released in response to cytolytic macrophages correlated well with those observed for the 51Cr and [3H]TdR radiolabels. Therefore, [111In]Ox can be utilized for relatively short-term (less than 20h) assays with lymphoma targets, as well as for longer-term assays with adherent cells. This should facilitate the testing, with the same radioisotope-release assay, of a wide range of tumor targets for susceptibility to macrophage-mediated cytotoxicity.
Assuntos
Citotoxicidade Imunológica , Hidroxiquinolinas/metabolismo , Índio/metabolismo , Macrófagos/imunologia , Neoplasias Experimentais/imunologia , Oxiquinolina/metabolismo , Animais , Adesão Celular , Sobrevivência Celular , Feminino , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , RadioisótoposRESUMO
In studies on the functional activity of macrophages isolated from murine sarcoma virus (MSV)-induced tumors, we have found that these cells may suppress immune responses as well as act as effector cells against the tumor. Previously, we reported that macrophages from the tumor could inhibit the antitumor response by suppressing proliferation-dependent immune functions. Here, we demonstrate that macrophages can also suppress the production of migration inhibition factor (MIF) and macrophage activation factor (MAF), two lymphocyte activities that are independent of cell proliferation. Conversely, we and others have found that macrophages from the tumor can exert an antitumor, cytolytic effect. In this study, using 1 g velocity sedimentation separation techniques, we have been able to identify 2-3 subpopulations of cytolytic macrophages in regressing tumors but in progressing tumors, only the smallest subpopulation of macrophages was active. T cells appeared to be required for activation of macrophages within the tumor, since MSV tumors induced in athymic, nude mice did not contain cytolytic macrophages.