RESUMO
The cytokine tumour necrosis factor alpha (TNF-alpha) is implicated in the regulation of diverse gynaecological cell types, its biological activity being potentially mediated by two distinct cell surface receptors (TNFR) of molecular weight 55 and 75 kDa, respectively. In this study the sensitivity to the growth regulatory properties of TNF-alpha of a panel of human cervical, endometrial and ovarian cancer cell lines was investigated in relation to the expression and biological activity of the 55- and 75-kDa receptor. There was no evidence of expression or function of the 75-kDa receptor in any of the cell lines tested. The expression and biological activity of the 55-kDa receptor was demonstrated in each TNF sensitive cell line, with one exception, the HOG-1 cervical cancer cell line. The data suggest that the 55-kDa receptor mediates the cellular response to TNF-alpha in sensitive gynaecological cancer cell lines but raises the possibility of the presence of a distinct receptor in HOG-1 cells.
Assuntos
Neoplasias dos Genitais Femininos/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Anticorpos/farmacologia , Divisão Celular/efeitos dos fármacos , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Feminino , Citometria de Fluxo , Neoplasias dos Genitais Femininos/patologia , Humanos , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Reação em Cadeia da Polimerase , Receptores do Fator de Necrose Tumoral/imunologia , Células Tumorais Cultivadas , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologiaRESUMO
Two conformationally distinct and stable forms of Qa-1b, one strongly associated with beta 2-microglobulin (beta 2m) and the other associated with a novel molecule, gp44, were observed during immunochemical studies on the expression of Qa-1b molecules in mouse spleen cells. Both forms are efficiently processed and expressed at the cell surface. However, a large proportion of Qa-1b was found to be disulfide linked to gp44 without any detectable beta 2m. In TAP1-deficient mice, both forms undergo carbohydrate processing and are expressed on the cell surface, suggesting that they may traffic using a pathway not requiring a TAP association step. Consistent with this, size exclusion chromatography of newly synthesized class I molecules shows that high molecular mass complexes containing H-2Kk do not contain Qa-1b. Although Qa-1b can be stably expressed without beta 2m, there was no maturation of either form in cells from beta 2m-deficient mice where heavy chains were rapidly degraded. These results suggest that Qa-1b, like most other class I molecules, requires beta 2m for an initial folding step. However, beta 2m is not essential for subsequent processing of Qa-1b molecules.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Microglobulina beta-2/fisiologia , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Células Cultivadas , Cromatografia em Gel , Antígenos H-2/análise , Antígenos de Histocompatibilidade Classe I/biossíntese , Antígenos de Histocompatibilidade Classe I/química , Substâncias Macromoleculares , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Modelos Moleculares , Conformação Proteica , Baço/citologia , Baço/metabolismoRESUMO
The peptide-binding properties of the nonclassical major histocompatibility complex (MHC) class 1b molecule Qa-1 were investigated using a transfected hybrid molecule composed of the alpha 1 and alpha 2 domains of Qa-1b and the alpha 3 domain of H-2Db. This allowed the use of a monoclonal antibody directed against H-2Db whilst retaining the peptide-binding groove of Qa-1b. By comparison with classical MHC class I molecules, intracellular maturation of the chimeric molecule was inefficient with weak intracellular association with beta 2-microglobulin. However, at the cell surface the hybrid molecules were stably associated with beta 2-microglobulin and were recognized by cytotoxic T lymphocyte (CTL) clones specific for the Qa-1b-presented peptide Qdm (AMAPRTLLL). A whole-cell binding assay was used to determine which residues of Qdm were important for binding to Qa-1b and CTL clones served to identify residues important for T cell recognition. Substitutions at position 1 and 5 did not reduce the efficiency of binding and had little effect on CTL recognition. In contrast, substitutions at position 9 resulted in loss of MHC class I binding. Mass spectrometric analysis of peptides eluted from immunopurified Qa-1b/Db molecules indicated that Qdm was the dominant peptide. The closely related peptide, AMVPRTLLL, which is derived from the signal sequence of H-2Dk, was also present, although it was considerably less abundant. The mass profile suggested the presence of additional peptides the majority of which consisted of eight to ten amino acid residues. Finally, the finding that a peptide derived from Klebsiella pneumoniae can bind raises the possibility that this non-classical MHC class I molecule may play a role in the presentation of peptides of microorganisms.
Assuntos
Antígenos de Histocompatibilidade Classe I/imunologia , Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Mapeamento de Epitopos , Antígenos H-2/imunologia , Camundongos , Dados de Sequência Molecular , Peptídeos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes/imunologia , Microglobulina beta-2/metabolismoRESUMO
Infection of dendritic cells (DC) by human immunodeficiency virus (HIV) has been disputed. Employing a fluorescence-activated cell sorter, DC, identified by the absence of membrane markers for T, B, natural killer (NK) and monocytic cells and by high levels of MHC class II DR antigen, were shown to express low levels of CD4. Immunomagnetic beads were used to separate blood low density cells, which are enriched for DC, into CD4-positive and -negative populations. Examination of these cells by electron microscopy showed an increase in the percentage of cells with DC morphology in the CD4-positive fraction and a reduction in the CD4-negative fraction. Electron microscopy of semi-purified DC preparations infected in vitro for 5 days with HIV-1 revealed morphologically distinct veiled DC with mature virions on the cell surface and virus budding through the cell membrane. Further evidence for the growth of HIV in DC was provided by experiments in which DC were extensively depleted of contaminating lymphocytes and monocytes prior to infection. Estimation of provirus load by a nested PCR indicated that after 5 days an infection level of one provirus copy per five cells could be achieved. After 7 days the provirus copy number could exceed the cellular genome copy number, suggesting that some cells had more than one provirus. Infectious virus could not be demonstrated in these cultures after 24 h but was detected after 5 or 7 days. Infection of DC in the presence of antibodies against CD4 was inhibited and suggests infection occurs via a CD4-dependent pathway. These results confirm that DC are susceptible to HIV infection in vitro. The immunological consequences of DC infection in vivo may be significant in the pathogenesis of AIDS.
Assuntos
Antígenos CD4/análise , Células Dendríticas/virologia , HIV/crescimento & desenvolvimento , Sequência de Bases , DNA Viral/análise , Células Dendríticas/imunologia , Humanos , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseAssuntos
Células Sanguíneas/virologia , Células Dendríticas/virologia , Infecções por HIV/virologia , Apresentação de Antígeno , Células Sanguíneas/imunologia , Antígenos CD4/metabolismo , Células Dendríticas/imunologia , HIV/genética , HIV/isolamento & purificação , HIV/patogenicidade , Humanos , Técnicas In Vitro , Reação em Cadeia da Polimerase , Provírus/genética , Provírus/isolamento & purificaçãoRESUMO
The ability of subcultured human vascular endothelial cells (EC) to provide immune accessory functions for proliferative responses of highly purified allogeneic CD4+ and CD8+ T cells has been examined. CD4+ T cells proliferated in response to IFN-gamma-pretreated EC which expressed class II molecules, but not to untreated EC. CD8+ T cells proliferated to MHC class I molecules expressed on both untreated and IFN-gamma-treated EC. Combined populations of CD4+ and CD8+ T cells showed synergistic, rather than additive, responses to both untreated and IFN-gamma-treated EC. Furthermore, CD8+ T cells were able to induce MHC class II expression on endothelial cells and this induction could be inhibited by an anti-IFN-gamma mAb. The synergistic response obtained by co-culturing CD4+ and CD8+ T cells with vascular EC was completely inhibited by the same anti-IFN-gamma mAb. These studies suggest that CD4+ and CD8+ T cells recognise and proliferate to allogeneic MHC molecules expressed by EC. CD4+ and CD8+ responses are synergistic under the conditions tested and this synergism appears to be due to induction of MHC class II antigens on EC by IFN-gamma secreted from CD8+ T cells.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Endotélio Vascular/imunologia , Ativação Linfocitária/imunologia , Linfócitos T Reguladores/imunologia , Células Apresentadoras de Antígenos/imunologia , Comunicação Celular , Células Cultivadas , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Interferon gama/imunologia , Subpopulações de Linfócitos T/imunologia , Veias UmbilicaisAssuntos
Células da Medula Óssea , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células de Langerhans/citologia , Fator de Necrose Tumoral alfa/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultura Livres de Soro , Sinergismo Farmacológico , Fatores de Crescimento de Células Hematopoéticas/farmacologia , Células-Tronco Hematopoéticas/citologia , HumanosAssuntos
Antígenos de Bactérias/análise , Chlamydia trachomatis/isolamento & purificação , Células Dendríticas/microbiologia , Monócitos/microbiologia , Antígenos de Superfície/análise , Células Cultivadas , Chlamydia trachomatis/imunologia , Células Dendríticas/imunologia , Citometria de Fluxo , Humanos , Linfócitos/imunologia , Linfócitos/microbiologia , Monócitos/imunologia , FagocitoseRESUMO
Colonies of CD1a+ HLA-DR+/DQ+ CD4+ cells with the functional and some of the structural attributes of Langerhans cells are observed in human bone marrow cultures in semi-solid media and are assumed to be the progeny of an early progenitor, the dendritic/Langerhans cell CFU (CFU-DL). The cytokine-regulated growth of these cells has been studied using a chemically defined serum-free system to culture both unfractionated and highly enriched bone marrow progenitor cell populations. Although unfractionated cell growth was optimal in serum replete cultures with PHA-stimulated leukocyte-conditioned medium (PHA-LCM) suboptimal proliferation of CFU-DL was observed in serum even in the absence of PHA-LCM. No colonies were observed under serum-free conditions when granulocyte-macrophage CSF (GM-CSF), IL-3, granulocyte CSF (G-CSF), and macrophage CSF (M-CSF) were present at levels optimal for granulocyte colony-forming unit (CFU-G) and macrophage colony-forming unit (CFU-M) growth. Addition of IL-1 alpha to these cytokines stimulated a small number of CFU-DL. However, in the presence of GM-CSF and IL-3, TNF-alpha or TNF-beta (5 U/ml) were both highly effective in promoting growth up to 82% of optimal and CFU-G growth was also enhanced at these concentrations. TNF was only active during the first 3 days of culture and higher concentrations of TNF-alpha but not TNF-beta were inhibitory for both CFU-DL and CFU-G. CD34+ cell-enriched populations were also enriched for both myeloid progenitors (CFU-G + CFU-M) and CFU-DL to 36- and 48-fold, respectively, and single cell cultures of CD34+ cells yielded single colonies containing both CD1a+ dendritic cells and CD1a- macrophages. Thus dendritic/Langerhans progenitors in the bone marrow expresses CD34, have a capacity for both macrophage and dendritic cell differentiation, and depend on hemopoietic growth factors and TNF for their further development in vitro.
Assuntos
Antígenos CD/análise , Células da Medula Óssea , Células Dendríticas/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Antígenos CD34 , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados , Células Dendríticas/imunologia , Células Dendríticas/fisiologia , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/fisiologia , Humanos , Interferon gama/farmacologia , Interleucina-1/farmacologia , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/fisiologia , Proteínas Recombinantes/farmacologiaRESUMO
The ability of dendritic cells (DC) to acquire and present antigen to T cells during antigen-induced nonresponsiveness (AINR) in contact sensitivity was examined by studying cells from lymph nodes draining the sites of antigen challenge. Mice were pretreated on the right flank with either vehicle (AOO), oxazolone (Ox), or fluorescein isothiocyanate (FITC) and challenged 5, 10, or 20 days later with FITC on the left flank. At 5, 10, and 20 days, compared with animals pretreated with vehicle and challenged with FITC, those pretreated and challenged with FITC showed reduced acquisition of antigen by DC and the DC showed a reduced ability to stimulate naive T cells in vitro. Proliferation of T cells immediately on isolation (reflecting in vivo activity) was also reduced. When the time between pretreatment and challenge was extended to 40 days, the proliferative responses and antigen acquisition returned to normal. Animals sensitized with Ox and challenged with FITC showed nonspecific inhibition of T cell proliferation at 5 days only and not at later times and antigen levels on the DC from these animals were normal. The results show that low T cell proliferation during specific AINR in contact sensitivity may be a consequence of reduced acquisition and presentation of antigen by DC.
Assuntos
Células Dendríticas/imunologia , Pele/imunologia , Linfócitos T/imunologia , Animais , Contagem de Células , Divisão Celular , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Fluoresceína-5-Isotiocianato/administração & dosagem , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos CBA , Oxazolona/administração & dosagemRESUMO
The triggering of the TCR/CD3 complex by anti-CD3 (OKT3) antibody leads to the formation of T cell clusters. In cultures of T lymphocytes from most normal individuals, the peak of cluster formation occurs at 24 h, but with cells from patients with common variable immunodeficiency (CVI) it was seen earlier at 4-9 h; in addition, the clusters were larger than normal, particularly at 9 h. Cluster formation by CVI and normal cells was dependent on temperature and divalent cations, but did not require Fc receptors. Since OKT3 clustering is known to be dependent on the LFA-1/ICAM-1 adhesion system, the effect of monoclonal antibodies directed against these molecules was tested. A potent inhibitor was the antibody against the common beta chain of the integrin family (CD18), but of four MoAbs against the alpha chains (CD11), three inhibited and one stimulated T cell aggregate formation. Increased expression of LFA-1 or ICAM-1 on CVI patients' T cells could not be demonstrated. The accelerated clustering was therefore probably due to an increase in the proportion of cells carrying the activated form of LFA-1. The formation of large numbers of homotypic lymphocyte clusters might reduce the effective interaction between B and T cells, thus contributing to the depression of immunoglobulin synthesis observed in this disease.
Assuntos
Síndromes de Imunodeficiência/imunologia , Antígeno-1 Associado à Função Linfocitária/fisiologia , Muromonab-CD3/imunologia , Linfócitos T/imunologia , Adolescente , Adulto , Idoso , Anticorpos Monoclonais/imunologia , DNA/biossíntese , Feminino , Imunofluorescência , Humanos , Masculino , Pessoa de Meia-Idade , Fito-HemaglutininasRESUMO
Suppression of cell-mediated immune responses by essential fatty acids (EFA) was demonstrated in mice maintained on a standard laboratory diet for rodents. Daily oral administration of EFA at doses ranging from 125 to 750 mg/kg body weight significantly suppressed local host-versus-graft and graft-versus-host reactions as measured by popliteal lymph node assay. Studies employing immune sera directed against E-type prostaglandin demonstrated that n-6 EFA-induced suppression was mediated through prostaglandin E1. In titration experiments the effect of n-6 EFA on the reactions was dose dependent with enhancement of the responses at low concentrations and suppression at high concentrations.
Assuntos
Ácidos Graxos Essenciais/imunologia , Imunidade Celular , Fenômenos Fisiológicos da Nutrição , Prostaglandinas E/imunologia , Alprostadil , Animais , Anticorpos/imunologia , Ácidos Graxos Essenciais/farmacologia , Reação Enxerto-Hospedeiro/efeitos dos fármacos , Reação Hospedeiro-Enxerto/efeitos dos fármacos , Imunidade Celular/efeitos dos fármacos , Terapia de Imunossupressão , Camundongos , Camundongos Endogâmicos , Baço/transplante , Transplante HomólogoRESUMO
Based on studies in lymphocyte cultures it has been suggested that endogenous prostaglandins (PG), especially those of the E series (PGE), suppress cell-mediated immune reactions during their induction phase. We have tested this theory in experimental animals using local host-versus-graft (HVG) and graft-versus-host (GVH) reactions in mice. These reactions were suppressed in a dose-dependent manner by treatment of the animals with PGE1. If, however, endogenous PGE was neutralized through treatment of the animals with immune sera directed against PGE (APSE1) then inhibition of the development of HVG and GVH reactions was seen. This inhibition could be abrogated in "add back" experiments by treatment of the animals with PGE1. We suggest that the action of PGE1 during the induction phase of CMI responses is governed by a bell-shaped dose-response curve with a response-enhancing effect at low PGE1 and a suppressive effect at high PGE1 concentrations. APSE1 has proved to be very effective in inhibiting responses, and thus treatment with anti-PG antibodies may not only represent a valuable tool for the study of the role of PG in immunoregulation, but may become useful in therapeutic interventions with the immune system--e.g., after bone marrow transplantation.
Assuntos
Reação Enxerto-Hospedeiro/efeitos dos fármacos , Reação Hospedeiro-Enxerto/efeitos dos fármacos , Imunidade Celular , Prostaglandinas E/fisiologia , Animais , Relação Dose-Resposta a Droga , Feminino , Soros Imunes , Imunidade Celular/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos , Prostaglandinas E/imunologia , Prostaglandinas E/farmacologia , Fatores de TempoRESUMO
The role of dendritic or veiled cells (VC) from lymph nodes or spleens of rats and mice in initiating immune responses in vivo has been investigated. Host-versus-graft responses were induced by injection of VC from spleens of (C57BL/10 X CBA) F1 mice into the footpads of parental strain (CBA) animals and measured by the increase in the weight of the draining popliteal lymph nodes. The potency of VC to induce the responses was 100-fold greater than that of unseparated spleen cells. The in vivo effect of VC was not limited to this direct allogeneic stimulation because autologous VC could also be used in the induction of an experimental autoimmune disease. In these studies, experimental allergic encephalomyelitis was produced in Lewis rats by injection of guinea pig brain and spinal cord material emulsified in Freund's complete adjuvant. Small numbers of VC from spleens or lymph nodes of rats showing clinical signs of experimental allergic encephalomyelitis induced a mild form of the disease when injected intravenously into normal Lewis rats. Thus, VC carrying antigen, either as an integral part of their surface membrane or acquired during exposure to antigenic substances, appeared to be very potent agents for the induction of immune responses.
Assuntos
Linfócitos T/imunologia , Animais , Encefalomielite Autoimune Experimental/imunologia , Reação Enxerto-Hospedeiro , Reação Hospedeiro-Enxerto , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos , Microscopia Eletrônica de Varredura , Ratos , Ratos Endogâmicos Lew , Baço/imunologia , Baço/transplante , Linfócitos T/transplante , Linfócitos T/ultraestruturaAssuntos
Imunidade Celular/efeitos dos fármacos , Prostaglandinas E/farmacologia , Animais , Anticorpos , Encefalomielite Autoimune Experimental/prevenção & controle , Rejeição de Enxerto/efeitos dos fármacos , Reação Enxerto-Hospedeiro/efeitos dos fármacos , Técnicas Imunológicas , Camundongos , Prostaglandinas E/imunologia , RatosAssuntos
Imunidade Celular/efeitos dos fármacos , Prostaglandinas/farmacologia , Animais , Encefalomielite Autoimune Experimental/tratamento farmacológico , Ácidos Graxos Essenciais/farmacologia , Ácidos Graxos Essenciais/uso terapêutico , Sobrevivência de Enxerto , Cobaias , Soros Imunes/farmacologia , Imunossupressores/farmacologia , Ácidos Linoleicos/uso terapêutico , Camundongos , Camundongos Endogâmicos CBA , Prostaglandinas/imunologia , Ratos , Ratos Endogâmicos Lew , Transplante de PeleAssuntos
Encefalomielite Autoimune Experimental/imunologia , Ácidos Graxos Essenciais/farmacologia , Imunidade Celular/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/tratamento farmacológico , Ácidos Graxos Essenciais/uso terapêutico , Feminino , Terapia de Imunossupressão , Indometacina/farmacologia , Masculino , Ratos , Ratos Endogâmicos Lew , EsplenectomiaRESUMO
In this paper we report a study of the effects of splenectomy on the immunosuppressive action of essential fatty acids (EFA) which is thought to be mediated through prostaglandins (PG) produced in the spleen. Experimental allergic encephalomyelitis (EAE) was induced in normal, splenectomized and sham splenectomized Lewis rats. EFA were administered orally, the animals in the control groups being treated with liquid paraffin. Treatment with EFA significantly suppressed clinical disease in those animals in which EAE was induced by the inoculation of central nervous system material of guinea-pigs or by passive transfer by Con A-stimulated spleen cells. Splenectomy abrogated the suppressive effect of EFA. This observation, together with previous results showing the abrogation of EFA immunosuppression by an inhibitor of the biosynthesis of PG from EFA, led us to postulate a close relationship between EFA, PG and a splenic factor suppressing immunopathological mechanisms in EAE.
Assuntos
Encefalomielite Autoimune Experimental/prevenção & controle , Ácidos Graxos Essenciais/uso terapêutico , Baço/imunologia , Animais , Peso Corporal , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Terapia de Imunossupressão , Prostaglandinas/biossíntese , Ratos , Baço/metabolismo , EsplenectomiaRESUMO
Experimental allergic encephalomyelitis in Lewis rats was suppressed by treatment with essential fatty acids (EFA) given perorally. This treatment effect could be abolished by administration of a drug (Indomethacin) known to inhibit biosynthesis of certain prostaglandins from EFA. This observation suggests that the suppressive effect of EFA on cell-mediated immune reactions is brought about by EFA-derived prostaglandins.