RESUMEN
The growth factors present during liver regeneration partially overlap with the regulators of the hepatic acute phase response. We analysed the acute phase reaction and changes in soluble cytokine receptors after partial hepatectomy, when tissue injury inducing acute phase reaction and major reduction of liver mass occur simultaneously. Three acute phase proteins and mRNAs were determined by ELISA and northern blot hybridisation in rats. Serum levels of IL-6 and three soluble cytokine receptors (sTNF-alpha R I and II, sIL-6R) were detected by ELIBA or dot-blot assay. Time-course profiles of fibrinogen, alpha(2)-macroglobulin and haptoglobin proteins and mRNA are presented. Elevation of IL-6, soluble TNF-alpha receptors and soluble IL-6 receptor levels were also detected. The time-course of changes in haptoglobin concentration and elevation of soluble cytokine receptors is described by this in vivo experimental system. The results show good correlation with (post)transcriptional activation of immediate and delayed early gene products. These data suggest the involvement of both acute phase proteins and soluble cytokine receptors in the regulation of liver regeneration.
Asunto(s)
Proteínas de Fase Aguda/biosíntesis , Reacción de Fase Aguda/metabolismo , Antígenos CD/biosíntesis , Regeneración Hepática , Receptores de Interleucina-6/biosíntesis , Receptores del Factor de Necrosis Tumoral/biosíntesis , Proteínas de Fase Aguda/genética , Animales , Fibrinógeno/biosíntesis , Fibrinógeno/genética , Haptoglobinas/biosíntesis , Haptoglobinas/genética , Hepatectomía , Interleucina-6/sangre , Cinética , Hígado/metabolismo , Masculino , ARN Mensajero/biosíntesis , Ratas , Receptores Tipo I de Factores de Necrosis Tumoral , Receptores Tipo II del Factor de Necrosis Tumoral , alfa-Macroglobulinas/biosíntesis , alfa-Macroglobulinas/genéticaRESUMEN
The in situ function of tumour-infiltrating leukocytes (TIL) in human colorectal adenocarcinoma (CRC) is unclear. Local cytokine expression probably regulates the anti-tumour immune response and tumour immune surveillance. We examined the distribution of mRNA for IFN-gamma, TNF-alpha, IL-10 and IL-4 in TIL, and tumour cells freshly isolated from 21 surgically removed primary CRC, using a semiquantitative RT-PCR. Lamina propria-infiltrating leukocytes (LPL) and epithelial cells from normal colon mucosa of 10 CRC patients served as negative controls. Median levels of IFN-gamma and TNF-alpha mRNA were higher in TIL than LPL (p = 0.0002 and 0.0001). IL-10 mRNA was generally observed in TIL and LPL, but no or very small amounts of IL-4 transcripts were detected in TIL and LPL. TNF-alpha and IL-10 mRNA were more abundant in colorectal tumour cells than in the normal epithelial cells (p = 0.0136 and 0.0036). The number of IFN-gamma transcripts in TIL correlated negatively (p = 0.039) and the number of TNF-alpha transcripts in tumour cells correlated positively with the Dukes' stages (p = 0.0147). Our results suggest that TIL are characterized by a type 1 (Th1/Tc1-like) pattern of cytokine expression and function as T cells (and macrophages) in the local, cell-mediated anti-tumour immune response in early stages of CRC. Changes in IFN-gamma and TNF-alpha mRNA in TIL and tumour cells could be related to tumour progress (e.g. by T cell anergy) or forming of metastases, respectively.
Asunto(s)
Adenocarcinoma/genética , Neoplasias Colorrectales/genética , Citocinas/genética , Linfocitos Infiltrantes de Tumor/metabolismo , ARN Mensajero/genética , Adenocarcinoma/patología , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Neoplasias Colorrectales/patología , Cartilla de ADN , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Cytokines are important regulators of lymphocyte function in SLE. However, the profile of Th1 and Th2 cytokines produced by circulating lymphocytes in human SLE has not been clearly elucidated. The aim of the present study was to characterize the gene expressions of the Th1-type cytokine IFN-gamma, and the Th2-type cytokines IL-10 and IL-4 in PBMC of 15 patients with SLE and 10 healthy individuals by a semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). Our results showed that expression of IFN-gamma (P = 0.0004) and IL-10 (P = 0.002) transcripts were significantly increased in PBMC of patients with SLE compared with healthy controls. By contrast, expression of IL-4 transcripts in PBMC of patients with SLE was significantly decreased compared with the healthy controls (P = 0.0008). Primary sources of IL-10 were B cells and monocytes, with variable contribution of T cells as detected in various fractions of PBMC of patients with SLE (P = 0.049). These findings support the hypothesis that the enhanced production of IFN-gamma by mononuclear cells may trigger inflammatory responses, together with the enhanced production of IL-10 resulting in autoantibody production by B cells in human SLE.
Asunto(s)
Interferón gamma/genética , Interleucina-10/genética , Interleucina-4/genética , Leucocitos Mononucleares/inmunología , Lupus Eritematoso Sistémico/genética , Adulto , Linfocitos B/inmunología , Femenino , Perfilación de la Expresión Génica , Humanos , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Interleucina-4/metabolismo , Leucocitos Mononucleares/citología , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/tratamiento farmacológico , Lupus Eritematoso Sistémico/inmunología , Masculino , Persona de Mediana Edad , ARN Mensajero/sangre , ARN Mensajero/genética , Linfocitos T/inmunologíaRESUMEN
Authors report here on a case presenting as B-CLL and complicated with cutaneous infiltration involving the legs and the trunk a year later. Immunohistochemic analysis and the immunoglobulin heavy chain gene rearrangement confirmed cell invasion into the skin identical with the underlying disorder. After failure of conventional chemotherapy, interferon alpha 2b therapy has been started with satisfactory result. Few cases presenting cutaneous infiltration in the course of B-CLL has already been reported in the literature. Secondary cutaneous B-cell lymphoma represents an entity of the poorest prognosis in comparison with primary cutaneous form treated with conventional therapy as well as with lymphomas lacking skin manifestations. Interferon alpha 2b therapy cleans up the skin and yields a favourable survival so it's introduction recommended in this entity. Authors summarise the characteristics of secondary cutaneous B-cell lymphomas on the basis of literature survey. According to authors investigations histidine decarboxylase activity was found to be absent from the lymphocytes infiltrating the skin in contrast to those remaining in the circulation. This seems to be a newly recognised feature of these cells. The changing character of the disease raises the possibility of an altered gene expression pattern of the cells invading the skin. Authors summarise data from the literature concerning suspected molecular mechanism of tissue invasion.
Asunto(s)
Antineoplásicos/uso terapéutico , Histidina Descarboxilasa/metabolismo , Interferón-alfa/uso terapéutico , Leucemia Linfocítica Crónica de Células B/patología , Neoplasias Cutáneas/secundario , Humanos , Inmunohistoquímica , Interferón alfa-2 , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/enzimología , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Proteínas Recombinantes , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/enzimologíaRESUMEN
We describe a patient presenting with B cell chronic lymphocytic leukemia (B-CLL) who subsequently developed cutaneous infiltrates. Specimens of the blood, bone marrow and cutaneous infiltrations all showed the same heavy-chain gene rearrangement. Following failure of conventional chemotherapy, and in view of the similarity of the disease to cutaneous T cell lymphoma, interferon-alpha therapy was employed with satisfactory results. Introduction of this cytokine to the therapeutic modalities for secondary cutaneous B-CLL would hopefully change the poor outcome of this entity, or at least could produce a better quality of life. Loss of histidine decarboxylase activity in the infiltrating cells - in contrast to circulating lymphocytes - may be associated with the transformation of B-CLL to a more aggressive infiltrative form, offering a possible explanation for tissue invasiveness. The changing character of the disease raises the possibility of a second mutational event in the course of B-CLL.
Asunto(s)
Leucemia Linfocítica Crónica de Células B/patología , Infiltración Leucémica , Linfoma de Células B/patología , Linfoma no Hodgkin/patología , Piel/patología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Clorambucilo/administración & dosificación , Ciclofosfamida/administración & dosificación , Doxorrubicina/administración & dosificación , Histidina Descarboxilasa/deficiencia , Humanos , Factores Inmunológicos/uso terapéutico , Interferón alfa-2 , Interferón-alfa/uso terapéutico , Leucemia Linfocítica Crónica de Células B/diagnóstico , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/enzimología , Linfoma de Células B/diagnóstico , Linfoma no Hodgkin/diagnóstico , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/deficiencia , Prednisolona/administración & dosificación , Prednisona/administración & dosificación , Proteínas Recombinantes , Terapia Recuperativa , Vincristina/administración & dosificaciónAsunto(s)
Terapia de Inmunosupresión/métodos , Trasplante de Riñón/inmunología , Transfusión de Plaquetas , Formación de Anticuerpos , Antígenos CD/biosíntesis , Antígenos CD/sangre , Antígenos de Histocompatibilidad Clase I/biosíntesis , Antígenos de Histocompatibilidad Clase I/sangre , Prueba de Histocompatibilidad , Humanos , Inmunidad Celular , Isoantígenos/análisis , Donadores Vivos , Prueba de Cultivo Mixto de Linfocitos , Complejo Mayor de HistocompatibilidadRESUMEN
OBJECTIVE AND DESIGN: Rats treated with aggregated IgG (Aggr.) become "refractory" to the hypotensive action of a second dose of Aggr. The objective of this study was to assess the responsiveness of animals pretreated with Aggr. to bacterial LPS and vice versa. MATERIAL OR SUBJECTS: Female Wistar rats (250-300 g) were used. Each experiment was carried on at least 4 animals. TREATMENT: A human IgG preparation containing 30% aggregates (10-16 mg/100 g) or E. coli serotype 0111.B4 (0.005-mg/100 g) was administered i.v. Certain groups of animals were pretreated with 1 mg/100 g GdCl3 or with 10 mg/100 g pentoxyphylline (PTX). METHODS: Arterial blood pressure was monitored in the carotis-using a polyethylene cannula and an electronic tension meter. Tumor necrosis factor alpha (TNF-alpha) activity was estimated by the use of an L-929 cell cytotoxicity assay. RESULTS: Pretreatment of rats with a sublethal dose of LPS impaired the hypotensive reaction of the animals to Aggr. Rats male "refractory" to Aggr. reacted to the injection of LPS with hypotension and a second phase milder than in the controls. Hypotension could not be elicited by Aggr. in rats pretreated with GdCl3. The same pretreatment had no effect on the first phase of hypotension induced by intravenous injection of LPS, whilst a mitigation of the second phase was observed. Infusion of PTX immediately prior to Aggr. administration prevented the drop of blood pressure. A sizeable level of TNF-alpha was detected only later than blood pressure had reached its minimum level following Aggr. administration. CONCLUSIONS: Hypotension induced by LPS may involve a macrophage population broader than that responsible for the vascular action of Aggr. The data presented do not support a primary role for TNF-alpha in Aggr. induced hypotension.
Asunto(s)
Complejo Antígeno-Anticuerpo/administración & dosificación , Hipotensión/inmunología , Inmunoglobulina G/administración & dosificación , Lipopolisacáridos/administración & dosificación , Animales , Femenino , Gadolinio/farmacología , Lipopolisacáridos/farmacología , Masculino , Ratones , Pentoxifilina/farmacología , Ratas , Ratas Wistar , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
A new flow cytometric method was developed to detect apoptotic cells with fragmented DNA and to determine cell cycle distribution of viable cells, in the same sample, by propidium iodide staining. Apoptosis, in HT58 human B lymphoma cells, was induced by etoposide and/or by staurosporine. Using appropriate alkaline solutions (between 1-10 mN NaOH in 150 mM saline) followed by neutralization with buffer solution, the fragmented DNA can be extracted quantitatively from ethanol fixed cells. Further, good resolution of the cell cycle distribution can be obtained in unimpaired cells without RNase treatment. Furthermore, unlike the widely used hypotonic-detergent extraction of unfixed cells, the suggested extraction method can prevent drug-induced disintegration of dead cells when karyorrhexis occurs.
RESUMEN
A long-term tissue culture line of highly metastatic IR202 immunocytoma of LOU rats, and five of its clones (B4, C2, C4, C5, D3) was examined for their ability to produce cytokines. A combination of cytokine detection bioassays was employed in order to compensate for differences in sensitivity and specificity, and the possibility of inhibitors masking an activity. All the IR202 variants tested were shown to secrete constitutively varying amount of interleukin (IL-6). In addition, most of the cells produce IL-10 and TGF-beta, except clones C4 and C2, respectively. Moreover, supernatants from clone B4, C2, C4, and D3 may contain low levels of soluble IL-1. Membrane-bound IL-1 was found on the surface of B4, C4, and D3 cells. None of the IR202 variants, however, produced IL-2, IL-4, tumor necrosis factor-alpha (TNF-alpha), or LT. These results are consistent with the concept of a new type of intratumor heterogeneity and the ability of immunocytoma tumors to generate different immunoregulatory molecules in tumor-bearing hosts.
Asunto(s)
Citocinas/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Animales , Bioensayo , Línea Celular Tumoral/metabolismo , Células Clonales/metabolismo , Interleucina-1/metabolismo , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Leucemia Linfocítica Crónica de Células B/metabolismo , Ratas , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The cell cycle has been shown to regulate the biological effects of human tumor necrosis factor (TNF), but to what extent that regulation is due to the modulation of TNF receptors is not clear. In the present report we investigated the effect of the cell cycle on the expression of surface and soluble TNF receptors in human histiocytic lymphoma U-937. Exposure to hydroxyurea, thymidine, etoposide, bisbensimide, and demecolcine lead to accumulation of cells primarily in G1/S, S, S/G2/M, G2/M, and M stages of the cell cycle, respectively. While no significant change in TNF receptors occurred in cells arrested in G1/S or S/G2 stages, about a 50% decrease was observed in cells at M phase of the cycle. Scatchard analysis showed a reduction in receptor number rather than affinity. In contrast, cells arrested at S phase (thymidine) showed an 80% increase in receptor number. The decrease in the TNF receptors was not due to changes in cell size or protein synthesis. The increase in receptors, however, correlated with an increase in total protein synthesis (to 3.8-fold of the control levels). A proportional change was observed in the p60 and p80 forms of the TNF receptors. A decrease in the surface receptors in cells arrested in M phase correlated with an increase in the amount of soluble receptors. The cellular response to TNF increased to 8- and 2-fold in cells arrested in G1 and S phase, respectively; but cells at G2/M phase showed about 6-fold decrease in response. In conclusion, our results demonstrate that the cell cycle plays an important role in regulation of cell-surface and soluble TNF receptors and also in the modulation of cellular response.
Asunto(s)
Ciclo Celular , Receptores del Factor de Necrosis Tumoral/metabolismo , Ciclo Celular/efectos de los fármacos , ADN/metabolismo , Demecolcina/farmacología , Inhibidores Enzimáticos/farmacología , Etopósido/farmacología , Fase G1/efectos de los fármacos , Fase G2/efectos de los fármacos , Células HeLa , Humanos , Hidroxiurea/farmacología , Leucemia Eritroblástica Aguda , Linfoma de Células B Grandes Difuso , Mitosis/efectos de los fármacos , Nocodazol/farmacología , Fase S/efectos de los fármacos , Inhibidores de Topoisomerasa II , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/metabolismo , Vinblastina/farmacologíaRESUMEN
Cord blood (CB) as a new source for bone marrow transplantation represents advantageous features concerning stem cell and leucocyte compartments and function. We attempted to get more information about the phenotypes and function of CB cells by investigating their cell surface markers and also the production of IL-2, IFN-gamma and IL-6 by mitogen and alloantigen stimulation. The CB cells were characterized by a low proportion of CD3+ T cells, CD4+ T subpopulation, activated T cells and CD3+CD16/CD56+ cytotoxic cells, suggesting reduced graft versus host potential. The significant increase of CD19/CD3 double positive cells and decrease of CD19/HLA-DR double positive mature B cells reflect that immature B cells exist in CB. In the functional studies, a 27- and 5-fold reduction was observed in the production of IFN-gamma by CB cells stimulated with PHA and allogeneic cells, respectively. The production of IL-2 in PHA-stimulated CB cells also showed a 50% determination. Decrease in the production of these cytokines by CB cells is supported by the decline of the proportion of CD3+ T cells. However, an increase was observed in the production of IL-6 by CB cells stimulated with allogeneic cells as compared with the controls. These results suggest a difference in the functional activity of the T helper cell subsets between the CB and peripheral blood and/or differences in the functional maturity of T helper cell subsets and B cells in these compartments.
Asunto(s)
Sangre Fetal/citología , Células Madre Hematopoyéticas/inmunología , Inmunofenotipificación , Células Cultivadas , Femenino , Sangre Fetal/inmunología , Humanos , Interferón gamma/biosíntesis , Interleucina-2/biosíntesis , Interleucina-6/biosíntesis , EmbarazoRESUMEN
Tumor necrosis factor (TNF) has been shown to inhibit the growth of some cell types and stimulate the proliferation of others by a mechanism that is not understood. In the present study, we investigated the effect of transfection of NIH-3T3 cells with either the basic fibroblast growth factor gene (bFGF) or the kaposi FGF gene (K-fgf) on the growth-modulatory effects of TNF. Our results show that transformation of cells with either gene leads to resistance to the growth-inhibitory effects of TNF. The K-fgf gene was found to be a more potent inducer of cellular resistance than the bFGF gene. The cellular resistance correlated with the inhibition of TNF-induced activation of phospholipase A2 and down-modulation of TNF receptors. Overall, our results indicate that both K-fgf and bFGF play an important role in suppression of antiproliferative effects of TNF.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/genética , Oncogenes , Proteínas Proto-Oncogénicas/genética , Receptores del Factor de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Células 3T3 , Animales , Recuento de Células/efectos de los fármacos , División Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación hacia Abajo , Factor 2 de Crecimiento de Fibroblastos/fisiología , Factor 4 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/fisiología , Ratones , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Proteínas Proto-Oncogénicas/fisiología , Transfección/genéticaRESUMEN
Tumor necrosis factor (TNF) functions both as a soluble molecule and as a cell surface 26 kDa transmembrane protein, from which the soluble form is proteolytically derived. The 26 kDa TNF molecules isolated from 32P labeled HeLa cells that had been transfected with the cDNA of a partially cleavable TNF mutant were found labeled. Phosphorylated 26 kDa TNF molecules could also be isolated from human LPS stimulated monocytic Mono Mac 6. Phosphoaminoacid analysis revealed that the labeled phosphate is bound to serine residues. No label was found incorporated in soluble 17 kDa TNF, indicating that the phosphorylated residue(s) of membrane-associated TNF occur in the cytoplasmic portion of the molecule. Phosphorylation of the intracellular domain of the 26 kDa TNF molecules may play a role in the regulation of expression or proteolytic processing of TNF, modulate TNF bioactivity, or take part in intracellular signaling by cell-surface TNF.
Asunto(s)
Células HeLa/metabolismo , Monocitos/metabolismo , Precursores de Proteínas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Expresión Génica , Humanos , Técnicas de Inmunoadsorción , Leucemia Monocítica Aguda , Peso Molecular , Mutación , Fosforilación , Transfección , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
The growth of cells is determined by the balance between growth-stimulatory and growth-inhibitory signals. In the present study, we demonstrate that the transfection of NIH 3T3 cells with a platelet-derived growth factor (PDGF-Blc-sis) gene induces resistance to the anticellular effects of tumor necrosis factor (TNF). Human tumor cell lines that express elevated levels of c-sis (e.g. epidermoid carcinoma, A-431) are also TNF resistant, whereas those that express no significant levels of this gene (e.g. breast adenocarcinoma, MCF-7) are TNF sensitive. Transfection of cells with the c-sis gene leads to down-modulation of TNF receptors and also a decrease in intracellular glutathione levels. Thus, our results demonstrate that over-expression of PDGF-Blc-sis by certain tumor cells can lead to their protection from the anticellular effects of TNF.
Asunto(s)
División Celular/fisiología , Factor de Crecimiento Derivado de Plaquetas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Células 3T3 , Animales , Glutatión/metabolismo , Humanos , Interferón gamma/farmacología , Ratones , Factor de Crecimiento Derivado de Plaquetas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-sis , Receptores del Factor de Necrosis Tumoral/metabolismo , Transfección , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
Tumor necrosis factor (TNF) is a growth-modulatory cytokine that inhibits the growth of certain cell lines, stimulates the growth of some, and has no effect on the growth of still others. The molecular basis for this differential regulation of growth by TNF is not understood. We postulate that the growth of normal or tumor cells is determined by the balance between growth-stimulatory and -inhibitory signals. In the present study, we demonstrate that the transfection of cells with the transforming growth factor (TGF)-alpha gene induces resistance to TNF. Colon carcinoma cell lines that express elevated levels of TGF-alpha were also found to be resistant to this cytokine. Exogenous addition of the growth factor was also effective in decreasing the antiproliferative effects of TNF. Transfection of cells with the TGF-alpha gene led to downmodulation of TNF receptors but an increase in intracellular glutathione levels. Thus, these results support our hypothesis that expression of growth factors by certain tumor cells can lead to resistance to antiproliferative agents such as TNF.
Asunto(s)
División Celular/efectos de los fármacos , Factor de Crecimiento Transformador alfa/fisiología , Factor de Necrosis Tumoral alfa/farmacología , Células 3T3/citología , Animales , Regulación hacia Abajo/efectos de los fármacos , Glutatión/biosíntesis , Humanos , Ratones , Receptores del Factor de Necrosis Tumoral/biosíntesis , Receptores del Factor de Necrosis Tumoral/metabolismo , Transfección , Factor de Crecimiento Transformador alfa/genética , Factor de Crecimiento Transformador alfa/farmacología , Células Tumorales Cultivadas/citología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
While some tumor cells are sensitive to the antiproliferative effects of tumor necrosis factor (TNF), others are resistant. The molecular basis for cellular resistance to TNF is not completely understood. Previously we have shown that transfection of cells with an oncogene HER2/neu/erb B2, a receptor tyrosine kinase, leads to resistance to the anticellular effects of TNF [(1988) Proc. Natl. Acad. Sci. USA 85, 5102-5106]. In the present study, we demonstrate that the overexpression of another oncogenic tyrosine kinase, pp60v-src also induces resistance to TNF. In contrast to HER2, however, pp60v-src transfection of cells did not lead to down-modulation of TNF receptors but rather to decreased intracellular glutathione levels. The pp60v-src-induced cellular resistance to TNF could be abrogated by interferon-gamma. Thus, these results indicate that the resistance of certain tumors to TNF may also be due in part to the overexpression of pp60v-src oncogene.
Asunto(s)
División Celular/efectos de los fármacos , Resistencia a Medicamentos/fisiología , Glutatión/metabolismo , Proteína Oncogénica pp60(v-src)/biosíntesis , Receptores del Factor de Necrosis Tumoral/biosíntesis , Factor de Necrosis Tumoral alfa/toxicidad , Células 3T3 , Animales , Regulación hacia Abajo , Receptores ErbB/biosíntesis , Receptores ErbB/genética , Humanos , Interferón gamma/farmacología , Cinética , Ratones , Proteína Oncogénica pp60(v-src)/metabolismo , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor ErbB-2 , Proteínas Recombinantes , Factores de Tiempo , TransfecciónRESUMEN
Tumor necrosis factor (TNF) is a multipotential cytokine known to regulate the growth of a wide variety of normal and tumor cells. It has been shown that the density of cells in culture can modulate the growth regulatory activities of TNF, the mechanism of which, however, is not understood. In this report, we investigated the effect of cell density on the expression of TNF receptors. The receptors were examined on epithelial cells (e.g., HeLa), which primarily express the p60 form, and on myeloid cells (e.g., HL-60) known to express mainly the p80 form. We observed that binding of TNF to both cell lines decreased with increase in cell density. Scatchard analysis of binding on HeLa and HL-60 cells revealed a 4- to 5-fold reduction in the number of TNF receptors without any significant change in receptor affinity in both cell types at high density. The decrease in TNF receptor numbers at high cell density was also observed in several other epithelial and myeloid cell lines. The downmodulation at high cell density was unique to TNF receptors, since minimum change in other cell surface proteins was observed as revealed by fluorescent activated cell sorter analysis. Neutralization of binding with antibodies specific to each type of the receptors revealed that both the p60 and p80 forms of the TNF receptor were equally downmodulated. A decrease in leucine incorporation into proteins was observed with increase in cell density, suggesting a reduction in protein synthesis. Since inhibition of protein synthesis by cycloheximide also leads to a decrease in TNF receptors, it is possible that the density-dependent reduction in TNF receptor number is due to an overall decrease in protein synthesis. The density-dependent decrease in TNF receptors was accompanied by a decrease in intracellular reduced glutathione levels. A reduction in the number of receptors on TNF sensitive tumor cells induced by cell-density correlated with increase in resistance to the cytokine.
Asunto(s)
Membrana Celular/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Recuento de Células , División Celular , Regulación hacia Abajo , Epitelio/metabolismo , Glutatión/metabolismo , Granulocitos/metabolismo , Células HeLa/metabolismo , Humanos , Leucemia Promielocítica Aguda/metabolismo , Biosíntesis de Proteínas , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Because human lymphotoxin (LT) was originally isolated from a lymphoblastoid cell line, we investigated the role of this molecule in three newly established Epstein-Barr virus (EBV)-infected human B cell lines. These lines were derived from acute lymphoblastic leukemia (Z-6), myelodysplastic syndrome (Z-43), and acute myelogenous leukemia (Z-55) patients who had a prior EBV infection. Each lymphoblastoid cell line had a karyotype that was different from that of the original parent leukemic cells, and all expressed B cell, but not T cell or myeloid surface markers. In all three lines, rearranged immunoglobulin heavy chain joining region (JH) bands were found, and the presence of EBV DNA was confirmed by Southern blotting. Z-6, Z-43, and Z-55 cell lines constitutively produced 192, 48, and 78 U/ml LT, respectively, as assessed by a cytotoxicity assay and antibody neutralization. Levels of tumor necrosis factor (TNF) were undetectable. Scatchard analysis revealed that all the cell lines expressed high-affinity TNF/LT receptors with receptor densities of 4197, 1258, and 1209 sites/cell on Z-6, Z-43, and Z-55, respectively. Furthermore, labeled TNF binding could be reversed by both unlabeled TNF, as well as by LT. Studies with p60 and p80 receptor-specific antibodies revealed that the three lines expressed primarily the p80 form of the TNF receptor. When studied in a clonogenic assay, exogenous LT stimulated proliferation of all three cell lines in a dose-dependent fashion at concentrations ranging from 25 to 500 U/ml. Similar results were obtained with [3H]TdR incorporation. Monoclonal anti-LT neutralizing antibodies at concentrations of 25-500 U/ml inhibited cellular multiplication in a dose-dependent manner. It is interesting that in spite of a common receptor, TNF (1,000 U/ml) had no direct effect on Z-55 cell growth, whereas it partially reversed the stimulatory effect of exogenous LT. In addition, TNF inhibited Z-6 and Z-43 cell proliferation, and its suppressive effect was reversed by exogenous LT. Both p80 and p60 forms of soluble TNF receptors suppressed the lymphoblastoid cell line proliferation and their inhibitory effect was partially reversed by LT. Our data suggest that (a) LT is an autocrine growth factor for EBV-transformed lymphoblastoid B cell lines; and (b) anti-LT antibodies, soluble TNF/LT receptors, and TNF itself can suppress the growth of lymphoblastoid cells, probably by modulating or competing with LT.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Linfocitos B/microbiología , Linfocitos B/patología , Sustancias de Crecimiento/análisis , Infecciones por Herpesviridae/patología , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/aislamiento & purificación , Leucemia Mieloide Aguda/patología , Linfotoxina-alfa/análisis , Síndromes Mielodisplásicos/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos/inmunología , Anticuerpos/farmacología , Anticuerpos Monoclonales , Linfocitos B/química , Southern Blotting , División Celular/fisiología , Células Cultivadas , ADN Viral/análisis , ADN Viral/genética , Relación Dosis-Respuesta a Droga , Sustancias de Crecimiento/metabolismo , Sustancias de Crecimiento/fisiología , Humanos , Cadenas Pesadas de Inmunoglobulina/análisis , Inmunofenotipificación , Cariotipificación , Leucemia Mieloide Aguda/genética , Linfotoxina-alfa/metabolismo , Linfotoxina-alfa/fisiología , Síndromes Mielodisplásicos/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Receptores de Superficie Celular/análisis , Receptores de Superficie Celular/metabolismo , Receptores del Factor de Necrosis Tumoral , Factor de Necrosis Tumoral alfa/análisis , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Tumor necrosis factor-alpha (TNF-alpha) induces the expression of human immunodeficiency virus type-1 (HIV-1) in vitro in chronically infected cells of T and monocytic origin. The tat protein from the HIV-1 virus has been shown to be essential for HIV replication and in the immunosuppression associated with the virus infection. Previous studies in our laboratory have shown that HIV-1 tat gene induces TNF-beta (lymphotoxin) in human B-lymphoblastoid cells (Sastry et al., 1990, J. Biol. Chem. 265, 20091-20093). In an attempt to characterize further the relationship between the host and HIV-1, we investigated the effect of the functional HIV-1 tat gene on the expression of TNF receptors in a human B lymphoblastoid cell line (Raji). We report here that Raji cells transfected with HIV-1 tat gene express fewer cell surface TNF receptors than control cells. At least a 5-fold decrease in the receptor number without any significant change in receptor affinity was observed. The decrease in TNF receptors in tat-transfected Raji cells (Raji-tat cells) was found not to be due to receptor occupancy by the autocrine production of TNF-beta. The decrease in the cell surface expression of TNF receptors in Raji-tat cells was also found to be not due to a decrease in the gene expression of the receptor. The kinetics, amount of TNF binding and its internalization were temperature dependent, and it was different in Raji-tat cells than in the control cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Genes tat , VIH-1/genética , Receptores de Superficie Celular/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Línea Celular , Membrana Celular/metabolismo , Regulación hacia Abajo , Expresión Génica , Productos del Gen tat/genética , Productos del Gen tat/inmunología , Productos del Gen tat/metabolismo , VIH-1/inmunología , Humanos , Tolerancia Inmunológica , Peso Molecular , Receptores de Superficie Celular/química , Receptores del Factor de Necrosis Tumoral , Transfección , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
Interferons are known to potentiate various biological effects of tumor necrosis factor (TNF). Recently, two different types of TNF receptors with molecular masses of 60 kDa (p60) and 80 kDa (p80), primarily expressed by epithelial cells and myeloid cells, respectively, have been identified. In the present report, we examined the effect of interferon-gamma (IFN-gamma) on each type of TNF receptor. Our results indicate that IFN-gamma induces TNF receptors on both myeloid (e.g. HL-60) and epithelial cells (e.g. HeLa). Furthermore, by using antibodies specific to each type of receptor, we demonstrate that both TNF receptors are equally inducible by IFN-alpha, IFN-beta and IFN-gamma. Thus, the increase in TNF receptors by interferons may play a role in their synergistic cellular response.