RESUMEN
BACKGROUND: T lymphocytes play a crucial role in the pathogenesis of inflammatory bowel disease. Achieving stable T-cell lines, rather than continuous bleeding of patients, is desirable in order to dissect their implication in the disease. METHODS: Long-lasting T-cell lines from patients with Crohn disease and ulcerative colitis and from healthy volunteers have been obtained by transformation of T lymphocytes using the lymphotropic Herpesvirus saimiri. Lines were subjected to phenotypic and functional analyses, and the results compared with freshly isolated peripheral blood mononuclear cells. RESULTS: Fresh cells revealed only minor differences between patients and controls, with regard to phenotype and proliferative capacity. In contrast, the use of T-cell lines showed that cells from Crohn disease patients, but not ulcerative colitis patients, over-responded to several membrane or cytoplasmic stimuli when compared to control T-cell lines. Thus, higher responses were found when stimulated with alphaCD3 and IL2, alphaCD3 and alphaCD28, IL2 alone, phorbol esters (PMA) and alphaCD3 and, finally, PMA and alphaCD2 (P < 0.05 in all instances). Further, lines from patients with Crohn disease responded more vigorously to alphaCD3 and alphaCD28 or alphaCD3 and PMA when compared to ulcerative colitis (P < 0.05 in both instances). CONCLUSIONS: The data obtained with these lines suggest that T cells from patients with Crohn disease differ in vivo in their proliferative capacity, as compared with those from ulcerative colitis patients, a finding that may reflect the clear Th-1 phenotype found in the former and absent in the latter.
Asunto(s)
Proliferación Celular , Colitis Ulcerosa/fisiopatología , Enfermedad de Crohn/fisiopatología , Leucocitos Mononucleares/fisiología , Linfocitos T/fisiología , Adulto , Anciano , Antígenos CD/metabolismo , Estudios de Casos y Controles , Línea Celular , Transformación Celular Viral , Colitis Ulcerosa/metabolismo , Enfermedad de Crohn/metabolismo , Femenino , Herpesvirus Saimiriino 2 , Humanos , Masculino , Persona de Mediana EdadRESUMEN
We have taken advantage of a recently described technique of transformation and immortalization of T lymphocytes using the lymphotropic Herpesvirus saimiri, to achieve long-lasting T-cell lines from gastric cancer patients and healthy volunteers. Blood samples were drawn and T lymphocytes were transformed. Once sustained growth was observed, lines were subjected to phenotypic and functional analyses, and the results compared with freshly isolated peripheral blood mononuclear cells. Cytofluorometric analysis revealed that CD3 and CD45 were found at lower proportion in primary cells from patients than from control individuals (54% vs 75%, p<0.001, 90% vs 96%, p<0.05, respectively), and in HVS-derived T-cell lines (90% vs 98%, p<0.05, 97% vs 100%, p<0.05, respectively). Proliferative analyses showed that primary isolated cells were unable to respond adequately to CD3-, CD2-, and PHA-mediated stimulation, as compared to controls. Similarly, T-cell lines from patients proliferated to a lesser extent when CD3- and CD2-mediated stimuli were considered, especially when simultaneous stimulation via CD3 and CD2 molecules was carried out (47,824 counts per minute [cpm] vs 121,478 cpm, p<0.05). Altogether these results show that the defects reported in T cells from patients with cancer are not exclusively due to tumour-derived factors, since the alterations persist in long-lasting, HVS-transformed, T-cell lines, suggesting that this model seems a suitable one to disclose them.
Asunto(s)
Adenocarcinoma/inmunología , Antígenos CD2/análisis , Complejo CD3/análisis , Neoplasias Gástricas/inmunología , Linfocitos T/inmunología , Línea Celular Transformada , Transformación Celular Viral , Femenino , Citometría de Flujo , Herpesvirus Saimiriino 2 , Humanos , Inmunofenotipificación , Activación de Linfocitos , MasculinoRESUMEN
Caco-2 is a colonic tumour cell line which, when cultured, spontaneously exhibits enterocyte-like characteristics. Given the difficulties in maintaining long-lasting cultures of enterocytes, this cell line may be a suitable in vitro model to carry out experiments trying to delineate the involvement of enterocytes in local immune responses, and their role in pathology. It seems then reasonable to obtain a detailed immune analysis of Caco-2, and compare it with available data on enterocytes. Cytofluorometry revealed several leukocyte markers on Caco-2, present also on human enterocytes. These markers include surface proteases (CD10, CD13 and CD26), antigen-presenting cell markers (CD13, CD14, CD35 and CD63), integrins (CD18 and CD61), epithelial/endothelial markers (CD21, CD31, CD47 and CD59) and finally, CD25 and CD28. In contrast to enterocytes, HLA-class 11 molecules are not found on Caco-2, whether resting or gamma-IFN-stimulated. Moreover, culture experiments with allogeneic lymphocytes revealed that Caco-2 cells were unable to induce their proliferation. Cytokine analysis showed an increased RANTES synthesis and IL-2 transcription upon stimulation with IL-1beta. Finally, amongst RANTES receptors, CCR1 is found on Caco-2 cells, whereas CCR3 and CCR5 are not.