RESUMEN
Activated natural killer (A-NK) cells, defined by immunophenotype and selected by adherence to the plastic, were cultured from murine splenocytes for up to 10 days with the addition of 1000 U/ml of recombinant human IL-2 at 48 h intervals. During culture days 2-4 with high DNA synthesis the initially non-granulated small cells established large granular lymphocyte (LGL) morphology and then differentiated further into giant hypergranulated cells with huge accumulations of glycogen. Timed EM observations indicated that specific dual-compartment (lytic) granules arose by a sequence of events starting with neo-synthesis of small progenitors with a dense core and a few membranous lamellae at one pole. Core and vesicular regions probably expanded independently to give the mature organization of the granule. Eventually, the vesicular region of granules contained large amounts of multi-lamellar material and probable debris, and the dense core could be multiplied. Intracellular proteoglycans, visualized with Cupromeronic Blue cytochemistry, were organized in a three-dimensional network within the dense cores. In contrast with earlier reports, and in spite of several-fold increased granularity, the in vitro cytotoxicity of the A-NK cells against YAC-1 and B16 cells decreased after the third day of culture. A-NK cells with glycogen accumulations caused focal clearing in melanoma monolayers whereas younger effectors adhered to the targets. It is concluded that high dose IL-2 stimulation causes more far-going progressive morphological and functional differentiations of the A-NK cells than has previously been observed with bearing for the use of these cells in experimental adoptive immunotherapy.
Asunto(s)
Comunicación Celular/inmunología , Gránulos Citoplasmáticos/metabolismo , Citotoxicidad Inmunológica , Glucógeno/metabolismo , Interleucina-2/farmacología , Células Asesinas Activadas por Linfocinas/metabolismo , Activación de Linfocitos , Animales , Adhesión Celular , Comunicación Celular/efectos de los fármacos , Compartimento Celular , División Celular/inmunología , Núcleo Celular/ultraestructura , Gránulos Citoplasmáticos/inmunología , Gránulos Citoplasmáticos/ultraestructura , Citotoxicidad Inmunológica/efectos de los fármacos , Relación Dosis-Respuesta Inmunológica , Glucógeno/biosíntesis , Glucógeno/ultraestructura , Humanos , Inmunofenotipificación , Cuerpos de Inclusión/ultraestructura , Células Asesinas Activadas por Linfocinas/inmunología , Células Asesinas Activadas por Linfocinas/ultraestructura , Metabolismo de los Lípidos , Activación de Linfocitos/efectos de los fármacos , Linfoma , Lisosomas/ultraestructura , Masculino , Melanoma Experimental , Ratones , Ratones Endogámicos C57BL , Proteoglicanos/metabolismo , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
An experimental set-up for estimating a) cellular migration under agarose and b) response to chemoattractant gradients built up in the agarose was used in order to explore the behavior of adherent interleukin-2 (IL-2)-activated natural killer (A-NK) cells on cell culture plastic and after coating with extracellular matrix (ECM) constituents. A-NK cells were deposited in wells in the agarose and directed migration, chemotaxis, towards aggregates and suspensions of B16F10 melanoma cells, suspensions of YAC-1 cells, and tumor-conditioned media, all deposited in wells at a 2.5 mm distance, was tested. A-NK cell chemotaxis was exclusively observed when B16F10 aggregates were used as attractants. The substrate influenced chemotaxis considerably, untreated plastic surface being most favorable for a chemotactic response, followed by laminin, fibronectin, and collagen IV pretreatments. Coating with reconstituted basement membrane matrix (Matrigel) gave lesser random movements, chemokinesis, of A-NK cells than coating with the purified components laminin and collagen IV, and the least motile response was obtained after collagen I pretreatment. These in vitro observations indicate that melanoma cell aggregates release humoral factors of a probably short-lived nature with a chemoattractant effect on A-NK cells, and that ECM composition influences migratory response, both conclusions with a bearing on the understanding of A-NK cell infiltration into tumors in vivo.
Asunto(s)
Interleucina-2/fisiología , Células Asesinas Naturales/citología , Melanoma Experimental/inmunología , Animales , Movimiento Celular , Quimiotaxis de Leucocito , Proteínas de la Matriz Extracelular/fisiología , Activación de Linfocitos , Melanoma Experimental/patología , Ratones , Células Tumorales CultivadasRESUMEN
Adoptively transferred activated natural killer (A-NK) cells infiltrate tumours in vivo. Two in vitro B16-F10 melanoma tumour models were used to study with fluorescence and electron microscopy the infiltration of adherent interleukin 2 (IL-2) A-NK cells: (1) substratum-bound sessile microtumours (MTs), and (2) three-dimensional cell growth on macroporous gelatinous microcarriers (Cultispheres). From 2 h and on increasing numbers of A-NK cells infiltrated the MTs regularly surrounded by a widened intercellular space. An IL-2-dependent disintegration of MTs began at 6-8 h resulting in a release of vital and dead cells. A-NK cell invasion into Cultispheres effectively displaced the melanoma cells from the highly convoluted substratum. Thus, A-NK cell infiltration had a protease-like effect on the tumour cell aggregates which might have a bearing on the interpretation of their cytolytic effect on target cells. Ultrastructural evidence was not obtained of specific A-NK/target conjugate formation or of granule-mediated target cell destruction in either model tumour.
Asunto(s)
Citotoxicidad Inmunológica , Interleucina-2/farmacología , Células Asesinas Activadas por Linfocinas/inmunología , Melanoma Experimental/inmunología , Traslado Adoptivo , Animales , Adhesión Celular/inmunología , Agregación Celular/inmunología , Células Asesinas Activadas por Linfocinas/efectos de los fármacos , Células Asesinas Activadas por Linfocinas/ultraestructura , Activación de Linfocitos , Masculino , Melanoma Experimental/ultraestructura , Ratones , Ratones Endogámicos C57BL , Células Tumorales CultivadasRESUMEN
To elucidate the role of tumor vascularization on the localization of adoptively transferred, interleukin 2-activated natural killer (A-NK) cells, pulmonary B16 melanoma metastases were analyzed with respect to location, morphological appearance, origin and density of microvessels, and infiltration by A-NK cells. The B16 melanoma metastases could be divided into four subtypes according to their location (superficial or deep in the lung parenchyma) and morphological appearance (compact or loose). Localization of adoptively transferred A-NK cells into the four subtypes of B16 pulmonary metastases differed significantly. More than 800 A-NK cells/mm2 were found in metastases of the deep-loose type, compared to approximately 400/mm2 A-NK cells in the superficial-loose metastases, and less than 200 A-NK cells/mm2 in the compact subtype, regardless of its location (deep or superficial). Although the origin (pulmonary or bronchial) of the blood supply to the metastatic subtypes (as revealed by electron microscopic analyses of lungs perfused with a lanthanum solution) did not account for this difference, the density of microvessels in the metastatic subtypes correlated with the number of A-NK cells that localized into these metastases. The resistance of metastases of the compact type to infiltration of adoptively transferred effector cells might explain, in part, why adoptive immunotherapy seldom results in complete eradication of disseminated cancer.