RESUMEN
Primary human monocytes and the human monocytic cell line THP-1 were induced to express receptors for interleukin-1 alpha (IL-1 alpha) and IL-1 beta. Treatment of primary monocytes with dexamethasone resulted in a 10-fold increase in receptor number over untreated cells, to approximately 2,000 receptors/cell. Treatment of THP-1 cells with phorbol ester followed by prostaglandin E2 and dexamethasone resulted in the expression of approximately 30,000 receptors/cell. Competitive binding assays on THP-1 cells showed that both IL-1 alpha and IL-1 beta bind to the same receptor. The monocyte IL-1R is significantly smaller (63 kDa) than the T cell IL-1R (80 kDa) and is immunologically distinct. However, induction of monocytes and monocytic cell lines leads to the appearance of an abundant mRNA of approximately 5,000 bases which hybridizes to a cDNA probe from the T cell-type IL-1R. Sequence data obtained from a cDNA clone of this mRNA indicate that the message is identical to the T cell IL-1R mRNA throughout the coding region. A smaller mRNA, also homologous to the T cell IL-1R mRNA, accumulated in induced THP-1 cells and has a shorter 3'-untranslated region than the larger. Data are presented which suggest that neither form of this message encodes the 63-kDa IL-1R, but rather that this protein is the product of a separate nonhomologous mRNA.
Asunto(s)
Interleucina-1/metabolismo , Monocitos/inmunología , ARN Mensajero/genética , Receptores Inmunológicos/genética , Linfocitos T/inmunología , Línea Celular , Dexametasona/farmacología , Dinoprostona/farmacología , Escherichia coli/genética , Humanos , Cinética , Monocitos/efectos de los fármacos , Receptores Inmunológicos/biosíntesis , Receptores Inmunológicos/metabolismo , Receptores de Interleucina-1 , Proteínas Recombinantes/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
We have designed and constructed an electroporation device capable of efficient transfer of DNA into both plant cell protoplasts and cultured murine lymphocytes. The electroporator design allows various combinations of voltage and capacitance to be used to optimize the electric pulse. Switching of large voltages and currents is accomplished with a silicon-controlled rectifier, yielding excellent reproducibility and long component life. A safety switch is provided to permit complete discharge of the device. Conditions suitable for high levels of transient expression and high frequencies of stable transformation for both plant and animal cell systems have been found.
Asunto(s)
Técnicas Citológicas/instrumentación , Plantas/ultraestructura , Protoplastos/ultraestructura , Animales , Células Cultivadas , Difusión , Linfocitos/ultraestructura , Ratones , Plantas Tóxicas , Plásmidos , Nicotiana/ultraestructuraRESUMEN
We have employed three different models of major histocompatibility complex (MHC) antigen to investigate the role of these antigens in some aspects of human T cell activation in in vitro; specifically the induction of lymphokine synthesis and receptors, and in antigen presentation. Those models were (1) allogeneic platelets (class I alone); (2) D/DR compatible lymphocytes (class I plus nonimmunogenic class II); and (3) allogeneic heat-treated lymphocytes. After heating at 45 C for 1 hr, the latter are completely viable and express both class I (HLA A and B) and class II (HLA, DR, MT/MB) MHC antigens but do not stimulate in the mixed lymphocyte reaction (MLR). These models were compared with conventional irradiated MLR stimulating or antigen-presenting cells. Only the conventional cells were able to stimulate the synthesis of interleukin-1 and interleukin-2. Also, irradiated but not heat-treated non-T cells could present soluble antigen to autologous T cells. This implies that intact and unmodified Ia molecules are required for those responses. On the other hand, heat treated allogeneic lymphocytes and D/DR-compatible lymphocytes but not allogeneic platelets could induce responsiveness to interleukin-2. This function appears to require a heat stable but nonallogeneic component of the class II molecules. Our previous observations employing these models have also shown that there is yet a third category of T cell responses, including memory cell priming and suppressor cell induction, that can occur in the presence of predominantly class I antigen. Taken together, these data illustrate the functional heterogeneity of T cell responses to Ia-like antigens. The possible relevance of these findings to clinical transplantation is discussed.