ImmunoMethods最新文献

The Kupffer cell (KC) is the resident hepatic macrophage, whose functions include local intrahepatic immune responses implicated in tolerance induction, participation in the septic state, and regulation of hepatic regeneration. The ability of the KC to participate In these biologically diverse functions is thought to be due to its release of pleiotropic cytokines, such as interleukin 6 (IL-6), which can act locally in a paracrine fashion or as hormones at distant sites. Many of the KC′s secretory responses are carefully regulated in an autocoid fashion by the eicosanoid prostaglandin E₂ (PGE₂). The degree of regulation depends on the particular cytokine and local environmental factors. In this review, we describe our method for isolating KCs by their adherence to plastic and for testing their IL-6 and PGE₂ secretory responses to lipopolysaccharide. In comparing the responses of KCs from normal and regenerating rat livers, we describe an in vitro KC secretory pattern of eicosanoid inhibition of IL-6, whereas both responses to LPS are augmented in the KC during hepatic regeneration. Such an enhancement is consistent with the shared putative supportive roles of IL-6 and PGE₂ in liver regeneration.

Prostaglandins are known to influence a variety of functional activities of T cells. Prostaglandins such as PGE₂ suppress T-cell function by mechanisms that depend on their capacity to stimulate adenylate cyclase and to increase cyclic AMP (cAMP) levels. PGE₂ inhibits both antigen- and mitogen-stimulated T-cell DNA synthesis and IL-2 production. Inhibition of IL-2 production is a primary inhibitory effect of PGE₂, as supplementation with IL-2 can overcome much of the PGE₂-mediated inhibition of T-cell DNA synthesis. Study of purified human T cells has revealed a number of mechanisms by which PGE₂ and increases in cAMP levels inhibit IL-2 production. PGE₂/cAMP inhibit activation of phospholipase C, one of the early biochemical steps involved in T-cell activation resulting from ligation of the T-cell receptor-CD3 complex. This can result in diminished mobilization of intracellular Ca²⁺ stores or activation of protein kinase C. This is not the sole mechanism by which PGE₂ inhibits IL-2 production, however, as phorbol esters, which directly activate protein kinase C, cannot prevent inhibition of IL-2 production. Even if initial activation is successful, elevation of cAMP levels can independently modulate gene transcription, leading to decreased mRNA levels for IL-2. In addition to suppressing T-cell responses, elevation of cAMP levels, which may also occur normally during T-cell activation, may be required for certain functional activities of T cells, including capping of the CD3-T-cell receptor complex or expression of the differentiation marker CD7. In this regard, PGE₂ or other cAMP-elevating agents can facilitate cell cycle progression after initial receptor-initiated biochemical events are accomplished. Thus, PGE₂ and other cAMP-elevating agents exert potent and specific regulatory influences on specific aspects of T-cell responsiveness to antigens and mitogens.

Prostaglandin E₂ (PGE₂) has selective effects on the production of murine helper-T-cell lymphokines. PGE₂ inhibits production of the Th1-associated lymphokines IL-2 and IFN-γ, but does not inhibit production of the Th2-associated lymphokines IL-4 and IL-5. This could have been due to differences in the Th1 and Th2 cells themselves or to differences in the cytokines. To discriminate between these models we first examined the effect of PGE₂ on IL-3 production, a lymphokine produced by both Th1 and Th2 cells. IL-3 production was inhibited by PGE₂ in some cells and enhanced in others, indicating that some property of the cell was critical. However, the effect on IL-3 production did not cleanly discriminate between Th1 and Th2 cells. Second, we examined the effect of PGE₂ on lymphokine production from Th0 cells. In some cells, production of IL-2, IL-3, and IL-4 was inhibited by PGE₂. In other cells, IL-2 and IL-3 were inhibited while IL-4 production was enhanced. These data again indicated that it was a property of the T cell, not necessarily the lymphokine itself, that determined the response to PGE₂. In Th1 and Th2 clones, both the mode of primary stimulation (antigen and antigen-presenting cells or calcium ionophore) and the presence of costimulation also were found to affect the response of IL-3 production to PGE₂. Therefore, the effect of PGE₂ on lymphokine production appears to depend upon the intracellular signaling pathways that are activated within a particular T cell.

The splenic focus assay is an extremely powerful technique for studying monoclonal B cell responses to in vitro T-cell-dependent antigenic stimulation. This method has been applied to studies of several aspects of B cell function, including that of B cell tolerance. Included here are descriptions of the splenic focus assay with its modifications that enable the analysis of B cell tolerance, the advantages and limitations of the assay, and the application of the in vitro assay to the study of various aspects of B cell tolerance, such as the parameters of B cell tolerance, the susceptibility of various B cell subpopulations, and the effect of tolerance on repertoire expression.

Basic research into the cellular and molecular mechanisms leading to transplantation tolerance has undergone a renaissance during the past decade. A number of elegant and ingenious experimental approaches have been developed and utilized to study, both in vitro and in vivo, the changes in T-lymphocytes that accompany tolerance induction. In this article, we emphasize mechanisms that accomplish T-cell-dependent transplantation tolerance via "passive" (clonal deletion/anergy) and "active" (suppression/priming of IL-4-producing T cells) mechanisms. Evidence is summarized from the recent literature describing several different and important experimental models of transplantation tolerance:intravenous injection of allogeneic cells, direct intrathymic injection of allogeneic cells, transgenic mice expressing genomically incorporated alloantigens, antibody-mediated depletion of T-cell subsets, and neonatal transplantation tolerance. At the present state of our understanding it is clear that only rarely does a single mechanism take sole responsibility for the tolerant condition; neonatal transplantation tolerance is an excellent example of a model that is induced and maintained by a coalition of tolerance-promoting processes. It is also apparent that induction of unresponsiveness among individual T-cells, once thought to occur exclusively in the thymus gland, can occur extrathymically, even among immunocompetent T-cells. This realization has revived optimism among basic and clinical transplanters who have long held the aspiration that prolonged, even indefinite, allograft survival can be achieved in adult human beings with only minimal perturbation of the immune system.

The complex mechanism by which an organism becomes immunologically self-tolerant and yet remains responsive to foreign antigens is not well understood. Transgenic technology, which makes possible the controlled expression of select genes in the developing mammal, provides us with a versatile tool for the study of tolerance in vivo. The permanent integration of selected genes into the mouse germline has allowed the expression of self-reactive T- and B-cell receptors, the ectopic expression of self-antigens, and the expression of modified self-antigens. In this review, we discuss methods for the production of transgenic mice and highlight experiments that have used transgenic animals to study tolerance.

This review describes several in vitro models of anergy in both murine and human CD4⁺ T cells, relating the role of lymphokine production to the induction and maintenance of tolerance. The division of CD4⁺ T cells into Th1 and Th2 sub-populations and the capacity of Th2 lymphokines to abrogate the response of T cells belonging to the Th1 subset are discussed. The model of nonresponsiveness is described in terms of phenotypic modulation, responses to IL2, requirement for co-stimulatory molecules, and lymphokine profiles. The various methods for measuring lymphokines using bioassays, immunoassays, or molecular techniques are compared and critiqued.

Despite the remarkable progress in clinical organ transplantation in the past two decades, significant problems remain to be solved. These problems include rejection, late opportunistic sepsis, spontaneous neoplasms, metabolic complications, and drug toxicity. In this article, various experimental models of tolerance and ways of inducing tolerance to tissue allographs are discussed.

The techniques of fetal thymus organ culture have been widely utilized for the study of thymocyte differentiation under carefully controlled conditions. Recent results suggesting a role for dendritic cells (DC) in selection of a competent T-cell repertoire have prompted attempts to construct chimeric thymus rudiments in vitro. Here, we describe a novel approach based on the migratory properties of mature lymphoid DC. Purified DC from adult thymus or spleen were found to migrate into fetal thymus rudiments in culture and localize specifically within the medulla. This distribution closely mirrors the situation in vivo, underlining the physiological relevance of the resulting microenvironment. Recolonization was shown to be selective, excluding cell types not normally represented in the thymus. To assess the extent of repertoire selection in recolonized fetal thymi, chimeric rudiments in which I-E determinants were expressed exclusively on the surface of immigrant DC were constructed. The failure of such rudiments to recruit a population of CD4⁺8⁻Vβ6⁺ thymocytes, restricted to antigen recognition in the context of I-E, argued against a role for DC in positive selection, in contrast, the widespread deletion of potentially autoreactive CD4⁺8⁻Vβ17a⁺ cells suggested an active role for DC in negative selection of the T-cell repertoire. These conclusions are consistent with the findings of various in vivo studies, endorsing the suitability of such a model for the study of tolerance induction in vitro.