Analysis of fluorescence decay curves by means of the Laplace transformation.

A computational procedure is described for the analysis of fluorescence decay data convolved with a lamp flash of finite width. The computer program calculates the ratio of the Laplace transforms of the decay and the lamp flash for different values of s to give the transforms of the impulse response for each value of s. These are set equal to the analytical Laplace transforms of the decay law involved. Solution of the nonlinear simultaneous equations yields the desired decay parameters. The method can be modified to analyze data that contains a component due to scattered light and can also provide essential information regarding transit time changes of the photomultiplier with changes in emission wavelength. The method was tested by the analysis of real and simulated data. The accuracy of the analysis depends on the degree of correlation among the parameters.     

A micromethod for the assay of cellular secretory physiology: application to rabbit parietal cells

A micromethod for investigating secretory physiology in isolated cells was evaluated. The method utilized a specially designed polycarbonate incubation chamber to provide constant oxygenation to cells incubating in a 96-well microtiter plate. Cells were rapidly separated from media by vacuum filtration. Isolated parietal cells were utilized to demonstrate the versatility of the method for assay of intracellular accumulation of [14C]-aminopyrine, secretion of intrinsic factor into the medium, and assay of intracellular cAMP. Histamine stimulated the uptake of [14C]aminopyrine and intrinsic factor secretion in a sustained and linear fashion. At the end of the 2-h period uptake of aminopyrine and secretion of intrinsic factor were increased 17- and 5-fold, respectively. This response to histamine was accompanied by a rapid and sustained 3-fold rise in intracellular cyclic AMP. In contrast, carbamylcholine caused a transient increase in [14C]aminopyrine accumulation and intrinsic factor secretion which was most pronounced during the first 10 min and had almost ceased by 30 min. Carbamylcholine had no effect on intracellular cAMP levels. This new method, which can handle 400 replicates using parietal cells from the fundic mucosa of a single rabbit, is suitable for studying the time course of intracellular events which accompany general secretory processes.     

In situ identification of cells in human leprosy granulomas with monoclonal antibodies to interleukin 2 and its receptor

Leprosy is a chronic granulomatous disease with an immunologic spectrum in which lepromatous leprosy patients have defective cell-mediated immune responses, in comparison to tuberculoid leprosy patients. Immunoregulatory aspects of this spectrum were investigated by using monoclonal antibodies to interleukin 2 (IL 2), IL 2 receptors (Tac), and T lymphocyte subpopulations with immunoperoxidase techniques on frozen sections of skin biopsy specimens from 10 tuberculoid and 10 lepromatous patients. A comparison of IL 2+ cells revealed markedly fewer IL 2+ cells in lepromatous specimens (lep. 0.028% +/- 0.02 vs tub. 0.46% +/- 0.28, p less than 0.001). These IL 2+ cells were large, exhibited cytoplasmic staining, and on double immunostaining were Leu-4+, Leu-3a+, Leu-2a-, Tac-, and OKT6-, consistent with the fact they are IL 2 producers. Equivalent numbers of Tac+ cells were observed in both lepromatous and tuberculoid granulomas (lep. 1.5% +/- 0.5 vs tub. 2.1% +/- 0.7, p, NS), suggesting that the responder cells are present in both conditions. The tuberculoid granuloma was highly organized, composed of a central core of mature macrophages, Leu-3a+ and Tac+ cells with a surrounding mantle of Leu-2a+, Leu-3a+, IL 2+, Tac+, and OKT6+ cells. In lepromatous granulomas, Leu-2a+, Leu-3a+, Tac+, and rare IL 2+ cells were randomly admixed with bacilli-laden macrophages. The defective cell-mediated immune responses in lepromatous leprosy appears to be associated with diminished IL 2 production and disorganization of the granuloma.     

Toll-like receptor 2 ligands as adjuvants for human Th1 responses

Bacterial lipopeptides (bLPs) are increasingly used as adjuvants to activate cell-mediated immune responses to foreign Ags. To explore mechanisms whereby bLPs adjuvant T cell responses, we stimulated human PBMCs with bLPs. We found that bLPs stimulate T cells to proliferate and produce IFN-gamma in an accessory cell-dependent manner and in the absence of exogenous protein Ags. The ability of bLPs to stimulate T cell proliferation was Toll-like receptor 2 dependent and required IL-12, interaction with costimulatory molecules, and MHC proteins. Our data suggest that bLPs adjuvant adaptive Th1 responses by enhancing Ag presentation of endogenous peptides.     

Overexpression of CD1d by keratinocytes in psoriasis and CD1d-dependent IFN-gamma production by NK-T cells

The MHC class I-like protein CD1d is a nonpolymorphic molecule that plays a central role in development and activation of a subset of T cells that coexpress receptors used by NK cells (NK-T cells). Recently, T cells bearing NK receptors were identified in acute and chronic lesions of psoriasis. To determine whether NK-T cells could interact with epidermal cells, we examined the pattern of expression of CD1d in normal skin, psoriasis, and related skin disorders, using a panel of CD1d-specific mAbs. CD1d was expressed by keratinocytes in normal skin, although expression was at a relatively low level and was generally confined to upper level keratinocytes immediately beneath the lipid-rich stratum corneum. In contrast, there was overexpression of CD1d in chronic, active psoriatic plaques. CD1d could be rapidly induced on keratinocytes in normal skin by physical trauma that disrupted barrier function or by application of a potent contact-sensitizing agent. Keratinocytes displayed enhanced CD1d following exposure to IFN-gamma. Combining CD1d-positive keratinocytes with human NK-T cell clones resulted in clustering of NK-T cells, and while no significant proliferation ensued, NK-T cells became activated to produce large amounts of IFN-gamma. We conclude that CD1d can be expressed in a functionally active form by keratinocytes and is up-regulated in psoriasis and other inflammatory dermatoses. The ability of IFN-gamma to enhance keratinocyte CD1d expression and the subsequent ability of CD1d-positive keratinocytes to activate NK-T cells to produce IFN-gamma, could provide a mechanism that contributes to the pathogenesis of psoriasis and other skin disorders.     

A prominent role for Sp1 during lipopolysaccharide-mediated induction of the IL-10 promoter in macrophages

IL-10 is an antiinflammatory cytokine secreted by activated macrophages and Th2 cells. IL-10 secretion promotes the down-regulation of proinflammatory cytokine synthesis and the development of Th2 responses. In macrophages, proinflammatory cytokines appear to be induced by similar mechanisms, but the IL-10 induction mechanisms have not been examined. We have analyzed the murine IL-10 promoter in the RAW264.7 macrophage line activated with LPS. A comprehensive mutant analysis revealed only one element upstream of the core promoter that was essential for promoter induction. A refined mutant analysis localized this element to nucleotides -89 to -78, and gel shift experiments revealed that it represents a nonconsensus binding site for Sp1. The functional relevance of Sp1 was supported by the high affinity of the interaction, the close correlation between the nucleotides required for Sp1 binding and promoter function, and the ability of an Sp1 consensus sequence to substitute for the -89/-78 promoter sequence. Evidence that Sp1 may be a target of signaling pathways involved in IL-10 induction was provided by the exclusive requirement for the Sp1 binding site, by the ability of the Sp1 site to confer induction to a heterologous promoter, and by the delineation of an Sp1 domain that can mediate induction. No relevant contribution from Rel, C/EBP (CCAAT/enhancer-binding protein), or AP-1 binding sites, which regulate most proinflammatory cytokine promoters, was observed. Together, these results demonstrate that IL-10 gene regulation is distinct from the regulation of proinflammatory cytokine genes, and suggest that Sp1 may be a central mediator of IL-10 induction.     

CTL-mediated killing of intracellular Mycobacterium tuberculosis is independent of target cell nuclear apoptosis

Two subsets of human CTL have been defined based upon phenotype and function: CD4(-) CD8(-) double-negative (DN) CTL lyse susceptible targets via Fas-Fas ligand interaction and CD8(+) CTL via the granule exocytosis pathway. CD8(+) CTL, but not DN CTL, can mediate an antimicrobial activity against Mycobacterium tuberculosis-infected target cells that is dependent on cytotoxic granules that contain granulysin. We investigated the role of nuclear apoptosis for the antimicrobial effector function of CD1-restricted CTL using the caspase inhibitor N:-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. We found that DN CTL-induced target cell lysis was completely dependent on caspase activation, whereas the cytolytic activity of CD8(+) CTL was caspase independent. However, both DN and CD8(+) CTL-induced nuclear apoptosis required caspase activation. More important, the antimicrobial effector function of CD8(+) CTL was not diminished by inhibition of caspase activity. These data indicate that target cell nuclear apoptosis is not a requirement for CTL-mediated killing of intracellular M. tuberculosis.     

Signaling lymphocytic activation molecule is expressed on CD40 ligand-activated dendritic cells and directly augments production of inflammatory cytokines.

Dendritic cells (DC) comprise a key part of the innate immune system that, upon activation, profoundly influences the nature of the adaptive T cell response. In this study, we present evidence that signaling lymphocytic activation molecule (SLAM), a molecule first identified in activated T and B cells, is strongly up-regulated in DC activated through CD40, as well as in response to inflammatory stimuli, including polyinosinic polycytidylic acid and LPS. mRNA encoding both membrane-bound and soluble secreted isoforms of SLAM was detected in CD40 ligand-activated DC, comprising two of the four known SLAM isoforms. Expression of membrane-bound SLAM protein peaked at 12 h poststimulation with CD40 ligand, gradually returning to baseline levels after 6 days. SLAM up-regulation appears to be a direct result of the induction of DC maturation, as inflammatory cytokines released during this process do not affect SLAM expression. Functionally, engagement of SLAM enhances DC production of IL-12 and IL-8, while having no effect on production of IL-10. Because SLAM is involved in the activation of T cells, the expression of SLAM on DC may provide a bidirectional signaling mechanism in which interacting DC and T cells are simultaneously and synergistically activated to mount proinflammatory Th1 responses.