A Leishmania protein that modulates interleukin (IL)-12, IL-10 and tumor necrosis factor-alpha production and expression of B7-1 in human monocyte-derived antigen-presenting cells.

LeIF, a gene homologue of the eukaryotic initiation factor 4A was first described as a leishmanial antigen that induced a Th1-type T cell response in peripheral blood mononuclear cells (PBMC) from leishmaniasis patients. Moreover, the interferon (IFN)-gamma production by PBMC was found to be interleukin (IL)-12 dependent. Herein, we characterize the effects of LeIF on cytokine production and expression of surface molecules by normal human monocytes as well as by monocyte-derived macrophages and dendritic cells (MoDC). LeIF was a strong inducer of IL-12 and, to a lesser extent, of IL-10 and tumor necrosis factor (TNF)-alpha in macrophages and MoDC. IL-12 production did not require CD40 triggering, confirming that the ability of LeIF to induce IL-12 was not mediated through an effect on T cells. However, addition of soluble CD40 ligand (L) synergistically augmented IL-12 production in macrophages and MoDC. The cytokine-inducing activity of LeIF is located in the N-terminal portion of the molecule and was both proteinase K sensitive and polymyxin B resistant. LeIF, lipopolysaccharide and fixed Staphylococcus aureus all induced comparable amounts of IL-12, validating the potent cytokine-inducing effects of LeIF. Moreover, of these stimuli, LeIF had the highest IL-12/IL-10 and IL-12/TNF-alpha ratio demonstrating the preference of LeIF for IL-12 induction. Studies investigating the expression of surface molecules showed that LeIF up-regulated B7-1 and CD54 (ICAM-1) on macrophages and MoDC. To our knowledge this is the first report describing IL-12 production, up-regulation of co-stimulatory and intercellular adhesion molecules by monocytic antigen-presenting cells in response to a protein from a pathogenic microorganism. These immunomodulatory characteristics of LeIF might be excellent properties for a Th1-type adjuvant.

In vitro recall of proliferative and cytolytic responses to minor histocompatibility antigens by dendritic cell enriched canine peripheral blood mononuclear cells.

It is difficult in vitro to demonstrate existent in vivo sensitization of dogs and humans to minor histocompatibility antigens. Using conventional one-way mixed leukocyte culture, when sensitized blood cells are stimulated with MHC antigen-matched sibling PBMC bearing the minor histocompatibility antigens, there is usually no proliferative or cytotoxic response detected. We reported previously that 0 of 17 dogs sensitized by transfusion of dog leukocyte antigen-identical littermate blood had proliferative responses in mixed leukocyte culture when unfractionated sibling PBMC were used as stimulator cells. We reasoned that this result might be due to the inability of unfractionated PBMC to efficiently present minor histocompatibility antigens to the in vivo-primed T cells, a function thought best performed by dendritic cells. When we used a low buoyant density Percoll fraction of canine PBMC, shown previously to be enriched in dendritic cells, as stimulator cells, we were able to generate cytotoxic and/or proliferative responses in mixed leukocyte culture in all 5 dogs that had been sensitized to minor histocompatibility antigens by transfusions of dog leukocyte antigen-identical sibling littermate blood. By contrast, using unfractionated PBMC as stimulator cells, we found evidence of sensitization in only 1 of the 5 dogs. These data support the concept that the presentation of minor histocompatibility antigens, in contrast to major histocompatibility antigens, to the immune system may be restricted to a subpopulation of professional APC.

Regulation of glutamic acid decarboxylase diabetes autoantigen expression in highly purified isolated islets from Macaca nemestrina.

Macaca nemestrina, which may have larger and more numerous pancreatic islets than other species, was used for large scale islet isolation by ductal collagenase perfusion and Ficoll gradient centrifugation. The average yield was 51,000 islet equivalents per pancreas, or 8,750 islets equivalents per g. The average purity was 91%, often exceeding 95%. These are the highest reported size, purity, and yield per g of any nonautomated primate islet series. Perifusion with glucose, arginine, and isobutylmethylxanthine showed appropriate biphasic insulin secretion. Unlike that in the rat, human islet glutamic acid decarboxylase (GAD) isoform expression is restricted. However, glycemic regulation of GAD expression has been shown only in rats. We, therefore, tested hypotheses that M. nemestrina islets also have restricted GAD expression, that GAD expression in primates is stimulated by glucose, and that this stimulation remains restricted to the 64,000 mol wt (GAD65) isoform. Immunoprecipitation of labeled islet extracts showed that GAD65 expression increased 16.7 +/- 0.6-fold during high glucose in vitro culture. After controlling for observed increases in protein synthesis, specific glucose stimulation was still 4.2 +/- 0.2-fold. Specific antisera revealed no GAD67 expression under basal conditions, and isoform restriction was maintained during stimulation. Increased GAD65 synthesis thus accounts for glucose stimulation of 64K expression. These time- and concentration-dependent effects of glucose suggest that hyperglycemia increases autoantigenicity and may accelerate beta-cell destruction in primates, supporting a role for beta-cell rest in insulin-dependent diabetes mellitus prevention.