Induction Of IFN-gamma Secretion Research Articles

Secretion of interferon‐γ by human macrophages demonstrated at the single‐cell level after costimulation with interleukin (IL)‐12 plus IL‐18

The interferon (IFN)-gamma component of the immune response plays an essential role in combating infectious and non-infectious diseases. Induction of IFN-gamma secretion by human T and natural killer (NK) cells through synergistic costimulation with interleukin (IL)-12 and IL-18 in the adaptive immune responses against pathogens is well established, but induction of similar activity in macrophages is still controversial, with doubts largely focusing on contamination of macrophages with NK or T cells in the relevant experiments. The possible contribution of macrophages to the IFN response is, however, an important factor relevant to the pathogenesis of many diseases. To resolve this issue, we analysed the production of IFN-gamma at the single-cell level by immunohistochemistry and by enzyme-linked immunosorbent spot (ELISPOT) analysis and unequivocally demonstrated that human macrophages derived from monocytes in vitro through stimulation with a combination of IL-12 and IL-18 or with macrophage colony-stimulating factor (M-CSF) were able to produce IFN-gamma when further stimulated with a combination of IL-12 and IL-18. In addition, naturally activated alveolar macrophages immediately secreted IFN-gamma upon treatment with IL-12 and IL-18. Therefore, human macrophages in addition to lymphoid cells contribute to the IFN-gamma response, providing another link between the innate and acquired immune responses.

Hsp110 and Grp170, members of the Hsp70 superfamily, bind to scavenger receptor‐A and scavenger receptor expressed by endothelial cells‐I

Heat shock protein 110 (hsp110) and glucose-regulated protein (grp170) act as anti-cancer vaccines when complexed to tumor antigens by heat shock. It has been proposed that receptors on antigen-presenting cells contribute to HSP-mediated immune responses. Here, we show that hsp110 binds in a receptor-mediated manner to RAW264.7 macrophages, as does grp170. This hsp110/grp170 binding is inhibited by scavenger receptor ligands, suggesting a role for scavenger receptors as binding structures. We examined scavenger receptor class A (SR-A) and scavenger receptor expressed by endothelial cells-I (SREC-I). We show that hsp110/grp170 binds to both SR-A- and SREC-I-expressing CHO cells in a saturable manner and scavenger receptor ligands inhibit binding. Hsp110 also saturably binds mouse bone marrow-derived dendritic cells (bmDC) and is inhibited by scavenger receptor ligands. When an hsp110-rat neu (intracellular domain) heat shock complex vaccine is used to pulse mouse bmDC in vitro, an induction of IFN-gamma secretion is observed by CD8+ T lymphocytes isolated from vaccine-immunized mice. This immune response is inhibited by the application of scavenger receptor ligands to bmDC. Thus, SR-A and SREC-I appear to contribute to the binding of hsp110 and grp170 on APC. Scavenger receptors, in general, contribute to the cross-presentation of hsp110-chaperoned protein antigen.

Workshop cluster 1+γδ T‐cell receptor+ T cells from calves express high levels of interferon‐γ in response to stimulation with interleukin‐12 and ‐18

Gammadelta T-cell receptor(+) T lymphocytes are an important element of the innate immune system. Early production of interferon (IFN)-gamma by gammadelta T cells may have a role in linking innate and adaptive immune responses and contribute to T helper-1 bias. We investigated the role of cytokines in the activation and induction of IFN-gamma secretion by bovine workshop cluster 1(+) (WC1(+)) gammadelta T cells. The effects of culture with interleukin (IL)-12, IL-18, IL-15 and IL-2 were investigated; these cytokines are known to influence murine and human gammadelta T cells. We report that bovine WC1(+)gammadelta T cells are synergistically stimulated by IL-12 and IL-18 to secrete large quantities of IFN-gamma. Neonatal calves were shown to have significantly higher numbers of circulating WC1(+)gammadelta T cells than adult animals. In addition, the response of peripheral blood WC1(+)gammadelta T cells was significantly higher in neonatal calves compared with adult animals. However, in adult animals the response of lymph node WC1(+)gammadelta T cells to IL-12/IL-18 was more pronounced than that of peripheral blood WC1(+)gammadelta T cells. We hypothesize that the induction of IFN-gamma secretion from WC1(+)gammadelta T cells by IL-12 and IL-18 is likely to be an important element of the innate response to pathogens such as Mycobacterium bovis. The high numbers of WC1(+)gammadelta T cells in neonatal calves, and their inherent ability to respond to inflammatory cytokines, could be a key factor in the enhanced responses seen in calves to BCG vaccination.

T-cell activation by recombinant immunoreceptors: impact of the intracellular signalling domain on the stability of receptor expression and antigen-specific activation of grafted T cells.

Recombinant immunoreceptors are modularily composed of extracellular antigen binding and intracellular signalling domains that are preferentially derived from CD3zeta or Fc epsilon RIgamma. The impact of the signalling domain on the stability of immunoreceptor expression and function is not completely understood. To address this issue, we generated and expressed a panel of recombinant zeta- and gamma-chain immunoreceptors, respectively, in human peripheral blood T cells. The expression level of zeta-chain immunoreceptors in human T cells is significantly lower than those of the homologous gamma-chain receptors. Low zeta-chain receptor expression in peripheral T cells is because of the intracellular signalling domain and independent of the Fc epsilon RIgamma or CD3zeta transmembrane region. Expression of both receptors decreases upon prolonged cultivation. Shortly after receptor engraftment, target cell lysis and induction of IFN-gamma secretion are mediated with similar efficiency by zeta- and gamma-chain immunoreceptors. Upon prolonged propagation, however, T-cell activation mediated by zeta-chain immunoreceptors is more efficient than by gamma-chain receptors, indicating that the initial high expression level of gamma-chain immunoreceptors compensates its lower activation capacity. Consequently, gamma-chain immunoreceptors exhibit a higher threshold value for specific activation and are more pronouncedly inhibited by soluble ligand antigen compared to the homologous zeta-chain receptor. These findings have substantial consequences for the design of recombinant immunoreceptors for use in adoptive immunotherapy.

Polarized porcine trophoblastic cell lines spontaneously secrete interferon-gamma.

Following the demonstration of high levels of interferon-gamma (IFN-gamma) synthesis by the pig trophectoderm around implantation, an adequate experimental system was established so as to study the regulation of endogenous IFN-gamma gene. Several stable cell lines have been isolated from Day 14 and 15 pig trophoblast, that could withstand indefinite growth when cultured on collagen-coated supports. Since no feeder cells were used for culture, and cell lines could be successfully cloned, these lines represent the first pure porcine trophoblastic (TB) cell lines isolated so far. These cells were shown to exhibit most differentiation markers of epithelial cells and in addition to express the porcine trophectoderm-specific antigen SN1-38. The TB cell line A (TBA) was characterized in more details upon culture on microporous filters, where a high apico-basal polarity could be obtained. In those conditions, a transient and acute interferon-gamma secretion was detected only in the apical medium. Moreover, the two trophoblast-specific mRNA of 1.3 and 1.4 kb that have been described in the blastocyst collected in vivo were shown to be synchronously transcribed. This polarized synthesis and secretion of IFN-gamma was correlated with the acquisition of maximal levels of electric resistance of TBA monolayers on filters, and was not observed on the same cells cultured on plastic. This differentiation was maintained over 30 passages, demonstrating that the induction of IFN-gamma secretion by pig conceptus is not maternally controlled. This cellular model will be of prime importance for studies on the developmental regulation of the IFN-gamma gene, and more generally for studies on relationship between secretion and polarity in transporting epithelia.

Functional consequences of the binding of MHC class II-derived peptides to MHC class II.

Three MHC class II-derived synthetic peptides (I-A beta (g7)1-16, I-A beta (g7)52-77 and I-A alpha (g7)63-82YC) were analyzed for their ability to bind to syngeneic and allogeneic MHC class II molecules using a whole cell, competitive peptide binding assay. These studies demonstrated that the A beta (g7)1-16 peptide was able to specifically bind to syngeneic as well as to four allogeneic MHC class II molecules. The A alpha (g7)63-82YC peptide bound to self MHC class II molecules with a lower relative affinity and was able to bind to three out of the four allogeneic cells tested. The binding of the three I-A(g7)-derived peptides to the self MHC class II was functionally significant. The A beta (g7)1-16 and A beta (g7)52-77 peptides inhibited the proliferation of a heat shock protein 60 peptide-specific Th1 clone by MHC blockade. Interestingly, the A alpha (g7)63-82YC peptide appeared to interact directly with T cells as pretreatment of the Th1 clone with this peptide resulted in inhibition of antigen-induced proliferation. This phenomenon was analyzed in more detail and it was found that this peptide could behave as a partial agonist. Incubation of T cells with the A alpha (g7)63-82YC peptide resulted in up-regulation of IL-2R alpha chain expression and induction of IFN-gamma secretion. In addition T cells pretreated with this peptide were rendered hyporesponsive to further antigenic stimulation. Thus, a peptide derived from MHC class II may be used in an immunoregulatory capacity.