The cross-linking of Fc receptors (FcR) on HL-60 cells inhibited the ability of recombinant IFN-gamma to induce HLA class II antigens. This appeared to be correlated with intracellular mRNA level. HL-60 lacked detectable HLA class II mRNA. IFN-gamma led to appearance of these transcripts, which were canceled by the cross-linking of FcR. Therefore, experiments were designed to investigate the intracellular signaling molecules regulating the appearance of HLA class II molecules or transcripts. The expression of HLA class II antigen induced by IFN-gamma was blocked by a calmodulin antagonist, W-7, but not by a protein kinase C (PKC) inhibitor, H-7. Furthermore, a direct activator of PKC, phorbol myristate acetate, was not able to induce the HLA class II antigen expression. These results suggest that IFN-gamma induces HLA class II antigens on HL-60 cells via a calcium-calmodulin pathway and not via a PKC pathway. Calmodulin is activated by a transient rise in the cytosolic free calcium. In fact, the measurement of calcium influx into HL-60 cells showed that a remarkable and time-dependent calcium accumulation was caused by IFN-gamma, and that depletion of Ca2+ from culture medium resulted in failure of IFN-gamma to induce class II antigen expression. Furthermore, calcium ionophore, A23187, by itself induced HLA class II antigen expression. These results suggest that IFN-gamma stimulates calcium influx and activates the calmodulin branch of the calcium messenger system, resulting in the induction of class II antigen expression on HL-60 cells. On the other hand, cross-linking of FcR elicited the accumulation of intracellular cAMP, which appeared to suppress the IFN-gamma-induced calcium influx, resulting in annulling HLA class II antigen-inducing activity of IFN-gamma. These intracellular events of HL-60 regulate the expression of HLA class II transcripts and molecules.
Both CD4(+) type 1 helper T (Th1) cells and CD8(+) cytotoxic T lymphocytes (CTL) play pivotal roles in protection against Mycobacterium tuberculosis infection. Here, we identified Th1 and CTL epitopes on a novel protective antigen, MPT51, in BALB/c and C57BL/6 mice. Mice were immunized with plasmid DNA encoding MPT51 by using a gene gun, and gamma interferon (IFN-gamma) production from the immune spleen cells was analyzed in response to a synthetic overlapping peptide library covering the mature MPT51 sequence. In BALB/c mice, only one peptide, p21-40, appeared to stimulate the immune splenocytes to produce IFN-gamma. Flow cytometric analysis with intracellular IFN-gamma and the T-cell phenotype revealed that the p21-40 peptide contains an immunodominant CD8(+) T-cell epitope. Further analysis with a computer-assisted algorithm permitted identification of a T-cell epitope, p24-32. In addition, a major histocompatibility complex class I stabilization assay with TAP2-deficient RMA-S cells transfected with K(d), D(d), or L(d) indicated that the epitope is presented by D(d). Finally, we proved that the p24-32/D(d) complex is recognized by IFN-gamma-producing CTL. In C57BL/6 mice, we observed H2-A(b)-restricted dominant and subdominant Th1 epitopes by using T-cell subset depletion analysis and three-color flow cytometry. The data obtained are useful for analyzing the role of MPT51-specific T cells in protective immunity and for designing a vaccine against M. tuberculosis infection.
It is generally held that one of the recessive genes controlling diabetes in the NOD mouse is linked to the major histocompatibility complex (MHC) . We therefore performed restriction fragment length polymorphism studies of MHC (class I, II, and III) in NOD mice in comparison with those of their nondiabetic sister strains, NON, CTS, and ILI mice which were derived from the same Jcl-ICR mice. When a minimum of four restriction enzymes were used, class II and III genes of NOD mice were indistinguishable from those of CTS and ILI mice but totally different from those of NON mice. While NON mice expressed the Eα gene, NOD, CTS, and ILI mice appeared to carry a deletion in the 5' end of the Eα gene resulting in failure to transcribe the Eα gene. When class I probe was used, CTS mice showed very different band patterns from those of the other ICR-derived mice.Unique substitution of Asp57 with Ser in the Aβ chain is considered to make the Aβ gene the MHC-linked susceptibility gene. We therefore analyzed the nucleotide sequences of the Aβ second exon in ILI, CTS, and NON mice. The DNA sequence analyses revealed that the Aβ second exon sequences in the ILI and CTS mice, but not in the NON mouse, are identical to that of the NOD mouse. Taken together, these data suggest that ILI and CTS mice possess a recessive diabetogenic gene linked to the MHC.We also examined the difference of Vβ usage between the NOD mouse and the ILI mouse spleen cells. No obvious difference, however, was evident.
A new cancer-treatment model, photodynamic therapy (PDT) combined with a type I topoisomerase inhibitor, camptothecin derivative (CPT-11), against HeLa cell tumors in BALB/c nude mice has been developed using a wide-band tunable coherent light source operated on optical parametric oscillation (OPO parametric tunable laser). The Photosan-3 PDT and CPT-11 combined therapy was remarkably effective, that is the inhibition rate (I.R.) 40 - 80%, as compared to PDT only in vivo. The analysis of HpD (Photosan-3) and CPT-11 effects on cultured HeLa cells in vitro has been studied by a video-enhanced contrast differential interference contrast microscope (VEC-DIC). Photosan-3 with 600 nm light killed cells by mitochondrial damage within 50 min, but not with 700 nm light. CPT-11 with 700 - 400 nm light killed cells within 50 min after nucleolus damage appeared after around 30 min. The localization of CPT-11 in cells was observed as fluorescence images in the nucleus, particularly the nucleoral area produced clear images using an Argus 100.
Mycobacterium tuberculosis is an intracellular bacterium that can replicate within infected macrophages. The intracellular parasitism by M. tuberculosis results from arresting phagosome maturation and inhibiting phagolysosome biogenesis in infected macrophages. It has been thought that M. tuberculosis arrests the maturation of its phagosome at the early stage. Several reports attended to the localization of Rab GTPases on mycobacterial phagosomes. Rab GTPases regulate membrane trafficking, but details of how Rab GTPases regulate phagosome maturation and how M. tuberculosis modulates their activities during inhibiting phagolysosome biogenesis remains elusive. Here, we introduce the new findings that M. tuberculosis alters the localization of Rab GTPases regulating phagosome maturation during inhibiting phagolysosome biogenesis.
Abstract: We investigated the cis ‐acting sequences that function in the B‐cell‐specific expression of the HLA‐DPB1 gene. Class II B major histocom‐patibility genes contain a conserved upstream sequence that is important in the expression of these genes. This region has been divided into three major elements, the W, X, and Y boxes. In this paper, we identified an additional positive regulatory element upstream from the DPB1 W box. Using 5′ deletion mutants and a substitution mutant, we mapped a positive element, called the W box, between‐184˜‐169 bp. Sequence comparison revealed that the W box shares homology with the W box. Electrophoretic mobility shift assay confirmed that the W box binds proteins that also recognize the W box. Furthermore, deletion and substitution mutants indicate that the W and W boxes effectively enhance CAT activities only when the X and Y boxes exist.
