We have established a novel TCRαβ (TCRVβ6)+CD4−CD8− T cell hybridoma designated B6HO3. When the B6HO3 cells were cocultured with bacterial-infected J774 macrophage-like cells, IFN-γ production by B6HO3 cells was triggered through direct cell-cell contact with dying J774 cells infected with Listeria monocytogenes (LM), Shigella flexneri, or Salmonella typhimurium that expressed the type III secretion system, but not with intact J774 cells infected with heat-killed LM, nonhemolytic lysteriolysin O-deficient (Hly−) LM, plasmid-cured Shigella, or stationary-phase Salmonella. However, the triggering of B6HO3 cells for IFN-γ production involved neither dying hepatoma cells infected with LM nor dying J774 cells caused by gliotoxin treatment or freeze thawing. Cycloheximide and Abs to H-2Kd, H-2Dd, Iad, CD1d, TCRVβ6, and IL-12 did not inhibit the contact-dependent IFN-γ response, indicating that this IFN-γ response did not require de novo protein synthesis in bacterial-infected J774 cells and was TCR and IL-12 independent. Thus, in an as yet undefined way, B6HO3 hybridoma recognizes a specialized form of macrophage cell death resulting from bacterial infection and consequently produces IFN-γ. Moreover, contact-dependent interaction of minor subsets of splenic αβ T cells, including NKT cells with dying LM-infected J774 and bone marrow-derived macrophage (BMM) cells, proved to provide an IFN-γ-productive stimulus for these minor T cell populations, to which the parental T cell of the B6HO3 hybridoma appeared to belong. Unexpectedly, subsets of γδ T and NK cells similarly responded to dying LM-infected macrophage cells. These results propose that innate lymphocytes may possess a recognition system sensing macrophage cell “danger” resulting from bacterial infection.
SUMMARYThe presence of human papillomavirus (HPV) type 16 DNA in biopsies from precancerous lesions and from early lesions of human cervical cancer, and the integration of virus DNA into host cell DNA were analysed by dot blot and Southern blot hybridizations. HPV 16 DNA was detected in 23~ of mild dysplasias, 329/o of moderate dysplasias, 55 ~ of severe dysplasias and 62 ~ of carcinomas in situ by dot blot hybridization. Digestion of the DNA with restriction enzymes PstI and BamHI followed by Southern blot analysis revealed the presence of some typical restriction fragments of HPV 16 DNA in most virus-positive samples. In addition, we detected submolar fragments which might represent virus-cell junction sequences in 869/oo of dysplasias, suggesting that the integration of HPV 16 DNA could occur in the precancerous stage.Human papillomavirus (HPV) type 16 and type 18 DNAs are frequently found in biopsies from precancerous and malignant cervical lesions (Boshart et al., 1984;Crum et al., 1985;Lehn et al., 1985;Tomita et al., 1986), with HPV 16 DNA being more common than HPV 18 DNA in these lesions (Diirst et al., 1983 ;Boshart et al., 1984;Yoshikawa et al., 1985). HPV 16 DNA has been cloned from an invasive cervical carcinoma (Dtirst et al., 1983) and the complete nucleotide sequence was determined (Seedorf et al., 1985). Recently, Diirst et al. (1985) reported the physical state of HPV 16 DNA in some malignant turnouts and showed that the integration of HPV 16 genome into the host cell DNA occurs with a head-to-tail viral genome arrangement. Their report includes a discussion of HPV integration in connection with the causative event in malignant transformation.Dysplasias are considered to be precancerous lesions and are classified as mild, moderate and severe (Koss, 1978). In order to see whether the integration of HPV 16 DNA into host cell DNA occurs in precancerous lesions, biopsy samples from mild, moderate and severe dysplasias and from cervical cancers were screened for the presence of HPV 16 DNA by dot blot hybridization. Virus-specific restriction fragments were analysed by Southern blot hybridization. We report here the detection of submolar fragments, which might be virus-cell junction sequences, in most dysplasias and carcinomas in situ that harbour HPV 16 DNA.Biopsy samples were collected under colposcopy and kept at -70 °C. High molecular weight DNA was extracted and purified from samples that had been histopathologically confirmed (Tomita et al., 1986). For dot blot hybridization, about 7.5 p.g of the purified DNA was denatured, neutralized, then spotted onto a nitrocellulose filter and hybridized with 32p-labelled cloned HPV 16 DNA in 6 x SSC at 68 °C for 24 h. The specific activity of the HPV 16 DNA was 108 to 2 x 108 c.p.m./~tg. The filter was washed extensively with 0
Interferon‐γ (IFN‐γ) activation of macrophages is a crucial step in the early innate defence against bacterial infection. This innate IFN‐γ is thought to be produced mainly by natural killer (NK) cells through activation with interleukin (IL)‐12p70 secreted by macrophages and dendritic cells (DCs) that have sensed bacterial products. However, a number of reports have shown that bacterial stimuli are unable to induce macrophages and/or DCs to produce sufficient amounts of IL‐12p70 unless these cells are primed by IFN‐γ. It remains, therefore, unsettled how initial IFN‐γ is produced. In a previous study, we reported a novel IFN‐γ production pathway that was associated with cell death in macrophages caused by intracellular bacteria like Listeria monocytogenes (LM) and Shigella flexneri. In this study, we showed that cell death of bone‐marrow‐derived macrophage (BMM) cells following in vitro infection with Staphylococcus aureus (SA), an extracellular bacterium, can also stimulate this IFN‐γ production pathway. We also unequivocally demonstrated by using BMM cells from IL‐12‐deficient mice that the bacterial‐infected macrophage cell death‐mediated IFN‐γ production can occur without IL‐12 although the magnitude of the response is much smaller than that in the presence of IL‐12. The enhancing effect of IL‐12 on this response proved to be attributable to the negligible amounts (0.5∼1.5 pg/ml) of IL‐12p70 but not to the large amounts of IL‐12p40 that were both secreted by SA‐ and LM‐infected macrophages. Taken all together, we propose that macrophage cell death caused by bacteria may trigger the initial IFN‐γ production at an early stage of bacterial infection.
Human papillomavirus type 16 (HPV16) genome DNA and its transcripts in biopsied cervical neoplasias were analyzed by simultaneous extraction of DNA and RNA from one biopsied sample. Southern blot analysis revealed that 5 of 20 cervical intraepithelial neoplasias (CINs) contained HPV16 DNAs existing primarily as episomes and two of seven invasive carcinomas harbored HPV16 genome sequences integrated into the host DNA. Northern (RNA) blot analysis showed that the HPV16 genome sequences were transcriptionally active in the five CINs, as well as in the two invasive carcinomas. The pattern of HPV16-specific transcripts in the CINs was uniform, and the major transcripts were 4.2, 2.2, 1.6, and 1.4 kilobases in size. However, the pattern of HPV16-specific transcripts in the invasive carcinomas was variable and different from that in CINs, suggesting that the alteration of transcriptional pattern might play a key role in the development of malignancy.
Production of innate interferon-γ (IFN-γ) is a crucial step in immunological defense against bacteria. However, there is little information regarding cellular mechanisms underlying IFN-γ production in vivo early after bacterial infection. Here we analyze innate IFN-γ production in the spleen of mice early after Listeria monocytogenes (LM) infection ex vivo by flow-cytometry and in situ by immunohistochemistry, and compare them with the IFN-γ-producing cells reported previously in our in vitro coculture system in which cell-cell interaction between lymphocytes and dying bacterial-infected macrophages is required for the production of IFN-γ. In the spleen at 20 h after LM infection, natural killer (NK) cells, a subset of αβ T cells, and subsets of NKT and γδ T cells produced IFN-γ with features similar to the IFN-γ-producing cells in our in vitro coculture system. Immunohistochemistry revealed that LM bacteria were first phagocytosed mainly by ER-TR9+ marginal zone macrophages (MZMs), then forming infectious foci in close vicinity of the marginal zone (MZ) at 20-h postinfection. At this time point, the IFN-γ-producing cells were accumulating at the same site of infectious foci, around which ER-TR9+ MZMs were clustered but most of bacteria were no longer associated with ER-TR9+ MZMs. These results indicate that innate IFN-γ production by innate lymphocytes takes place at infectious foci formed in close vicinity of the MZ, and they also suggest an important role for the microenvironment of the cells accumulated at infectious foci in inducing the production of innate IFN-γ.
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