Emergence, origin, and function of neutrophil-dendritic cell hybrids in experimentally induced inflammatory lesions in mice.

Although unusual neutrophils expressing major histocompatibility complex class II (MHC II) and costimulatory molecules have been detected at inflammatory sites in mice and humans, their identity, origin, and function remain unclear. We have demonstrated that, when cultured with granulocyte macrophage-colony-stimulating factor, neutrophils can give rise to a unique hybrid population exhibiting dual phenotypic and functionality of neutrophils and dendritic cells (DCs). Here we report that hybrid cells expressing surface markers of neutrophils (Ly6G, L-selectin, CXC chemokines receptor 2, and 7/4) and DCs (CD11c, MHC II, CD80, and CD86) become detectable in the peritoneal cavity, skin, lung, and lymph nodes under inflammatory conditions. Importantly, 20% to 30% of the adoptively transferred neutrophils acquired CD11c and MHC II expression when recovered from inflammatory lesions, demonstrating neutrophil → hybrid conversion in living animals. Using Escherichia coli strains expressing green fluorescent protein and ovalbumin, we further show hybrids play dual protective roles by rapidly clearing bacteria and presenting bacterial antigens to CD4 T cells. These results indicate that some of the neutrophils recruited to inflammatory lesions can differentiate into neutrophil-DC hybrids, thus challenging the classic view of neutrophils as terminally differentiated leukocytes destined to die or to participate primarily in host innate immunity.

Assignment of the receptor for ecotropic murine leukemia virus to mouse chromosome 5.

The gene for the receptor for ecotropic murine leukemia virus (Rev) has been assigned to mouse chromosome 5. This determination was made possible by an analysis of somatic cell hybrids between mouse and Chinese hamster cells. The parents of these hybrids were A/HeJ or Mus poschiavinus peritoneal exudate cells or BALB/c primary embryo fibroblasts and E36, a Chinese hamster lung fibroblast deficient in hypoxanthine guanine phosphoribosyltransferase. Segregation of mouse chromosomes in these hybrids was analyzed by chromosome banding and isozyme expression. Cells were tested for their ability to absorb and replicate vesicular stomatitis virus (murine leukemia virus [MuLV]) pseudotype particles and ecotropic MuLV as measured by the XC test. The presence of chromosome 5 was essential for receptor expression as determined by three statistical procedures. Segregation of the receptor for ecotropic murine leukemia virus was also followed in two series of subclones. In both, receptor expression was syntenic with phosphoglucomutase-1, an isozyme which has been mapped to mouse chromosome 5.

Genetic mapping of the ecotropic murine leukemia virus-inducing locus of BALB/c mouse to chromosome 5.

By means of an approach that combined the techniques of somatic cell genetics and Mendelian breeding studies, the inducibility locus, designated Cv, for ecotropic murine leukemia virus in BALB/c mice, was mapped to chromosome 5, 23 units from the locus for phosphoglucomutase-1, with gene order Cv-Pgm-1-Gus. This low-efficiency inducibility locus is therefore not allelic with the chromosome 7 loci previously described for two other mouse strains with high virus inducibility. These studies provide further evidence that endogenous ecotropic viruses represent viral genomes inserted at different chromosomal sites in the various mouse strains.

Human T cell leukemia viruses use a receptor determined by human chromosome 17.

Human T cell leukemia viruses (HTLV-I and HTLV-II) can infect many cell types in vitro. HTLV-I and HTLV-II use the same cell surface receptor, as shown by interference with syncytium formation and with infection by vesicular stomatitis virus (VSV) pseudotypes bearing the HTLV envelope glycoproteins. Human-mouse somatic cell hybrids were used to determine which human chromosome was required to confer susceptibility to VSV(HTLV) infection. The only human chromosome common to all susceptible cell hybrids was chromosome 17, and the receptor gene was localized to 17cen-qter. Antibodies to surface antigens known to be determined by genes on 17q did not block the HTLV receptor.

Murine leukemia virus: restriction in fused permissive and nonpermissive cells.

Cultures of human cells nonpermissive for mouse leukemia virus replication could not be induced to support virus replication by homologous fusion in the presence of Moloney leukemia virus. Human cells were also fused with permissive mouse cells, and the fate of the virus in heterokaryons was determined by a simultaneous autoradiography and fluorescent antibody technique. Heterokaryons containing the full chromosome complement of both cells were likewise nonpermissive for virus synthesis, but hybrids of human and mouse cells, which lacked up to half of the human chromosome complement, were permissive for virus synthesis. The results suggest that human cell genes can direct a repressive control over mouse leukemia virus replication.

Epstein-Barr virus: detection of genome in somatic cell hybrids of Burkitt lymphoblastoid cells.

Somatic cell hybrids of Burkitt lymphoblastoid cells, from which Epstein-Barr virus can be recovered, were examined for the presence of virus DNA by DNA-RNA hybridization. Four clones of hybrid cells, each negative for virus antigens by immunofluorescence, contained virus DNA in varying genomic equivalents. The number of virus genome equivalents increased in the hybrid cells after induction of virus with iododeoxyuridine.

Simian virus 40 (SV40) production from SV40-transformed human amnion cells of established lines.

Sixteen established cell lines of simian virus 40 (SV40)-transformed human amnion cells were examined for SV40 production. Many of these lines produced SV40 for extensive periods. Virus production had not ceased for 2 lines after 18 months, for 3 lines after 12 months, and for 3 lines at 3 months after recovery from "crisis". Three lines became virus-free in the first month, 1 line in the second month, 1 in the third month, and 1 in the fourth month, and 2 lines stopped virus production between 6 and 11 months after recovery. The virus titers were relatively low. Inclusion body-containing cells were infrequent. In contrast, in most cultures of SV40-transformed human fibroblasts rescued from crisis, no infectious virus was demonstrated, although exceptions have been reported. Virus was produced after heterokaryon formation of cells of the virus-free amnion lines with CV-1 cells in the presence of inactivated Sendai virus, as observed for SV40-transformed human fibroblasts. During the crisis period, some of the SV40-transformed amnion cells produced substantial amounts of virus. Titers decreased during the later periods of crisis. The most pronounced decrease in titers was in cultures from which established lines were recovered.

Sarcoma in a hamster inoculated with BK virus, a human papovavirus.

An undifferentiated sarcoma occurred in 1 of 52 hamsters inoculated when newborn with BK virus (BKV), A SIMIAN VIRUS 40 (SV40)-related human papovavirus. It was transplantable and grew in tissue culture. Sera of tumor-bearing hamsters were without antibodies reactive to BKV virion antigens in hemagglutination-inhibition and neutralizationtexts, but contained antibodies reactive in immunofluorescence (IF) tests to SV40 T antigen in SV40-TRANSFORMED CELLS AND TO ANTIGENS IN CELLS ACUTELY INFECTED WITH BKV or SV40. The IF reaction between tumor cells and sera of tumor-bearing hamsters was minimal in texts of tumor cells of an early passage but satisfactory in tests of cells from a later passage. Fusion of tumor cells with permissive (monkey and human) cells, induced by inactivated Sendai virus, did not lead to recovery of infectious BKV. Immunization of hamster with BKV or SV40 failed to protect against challenge with tumor cells. There was considerable cross-reactivity between the T antigens of BKV and SV40 viruses

Activation of Epstein-Barr virus in hybrid cells.

Human-primate hybrid cell lines were established by fusion of African green monkey kidney cells (VERO) with lymphoblastoid cells from patients with infectious mononucleosis (IM)(IMK101) and from Burkitt's lymphoma culture (HR1K). Both Epstein-Barr virus (EBV)-specific antigens and EBV particle-containing cells increased in the hybrid lines (IMK1-1/VERO,HR1K/VERO). Treatment of the hybrids with 5-bromodeoxyuridine induced more antigen-positive and more virus-containing cells. EBV could be activated from IM lymphoblastoid cells by fusion of the lymphoblastoid cells with the VERO cells. The increase of viral antigens and virus particles may have been due to the cellular interaction between VERO cells and the lymphoblastoid cells or to a possible derepressor supplied by the VERO component of the hybrid. Virus derived from the HR1K cell line was replicated in the human-primate hybrid, but further investigation may be necessary to determine if it was identical to the EBV derived from the human cell line.

Tumorigenicity and intracisternal A-particle expression of hybrids between murine myeloma and lymphocytes.

Hybrids of BALB/c lymphocytes and a murine myeloma, a tumor that expresses intracisternal A-particles, were obtained with polyethylene glycol as the fusogen. The karyotype, tumorigenicity, and A-particle expression of the hybrid clones were assessed. All the hybrid clones analyzed were tumorigenic and expressed intracisternal A-particles even when they were the result of a fusion event between two lymphocytes and one myeloma cell in which no loss of chromosomes was detected. The tumors that developed following inoculation of hybrid cells into BALB/c mice (1 x 10(6) cells/mouse) were karyotypically identical to the inoculated cells. It appears that the two myeloma cell phenotypic traits analyzed (tumorigenicity and A-particle expression) are dominant.

Superinfection of epithelial hybrid cells (D98/HR-1, NPC-KT, and A2L/AH) with Epstein-Barr virus and the relationship to the C3d receptor.

Three Epstein-Barr virus (EBV) genome-positive epithelial/hybrid cell lines (D98/HR-1, NPC-KT, and A2L/AH) were superinfected with EBV derived from P3HR-1 (HR-1), NPC-KT, and B95-8 cells. The HR-1 virus is lytic and induces early antigen in superinfected Raji cells; the virus is not capable of transforming B-lymphocytes. Virus preparations from NPC-KT cells have both transforming and early antigen-inducing properties, while B95-8 virus can only transform B-lymphocytes. It was possible to demonstrate EBV antigens after superinfection of D98/HR-1 cells with both HR-1 virus and NPC-EBV. The NPC-KT hybrid cells, which were originally prepared by fusing human adenoid epithelial cells (Ad-AH) and EBV genome-positive NPC explanted epithelial cells, were susceptible to superinfection with HR-1 virus but not to NPC-EBV. The A2L/AH hybrid cells, which were prepared by fusion between Ad-AH cells and lymphocytes transformed by B95-8 virus, could not be superinfected with any of the virus preparations. In order to further investigate the nature of the EBV receptor as it relates to other cell membrane components, we examined cell surface markers on Ad-AH, NPC-KT, A2L/AH, and D98/HR-1 cells using monoclonal antibodies and by rosette formation. We found that the NPC-KT, A2L/AH, and Ad-AH cell lines express the OKB2 antigen and that the common acute lymphoblastic leukemia antigen is expressed on the A2L/AH cells. We also found that NPC-KT parental cells and a clone of NPC-KT cells express erythrocyte antibody complement b and erythrocyte antibody complement d, as determined by rosette formation, but were negative for C3b and C3d when monoclonal antibodies against these two markers were used. The D98/HR-1 cells were also confirmed to be negative for C3b and C3d. The data suggest that the C3d receptor may be part of the EBV receptor but that the C3d receptor, by itself, is not the only receptor to which EBV can bind.

Electrofusion of individual animal cells directly to intact corneal epithelial tissue.

An electromechanical process was developed to electrofuse human and nonhuman cultured cells directly to rabbit corneal epithelial tissue in vitro and in situ. This new process was utilized successfully to incorporate functional gonococcal membrane attachment receptors from human lymphoma cells into superficial rabbit corneal epithelium. Thus, cell-tissue electrofusion biotechnology may be employed to establish unique and novel animal models for investigating receptor-mediated processes in vivo.

Restriction of human adenovirus replication in Chinese hamster cell lines and their hybrids with human cells.

We have found that the replication of human adenovirus (Ad2) is restricted in multiple Chinese hamster cell lines including CHO and V79. The major site of restriction involves differential accumulation of late viral proteins as demonstrated by immunofluorescence assay and polyacrylamide gel electrophoresis with and without prior immunoprecipitation. Synthesis of fiber and penton base are markedly reduced, whereas others, such as the 100K polypeptide, are synthesized efficiently. This pattern of restriction is similar to that previously reported for Ad2 infection of several monkey cell lines; however, the restriction is more marked in the Chinese hamster cell lines. The restriction is most likely due to a deficient cellular function since stable cell hybrids between V79 or CHO and human cells are permissive for virus replication. By analysis of a series of hybrids with reduced numbers of human chromosomes, fiber synthesis was correlated with the presence of the short arm of human chromosome 3. More hybrids showed restoration of fiber synthesis than production of progeny virus, suggesting that more than one unlinked function is required for the latter.

A lack of correlation between 'T' antigen and any particular human chromosome in hybrids made between SV40 transformed human fibroblasts and mouse LMTK cells.

Lines of hybrid cells were established by fusion of mouse LMTK- cells with SV40 transformed human fibroblasts. The lines were examined for the presence of SV40 'T' antigen and the human chromosomes they carried identified by Giemsa banding. No significant correlation could be found between any particular human chromosome and the presence of SV40 virus.

Spontaneous and induced patterns of the Epstein-Barr virus (EBV) cycle in a new set of somatic cell hybrids.

A somatic cell hybrid line and its subclones obtained by fusing two Burkitt lymphoma lines (Raji and Namalwa) were examined for the expression of EBV-specific antigens both spontaneously and after induction. The hybrids retained spontaneous early antigen (EA) production at the level characteristic of Raji. Similarly the more permissive Raji pattern dominated the induction of EA by IUDR treatment or P3HR-1 virus superinfection. These findings accord with our previous results on independently derived Raji/Namalwa hybrids. Virus capsid antigen was induced in the hybrids by P3HR-1 virus superinfection though at a lower level than in the Raji parental cell.

Susceptibility of human-mouse T cell hybrids to HIV-productive infection.

Interspecies human x mouse cell hybrids were used to investigate the genetic basis of human permissivity to HTLV-IIIB infection. T cell hybrids between the mouse BW 51.47 T lymphoma line and normal, PHA-IL-2 activated, human peripheral mononuclear cells (PBMCs) were generated. These hybrids preferentially segregated human chromosomes, as assessed by phenotype and karyotype analysis. Viral integration occurred only in those hybrids expressing CD4+ at the cell surface. However, infectious progeny production was demonstrated only in two of the three CD4+ hybrids tested. By segregation analysis, we could correlate the absence of human chromosomes 1, 3, and 9 with the lack of infectious viral progeny.

Epstein-Barr virus lytic cycle spreads via cell fusion in a nasopharyngeal carcinoma hybrid cell line.

NPC-KT cl.S61, a subclone derived from an epithelial-nasopharyngeal carcinoma hybrid cell line NPC-KT, showed extensive Epstein-Barr virus (EBV) production and cell fusion when the EBV replicative cycle was induced by 5-iodo-2'-deoxyuridine. On the contrary, parental NPC-KT cells produced virus at a lower level and did not show cell fusion. Cell fusion in cl.S61 cells was blocked by 2-deoxyglucose and acyclovir, inhibitors of glycosylation and EBV DNA polymerase, respectively, with a concomitant decrease in the number of cells expressing EBV growth-associated antigens. However, the frequency of virus antigen expression in parental NPC-KT cells was not significantly affected by these drugs. This result suggests that efficient production of EBV from cl.S61 cells is due to the spreading of viral replicative cycle via cell fusion. It was also demonstrated by in situ autoradiography that cl.S61 cells producing virus fused to not only EBV receptor/CR2 positive Raji and BJAB cells, but also to receptor-negative Jurkat cells. The possible mechanism of EBV entry into cells devoid of virus receptor by cell fusion is discussed.

Epstein-Barr virus nuclear antigen-positive nasopharyngeal hybrid cells.

An epithelial-like hybrid cell line was established by cell fusion of 8-azahypoxanthine-resistant epithelial cells (Ad-AH) with lymphoblastoid cells (A2L), derived from the human nasopharynx. The nasopharyngeal hybrid cells, designated as A2L/AH, were Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA)-positive by the anticomplement immunofluorescence method. Furthermore, the treatment of the hybrid cells with 5-Iodo-2' -deoxyuridine (IUDR) induced early antigen (EA) and viral capsid antigens (VCA), while the treatment of nonproducer lymphoblastoid cells, A2L, induced EA but not VCA. The appearance of IUDR-induced VCA in the hybrid cells suggests that some factor produced by the Ad-AH cells might neutralize a repressed state of VCA and thus activate these antigens with the treatment of IUDR. These established nasopharyngeal hybrid cells might be useful for studies of in vitro nasopharyngeal carcinoma (NPC) since no EBV-carrying NPC cell lines have been established.

Effects of temperature on Epstein-Barr virus replication in epithelial/nasopharyngeal carcinoma hybrid cells.

The authors studied the replication of Epstein-Barr virus (EBV) in the epithelial hybrid cells derived from nasopharyngeal carcinoma (NPCKT) caused by the effects of sub-optimal incubation at 34, 32 and 28 degrees C, respectively, using an immunofluorescence technique. The intensity of EBV induction by sub-optimal temperatures was in the order of 32 degrees C----28 degrees C----34 degrees C. In particular, 18-24% positive cells for early antigen and viral capsid antigen were observed at around 10 to 14 days after shift-down of the incubation temperature from 37 to 32 degrees C.

Production of bacterial blight resistant lines from somatic hybridization between Oryza sativa L. and Oryza meyeriana L.

Novel bacterial blight (BB) resistance gene(s) for rice was (were) introduced into a cultivated japonica rice variety Oryza sativa (cv. 8411), via somatic hybridization using the wild rice Oryza meyeriana as the donor of the resistance gene(s). Twenty-nine progenies of somatically hybridized plants were obtained. Seven somatically hybridized plants and their parents were used for AFLP (amplified fragment length polymorphism) analysis using 8 primer pairs. Results confirmed that these plants were somatic hybrids containing the characteristic bands of both parents. The morphology of the regenerated rice showed characters of both O. sativa and O. meyeriana. Two somatic hybrids showed highest BB resistance and the other 8 plants showed moderate resistance. The new germplasms with highest resistance have been used in the rice breeding program for the improvement of bacterial blight resistance.