Mouse NK cells express at least seven inhibitory Ly49 receptors. Here we employ a semiquantitative cell-cell adhesion assay as well as class I/peptide tetramers to provide a comprehensive analysis of specificities of Ly49 receptors for class I MHC molecules in eight MHC haplotypes. Different Ly49 receptors exhibited diverse binding properties. The degree of class I binding was related to the extent of functional inhibition. The tetramer studies demonstrated that neither glycosylation nor coreceptors were necessary for class I binding to Ly49 receptors and uncovered peptide-specific recognition by a Ly49 receptor. The results provide a foundation for interpreting and integrating many existing functional studies as well as for designing tests of NK cell development and self-tolerance.

We report an in vitro stroma-dependent system for the clonal growth and differentiation of natural killer (NK) cells from lymphoid-restricted bone marrow progenitors or bone marrow NK1.1+ cells. Strikingly, the potential to initiate expression of specific Ly49 receptors becomes increasingly restricted as NK cells develop. Moreover, when NK cells express a Ly49 receptor specific for stromal cell class I MHC, they are less likely to initiate expression of another Ly49 receptor in the clonal culture system. The results indicate multiple roles for stromal cells in NK cell development, in supporting clonal growth, in initiation of Ly49 receptor expression, and in formation of the NK cell receptor repertoire.

We have studied the transcriptional regulatory mechanism of the human histo-blood group ABO genes, and identified DNA cis-elements and trans-activating protein that control the expression of these genes which are important in blood transfusion and organ transplantation. We introduced the 5 -upstream sequence of ABO genes into the promoterless reporter vector and characterized the promoter activity of deletion constructs using transient transfection assays with gastric cancer cell line KATO III cells. The sequence just upstream of the transcription start site (cap site), and an enhancer element, which is located further upstream (between ؊3899 and ؊3618 base pairs ( Histo-blood group ABH(O) antigens, the major alloantigens in humans (1), have been characterized as defined trisaccharide determinants GalNAc␣133(Fuc␣132)Gal␤13 R, Gal␣133(Fuc␣132)Gal␤13 R, and disaccharide determinant Fuc␣132Gal␤13 R for A, B, and H, respectively (2, 3). These structures represent the secondary gene products which are synthesized from the precursor H substrate by ␣133GalNAc (A transferase) and ␣133Gal transferase (B transferase), the primary gene products coded by the functional alleles at the ABO locus (4, 5). Molecular genetic studies of the ABO genotypes have identified two critical single-base substitutions between A and B genes, the resultant 2-amino acid substitutions being responsible for the different donor nucleotide-sugar substrate specificity between A and B transferases. A single base deletion, which shifts the codon reading frame and abolishes the function of A transferase, has been identified in O allelic cDNAs (6, 7).ABH antigens are known to undergo drastic changes during development, differentiation, and maturation. Studies of these antigens in stratified squamous epithelia provided one of the clearest examples of differential expression during cell maturation (8). In non-keratinized stratified squamous epithelia, the immature cells in the basal layers are characterized by the expression of sialylated or unsubstituted precursor peripheral cores, while differentiated and mature cells in the upper layers sequentially express ␣132-fucosylated H structures, and A and B antigens depending on the ABO genotype of the individual. This sequential expression of carbohydrate antigens is associated with the differentiation pattern of the epithelium. An interesting question is how these changes are controlled during cell differentiation. Since keratinocytes are known to greatly change their gene expression during terminal cell differentiation (9), the switch-on of the ABO genes during the maturation may be governed by the same factor(s). To fill in the gap between the expression of the ABO genes and the appearance of the ABO phenotypes in the terminal differentiation of epithelial cells, it is essential to understand the transcriptional regulatory mechanism of the ABO genes. In addition to the normal cell differentiation process, the changes of ABH antigen expression have also been documented in abnormal processes such as tumorig...

Accumulating evidence suggests an important role of interleukin-8 (IL-8) in Helicobacter pylori infectionassociated chronic atrophic gastritis and peptic ulcer. We observed in this study that a gastric cancer-derived cell line, MKN45, produced a massive amount of IL-8 upon coculture with live H. pylori but not with killed H. pylori, H. pylori culture supernatants, or live H. pylori separated by a permeable membrane, indicating that IL-8 production requires a direct contact between the cells and live bacteria. Moreover, the tyrosine kinase inhibitor herbimycin but neither a protein kinase C inhibitor (staurosporine) nor a protein kinase A inhibitor (H89) inhibited IL-8 production by MKN45 cells cocultured with live bacteria, suggesting the involvement of a tyrosine kinase(s) in H. pylori-induced IL-8 production. In addition, coculture of H. pylori induced IL-8 mRNA expression in MKN45 cells and an increase in luciferase activity in cells which were transfected with a luciferase expression vector linked with a 5-flanking region of the IL-8 gene (bp ؊133 to ؉44), indicating that the induction of IL-8 production occurred at the transcriptional level. This region contain three cis elements important for induction of IL-8 gene expression: AP-1 (؊126 to ؊120 bp), NF-IL6 (؊94 to ؊81 bp), and NF-B (؊80 to ؊70 bp) binding sites. Mutation of the NF-B binding site abrogated completely the induction of luciferase activity, whereas that of the AP-1 site partially reduced the induction. However, mutation of the NF-IL6 binding site resulted in no decrease in the induction of luciferase activity. Moreover, specific NF-B complexes were detected in the nuclear proteins extracted from MKN45 cells which were infected with H. pylori. Collectively, these results suggest that H. pylori induced the activation of NF-B as well as AP-1, leading to IL-8 gene transcription.

We have investigated the regulatory role of DNA methylation in the expression of the human histo-blood group ABO genes. The ABO gene promoter region contains a CpG island whose methylation status correlates well with gene expression in the cell lines tested. The CpG island was found hypomethylated in some cell lines that expressed ABO genes, whereas the other cell lines that did not express ABO genes were hypermethylated. The ABO blood group system discovered by Karl Landsteiner (1) at the beginning of this century is of great importance in blood transfusions and organ transplantations. Two carbohydrate antigens, A-and B-antigens, and their antibodies constitute this system. The A and B functional alleles at the ABO genetic locus encode glycosyltransferases ␣133GalNAc transferase (designated A-transferase) and ␣133Gal transferase (designated B-transferase), respectively. A-transferase transfers a GalNAc residue from UDP-GalNAc to the precursor H substrate, producing A antigens as defined by the trisaccharide determinant structure, GalNAc␣133(Fuc␣132)Gal␤13 R. Similarly, B-transferase catalyzes the transfer of a Gal from UDP-Gal to the same H substrate, producing B antigens defined by Gal␣133(Fuc␣132)Gal␤13 R (2-5). Molecular genetic studies of the human ABO genes have identified two critical single base substitutions that result in amino acid substitutions responsible for the different donor nucleotidesugar substrate specificity between A-and B-transferases. A single base deletion, which shifts the reading frame of codons and abolishes the function of A-transferase, has been identified in most O alleles (6, 7).The ABO genes are expressed in a cell type-specific manner; the isoantigens A, B, and H of blood groups A, B, and O are not confined to red cells only but are also found in most secretions and on some epithelial cells. However, they are absent in connective tissues and the central nervous system (8). ABH antigens are known to undergo drastic changes during development, differentiation, and maturation of normal cells (9). In addition to these physiological processes, profound changes have also been documented in pathological processes such as tumorigenesis. Reduction or complete deletion of A/B antigen expression in bladder and oral cancers has been documented, as well as the apparent onco-developmental expression of the ABH antigens in gastric and distal colon tumors (10 -12). Moreover, the loss of ABH antigens has been correlated with tumor progression of various carcinomas including lung and bladder carcinomas (13-16). Thus, delineation of regulatory mechanism is essential to understand these complicated expression patterns of the ABO genes.In an initial attempt to elucidate the molecular mechanism controlling the expression of the human ABO genes, we isolated several genomic clones that covered the ABO genes over 18 kb 1 (17). A 4.7-kb EcoRI/NcoI 5Ј-upstream fragment flanking the coding sequence in exon 1 of the human ABO gene was subcloned into the promoterless pGL3-basic vector upstream of the lucifer...