Polarized expression and function of the costimulatory molecule CD58 on human intestinal epithelial cells.

BACKGROUND & AIMS: Intestinal epithelial cells (IECs) can process foreign protein antigens and display antigenic peptides to CD4(+) T lymphocytes via HLA class II molecules. The purpose of this study was to determine the nature of the second, or costimulatory, signal provided by IECs. METHODS: We investigated surface expression of the costimulatory molecules CD58 (LFA-3), CD80 (B7-1), and CD86 (B7-2) by using flow cytometry, confocal microscopy, and vectorial biotinylation. Antibodies specific for CD58, CD80, and CD86 were used in blocking experiments to assess the role of these molecules in providing a costimulatory signal to CD4(+) T cells by IECs. RESULTS: CD58, but not CD80 or CD86, was observed to be expressed constitutively on both native IECs and in the IEC lines T84 and HT-29. The surface expression of CD58 was highly polarized and restricted to the basolateral surface of the cell. Antibodies against CD58, but not CD80 or CD86, inhibited the stimulation of CD4(+) T-cell proliferation mediated by IECs. CONCLUSIONS: CD58 is expressed by polarized IECs in a topologically restricted manner at the region of T-cell contact and can function as a costimulatory molecule in HLA class II-mediated antigen presentation.

Highly polarized HLA class II antigen processing and presentation by human intestinal epithelial cells.

The high concentration of foreign antigen in the lumen of the gastrointestinal tract is separated from the underlying lymphocytes by a single cell layer of polarized epithelium. Intestinal epithelial cells can express HLA class II antigens and may function as antigen-presenting cells to CD4(+) T cells within the intestinal mucosa. Using tetanus toxoid specific and HLA-DR-restricted T lymphocytes, we show that polarized intestinal epithelial cells directed to express HLA-DR molecules are able to initiate class II processing only after internalization of antigen from their apical surface. Coexpression of the class II transactivator CIITA in these cells, which stimulates highly efficient class II processing without the characteristic decline in barrier function seen in polarized monolayers treated with the proinflammatory cytokine gamma-IFN, facilitates antigen processing from the basolateral surface. In both cases, peptide presentation to T cells via class II molecules was restricted to the basolateral surface. These data indicate a highly polarized functional architecture for antigen processing and presentation by intestinal epithelial cells, and suggest that the functional outcome of antigen processing by the intestinal epithelium is both dependent on the cellular surface at which the foreign antigen is internalized and by the underlying degree of mucosal inflammation.

New genetic techniques for group B streptococci: high-efficiency transformation, maintenance of temperature-sensitive pWV01 plasmids, and mutagenesis with Tn917.

Three techniques were developed to improve the genetic manipulation of group B streptococci (GBS). We first optimized a protocol for transformation of GBS by electroporation, which provided transformation efficiencies of 10(5) CFU/microgram. Variables that influenced the transformation efficiency were the glycine content of the competent cell growth media, the electric field strength during electroporation, the electroporation buffer composition, the host origin of the transforming plasmid, and the concentration of selective antibiotic at the final plating. Our transformation protocol provides an efficiency sufficient for cloning from ligation reactions directly into GBS, obviating an intermediate host such as Escherichia coli. Second, temperature-sensitive plasmids of the pWV01 lineage were shown to transform GBS, and their temperature-sensitive replication was confirmed. Lastly, the temperature-sensitive pWV01 plasmid pTV1OK, which contains Tn917, was used as a transposon delivery vector for the construction of genomic Tn917 mutant libraries. We have shown, for the first time, that Tn917 transposes to the GBS chromosome and at a frequency of 10(-3)/CFU. Furthermore, representative clones from a Tn917 library contained single transposon insertions that were randomly located throughout the chromosome. These techniques should provide useful methods for cloning, mutagenesis, and characterization of genes from GBS.

Intestinal epithelial cells use two distinct pathways for HLA class II antigen processing.

Intestinal epithelial cells express a low level of HLA class II molecules constitutively, with elevated levels seen in the setting of mucosal inflammation including inflammatory bowel disease. The ability of intestinal epithelial cells to act as antigen presenting cells for alphabeta CD4(+) T lymphocytes was examined through a molecular analysis of the HLA class II antigen processing pathway. We have shown that intestinal epithelial cells contain abundant constitutive levels of the cathepsin proteases proven to function in HLA class II mediated antigen presentation. Activation of these cells by gamma-IFN induced the expression of invariant chain and HLA-DM alphabeta, thus facilitating the formation of compact, SDS-stable HLA- DR alphabeta heterodimers. Using HLA-DR-restricted T cells and retroviral mediated gene transfer of HLA-DR alleles into the intestinal epithelial cell lines HT-29 and T84, we demonstrated efficient antigen processing and presentation to CD4(+) T lymphocytes in the presence of the proinflammatory cytokine gamma-IFN. The class II processing pathway and presentation in the presence of gamma-IFN was indistinguishable from that observed with a conventional antigen presenting cell. Antigen processing also occurred in intestinal epithelial cells in the absence of gamma-IFN, and in contrast to that seen after stimulation with gamma-IFN, required high concentration of antigen and was not inhibited by the protease inhibitor leupeptin. These data suggest the use of two distinct pathways of HLA class II antigen processing in enterocytes with differential immunomodulatory properties in the presence or absence of mucosal inflammation.

Group B streptococcal beta-hemolysin expression is associated with injury of lung epithelial cells.

Group B streptococci (GBS) are the leading cause of serious bacterial infection in newborns. Early-onset disease is heralded by pneumonia and lung injury, and the lung may serve as a portal of entry for GBS into the bloodstream. To examine a potential role for GBS beta-hemolysin in lung epithelial injury, five wild-type strains varying in beta-hemolysin expression were chosen, along with five nonhemolytic (NH) and five hyperhemolytic (HH) variants of these strains derived by chemical or transposon mutagenesis. Monolayers of A549 alveolar epithelial cells were exposed to log-phase GBS or stabilized hemolysin extracts of GBS cultures, and cellular injury was assessed by lactate dehydrogenase (LDH) release and trypan blue nuclear staining. Whereas NH strains produced no detectable injury beyond baseline (medium alone), hemolysin-producing strains induced LDH release from A549 cells in direct correlation to their ability to lyse sheep erythrocytes. HH strains were also associated with marked increases in trypan blue nuclear staining of A549 monolayers. The extent of LDH release produced by HH strains was significantly reduced in the presence of dipalmitoyl phosphatidylcholine, a known inhibitor of hemolysin and the major phospholipid component of human surfactant. Electron microscopic studies of A549 cell monolayers exposed to HH GBS mutants revealed global loss of microvillus architecture, disruption of cytoplasmic and nuclear membranes, and marked swelling of the cytoplasm and organelles. We conclude that GBS hemolysin expression correlates with lung epithelial cell injury and may be important in the initial pathogenesis of early-onset disease, particularly when pulmonary surfactant is deficient.

Yeast ARS function and nuclear matrix association coincide in a short sequence from the human HPRT locus.

A sequence that supports extrachromosomal replication of plasmids in yeast has been identified within the first intron of the human hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene. This represents the first isolation of such an autonomously replicating sequence (ARS) from an exactly known position in the human genome. This ARS shares similarities of imparted yeast phenotype and DNA sequence with other heterologous ARSs. In addition, this sequence is found to be a matrix association region (MAR) on the basis of specific binding to nuclear matrices prepared from several mammalian cell types. It also exhibits anomalous electrophoretic behavior, characteristic of bent DNA, on polyacrylamide gels. The coincidence of these properties supports the possibility that this region may play a role in DNA replication within its normal chromosomal context.

Fine structure of the human hypoxanthine phosphoribosyltransferase gene.

The human hypoxanthine phosphoribosyltransferase (HPRT) gene has been characterized by molecular cloning, mapping, and DNA sequencing techniques. The entire gene, which is about 44 kilobases in length, is composed of nine exon elements. The positions of the introns within the coding sequence are identical to those of the previously-characterized mouse HPRT gene, although there are significant differences between intron sizes for the two genes. HPRT minigenes have been used in a transient expression assay involving microinjection into HPRT- cells to demonstrate functional promoter activity within a 234-base-pair region upstream from the ATG codon. The promoter of this gene resembles those of other recently characterized "housekeeping" genes in that it lacks CAAT- and TATA-like sequences, but contains several copies of the sequence GGGCGG. Both RNase protection and primer extension analysis indicate that human HPRT mRNA is heterogeneous at the 5' terminus, with transcription initiation occurring at sites located congruent to 104 to congruent to 169 base pairs upstream from the ATG codon. Comparison of the mouse and human HPRT 5'-flanking sequences indicates that there are only limited stretches of conserved sequence, although there are other shared features, such as an extremely high density of potential methylation sites, that may have functional significance.