A non-class I MHC intestinal epithelial surface glycoprotein, gp180, binds to CD8.

The activation of CD8(+) T cells by normal intestinal epithelial cells in antigen-specific or allogeneic mixed cell culture systems has significant implications for the modulation of mucosal immune responses due to the fact that these T cells appear to have regulatory rather than cytolytic activity. A 180-kDa glycoprotein (gp180) has been identified and shown to be important in CD8(+) T cell activation by intestinal epithelial cells. In this study, we examine, in further detail, the role that the CD8 molecule plays in this interaction. It has been previously shown that monoclonal antibodies against gp180 inhibited the activation of CD8-associated p56(lck) in T cells. Although indirectly suggested by these data, there was no evidence that the activation of this protein tyrosine kinase was a direct result of gp180 interacting with the CD8 molecule. In this study, we document that soluble gp180 is able to bind to CD8-Fc fusion proteins and is absorbed by human CD8 alpha but not CD4 transfected murine T cells and that this interaction is dependent upon carbohydrate on the gp180 molecule. Furthermore, the sites used for binding by gp180 are distinct from those used by the conventional CD8 ligand, class I MHC. Thus, gp180 appears to be a novel CD8 ligand that plays an important role in the activation of CD8-associated kinases and of CD8(+) T cells.

The intestinal epithelial cell: processing and presentation of antigen to the mucosal immune system.

The immunologic tone of the intestinal tract is one of suppressed or highly regulated responses. While there are several components (intrinsic and extrinsic to the gut-associated lymphoid tissue) responsible for this immunologically suppressed tone, the intestinal epithelial call (IEC) has been proposed as a key player in this process. IECs can take up and process antigen but distinct surface molecules and restriction elements allow them to present these antigens to unique regulatory T cells. These include the expression of the class Ib molecule CD1d as well as a novel CD8 ligand, gp180. These molecules come together to activate a subpopulation of CD8+ regulatory cells whose function is to suppress immune responses in an antigen non-specific fashion most likely through cognate interactions. This form of regulation may be unique to the gut-associated lymphoid tissue which is consistent with the unusual demands upon this part of the immune system.

Human airway epithelial cells stimulate T-lymphocyte lck and fyn tyrosine kinase.

Previous studies have shown that human airway epithelial cells (AEC) can stimulate allogeneic peripheral blood T-lymphocyte (PBT) proliferation. We now sought to determine which AEC surface molecule/T-cell coreceptors are involved in this process. AEC-induced PBT proliferation was inhibited by 25 microM genestein or herbamycin A (0.9 and 1.8 microM), both tyrosine kinase inhibitors. Anti-phosphotyrosine immunoblots performed on PBT lysates after coculture with AEC demonstrated phosphorylation of 56kD and 60kD bands. To determine whether CD3 associated p59fyn, or CD4 and CD8 associated p56lck phosphotyrosine kinases (PTK) were involved, we assayed kinase activity in lymphocyte lysates immunoprecipitated with anti-p56lck and p59fyn mAbs. PBT cells or murine T-cell line transfectants expressing human CD4 (3G4) or human CD8alpha (3G8) were cocultured with AEC or A549, an alveolar-like cell line lacking class II Ag expression. After A549 or AEC coculture, p56lck activity in PB T-cells peaked at 2 min whereas p59fyn kinase activity continued to rise at 8 min. AEC (expressing class II Ags) stimulate PTK activity in both 3G8 and 3G4 cells. A549 stimulated p56lck in 3G8, but not in 3G4 cells. This activation of p56lck was not blocked by preincubation of A549 with anti-class I or anti-CD1d mAbs. An antibody generated in our laboratory, which recognizes an epithelial specific surface molecule (mAb L12) and which blocks AEC driven PBT proliferation, was shown to block PTK activity of peripheral blood T-cell lysates, though not of 3G8 lysates. These studies suggest that AEC are capable of stimulating CD4 and CD8 associated lck and CD3 associated fyn kinases through class II dependent and independent pathways.

Defective expression of gp180, a novel CD8 ligand on intestinal epithelial cells, in inflammatory bowel disease.

Previous studies support a role for intestinal epithelial cells (IEC) as antigen-presenting cells in mucosal immune responses. T cells activated by IEC are CD8+, suppressor in function, and dependent upon CD8-associated p56lck activation. A 180-kD glycoprotein (gp180) recognized by mAbs B9 and L12 has been identified and shown to be important in CD8+ T cell activation by IEC. Since IEC derived from patients with inflammatory bowel disease (IBD) are incapable of activating CD8+ T cells, we asked whether this correlated with gp180 expression. While frozen sections of normal bowel revealed bright gp180 staining on all IEC, both inflamed and uninflamed ulcerative colitis (UC) specimens showed patchy staining. In Crohn's disease (CD), staining was faint to absent. Flow cytometry confirmed immunohistochemical data. The staining patterns correlated with the ability of IEC to activate CD8-associated p56lck. Normal IEC induced phosphorylation of p56lck in CD8alpha but not CD4+ transfectants. In contrast, both UC and CD IEC activated CD4 and, to a much lesser extent, CD8-associated p56lck. Thus, gp180 expression by IBD IEC appears to be altered, and correlates with a functional alteration of lck activation. This defect may reflect a more proximal event in the pathogenesis of IBD.

Characterization of a 180-kDa intestinal epithelial cell membrane glycoprotein, gp180. A candidate molecule mediating t cell-epithelial cell interactions.

Previous studies have shown that normal human intestinal epithelial cells stimulate CD8(+) suppressor T cell proliferation in an allogeneic mixed epithelial/T cell co-culture system, which is neither restricted by class I or class II major histocompatibility complex antigens nor by any soluble factors from epithelial cells. Two epithelial specific monoclonal antibodies (mAb), mAb B9 and mAb L12, are potent inhibitors of this mixed epithelial/T cell reaction but not of conventional mixed lymphocyte reactions. While phenotypically distinct by tissue staining, both mAbs recognize a 180-kDa epithelial membrane glycoprotein (gp180). Further characterization of gp180 revealed the following. 1) The protein migrated between 150 and 180 kDa in SDS-polyacrylamide gel electrophoresis and could be resolved by Western blot using mAb B9 or mAb L12. 2) The molecule has two forms, an apically sorted glycosylphosphatidylinositol-anchored form and a basolateral transmembrane form. 3) gp180 is heavily N-glycosylated, since N-glycanase treatment results in a >50% reduction in size. 4) Purified gp180 can bind to peripheral blood T cells and activate p56(lck). 5) gp180 can activate p56(lck) in 3G8 (a murine T cell hybridoma transfected with human CD8alpha cDNA) but not in 3G4 (CD4 transfectant), suggesting that gp180 binds to CD8. Thus, gp180 appears to be a novel regulator of mucosal immune responses.

Human intestinal epithelial cell-induced CD8+ T cell activation is mediated through CD8 and the activation of CD8-associated p56lck.

The activation of CD8+ suppressor T cells by normal intestinal epithelial cells in antigen-specific or allogeneic mixed cell culture systems has significant implications for the regulation of mucosal immune responses. In this study, we found that the capacity of epithelial cells to induce CD8+ suppressor T cell activation appeared to be linked to the binding of CD8 molecules on the T cell surface. This appears to be mediated by a non-class I molecule expressed on the epithelial cell surface, which binds to CD8 and results in the activation of the CD8-associated src-like tyrosine kinase, p56lck. Epithelial cell-stimulated p56lck activation is an early event (in contrast to monocytes) and is essential for T cell activation, since proliferation could be completely abrogated by pretreatment of T cells with genestein or herbamycin, both of which are protein tyrosine kinase inhibitors. Pretreatment of T cells with anti-CD8 or of intestinal epithelial cells with an anti-epithelial cell mAb B9 inhibited p56lck activation and further confirmed that CD8 on the T cell and a CD8 ligand on the epithelial cell were involved in this T cell activation event. The specificity of this reaction was confirmed in experiments in which murine transfectants 3G4 and 3G8, expressing CD4 or CD8, respectively, were used. Coculture of 3G8 with epithelial cells but not with monocytes activated p56lck in this cell line, whereas p56lck was preferentially activated in 3G4 cells when monocytes were used as the stimulator cells. Although stimulation through CD8- and CD8-associated p56lck was important for epithelial cell-induced T cell activation, T cell proliferation could not be induced by cross-linking CD8 alone with monoclonal antibody anti-CD8. These data suggest that a second signal, possibly through the T cell antigen receptor since activation of the T cell receptor-associated kinase fyn was also seen, is required for epithelial cell-driven T cell proliferation.

Molecular characterization of J558 genes encoding tight-skin mouse autoantibodies: identical heavy-chain variable genes code for antibodies with different specificities.

Tight-skin mouse, a mutant strain with a single gene defect, develops cutaneous hyperplasia and specific autoantibodies, like humans affected by scleroderma. The autoantibodies produced in the tight-skin mouse are encoded primarily by heavy-chain variable (VH) genes from the J558 family. To understand the genetic basis of production of autoantibodies, we have analyzed the structure of J558 genes encoding these autoantibodies. The results showed that J558 genes encoding these antibodies were not derived from a selected germ-line gene(s) or a single subfamily but were derived from genes belonging to diverse J558 subfamilies. However, two prototype VH genes representing two new subfamilies were found to be repeatedly expressed in their germ-line form in eight independent clones. Autoantibodies with distinct specificities appear to be generated by pairing of similar/identical VH genes with different V kappa genes derived from the same or different families. Fourteen of 18 autoantibodies shared a conserved heptapeptide sequence motif, YNEKFKG, in the second complementarity-determining region of heavy chains. Usage of germ-line genes from diverse J558 subfamilies bearing a common motif to encode autoantibodies suggests a regulatory role for this motif. Thus, selection and expansion of the autoreactive B-cell repertoire in the tight-skin mouse appear to be VH-gene mediated. The frequency of N nucleotide addition at diversity-joining (D-JH) junctions was lower, whereas the frequency of usage of the DFL16 segment was higher. Finally, in contrast to normal and other autoimmune mouse strains, the frequencies of D-D fusions and D inversions were higher in tight-skin mouse total immunoglobulin as well as autoantibody repertoires.

Highly sensitive sandwich enzyme immunoassay for alpha-fetoprotein in human saliva.

To determine alpha-fetoprotein (AFP) in human saliva, a highly sensitive sandwich enzyme immunoassay for saliva AFP was developed. AFP standards and saliva samples were added into the wells of a polystyrene plate coated with goat IgG antibody against human AFP. After incubation, the wells were washed and horseradish peroxidase-labelled antibody was added. The enzyme activity specifically bound to the well was assayed using 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide as substrate. The reaction was stopped by addition of 2 M sulphuric acid and the AFP concentration was determined from the absorbance at 450 nm. The minimum detectable concentration was 8 ng/L. The recovery of AFP mixed with human saliva was 91.1-102.4%. The within-assay and between-assay coefficients of variation were 6.5-8.9% and 7.6-10.8%, respectively. The assay correlated well with a radioimmunoassay for human AFP (r = 0.985, n = 13, P less than 0.001). The mean concentration of AFP in normal human saliva was 14.3 ng/L (SEM = 4.9 ng/L, n = 10) and significantly higher levels of saliva AFP were observed in hepatocellular carcinoma patients with positive serum AFP (mean 1367.8 ng/L, SEM 595.4 ng/L, n = 6; P less than 0.001). Strong correlation was observed between saliva AFP and serum AFP (r = 0.978, P less than 0.01, n = 13).

Bile secretory immunoglobulin A in biliary infection and cholelithiasis.

Bile samples from 71 patients with cholelithiasis and a control group of 10 subjects without hepatobiliary diseases were cultured for bacteria and measured for secretory immunoglobulin A (SIgA) using enzyme immunoassay specific for SIgA. The results of bile bacterial culture were all positive in patients with primary bile duct pigment stones, and significantly lower bile SIgA levels were observed than in normal controls (P less than 0.005). It was also shown that the constitutent ratios of SIgA to total bile immunoglobulin and the bile-serum ratio of SIgA were markedly lower in these patients than in normal controls (P less than 0.001, P less than 0.001). In patients with cholecystolithiasis, bile SIgA concentrations of patients with biliary infections were remarkably lower than those of patients without biliary infection (P less than 0.01) and those of normal controls (P less than 0.01). These results suggest a close relationship between biliary tract infection and low concentrations of bile SIgA.