Regulation of human intestinal intraepithelial lymphocyte cytolytic function by biliary glycoprotein (CD66a).

Human small intestinal intraepithelial lymphocytes (iIEL) are a unique population of CD8alphabeta+ TCR-alphabeta+ but CD28- T lymphocytes that may function in intestinal epithelial cell immunosurveillance. In an attempt to define novel cell surface molecules involved in iIEL function, we raised several mAbs against activated iIELs derived from the small intestine that recognized an Ag on activated, but not resting, iIELs. Using expression cloning and binding studies with Fc fusion proteins and transfectants, the cognate Ag of these mAbs was identified as the N domain of biliary glycoprotein (CD66a), a carcinoembryonic Ag-related molecule that contains an immune receptor tyrosine-based inhibitory motif. Functionally, these mAbs inhibited the anti-CD3-directed and lymphokine-activated killer activity of the P815 cell line by iIELs derived from the human small intestine. These studies indicate that the expression of biliary glycoprotein on activated human iIELs and, potentially, other mucosal T lymphocytes is involved in the down-regulation of cytolytic function.

Lymphocyte adhesion to epithelia and endothelia mediated by the lymphocyte endothelial-epithelial cell adhesion molecule glycoprotein.

Upon encountering the relevant vascular bed, lymphocytes attach to endothelial adhesion molecules, transmigrate out of circulation, and localize within tissues. Lymphocytes may then be retained at microanatomic sites, as in tissues, or they may continue to migrate to the lymphatics and recirculate in the blood. Lymphocytes also interact transiently, but with high avidity, with target cells or APC that are infected with microbes or have taken up exogenous foreign Ags. This array of adhesive capabilities is mediated by the selective expression of lymphocyte adhesion molecules. Here, we developed the 6F10 mAb, which recognizes a cell surface glycoprotein designated lymphocyte endothelial-epithelial cell adhesion molecule (LEEP-CAM), that is distinct in biochemical characteristics and distribution of expression from other molecules known to play a role in lymphocyte adhesion. LEEP-CAM is expressed on particular epithelia, including the suprabasal region of the epidermis, the basal layer of bronchial and breast epithelia, and throughout the tonsillar and vaginal epithelia. Yet, it is absent from intestinal and renal epithelia. Interestingly, it is expressed also on vascular endothelium, especially high endothelial venules (HEV) in lymphoid organs, such as tonsil and appendix. The anti-LEEP-CAM mAb specifically blocked T and B lymphocyte adhesion to monolayers of epithelial cells and to vascular endothelial cells in static cell-to-cell binding assays by approximately 40-60% when compared with control mAbs. These data suggest a role for this newly identified molecule in lymphocyte binding to endothelium, as well as adhesive interactions within selected epithelia.

Direct and regulated interaction of integrin alphaEbeta7 with E-cadherin.

The cadherins are a family of homophilic adhesion molecules that play a vital role in the formation of cellular junctions and in tissue morphogenesis. Members of the integrin family are also involved in cell to cell adhesion, but bind heterophilically to immunoglobulin superfamily molecules such as intracellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, or mucosal addressin cell adhesion molecule (MadCAM)-1. Recently, an interaction between epithelial (E-) cadherin and the mucosal lymphocyte integrin, alphaEbeta7, has been proposed. Here, we demonstrate that a human E-cadherin-Fc fusion protein binds directly to soluble recombinant alphaEbeta7, and to alphaEbeta7 solubilized from intraepithelial T lymphocytes. Furthermore, intraepithelial lymphocytes or transfected JY' cells expressing the alphaEbeta7 integrin adhere strongly to purified E-cadherin-Fc coated on plastic, and the adhesion can be inhibited by antibodies to alphaEbeta7 or E-cadherin. The binding of alphaEbeta7 integrin to cadherins is selective since cell adhesion to P-cadherin-Fc through alphaEbeta7 requires >100-fold more fusion protein than to E-cadherin-Fc. Although the structure of the alphaE-chain is unique among integrins, the avidity of alphaEbeta7 for E-cadherin can be regulated by divalent cations or phorbol myristate acetate. Cross-linking of the T cell receptor complex on intraepithelial lymphocytes increases the avidity of alphaEbeta7 for E-cadherin, and may provide a mechanism for the adherence and activation of lymphocytes within the epithelium in the presence of specific foreign antigen. Thus, despite its dissimilarity to known integrin ligands, the specific molecular interaction demonstrated here indicates that E-cadherin is a direct counter receptor for the alphaEbeta7 integrin.

p126 (CDw101), a costimulatory molecule preferentially expressed on mucosal T lymphocytes.

Intestinal mucosal lymphocytes are defined by their anatomic location within the epithelium (intraepithelial lymphocytes), the interstitium between the epithelial basement membrane and the underlying muscularis mucosa (lamina propria lymphocytes), or in organized lymphoid tissues (Peyer's patches). Although intestinal intraepithelial lymphocytes have a distinct localization, their function has not been determined. To define cell surface proteins that are involved in intestinal intraepithelial lymphocyte localization or function, cultured human mucosal lymphocytes were used as immunogens to develop mAbs that react predominantly with this cell population in an immunohistochemical screening assay. Three mAbs were selected that subsequently were found by biochemical analysis to identify a 200-kDa homodimeric polypeptide on 88 to 98% of CD3+ mucosal lymphocytes but only 18 +/- 13% of PBLs. Expression on granulocytes and monocytes was also observed. This polypeptide has been termed p126 based on its SDS-PAGE-determined M(r) under reducing conditions. Cleveland digest maps demonstrated similarity between the p126 and CDw101 polypeptides. Determined amino acid sequence analysis of the purified p126 polypeptide revealed that it is the protein product of the recently identified V7 gene, which has structural similarities to members of the Ig gene superfamily. Two of the anti-p126 mAbs were costimulatory with suboptimal concentrations of anti-CD3 mAb inducing proliferation of cultured intestinal intraepithelial lymphocytes. Thus, we conclude that p126 is CDw101 encoded by a gene that predicts a seven-Ig domain chain-like structure. It has restricted expression predominantly on mucosal T lymphocytes and appears to have a costimulatory function of special relevance for CD28- T cells and for mucosal lymphocytes.

The future of biodiversity.

Recent extinction rates are 100 to 1000 times their pre-human levels in well-known, but taxonomically diverse groups from widely different environments. If all species currently deemed "threatened" become extinct in the next century, then future extinction rates will be 10 times recent rates. Some threatened species will survive the century, but many species not now threatened will succumb. Regions rich in species found only within them (endemics) dominate the global patterns of extinction. Although new technology provides details of habitat losses, estimates of future extinctions are hampered by our limited knowledge of which areas are rich in endemics.

Inhibition of [3H]mebendazole binding to tubulin by structurally diverse microtubule inhibitors which interact at the colchicine binding site.

A rapid and convenient radioligand assay was used to characterise the interaction of several structurally diverse microtubule inhibitors with the colchicine binding domain of tubulin. Values determined for the inhibition of [3H]mebendazole binding to tubulin by colchicine, combretastatin A4, NSC 181928, NSC 321567, podophyllotoxin and tubulozole-C provided an independent measure of the relative potency of these compounds. This methodology has several advantages over the inhibition of [3H]colchicine binding as a technique for investigating the molecular mechanisms involved in determining tubulin-ligand interactions.

Adhesion between epithelial cells and T lymphocytes mediated by E-cadherin and the alpha E beta 7 integrin.

In contrast to sessile cell types, lymphocytes migrate through the vasculature to become diffusely distributed in tissues or organized in lymphoid structures. A complex array of adhesion molecules including selectins, integrins and their counter-receptors mediate lymphocyte homing and migration into tissues and may be constitutively expressed or induced. However, the molecules that mediate the tissue-specific retention of lymphocytes within the parenchyma have not been identified. Along the epithelium at the basolateral surface of enterocytes, intestinal intraepithelial lymphocytes are found. These T cells of the mucosal immune system serve as a model for the tissue-specific compartmentalization of lymphocytes. We investigated whether the localization of these intestinal intraepithelial lymphocytes could be mediated by specific interactions between adhesion molecules expressed selectively on this subpopulation of T cells and tissue-restricted adhesion molecules on epithelial cells. Here we show that heterotypic adhesive interactions between epithelial cells and intraepithelial lymphocytes in vitro are mediated by E-cadherin and the alpha E beta 7 integrin.

Distinct structural and functional epitopes of the alpha E beta 7 integrin.

Intestinal intraepithelial lymphocytes (iIEL) are predominantly CD3+, CD8+ T lymphocytes located above or adjacent to the mucosal basement membrane. Although they are positioned to interact with intercellular luminal antigen or with enterocytes, the function of iIEL remains unknown. Most (> 85%) of the iIEL express the alpha E beta 7 integrin which appears to be involved in the adhesion of lymphocytes to epithelial cells. We report the characterization of three monoclonal antibodies (mAb) termed alpha E7-1, alpha E7-2, and alpha E7-3, that react with the alpha E beta 7 integrin recognized by the previously described mAb HML-1 as demonstrated by identical sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility and charge. Flow cytometric analysis of antibody cross-blocking indicated that these mAb recognize distinct epitopes of alpha E beta 7. While all of the mAb were capable of blocking the adhesion of cultured iIEL to a breast epithelial cell line, only HML-1 and alpha E7-1 (which recognize an identical or closely related epitope) were co-stimulatory with suboptimal concentrations of anti-CD3 mAb in inducing proliferation of cultured iIEL. Thus, these mAb appear to recognize functionally distinct epitopes of alpha E beta 7 and will be useful to study relationships between the structure and function of this integrin.

Evidence for a structural difference in the CD7 polypeptide on human thymocytes and intraepithelial lymphocytes defined by a new monoclonal antibody, 3D9.

Intestinal intraepithelial lymphocytes (IEL) represent a distinct subpopulation of lymphocytes located on or above the basement membrane and adjacent to the basolateral membrane of enterocytes. Thus IEL are strategically positioned to mediate a response to the uptake of foreign antigens or to the alteration of enterocytes by injury or infection. Because of their unique location, we hypothesized that IEL might selectively express specialized cell surface proteins important in their site-specific localization or function. To identify such proteins, we immunized mice with purified human IEL and identified one monoclonal antibody, 3D9, which was found to react with a majority of IEL but with very few lamina propria lymphocytes (LPL) and weakly with most peripheral blood lymphocytes (PBL). Evaluation of this antibody with two-dimensional gels demonstrated that it reacts with CD7, previously known as an early thymocyte differentiation antigen. Interestingly, unlike all other anti-CD7 monoclonal antibodies, 3D9 identified only occasional thymocytes by immunohistochemistry suggesting that CD7 is structurally different on IEL and thymocytes. However, the CD7 polypeptide immunoprecipitated from IEL and thymocytes appeared identical in SDS-PAGE mobility and in charge by two-dimensional gels, despite being recognized differently by monoclonal antibodies. These studies emphasize the expression of CD7 by IEL T cells and reveal the existence of an undefined structural difference between CD7 molecules on IEL compared to thymocytes.

Molecular cloning of the human mucosal lymphocyte integrin alpha E subunit. Unusual structure and restricted RNA distribution.

The human mucosal lymphocyte-1 (HML-1) antigen is expressed on a subclass of T-lymphocytes known as intra-epithelial lymphocytes which are located between mucosal epithelial cells. The HML-1 complex is known to mediate adhesion of intra-epithelial T-lymphocytes to epithelial cell monolayers in vitro. We and others have shown that the HML-1 antigen is an integrin composed of the beta 7 subunit in association with a novel alpha subunit, alpha E. Here we report the cloning of the alpha E cDNA and its primary amino acid sequence. alpha E contained an inserted or I domain and was more homologous to the other I domain containing integrins than to the cleaved group of integrin alpha subunits. However, alpha E contained a unique extra domain of 55 amino acids located just NH2-terminal to the I domain without counterpart in other integrins. This extra domain contained a stretch of 18 consecutive charged residues and included a proteolytic cleavage site. Thus alpha E is the only I domain containing integrin alpha subunit that is also cleaved, and the cleavage site is distinct from that of members of the cleaved group of integrin alpha subunits. These structural features mark alpha E as an unusual member of the integrin family. High levels of alpha E and beta 7 mRNA were restricted to mucosal lymphocytes supporting the hypothesis that alpha E beta 7 plays a role in the localization or site-specific functions of intra-epithelial T-lymphocytes.

Times to extinction for small populations of large birds.

A major practical problem in conservation biology is to predict the survival times-"lifetimes"-for small populations under alternative proposed management regimes. Examples in the United States include the 'Alala (Hawaiian Crow; Corvus hawaiiensis) and Northern Spotted Owl (Strix occidentalis caurina). To guide such decisions, we analyze counts of all crow, owl, and hawk species in the most complete available data set: counts of bird breeding pairs on 14 European islands censused for 29-66 consecutive years. The data set yielded 129 records for analysis. We define the population ceiling as the highest number of breeding pairs observed from colonization to extinction, within a consecutive series of counts for a given species on a given island. The resulting distributions of population lifetimes as a function of population size prove to be highly skewed: most small populations disappear quickly, but a few last for a long time. Median (i.e., 50th percentile) lifetimes are calculated as only 1-5 yr for hawk, owl, and crow populations with ceilings of one or two breeding pairs. As expected if demographic accidents are the main cause of extinction for small populations, lifetimes rise by a factor of 3-4 for each additional pair up to three pairs. They rise more slowly thereafter. These observations suggest that lifetimes of the 'Alala (now reduced to about three pairs in the wild), and of populations of Northern Spotted Owl in the smallest forest fragments, will be short unless active management is implemented.

Cytotoxic potential of intraepithelial lymphocytes (IELs). Presence of TIA-1, the cytolytic granule-associated protein, in human IELs in normal and diseased intestine.

Human intestinal intraepithelial lymphocytes (IELs) have phenotypic characteristics of cytotoxic T cells, yet a cytotoxic function has not been demonstrated in redirected lysis assays. A monoclonal antibody that reacts with a cytotoxic granule-associated protein, TIA-1, was used in this study to identify this protein in many, but not all, IELs of normal human proximal small intestine. Furthermore, in active celiac disease, in which the number of IELs is significantly increased, a corresponding increase in the number of TIA-1 cells was found. These results indicate that whereas cytotoxicity of human IELs has been difficult to demonstrate, they contain at least one of the proteins associated with cytotoxicity, and a failure to demonstrate this function may be related to the in vitro assay system used.

Anti-KCA-3, a monoclonal antibody reactive with a rat complement C3 receptor, distinguishes Kupffer cells from other macrophages.

A new mAb, designated anti-KCA-3, was developed against rat Kupffer cells. The reactivity of anti-KCA-3 was restricted to macrophages with preferential binding to Kupffer cells; only a few macrophages in the spleen, lymph nodes, lungs, and intestine stained with the antibody. A very small number of peritoneal resident and exudate macrophages reacted with the antibody and no reactivity was seen within the thymus, skin, heart, kidneys, brain, peripheral blood, and bone marrow. KCA-3 was expressed predominantly by the Kupffer cells in the periportal region rather than in the centrilobular region of the hepatic lobules. The cells in the portal tract did not stain with the antibody. The staining of the cytosmears and FACS analysis of the Kupffer cell fraction isolated from hepatic sinusoidal cells by centrifugal elutriation revealed that as many as 62% and 49% of the cells were stained with anti-KCA-3, respectively. Immunoelectron microscopic study of the liver indicated that expression of KCA-3 on Kupffer cells was limited to the plasma membrane facing the sinusoid rather than the space of Disse. Immunoprecipitation and SDS-PAGE analysis demonstrated KCA-3 to have a m.w. of approximately 50 kDa under both reducing and nonreducing conditions. After treatment of KCA-3 with N-glycanase, there was no significant change in the m.w., indicating KCA-3 was not highly glycosylated. C3b- and iC3b-mediated rosette formation between Kupffer cells and sensitized SRBC was inhibited by the antibody, implying that KCA-3 functioned as a complement C3 receptor or complement receptor-associated molecule. Furthermore, KCA-3 was eluted from C3b-Sepharose but not HSA-Sepharose after incubation with Kupffer cell lysate, indicating that KCA-3 directly binds C3b. The cell distribution, ligand-binding specificity, and biochemical properties of the protein were found to be different from the complement C3 receptors previously described. Because OX42 (antibody reactive with the rat CR3 receptor) inhibited complement C3-mediated rosette formation with peritoneal resident macrophages but not with Kupffer cells, the findings suggest that C3-mediated binding to Kupffer cells and to peritoneal macrophages is mediated by two different receptors. We conclude that anti-KCA-3 recognizes a novel type of complement C3 receptor preferentially expressed on Kupffer cells.

Differential stability of the benzimidazole (BZ)-tubulin complex in BZ-resistant and BZ-susceptible isolates of Haemonchus contortus and Trichostrongylus colubriformis.

The binding of [3H]mebendazole ([3H]MBZ) to tubulin from BZ-susceptible (BZ-S) and BZ-resistant (BZ-R) isolates of Haemonchus contortus and Trichostrongylus colubriformis was investigated using charcoal extraction and gel filtration techniques. The amount of [3H]MBZ bound at infinite free ligand concentration (Bmax) was significantly reduced for the BZ-R isolate compared with the BZ-S isolate in both species when assayed by charcoal extraction. However, Bmax was increased to comparable levels for both BZ-S and BZ-R isolates of each species when assayed by the less stringent gel filtration technique. These results indicate that the BZ-tubulin interaction in trichostrongylid nematodes is comprised of a minimum of two components. As similar levels of total [3H]MBZ binding were observed for both BZ-S and BZ-R isolates of each species when assayed by gel filtration, it is suggested that the reduction in the pseudo-irreversible BZ binding component in BZ-R isolates results in an increase in the level of reversible BZ binding and therefore provides a survival advantage to BZ-R nematodes.

Binding of [3H]benzimidazole carbamates to mammalian brain tubulin and the mechanism of selective toxicity of the benzimidazole anthelmintics.

The binding of tritiated benzimidazole carbamates ([3H]BZCs) to mammalian brain tubulin was examined to investigate the kinetics of the BZC-tubulin interaction and to establish the mechanism of the selective toxicity of the BZC based anthelmintics. [3H]BZC binding to tubulin was markedly greater at 4 degrees than at 37 degrees for all ligands. The association constant (Ka) and maximum amount of [3H]BZC bound (Bmax) were temperature dependent for [3H]mebendazole ([3H]MBZ), [3H]oxibendazole ([3H]-OBZ) and [3H]oxfendazole ([3H]OFZ). The Ka and Bmax values obtained for [3H]MBZ, [3H]OBZ and [3H]OFZ, and the comparatively weak binding of [3H]carbendazim, reflected the known in vitro potency of these compounds as microtubule inhibitors. Dissociation of the [3H]MBZ-tubulin complex was also temperature dependent, the first order dissociation rate constant being reduced by two orders of magnitude at 4 degrees compared with that observed for 37 degrees. These results indicate that the binding of BZCs to mammalian brain tubulin is temperature dependent and suggest that temperature induced conformational changes in the tubulin dimer influence the ability of the BZCs to form a stable BZC-tubulin complex. The temperature dependence of BZC binding and the affinity of the BZCs for mammalian tubulin are therefore unlike the BZC-tubulin interaction observed for parasitic nematodes, where optimum BZC binding occurs at 37 degrees and results in the formation of a pseudo-irreversible complex.

A family of beta 7 integrins on human mucosal lymphocytes.

The heterodimeric protein complex recognized by the human mucosal lymphocyte 1 (HML-1) monoclonal antibody is expressed on 95% of intraepithelial lymphocytes but on only 1-2% of peripheral blood lymphocytes [Cerf-Bensusson, N., Jarry, A., Brousse, N., Lisowska-Grospierre, B., Guy-Grand, D. & Griscelli, C. (1987) Eur. J. Immunol. 17, 1279-1285]. We purified the smaller HML-1 subunit (105 kDa under nonreducing conditions) from hairy-cell leukemia cells and determined the N-terminal amino acid sequence of this chain. The 17 residues determined were identical to the deduced amino acid sequence encoded by an integrin beta 7 cDNA clone [Yuan, Q., Jiang, W.-M., Krissansen, G.W. & Watson, J.D. (1990) Int. Immunol. 2, 1097-1108]. Biochemical analysis of the larger HML-1 subunit (175 kDa under nonreducing conditions) suggested that it was a distinct member of the cleaved group of integrin alpha chains, which we designated alpha E. The beta 7 chain also was associated with the integrin alpha 4 subunit, suggesting that the HML-1 antigen (alpha E beta 7) and alpha 4 beta 7 constitute a beta 7 integrin family on mucosal lymphocytes. Interestingly, regulation of the expression of the HML-1 antigen was reciprocal to that of lymphocyte function-associated molecule 1 in the presence of transforming growth factor beta 1. We suggest that these beta 7 integrins may play a specific role in mucosal localization or adhesion and that the expression of the HML-1 antigen might be regulated by transforming growth factor beta 1 produced at or near epithelial tissues.

Oligoclonality of human intestinal intraepithelial T cells.

T cells bearing the T cell receptor alpha/beta (TCR-alpha/beta) are the predominant lymphocyte population in the human intestinal epithelium. To examine if normal intestinal intraepithelial lymphocytes (IEL) have a TCR repertoire distinct from the TCR-alpha/beta repertoire in peripheral blood lymphocytes (PBL), comparative analysis of relative V beta gene usage in IEL and PBL was performed by quantitative polymerase chain reaction. In each of the six individuals examined, one to three V beta families made up more than 40% of the total V beta transcripts detected in the IEL, whereas there was a more even distribution of V beta gene usage in the paired PBL. The predominant V beta families, especially V beta 1, V beta 2, V beta 3, and V beta 6, were frequently shared among IEL of different individuals. PCR cloning and sequence analysis of the predominant V beta 6 family in two individuals revealed an identical V-D-J-C sequence in 13 of 21 clones obtained from one donor, and a different repeated sequence in 18 of 27 clones examined in the second donor. These data suggest that the V beta skewing in IEL is due to an oligoclonal T cell expansion and may reflect the response of the intestinal mucosal immune system to a restricted set of as yet undefined antigens present in the gut.

Temperature dependent binding of mebendazole to tubulin in benzimidazole-susceptible and -resistant strains of Trichostrongylus colubriformis and Caenorhabditis elegans.

The binding of [3H]mebendazole ([3H]MBZ) to tubulin in benzimidazole-susceptible (BZ-S) and benzimidazole-resistant (BZ-R) strains of Trichostrongylus colubriformis and Caenorhabditis elegans was examined in order to investigate the biochemical changes to tubulin that result in BZ resistance in parasitic and free-living nematodes. In both species the extent of [3H]MBZ binding to tubulin was significantly reduced in the BZ-R strain compared with the BZ-S strain. The decrease in [3H]MBZ binding in the BZ-R strain of each species was the result of a significant reduction in the amount of charcoal stable [3H]MBZ-tubulin complexes and was not related to a change in the association constant of the [3H]MBZ-tubulin interaction. [3H]MBZ binding to tubulin was temperature dependent, reaching maximum levels at 37 degrees C in BZ-S T. colubriformis and 10 degrees C in BZ-R T. colubriformis. Both the BZ-S and BZ-R strains of C. elegans displayed maximum [3H]MBZ binding at 4 degrees C. Resistance ratios derived from the amount of [3H]MBZ binding in the BZ-S and BZ-R strains and in vitro development assays demonstrated that the temperature dependence and extent of drug binding was indicative of BZ resistance status and was species specific in the BZ-S isolates. These results indicate that biochemical differences exist in the binding of benzimidazole carbamates to tubulin in nematode species, and suggest that the susceptibility of the parasitic nematodes to the benzimidazole anthelmintics is the result of a unique high affinity and/or high capacity interaction of benzimidazole carbamates with tubulin.