MEETING BRIEFBy Lloyd Mayer
(Issue 8 · posted May 16, 1997; archived May 30, 1997)There is a growing interest in developing new vaccines that can be orally administered and targeted to the mucosal inductive sites in the gut. To achieve this goal requires a more thorough understanding of the interactions between mucosal immune cells resident in the gut, the gut epithelium, and the microorganisms, both benign and pathogenic, that may be present in the lumen. Recently Wyeth-Lederle Laboratories sponsored a conference to address the issue of cellular and molecular cross-talk at mucosal surfaces. Held March 23-28 in Santa Fe, New Mexico, the conference was chaired by Drs. Marian R. Neutra and Martin F. Kagnoff.
The mucosal immune system has evolved as a distinct arm of the adaptive immune system due to unique demands upon its functional components. Luminal bacteria, viruses, and dietary antigens serve as constant reminders of what exists in the external environment. Immunocompetent cells in this compartment must respond selectively, defending against pathogens but showing restraint against normal constituents. The key to these responses is the recruitment of specialized lymphocytes and the establishment of organized lymphoid tissues to interact with luminal bacteria and viruses. The effector functions, positive or negative, reflect all of the various components talking to each other and creating an appropriate environment.
Recognition and interaction of lymphocytes and antigen-presenting cells with epithelial cells
The mucosal lymphocyte populations are characterized by two quite unusual populations, intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL). LPLs gain access to the lamina propria using a distinct set of homing molecules, MadCAM-1, that binds to its ligand, the integrin alpha-4/beta-7. Peripheral lymphocytes, after activation, frequently express this integrin. One concept is that the gut is a sink for such activated cells since the mucosa serves as the primary site for exposure to pathogens. LPLs are activated lymphocytes whose typical activation pathway has not been clearly defined. The other unique cell population is that of the intraepithelial lymphocytes. In the mouse these cells frequently express the gamma/delta T-cell receptor (TcR) and a percentage of these cells are thought to be derived extrathymically. IELs are notoriously difficult to activate and their antigen reactivity has not been clearly defined. Dr. Michael Brenner of Brigham and Women's Hospital, Boston, has cloned an integrin [alpha-E/beta-7 (HML-1)] expressed on IELs that may aid in our understanding of these cells. The ligand for this integrin appears to be E-cadherin, which is expressed on intestinal epithelial cells and is linked to beta-catenins within the epithelial cell. Alpha-E knockout mice have been generated by Dr. Christina Parker, also of Brigham and Women's Hospital. These animals have reduced numbers of IELs (40% of control littermates) and reduced bowel inflammation in a mouse model of inflammatory bowel disease (the CD45RBhi-SCID transfer model of Powrie). The knockouts are not otherwise abnormal, suggesting that IELs may regulate inflammation but are not the major mediators.
These cells were further characterized in studies by Drs. Mitchell Kronenberg (University of California at Los Angeles) and Wendy L. Havran (Scripps Research Institute). In the former case, IEL subpopulations were defined. TcR- IEL were capable of expressing both Tdt (allowing for N region additions) and RAG-1 (recombination enzyme found in thymic tissue). The presence of these two enzymes supports the possibility that the gut might serve as an extrathymic site for T-cell differentiation. Furthermore, the IEL compartment is the only site where CD4/CD8 double positive cells exist other than the thymus. Transfer of these TcR- cells into SCID mice fails to result in the rearrangement of TcR genes. Potentially other factors are involved.
The IELs also serve to create a positive environment for the epithelial cells. Havran and colleagues have documented that IELs produce fibroblast growth factor 7 (FGF-7), promoting epithelial cell growth. The FGF-7 secreting cells appear to be a subset of intestinal gamma/delta T cells and not peripheral or skin gamma/delta T cells. Dr. Havran has proposed that these factors serve to maintain and repair epithelial surfaces in the GI tract.
Epithelial growth and differentiation is regulated by luminal bacteria as well. Jeffrey Gordon of Washington University School of Medicine, St. Louis, presented data supporting a role for normal bacterial flora to aid in the growth and differentiation scheme of normal enterocytes. Bacteriodes thetaiotamicron induces the fucosylation of enterocytes, which allows for their binding to IEC. The benefit to the enterocyte is unclear, but in the absence of these bacteria, normal epithelial growth patterns are not observed. This and two other models support a role for normal luminal constituents in bowel integrity.
Development and assembly of organized mucosal lymphoid tissues
The theme of epithelial cell-lymphocyte interaction and regulation was further studied in two intriguing model systems. Dr. Tor Savidge of the Babraham Institute, United Kingdom, has established a model of human fetal gut development in SCID mice. Epithelial cell components are regenerated over a several-week period, although human lymphocytes do not necessarily home to this site. This fetal tissue is not vascularized, which is a limitation of the model. It will more likely serve as a model of human epithelial cell differentiation rather than lympho-epithelial interactions. However, antigen added to the lumen can define the interaction of bacteria and enterocytes as described above and will help to define sites of antigen sampling [follicle associated epithelium (M cells) versus epithelium]. A more direct approach to lympho-epithelial interaction was defined by Dr. Eric Pringault of the Institut Pasteur, Paris. He has previously shown that transfer of Peyer's patch (PP) lymphocytes to regions where PPs do not normally exist results in the induction of M cell development. In studies presented here he has shown that this event is cell contact dependent and that B cells may play a role in this process. Thus lymphocytes clearly regulate epithelial cell differentiation.
Roles of epithelial antigen transport and
epithelium-lymphocyte interactions
The direct interaction of epithelial cells and lymphocytes was addressed by Drs. Rich ard Blumberg (Brigham and Women;s Hospital) and Lloyd Mayer (Mount Sinai Medical School, New York). Dr. Blumberg documented the expression of non-classical class I molecules by human IEC. These include CD1d, an intracellular-transporters (TAP) independent, ?/B2 microglobulin independent nonpolymorphic restriction element. Studies by this group have shown that IEL as well as PB T cells can recognize CD1d bearing IECs. Another member of the class Ib family is the neonatal Fc receptor. This molecule binds IgG at low pH (pH 5-6) and may serve as a transporter of antigen in the neonate as well as in the adult. Whether it can also serve as a restriction element remains to be determined. Dr. Mayer's lab has documented that normal IEC can process and present antigen to primed T cells. Interestingly, the T cells proliferating in this co-culture system are antigen nonspecific CD8+ suppressor T cells. They appear to be CD1d restricted, but also appear to require the engagement of the CD8-gamma chain expressed on these cells and the activation of CD8 associated p56lck. Mayer's group has identified a novel glycoprotein, gp180, expressed on normal IEC that not only binds to CD8-gamma but also associates with CD1d. The CD1d:gp180 complex can bind peptide and be recognized by the TcR:CD8 co-receptor complex, allowing for T-cell activation. This selective activation of CD8+ suppressor cells may be partly responsible for local controlled (or physiologic) inflammation as well as some forms of oral tolerance.
Tolerance in mice has been well defined. Dr. Howard Weiner's group (Brigham and Women's Hospital) has been instrumental in defining several of the regulatory parameters involved in the induction of oral tolerance. The type of tolerance induced is dose dependent. High doses of antigen lead to clonal deletion or anergy. Lower doses activate "regulator" T cells. Weiner's group has demonstrated that these regulatory CD4+ cells secrete transforming growth factor beta (TGFB) predominantly with some IL10 and require IL4 for growth. He has coined these cells "Th 3" cells. The secretion of TGFB, a potent immunosuppressive cytokine, may account for the phenomenon of bystander suppression, that is, an antigen-specific induction of response with an antigen-nonspecific effector phase. If one were to use oral tolerance to treat a chronic inflammatory or autoimmune response, it would not require the inciting antigen to be the one that is fed. Rather, if the antigen is in the vicinity of the true autoantigen, it could trigger TGFB-secreting cells that would then nonspecifically inhibit the autoreactive T cells as well. Trials are ongoing in the use of oral tolerance to treat a variety of human autoimmune disorders, but it is too early to tell whether this therapeutic approach will be effective.
Cellular and molecular basis of toxin/adjuvant action in the mucosal system
Some antigens fail to induce tolerance. One example is cholera toxin (CT). This toxin is composed of two subunits, A and B. The A subunit is the active moiety stimulating cAMP production by the enterocyte. The B subunit is the binding moiety. CT has been defined as a potent mucosal adjuvant, stimulating local and systemic antibody responses if coupled to or administered simultaneously with antigen via the oral route. CT binds to both M cells and IEC via GM1 ganglioside. Interestingly, purified recombinant CTB is a mucosal tolerogen and enhances tolerance to antigen if given orally when coupled to or administered simultaneously with antigen. The mechanism of this tolerizing potential has not been defined but it is abrogated by the smallest contamination with the A subunit. Conceivably the A subunit alters the manner whereby antigen is handled or which cells are involved in the process.
Cross-talk between bacterial pathogens and epithelial, lymphoid and antigen-presenting cells
The effect of bacteria and viruses upon the mucosal immune system was also discussed. Several laboratories, including those of Drs. Martin Kagnoff (University of California at San Diego) and Catharina Svanborg (University of Lund, Sweden) have demonstrated that bacterial interaction with epithelial cells (GI and urinary tract epithelium respectively) results in the secretion of proinflammatory cytokines such as IL8, ENA-78, MCP-1, and MIP-1 alpha. These interactions help to promote neutrophil as well as monocyte infiltration into the tissue.
Epithelial cells also secrete stem cell factor (SCF) and IL7, which act on IEL through C-kit and the IL7R respectively. These cytokines promote the growth of gamma/delta IELs especially, but generally all IEL (IL7). C-kit- and IL7-deficient mice have no IEL.
Listeria monocytogenes infection of the epithelium utilizes the E-cadherin pathway. Internalin A, produced by Listeria, binds to E-cadherin and induces PI3 kinase (p85/110). Inhibitors of this kinase (e.g., wartmanin) inhibit Listeria infection. Furthermore, a dominant negative (p85-) decreases invasion by this organism, documenting the use of normal cell surface constituents to aid in bacterial infection.
Interaction of viruses with epithelial cells and the mucosal immune system
Dr. Harry Greenberg of Stanford University Medical School discussed the immune response to rotavirus, a prominent intestinal infection affecting large areas of the globe. The major antibody response is against VP4 (penetration and attachment protein) and VP6 (a structural protein). Antibodies against these two epitopes are protective for reinfection. CD8+ CTLs are required for short-term immunity (viral clearance) but not long-term immunity. CD8-/- and perforin-/- mice clear but more slowly than do wild-type mice. Ig-/- mice clear the infection normally but can be reinfected. The double CD8/Ig knockout develops a persistent infection. Thus a combination of an antiviral cytolytic and antibody-mediated clearance and long-term protection are involved.
Finally, Dr. Marian Neutra of Children's Hospital, Boston, discussed advances in our understanding of M cells or FAE. M cells in contrast to IEC have a minimal glycocalyx. This glycoprotein coat is a series of integral membrane proteins with a negative charge and mucin-like characteristics. This serves as a physical barrier to pathogens. M cells in contrast take up particulate antigens and microorganisms at least 100 times more efficiently than they do soluble proteins. M cells express distinct oligosaccharides, which provide greater access for glycoproteins and glycolipids. Thus many bacteria and viruses target the M cell and result in an active immune response rather than tolerance.
In summary, this symposium stressed the critical interactions between the mucosal immune system, luminal contents and the epithelium. Without effective cross-talk, normal homeostasis may not be maintained.
Lloyd Mayer, M.D. is Professor of Medicine and Microbiology, and Division Chief of Clinical Immunology, at Mount Sinai School of Medicine, New York City. He is also Director of the Immunobiology Center at Mount Sinai.
Endlinks
Mucosal Immunity by Stephen P. James is a basic, introductory presentation comprising lecture notes and slides.
The R&D Systems Web site is a rich source for information about cytokines and antibodies. The site includes a Sourcebook (catalog) containing technical and product information as well as relevant mini-reviews. The Cytokine Bulletin, issued quarterly, has original articles on new developments and applications in cytokine research. Also listed here are links to other Web sites on cytokines and related topics. The Cytokine Database, also from R&D Systems, can be accessed through BioMedNet.
Additional databases of interest to immunological researchers are The Cytokine Family cDNA Database and The Gene Knockout Database.
The DNA Vaccine Web, "A Global Platform for Vaccine Research & Development," explains the development and application of DNA vaccines for treating various infections. The site provides laboratory protocols, governmental guidelines for preclinical testing, links to the full text of issued patents, and more.
Mucosal
Immunology Update is the quarterly official publication of
the Society for Mucosal Immunology. A sample copy of
subscription may be ordered at the journal's Web site. The
journal Immunity may also
have relevant articles.
Several books relating to mucosal immunity have been published recently. Online abstracts are provided for two: