Initiation of adaptive mucosal immunity occurs in organized mucosal lymphoid tissues such as Peyer’s patches of the small intestine. Mucosal lymphoid follicles are covered by a specialized follicle-associated epithelium (FAE) that contains M cells, which mediate uptake and transepithelial transport of luminal Ags. FAE cells also produce chemokines that attract Ag-presenting dendritic cells (DCs). TLRs link innate and adaptive immunity, but their possible role in regulating FAE functions is unknown. We show that TLR2 is expressed in both FAE and villus epithelium, but TLR2 activation by peptidoglycan or Pam3Cys injected into the intestinal lumen of mice resulted in receptor redistribution in the FAE only. TLR2 activation enhanced transepithelial transport of microparticles by M cells in a dose-dependent manner. Furthermore, TLR2 activation induced the matrix metalloproteinase-dependent migration of subepithelial DCs into the FAE, but not into villus epithelium of wild-type and TLR4-deficient mice. These responses were not observed in TLR2-deficient mice. Thus, the FAE of Peyer’s patches responds to TLR2 ligands in a manner that is distinct from the villus epithelium. Intraluminal LPS, a TLR4 ligand, also enhanced microparticle uptake by the FAE and induced DC migration into the FAE, suggesting that other TLRs may modulate FAE functions. We conclude that TLR-mediated signals regulate the gatekeeping functions of the FAE to promote Ag capture by DCs in organized mucosal lymphoid tissues.
Epithelial cells of the respiratory and gastrointestinal tracts are extremely vulnerable to the cytotoxic effects of ricin, a Shiga-like toxin with ribosome-inactivating properties. While mucosal immunity to ricin correlates with secretory immunoglobulin A (IgA) antibody levels in vivo, the potential of IgA to protect epithelial cells from ricin in vitro has not been examined due to the unavailability of well-defined antitoxin IgA antibodies. Here we report the characterization of four monoclonal IgA antibodies (IgA MAbs) produced from the Peyer's patches and mesenteric lymph nodes of BALB/c mice immunized intragastrically with ricin toxoid. Two IgA MAbs (33G2 and 35H6) were active against ricin's lectin subunit (RTB), and two (23D7 and 25A4) reacted with the toxin's enzymatic subunit (RTA). All four IgA MAbs neutralized ricin in a Vero cell cytotoxicity assay, blocked toxin-induced interleukin-8 release by the human monocyte/macrophage cell line 28SC, and protected polarized epithelial cell monolayers from ricin-mediated protein synthesis inhibition. 33G2 and 35H6 reduced ricin binding to the luminal surfaces of human intestinal epithelial cells to undetectable levels in tissue section overlay assays, whereas 23D7 had no effect on toxin attachment. 23D7 and 25A4 did, however, reduce ricin transcytosis across MDCK II cell monolayers, possibly by interfering with intracellular toxin transport. We conclude that IgA antibodies against RTA and RTB can protect mucosal epithelial cells from ricin intoxication.Recent bioterrorism incidents in the United States and abroad have alerted public health officials to the need for vaccines and therapies against pathogens and toxins previously deemed to be of little concern (2, 7, 27). Ricin, for example, is an extremely potent toxin that is easily purified in high concentrations from its natural source, the castor bean (Ricinus communis). Ricin (molecular weight, 64,000) is a relatively simple toxin consisting of an enzymatic A subunit (RTA) and a binding B subunit (RTB) joined by a disulfide bond (32). Its potency is attributed in part to the fact that RTB is a bivalent lectin with specificities for ubiquitous glycoproteins and glycolipids containing (1-3)-linked galactose or N-acetylgalactosamine residues (4), which enable the toxin to bind and be internalized by all known cell types. The toxic properties of ricin are further compounded by RTA, which is an extremely efficient N-glycosidase specific for a highly conserved adenine residue in the so-called sarcin/ricin loop of eukaryotic 28S rRNA (9). Due to the absence of a specific vaccine or therapy, treatment of individuals intoxicated with ricin is strictly supportive (3).As an agent of bioterrorism, it is feared that ricin will be disseminated by aerosol or food/water supplies, thereby potentially exposing humans via the respiratory or gastrointestinal tract. It is known from animal studies that both mucosal compartments are susceptible to ricin intoxication. Monkeys or rodents exposed to aerosolized ricin (ϳ20 to 40 g/k...
Secretory IgA (sIgA) Abs are polymeric Igs comprised of two or more IgA monomers joined together at their C termini and covalently associated with a 70-kDa glycoprotein called secretory component. As the predominant Ig type in gastrointestinal sections, sIgA Abs are centrally important in adaptive immunity to enteropathogenic bacteria, viruses, and toxins. In this study, we demonstrate that sIgA Abs may also function in innate defense against ricin, a naturally occurring, galactose-specific plant lectin with extremely potent shiga toxin-like enzymatic activity. In lectin blot overlay assays, we found that ricin bound to secretory component and the H chain of human IgA, and this binding was inhibited by the addition of excess galactose. The toxin also recognized IgM (albeit with less affinity than to IgA), but not IgG. Ricin bound to both human IgA1 and IgA2, primarily via N-linked oligosaccharide side chains. At 100-fold molar excess concentration, sIgA (but not IgG) Abs inhibited ricin attachment to the apical surfaces of polarized intestinal epithelial cells grown in culture. sIgA Abs also visibly reduced toxin binding to the luminal surfaces of human duodenum in tissue section overlay assays. We conclude that sIgA Abs in mucosal secretions may serve as receptor analogues for ricin, thereby reducing the effective dose of toxin capable of gaining access to glycolipid and glycoprotein receptors on epithelial cell surfaces.
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