Immunoglobulin A Antibodies against Ricin A and B Subunits Protect Epithelial Cells from Ricin Intoxication

Abstract

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...

Evidence for Widespread Epithelial Damage and Coincident Production of Monocyte Chemotactic Protein 1 in a Murine Model of Intestinal Ricin Intoxication

“…Nonetheless, even small amounts of toxin can have detrimental consequences to intestinal epithelial cells. Application of ricin to the apical surfaces of polarized epithelial cell monolayers grown in vitro results in rapid toxin internalization, transcytosis, and arrest in protein synthesis (29,42).…”

“…Serum and fecal pellets were collected as described previously (28) 2 days before the first immunization and 7 days after each immunization. The antiricin IgA and IgG antibody titers in serum and the antiricin IgA antibody titers in fecal pellets were determined by enzyme-linked immunosorbent assay (ELISA), as done previously (29). Briefly, NUNC Maxisorb F96 microtiter plates (Krackeler Scientific, Albany, NY) were coated overnight at 4°C with 0.1 g of ricin (or RTA or RTB) per well in a volume of 0.1 ml in PBS (pH 7.4).…”

“…ELISA plates were developed with a one-component TMB colorimetric substrate (Kirkegaard and Perry, Gaithersburg, MD) and were read using a SpectraMax 250 microtiter plate reader equipped with Softmax software (Molecular Devices, Union City, CA). Mouse monoclonal IgGs and IgAs specific for RTA or RTB were used as controls for these assays (29,30).…”

A Neutralizing Antibody to the A Chain of Abrin Inhibits Abrin Toxicity both In Vitro and In Vivo

“…Several neutralizing antibodies for biological toxins have been described earlier (5,20,24,39). Antibodies to the galactose binding domain of the B chain have been shown to inhibit ricin binding to cells, thereby inhibiting toxin activity (19,20). Antibodies to the A chain of ricin also protect cells effectively from ricin toxicity (18).…”

“…The passive administration of antibodies has proven to be a specific and effective mode of defense against poisoning by various biological toxins (29). Although both anti-A chain and anti-B chain antibodies are able to neutralize toxins in vitro and in vivo, antibodies against the A chain of ricin have better protective efficacy than anti-B chain antibodies (14,19).…”