IgA antibodies in rat bile inhibit cholera toxin-induced secretion in ileal loops in situ.

The biological actions of IgA antibodies in bile are largely undefined. We therefore tested whether biliary IgA antibodies could specifically inhibit cholera enterotoxin (CT)-induced secretion in the rat ileum. Rats were immunized by CT given orally or by injection into Peyer's patches. Bile was collected by bile duct cannulation, and anti-CT antibodies in the bile were measured by ELISA. CT plus bile from either immunized or unimmunized rats, or CT plus anti-CT-containing bile which had been absorbed by a CT immunosorbent, were instilled into in situ ileal loops in unimmunized rats; CT alone, or buffer was instilled into other loops. The bile used from the immunized rats contained IgA, but neither IgG nor IgM, anti-CT antibodies. It was found that bile containing IgA anti-CT antibodies almost totally inhibited the secretory effect of CT, and this inhibition was abrogated by absorption of the IgA anti-CT antibodies. Thus, IgA antibodies to an enterotoxin, secreted into bile, are effective against the enterotoxin in the rat intestine in vivo.     

Immunological control mechanism against cholera toxin: interference with toxin binding to intestinal receptors.

The immunological control mechanism against cholera toxin (CT) in the small intestine of rats was studied in vivo. CT binding to intestinal receptors was determined by injected radiolabeled CT into the loops of rat small intestine and subsequently separating purified microvillus membranes from mucosal scrapings of those loops. substantial radioactivity (10(5) cpm/mg of microvillus protein) was present in microvillus fractions of small intestine exposed to 125I-labeled CT compared to radioactivity (10(2) cpm/mg) in fractions from intestine exposed to radiolabeled bovine serum albumin (BSA) used as a control. CT binding to intestinal receptors was significantly inhibited (P less than 0.02) in rats immunized with crude toxin by a combined intraperitoneal and oral method compared to CT binding in animals immunized with BSA or controls, suggesting a specific relationship between intestinal antitoxin and inhibition of binding. Furthermore, ligated ileal loops from CT-immunized animals showed a significant decrease in fluid accumulation when exposed to CT compared to loops from control or BSA-immunized animals, suggesting that antitoxins also interfered with the biological action of CT under conditions of immunization. These studies provide direct evidence that intestinal antitoxins protect against CT-induced diarrhea by interfering with the attachment of the toxin to the intestinal microvillus surface.     

The role of leucocytes in the induction of fluid secretion by Salmonella typhimurium

Nitrogen mustard (N2M) treatment of rabbits induced neutropenia, and, in ligated ileal loops, it inhibited fluid secretion induced by salmonella or by cholera toxin (CT). Pretreatment of rabbits with indomethacin almost abolished salmonella-induced fluid secretion and significantly reduced that induced by CT. Similar effects of N2M and indomethacin on fluid secretion induced by salmonella, but not by CT, have been reported by other workers and used to implicate prostaglandins, from the salmonella-induced inflammation, as mediators of fluid secretion. In contrast, we show that N2M treatment, in addition to reducing CT-induced secretion, caused severe morphological alterations to ileal mucosa. Irradiation techniques were developed for inducing neutropenia, but they did not totally inhibit salmonella-induced leucocyte influx into ileal mucosa. We propose an alternative mechanism for the inhibitory effect of N2M on salmonella- and CT-induced secretion, based on the known anti-mitotic activity of N2M. Also, the anti-secretory effect of indomethacin cannot be attributed uniquely to its anti-inflammatory activity because it depressed CT-induced secretion as well as salmonella-induced secretion. These results support the concept of pathophysiological secretion in infectious diarrhoea, developed previously for rotavirus and extended to bacterial infections.     

Protection of rat intestine against cholera toxin challenge by monoclonal anti-idiotypic antibody immunization via enteral and parenteral routes.

A mouse monoclonal anti-idiotypic (anti-id) immunoglobulin M (IgM) antibody, called MAb2, was raised against a mouse monoclonal anti-cholera toxin (anti-CT) antibody (MAb1). The MAb2 was shown, by competition with CT for MAb1, to bear the internal image of an epitope of CT. MAb2 immunization of rats was performed via the intraperitoneal, intragastric, and intrajejunal routes and compared with immunization of rats with either a control, isotype- and allotype-matched MAb or with CT via the same routes. Both serum IgG and bile IgA anti-CT Ab3's were detected by enzyme-linked immunosorbent assay in anti-id MAb2-immunized rats, although their titers were lower than those in CT-immunized rats. No anti-CT antibodies were detected in sera and bile of rats immunized with the control MAb. When tested for degree of gut protection against a CT challenge, rats immunized with MAb2 by the intrajejunal route showed a rather high degree of protection, which was only slightly lower than that of rats immunized with CT via the same route; all rats but one immunized with the control MAb were unprotected. There was, however, no correlation between serum or bile anti-CT titers and degree of gut protection in MAb2-immunized rats. Their serum anti-CT Ab3's were purified by adsorption and elution from a CT immunosorbent and resembled anti-CT MAb1 in their unique reactivity with MAb2. This constitutes to our knowledge the second report of protection against a pathogen by anti-id immunization via the enteric route.     

Cholera toxin protects against action by Clostridium difficile toxin A. The role of antisecretory factor in intestinal secretion and inflammation in rat

The protein antisecretory factor (AF) inhibits intestinal fluid secretion induced by the cholera toxin (CT) and Clostridium difficile toxin A (CDA). The present work investigated whether CT-induced AF protects against the enterotoxin action by CDA. Rats were pretreated perorally with CT or buffer as control, whereafter CDA-induced fluid secretion and cytotoxicity was tested in vivo in ligated intestinal loops; the mucosal level of AF was estimated using the Western blot technique. Rats given repeated peroral doses of CT became tolerant to CDA, the inhibition of fluid secretion and of cytotoxicity being 79% in eight out of nine animals. The repeated CT-treatment also induced long-lasting rise of AF in the mucosal epithelium. Recombinant AF given either perorally or intravenously inhibited both fluid secretion and cytotoxicity by CDA; similar results were obtained with a truncated 16-mer AF peptide. In conclusion: peroral CT-treatment induced tolerance to CDA in rat small intestine. The tolerance was probably mediated by AF induced via action of cholera toxin on the enteric nervous and immune system.     

Potentiating effect of bile on enterotoxin-induced diarrhea.

The influence of bile acids on adenosine 3',5'-phosphate-induced intestinal secretion was studied in mice. Bile flow was stopped by ligation of the common bile duct, and secretion was induced in ligated loops of the small intestine. The decrease of bile led to inhibition of hypersecretion after challenge with heat-labile enterotoxins from Vibrio cholerae and Escherichia coli, as well as with prostaglandin E1. In contrast, the fluid response induced by dibutyryl-adenosine 3',5'-phosphate was unaffected by intestinal bile. Injection of bile or bile acids into intestinal loops before cholera toxin challenge restored the toxin-induced secretion in the bile-depleted intestine. At the subcellular level the decrease of intestinal bile led to inhibition of cholera toxin-activated adenylate cyclase, whereas the bile concentration did not influence the binding of 125I-labeled toxin to the intestinal epithelial cells. The results suggest that intestinal bile interacts with adenylate cyclase in the induction of fluid secretion by enterotoxins and prostaglandin E1.     

Bile and milk from cholera toxin treated rats contain a hormone-like factor which inhibits diarrhea induced by the toxin

Protection against intestinal secretion induced by cholera toxin (CT) was studied in adult and suckling rats. Peroral CT treatment of lactating females protected their suckling offspring against diarrhea. This milk contained a protective antisecretory factor (ASF) as shown by passive peroral or intravenous transfer of the milk to adult, nontreated recipients in which the cholera response was tested in ligated jejunal loops. Bile from CT-treated rats also contained ASF, which was still present 100 days after the last treatment. Although milk and bile from untreated rats contained no ASF, bile from very old untreated rats did. Chemical characterization showed that ASF in bile and milk both had isoelectric points of about 5.0 and molecular weights of about 25,000. These data are similar to those previously obtained for ASF isolated from porcine pituitary glands, and show that the antisecretory activity is unrelated to immunoglobulins.     

Intestinal resistance to cholera toxin in mouse. Antitoxic antibodies and desensitization of adenylate cyclase

Mice were immunized perorally with cholera toxin (CT), cholera B-subunit (CB), or buffer as control. The response of anti-CT antibodies of the IgG, IgA and IgM class in bile, IgA being predominating, were similar in both immunized groups. The same number of anti-CT containing plasma cells (ACC) were determined in the intestinal lamina propria of CT - as well as of CB-immunized mice 20 days after the last immunization, while ACC at day 4 in the CB group were 50% higher than in the CT group. In contrast to the vigorous antibody response to CT in both groups of immunized mice, only animals immunized with CT displayed resistance to CT-induced intestinal hypersecretion and to CT stimulation of adenylate cyclase. The CB-treated group responded to CT with fluid accumulation and enzyme activation similar to controls. The results suggest that intestinal resistance to CT in mouse is due to desensitization of adenylate cyclase rather than to CT-neutralizing antibodies.     

Relevance of Biliary IgA Antibodies in Rat Intestinal Immunity

Two intraperitoneal administrations of foreign red cells in Freund's complete adjuvant, or two intragastric intubations of erythrocytes, given to rats at 15 days interval, both elicit the appearance of specific antibodies in bile and serum. When bile was compared to serum, the selective predominance of IgA antibodies in this secretion was observed for both the intraperitoneally and intragastrically immunized groups, being more pronounced for the orally immunized group when related to IgG or IgM antibodies. The IgA content of upper intestinal washings was roughly ten-fold smaller in rats with bile duct cannulation than in sham-operated controls. Altogether, the data demonstrate that bile IgA may significantly contribute to the secretory IgA system of the gut.     

Role of cyclooxygenase enzymes in a murine model of experimental cholera

Nonsteroidal anti-inflammatory drugs (e.g., indomethacin) inhibit and reduce the fluid secretion responses elicited by cholera toxin (CT), but it has not been conclusively determined which cyclooxygenase (COX) isoform is involved in CT's action. This study evaluated the role of the COX enzymes and their arachidonic acid metabolites in experimental cholera. Swiss-Webster mice were dosed with celecoxib and rofecoxib and challenged with CT in ligated small intestinal loops, and intestinal segments from mice deficient in COX-1 and COX-2 were challenged with CT. The effects of CT on fluid accumulation, prostaglandin E(2) production, mucosal tissue injury, and markers of oxidative stress were measured. Celecoxib and rofecoxib given at 160 micro g per mouse inhibited CT-induced fluid accumulation by 48% and 31%, respectively, but there was no significant difference among cox-1(-/-) and cox-2(-/-) mice in response to CT compared to wild-type controls. CT elevated tissue levels of oxidized glutathione and lipid peroxides and elicited small intestinal tissue injury in two of five cox-1(-/-) and four of five cox-2(-/-) mice. A role for COX-2 in CT's mechanism of action has previously been suggested by the effectiveness of COX-2 inhibitors in reducing CT-induced fluid secretion, but CT challenge of COX-1 and COX-2 knockout mice did not corroborate the pharmacological data. The results of this study show that CT induced oxidative stress in COX-deficient mice and suggest a tissue-protective role for arachidonic acid metabolites in the small intestine against oxidative stress.     

Further evidence that hepatic sources confer biliary antibody in the rat.

The notion that bile-dedicated antibody is made within the liver by migratory antibody-forming cells (AFC) was examined further in rats. Livers from immunized animals were removed to perfusion in isolation so that plasma influences on bile antibody would be obviated. Antibody was secreted for at least 5 hr by the livers of rats that had received intravenous (i.v.) or intra-Peyer's patch (IPP) immunization with horse erythrocytes. After initially declining, the titres stabilized at 5-8% of the starting value for IPP-immunized rats and at 0.8% for i.v.-immunized animals, levels that were then sustained. In other experiments, the biliary antibody output was measured in immunized rats in the period immediately following splenectomy, an expedient that would deny the liver any newly formed AFC. Splenectomy during spleen-based, IgM antibody responses led to bile titres falling, over about 12 hr, to 21% of initial values. This level was then maintained for at least another 12 hr. Serum titres over this period remained static. Lastly, the bile ducts of immunized rats were ligated to test whether locally made antibody that was destined for bile could be forced instead to reflux to blood. Biliary obstruction during IgM responses to horse erythrocytes and pneumococcal polysaccharide, type 3, was found to raise significantly serum antibody titres. For pneumococcal polysaccharide, the serum response was also noticeably prolonged. These findings are consistent with the biliary antibody of immunized rats being constituted, in part, from local sources and not from plasma alone.     

Pathogenic properties of Campylobacter jejuni: assay and correlation with clinical manifestations.

The pathogenic properties of 20 strains of Campylobacter jejuni isolated from persons with clearly defined clinical manifestations were determined. Cell-free broth filtrates were examined for (i) enterotoxin production by Chinese hamster tissue culture assay and an enzyme-linked immunosorbent assay (ELISA) employing GM1 ganglioside and affinity-purified antiserum to Escherichia coli heat-labile toxin, (ii) cytotoxin production by Vero and HeLa cell tissue culture lines, and (iii) their ability to cause fluid secretion in rat ligated ileal loops. Viable bacteria were examined for invasive properties by an ELISA with the immunoglobulin fraction of antiserum to Formalin-killed bacteria of an invasive strain, and by their effect on fluid secretion and morphology in rat ligated ileal loops. None of the eight isolates obtained from asymptomatic carriers had any detectable pathogenic properties. All six strains isolated from persons with bloody invasive-type diarrhea elaborated a cytotoxin; their viable bacteria had high titers in the ELISA for invasive properties and caused fluid secretion in ligated ileal loops, although consistent morphologic abnormalities and evidence of mucosal invasion, examined by immunofluorescence techniques, were not detected. All six strains isolated from persons with watery secretory-type diarrhea produced an enterotoxin, one elaborated a cytotoxin, and broth filtrates of all strains caused fluid secretion in ligated ileal loops; viable bacteria had low titers in the ELISA for invasive properties and evoked fluid secretion in ligated loops by means of enterotoxin production. These observations show (i) that a correlation exists between the pathogenic properties of the infective C. jejuni strain and gastrointestinal manifestations in the infected host, and (ii) that these pathogenic properties can be identified by in vitro assays, including ELISAs.     

Biliary lipid secretion in the regenerating liver of the rat.

The effect of two-thirds hepatectomy on biliary lipid secretion was studied in fasted rats. Bile acids, cholesterol and phospholipids were determined in the bile of rats with biliary drainage. Bile acid and phospholipid concentrations were not significantly modified during liver regeneration but there was a significant decrease in cholesterol concentrations in bile at 12 h after hepatectomy, with a gradual return to normal values. The molar percentage of cholesterol and the calculated lithogenic index of bile were also significantly decreased by 12 h. The biliary secretion of bile acids and phospholipids, expressed per gram of liver, showed a tendency to increase regeneration, but cholesterol secretion was significantly decreased from 12 to 48 h following liver resection. Some possibilities to explain the uncoupling between cholesterol secretion and bile acid secretion are discussed.     

Biliary lipid secretion in the regenerating liver of the rat

The effect of two-thirds hepatectomy on biliary lipid secretion was studied in fasted rats. Bile acids, cholesterol and phospholipids were determined in the bile of rats with biliary drainage. Bile acid and phospholipid concentrations were not significantly modified during liver regeneration but there was a significant decrease in cholesterol concentrations in bile at 12 h after hepatectomy, with a gradual return to normal values. The molar percentage of cholesterol and the calculated lithogenic index of bile were also significantly decreased by 12 h. The biliary secretion of bile acids and phospholipids, expressed per gram of liver, showed a tendency to increase regeneration, but cholesterol secretion was significantly decreased from 12 to 48 h following liver resection. Some possibilities to explain the uncoupling between cholesterol secretion and bile acid secretion are discussed.     

Effects of the antisecretory factor in pigs

The effect of the antisecretory factor (ASF) on experimental porcine enterotoxin-induced jejunal secretion was tested. The heat-labile enterotoxin (LT) fromEscherichia coli and cholera toxin (CT) was used for challenge in ligated intestinal loops. Less than 10 units of ASF inhibited the LT-induced secretion, while that due to CT required more than 10 units of ASF. ASF was effective only when administered prior to toxin challenge, and could be given either intravenously or intra-intestinally. Mixing of ASF with specific anti-ASF antibodies prior to injection abolished its antisecretory effect. LT- and CT-induced secreted fluid contained equal concentrations of Na⁺, K⁺ and Cl^–, and the ionic concentration was not affected by ASF. Less than 0.1 units of ASF per pituitary gland was present in 3- and 5-week old pigs, while it increased to 4.5 units in 28-week old animals, and to 12.2 units in pigs older than two years. However, after intra-intestinal vaccination with 2.0 mg CT, the pituitary ASF content in the 5-week old animals increased to 2.0 units within 24h.Abbreviations used ASF antisecretory factor - CT cholera toxin - LT the heat-labile enterotoxin fromEscherichia coli     

Protection in rabbits immunized with a vaccine of Escherichia coli heat-stable toxin cross-linked to the heat-labile toxin B subunit.

Rabbits and rats were immunized with a vaccine consisting of synthetically produced Escherichia coli heat-stable toxin cross-linked by the carbodiimide reaction to the B subunit of biologically produced porcine heat-labile toxin. The vaccine contained 50% of each toxin component by weight and antigenicity; the toxicity of the heat-stable enterotoxin component was reduced by greater than 600-fold. Two or three peroral immunizations with vaccine containing 1,000 antigen units of each component raised greater-than-threefold increases in specific mucosal immunoglobulin A antitoxin titers to each component in all animal groups. Protection index values for challenge with either heat-labile or heat-stable toxins in ligated ileal loops were 3.4 to 4.0 in rats immunized by a parenteral primary immunization followed by two peroral booster immunizations, greater than 9 in rabbits immunized by these routes, and greater than 8 in rabbits given just three peroral immunizations. The antigenicity of the B-subunit component of the peroral vaccine was protected equally well against gastric acidity either by pretreatment with cimetidine or by delivery of the vaccine encapsulated in pH-dependent microspheres. The vaccine did not cause diarrhea when given perorally to any of the experimental animals or evoke fluid secretion when instilled into rabbit ligated ileal loops. These observations (i) confirm the effectiveness of this vaccine as an immunogen in a second animal model, (ii) establish that it is effective when given exclusively by the peroral route, and (iii) provide further evidence regarding its lack of toxicity.     

Host defense against cholera toxin is strongly CD4+ T cell dependent.

This study investigates the role of CD4+ T cells in host defense against cholera enterotoxin-induced diarrhea. Antitoxin immunoglobulin A formation and gut protection against cholera toxin (CT) following oral immunizations with CT were evaluated in normal mice and mice that had been depleted of CD4+ T cells by in vivo treatment with specific anti-CD4 monoclonal antibodies. Flow cytometer analysis demonstrated that anti-CD4 monoclonal antibody effectively eliminated CD4+ T cells in the spleen, mesenteric lymph nodes, and Peyer's patches. In contrast, lamina propria lymphocytes demonstrated only some decrease in CD4+ T-cell numbers following antibody treatment. However, CD4 expression of individual lamina propria lymphocytes was strongly down-regulated. Depletion of CD4+ T cells performed prior to oral immunization with CT completely inhibited the ability to respond to CT. No antitoxin production, as detected at the single-cell level by the ELISPOT technique, was found in the spleen, mesenteric lymph nodes, or Peyer's patches, nor did we observe serum antitoxin responses in these mice. Control mice demonstrated strong antitoxin responses in all locations following oral immunization with CT. Anti-CD4 antibody treatment also effectively inhibited the antitoxin immunoglobulin A response in the lamina propria to CT as well as blocked the ability to develop gut protection against CT challenge of ligated intestinal loops after oral CT immunization. Thus, in vivo CD4+ T-cell depletion rendered these mice unable to develop protective immunity in the gut following oral immunization with CT. Moreover, CD4+ T-cell depletion effectively inhibited the antitoxin immune response in the gut lamina propria, mesenteric lymph nodes, Peyer's patches, and spleen when performed prior to both priming and booster immunizations with CT. This study clearly demonstrates the requirement of functional CD4+ T cells in the gut immune system for the development of host defense against CT-induced disease. Our data also reinforce the concept of a strong association between gut protection against CT and local production of neutralizing immunoglobulin A antitoxin.     

Immune response of the female rat genital tract after oral and local immunization with keyhole limpet hemocyanin conjugated to the cholera toxin B subunit.

The immune response of the female rat genital tract was evaluated with Lewis rats given primary and secondary immunizations with keyhole limpet hemocyanin (KLH) alone or coupled to the cholera toxin (CT) B subunit (CTB) by the oral or intravaginal-uterine route or a combination of routes. CT (2 to 5 micrograms) was administered as an adjuvant with the KLH-CTB conjugate. While a significant mucosal immunoglobulin A (IgA) response was induced by KLH, there were no significant differences among the immunized groups in the levels of IgA antibodies in salivary gland, gut, vaginal, and uterine secretions, with the exception that rats immunized only orally with the KLH-CTB conjugate lacked a detectable vaginal response. Levels of IgA antibodies to CT, however, were significantly increased in genital tract secretions of rats immunized locally versus orally with the KLH-CTB conjugate. Antibody activity of the IgG isotype against both KLH and CT was significantly elevated in genital tract secretions of rats immunized with KLH-CTB by the oral or intravaginal-uterine route and given genital tract boosters, in comparison with the results for the other groups. IgM antibody titers were generally negligible in the different secretions. An enzyme-linked spot-forming assay revealed IgA and IgG antibody-secreting cells in salivary gland and uterine tissues. A highly significant correlation between the numbers of antibody-secreting cells and antibody titers existed for uterine IgG but not IgA responses to KLH among the different groups of rats. In conclusion, a vigorous local immune response was induced after immunization of the female rat reproductive tract alone or in combination with peroral challenge with the KLH-CTB conjugate.     

Cholera toxin and Salmonella typhimurium induce different cytokine profiles in the gastrointestinal tract.

Salmonella infection of the gastrointestinal tract (GT) results in fluid secretion and inflammation. In contrast, cholera toxin (CT) induces fluid secretion but no inflammation. Using a murine ligated intestinal loop model, we investigated cytokine production (interleukin-1 [IL-1], IL-2, IL-4, IL-6, IL-10, gamma interferon, and tumor necrosis factor alpha) in the GT following exposure to these agents. Salmonella typhimurium induced a Th1-like cytokine profile in loops obtained from either nonimmune mice or Salmonella-immunized mice. CT induced only IL-6 and IL-10 production in ligated loops from nonimmune mice but induced a Th2-like cytokine profile in ligated loops obtained from CT-immunized mice. These results show that CT and S. typhimurium induce very different cytokine profiles in the GT.     

Protection against Cholera Toxin after Oral Immunization is Thymus-Dependent and Associated with Intestinal Production of Neutralizing IgA Antitoxin

We studied whether gut mucosal IgA antitoxin production as well as the acquired protection against cholera toxin (CT) after oral immunization with CT are both thymus-dependent immune manifestations. In contrast to normal BALB/c mice, nude, athymic mice did not respond to oral immunizations with CT with either IgA antitoxin-producing cells (SFC) in the lamina propria or protection against challenge with CT in ligated intestinal loops. However, when nude mice were first reconstituted by grafting of syngeneic thymus glands, both IgA antitoxin SFC in the lamina propria and protection were stimulated by oral immunizations with CT and the response were of similar magnitude to those of normal mice after immunizations. During in vitro culture, isolated lamina propria lymphocytes from immunized but not from control mice concomitantly and proportionally produced IgA antitoxin and CT-neutralizing activity. We conclude that intestinal antitoxin formation and protection against toxin challenge after oral immunization with CT are both critically thymus-dependent and therefore likely to be under T-cell control.