Macrophage-dependent stimulation of T cell-depleted spleen cells by Clostridium difficile toxin A and calcium ionophore

Miller, P.David; Pothoulakis, Charalabos; Baeker, Thomas R.; LaMont, J. Thomas; Rothstein, Thomas L.

doi:10.1016/0008-8749(90)90308-e

Cited by 37 publications

(23 citation statements)

References 30 publications

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“…Since pertussis toxin inactivates guanine nucleotide-binding proteins, these findings suggest that toxin A stimulation of neutrophils is mediated via a G proteindependent mechanism (10). We have also shown that toxin A is chemotactic to human neutrophils (9) and stimulates cytokine release from macrophages in vitro ( 11). These findings lead us to hypothesize that the ability of toxin A to directly activate inflammatory cells may underlie its potent proinflammatory effects in vivo.…”

Section: Introductionmentioning

confidence: 69%

Neutrophil recruitment in Clostridium difficile toxin A enteritis in the rabbit.

et al. 1994

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Neutrophil infiltration is a prominent feature of Clostridium difficile-associated enteritis and colitis. The aim of this study was to examine the importance of neutrophil recruitment and neutrophil-mediated tissue damage in C. difficile toxin A-induced enteritis. Competitive binding experiments using purified 3H-toxin A demonstrated the presence of a single class of medium affinity receptors on rabbit neutrophils (Kd 7 X 10-8 M). Pertussis toxin and the nonhydrolyzable GTP analog GTPgamma S both inhibited 3H-toxin A binding (by 56 and 65%, respectively), indicating that the rabbit neutrophil toxin A receptor is G protein linked. Toxin A elicited a dose-dependent (25-200 gg/ml) stimulation of neutrophil migration in vitro, and this functional effect was also pertussis toxin sensitive (69% inhibition). Treatment of neutrophils with R15.7, a blocking monoclonal antibody to the leukocyte adhesion molecule CD18, inhibited toxin A-stimulated neutrophil migration by 85% in vitro. Pretreatment of rabbits with R15.7 also prevented neutrophil infiltration of toxin A-exposed ileal loops in vivo as determined by histologic examination and by ileal tissue myeloperoxidase levels. Furthermore, R15.7 effected a substantial inhibition of fluid secretion (by 65%), mannitol permeability (by 66%), and histologic damage in toxin A-exposed ileal loops. Anti-CD18 (R15.7) had no inhibitory effect on cholera toxin enterotoxicity. These data demonstrate that C. difficile toxin A is a proinflammatory toxin whose enterotoxic effects are substantially dependent upon neutrophil recruitment. (J.

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Section: Introductionmentioning

confidence: 69%

Neutrophil recruitment in Clostridium difficile toxin A enteritis in the rabbit.

et al. 1994

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“…When exposed to toxin A these cells express an immediate pertussis toxin-sensitive calcium transient and exhibit chemotaxis (17). Another example of cellular activation by toxin A occurs in mouse spleen lymphocytes which undergo proliferation when exposed to toxin A and a calcium ionophore (18). In contrast to these activation events, exposure ofintestinal epithelial cells to toxin A in vivo (6)(7)(8) and in vitro (15,43), and exposure of cultured Tg4 colon cancer cells to toxin A (16) results in cellular toxicity as manifested by necrosis, increased paracellular permeability, and inhibition of protein synthesis.…”

Section: Resultsmentioning

confidence: 99%

“…For example, we have reported that toxin A induces chemotaxis and a pertussis toxin-sensitive cytosolic calcium transient in human neutrophils (17), and elicits a proliferative response in mouse spleen lymphocytes (18). These observations suggest that toxin A binding to membrane receptors on inflammatory cells activates signal transduction pathways which may exert cellular effects independent of the catalytic action of the toxin.…”

mentioning

confidence: 99%

Characterization of rabbit ileal receptors for Clostridium difficile toxin A. Evidence for a receptor-coupled G protein.

et al. 1991

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The purpose of this study was to characterize the surface receptor for toxin A, the enterotoxin from Clostridium difficik, on rabbit intestinal brush borders (BB) and on rat basophilic leukemia (RBL) cells. Purified toxin A was radiolabeled using a modified Bolton-Hunter method to sp act 2 tCi/4g, with retention of full biologic activity. 3H-Toxin A bound specifically to a single class of receptors on rabbit BB and on RBL cells with dissociation constants of 5.4 X 10-8 and 3.5 X 10' M, respectively. RBL cells were highly sensitive to toxin A (cell rounding) and had 180,000 specific binding sites per cell, whereas IMR-90 fibroblasts were far less sensitive to toxin A and lacked detectable specific binding sites. Exposure of BB to trypsin or chymotrypsin significantly reduced 3H-toxin A specific binding. Preincubation of BB with Bandeirea simplicifoia (BS-1) lectin also reduced specific binding, and CHAPS-solubilized receptors could be immobilized with WGA-agarose. The addition of 100 nM toxin A accelerated the association of 35SGTP-yS with rabbit ileal BB, and preincubation of BB with the GTP analogues GTP'yS or Gpp(NH)p, significantly reduced 3H-toxin A specific binding. Our data indicate that the membrane receptor for toxin A is a galactose and N-acetyl-glucosamine-containing glycoprotein which appears to be coupled to a G protein.

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“…Direct toxicity occurs when cultured cells are exposed to toxins A and B, and presumably occurs via toxin binding, internalization (17) and disruption of the cytoskeleton with eventual inhibition of protein synthesis (18) and cell division. Part of the intestinal effects of toxin A described in ileal loop studies results from indirect activation of lamina propria neuroimmune cells (23,24,(32)(33)(34)(35) and infiltration of the ileal submucosa by neutrophils (23,24,35,36). In the experiments reported here we assume that the immediate onset of electrophysiological changes after luminal toxin exposure suggests direct damage, although we can not exclude the release of cytokines, prostanoids or other pro-inflammatory mediators from lamina propria immune cells present in these mucosal strips.…”

Section: Discussionmentioning

confidence: 99%

Clostridium difficile toxin B is more potent than toxin A in damaging human colonic epithelium in vitro.

Riegler¹,

Sedivy²,

Pothoulakis³

et al. 1995

J. Clin. Invest.

312

207

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Permeability to [3H]mannitol increased 16-fold after exposure to 32 nM of toxin A and to 3 nM of toxin B when compared with controls (P < 0.05). Light and scanning electron microscopy after exposure to either toxin revealed patchy damage and exfoliation of superficial epithelial cells, while crypt epithelium remained intact. Fluorescent microscopy of phalloidin-stained sections showed that both toxins caused disruption and condensation of cellular F-actin. Our results demonstrate that the human colon is -10 times more sensitive to the damaging effects of toxin B than toxin A, suggesting that toxin B may be more important than toxin A in the pathogenesis of C. difficUe colitis in man. (J.

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Macrophage-dependent stimulation of T cell-depleted spleen cells by Clostridium difficile toxin A and calcium ionophore

Cited by 37 publications

References 30 publications

Neutrophil recruitment in Clostridium difficile toxin A enteritis in the rabbit.

Neutrophil recruitment in Clostridium difficile toxin A enteritis in the rabbit.

Characterization of rabbit ileal receptors for Clostridium difficile toxin A. Evidence for a receptor-coupled G protein.

Clostridium difficile toxin B is more potent than toxin A in damaging human colonic epithelium in vitro.

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