Effects of toxin A from Clostridium difficile on mast cell activation and survival - PubMed (original) (raw)

Effects of toxin A from Clostridium difficile on mast cell activation and survival

G M Calderón et al. Infect Immun. 1998 Jun.

Abstract

Toxins A and B from Clostridium difficile are the main cause of antibiotic-associated diarrhea and pseudomembranous colitis. They cause fluid accumulation, necrosis, and a strong inflammatory response when inoculated in intestinal loops. Since mast cells are a rich source of inflammatory mediators, abundant in the gut, and known to be involved in C. difficile-induced enteritis, we studied the in vitro effect of toxin A on isolated mast cells. Normal rats sensitized by infection with Nippostrongilus brasiliensis were used to isolate peritoneal mast cells (PMC). PMC from naive rats were stimulated with calcium ionophore A23187 as a model of antigen-independent activation, and PMC from sensitized rats were stimulated with N. brasiliensis antigens to study immunoglobulin E-dependent mast cell activation. After 4 h, toxin A did not induce release of nitric oxide or histamine in naive PMC. However, 10 ng of toxin per ml caused a significant release of tumor necrosis factor alpha (TNF-alpha). In contrast, 1 microg of toxin per ml inhibited antigen or A23187-induced histamine release by PMC. Toxin A at 1 microg/ml for 4 h caused disruption of actin which aggregated in the cytoplasm and around the nucleus. After 24 h, chromatin condensation, cytoplasmic blebbing, and apoptotic-like vesicles were observed; DNA fragmentation was documented also. These results suggest that mast cells may participate in the initial inflammatory response to C. difficile infection by releasing TNF-alpha upon interaction with toxin A. However, longer exposure to toxin A affects the release of inflammatory mediators, perhaps because of the alteration of the cytoskeleton and induction of apoptosis. The impaired functions and survival of mast cells by C. difficile toxin A could hamper the capacity of these cells to counteract the infection, thus prolonging the pathogenic effects of C. difficile toxins.

PubMed Disclaimer

Figures

FIG. 1

FIG. 1

(A) Effects of toxin A (Tox A) on TNFα production by rat PMC. Cells were incubated with toxin A for 4 h, washed, and then cultured for an additional 6 h before the supernatants were assayed for TNF-α or NO. Results are the means ± SE for five experiments. Shown are effects on TNF-α production (∗, P < 0.05 by comparison with cells not treated with toxin A). (B) Effect on NO production. PMC were treated under same conditions as above (_P_ > 0.05 by comparison with cells not treated with toxin A).

FIG. 2

FIG. 2

Effect of C. difficile toxin A on NbAg and on A23187 induction of histamine release by PMC. The cells were incubated for 4 h with different concentrations of toxin A (Tox) and further stimulated with NbAg (Ag; 10 worm equivalents/ml) or A23187 (Ion; 2.5 μM) for 20 min. A significant inhibition (∗, P < 0.01) was observed on mast cells treated with 1 μg of toxin A per ml plus antigen (77%) or toxin A plus A23187 (41%). Results are the mean ± SE of five experiments with duplicates in each experiment.

FIG. 3

FIG. 3

Effect of toxin A on PMC actin microfilaments. (A) Mast cells showed a normal distribution of actin filaments without toxin A treatment. (B) Mast cells treated with 1 μg of toxin A per ml showed cytoskeletal rearrangements with actin-forming dense deposits in the cytoplasm and cytoplasm retraction.

FIG. 4

FIG. 4

TEM and SEM of control PMC (A and B) and PMC treated with 1 μg of toxin A per ml for 4 h (C and D). TEM showed that mast cells exposed to toxin A underwent loss of membrane microvilli and nuclear chromatin condensation (C). With SEM, some cells showed signs of alteration: surface blebbing, loss of rounding and normal granule organization, cell membrane disorganization, and changes in morphology (D).

FIG. 5

FIG. 5

Analysis of DNA fragmentation of PMC treated with 1 μg of toxin A per ml for 4 h. Ten micrograms of DNA was separated in a 1% agarose gel and stained with SYBR green. Lane A, molecular weight marker profile from a 123-bp ladder from Gibco-BRL; lane B, DNA from PMC incubated in RPMI 1640–5% FBS for 24 h; lane C, fragmented genomic DNA from PMC treated with toxin A for 24 h.

Similar articles

Cited by

References

    1. Bartlett, J. G. 1994. Clostridium difficile: history of its role as an enteric pathogen and the current state of knowledge about the organism. Clin. Infect. Dis. 18(Suppl. 4):S265–S272. - PubMed
    1. Befus A D, Pearce F L, Gauldie J, Horsewood P, Bienenstock J. Mucosal mast cells. I. Isolation and functional characteristics of rat intestinal mast cells. J Immunol. 1982;128:2475–2480. - PubMed
    1. Benyon R C, Enciso J A, Befus A D. Analysis of human skin mast cell proteins by two-dimensional gel electrophoresis: identification of tryptase as a siaylated glycoprotein. J Immunol. 1993;151:2699–2706. - PubMed
    1. Bissonnette E Y, Enciso J A, Befus A D. TGF-β1 inhibits the release of histamine and tumor necrosis factor-α from mast cells through an autocrine pathway. Am J Respir Cell Mol Biol. 1997;16:275–282. - PubMed
    1. Bissonnette E Y, Hogaboam C M, Wallace J L, Befus A D. Potentiation of tumor necrosis factor-alpha-mediated cytotoxicity of mast cells by their production of nitric oxide. J Immunol. 1991;147:3060–3065. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources