Dec 29, 1986 - Brown, J. E., D. E. Griffin, S. W. Rothman, and B. P. Doctor. 1982. Purification and biological ... Heinegard, A. Lundblad, and S. Svensson (ed.), Proceedings of .... Vicari, G., A. L. Olitzki, and Z. Olitzki. 1960. The action of the.
INFECTION AND IMMUNITY, June 1987, p. 1533-1535 0019-9567/87/061533-03$02.00/0 Copyright C 1987, American Society for Microbiology
Vol. 55, No. 6
Cytotoxicity of Shiga Toxin for Primary Cultures of Human Colonic and Ileal Epithelial Cells MARY PAT MOYER,'* PATRICIA S. DIXON,' SARA W. ROTHMAN,2 AND J. EDWARD BROWN2 Department of Surgery, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284-7842,1 and Department of Biological Chemistry, Division of Biochemistry, Walter Reed Army Institite of Research, Washington, D.C. 20307-51002 Received 29 December 1986/Accepted 24 February 1987
Shiga toxin purified from Shigella dysenteriae 1 was cytotoxic to cultured epithelial cells from human colon and ileum. The cytotoxicity, which affected only about 50% of treated cells, was neutralized by rabbit antiserum monospecific for Shiga toxin and mediated by protein synthesis inhibition.
Shigella dysenteriae 1 produces Shiga toxin, a protein toxin which is lethal to mice and rabbits (7), is cytotoxic for certain epithelial cells (24), and causes accumulation of fluid in ligated segments of rabbit ileum (13). Shiga toxin has been purified to homogeneity (2, 20, 22). It is composed of two types of subunits designated A and B. The A subunit (Mr, 31,500) inhibits protein synthesis. The B subunit (Mr, 7000), present in multiple copies in holotoxin, is believed to facilitate binding to susceptible cells (22). In susceptible cells in vitro, the toxin inhibits protein synthesis (4) by enzymatic inactivation of the 60S ribosomal subunit (23), leading to an inhibition of aminoacyl tRNA binding of peptide elongation (3a, 4, 5; T. G. Obrig, T. P. Moran, and J. E. Brown, submitted for publication). The role of Shiga toxin in the pathophysiology of disease has not been delineated. The presence of a similar cytotoxin in Escherichia coli strains associated with hemorrhagic colitis (A. D. O'Brien, T. A. Lively, M. E. Chen, S. W. Rothman, and S. B. Formal, Letter, Lancet i:702, 1983), infant diarrhea (19), and hemolytic uremic syndrome (12) has drawn additional attention to the role of Shiga toxin in disease (summarized in reference 6). Various cell lines have been tested for their susceptibility to Shiga toxin (8, 11, 24). Several strains of HeLa (cervical carcinoma) cells have been shown to be particularly sensitive, reacting to picomolar or even to femtomolar concentrations. Other cell lines, such as the colon carcinoma cell line HT29, are moderately sensitive, whereas nonepithelial cells and some other cell types are insensitive to the toxin (8, 24). Although studies with established cell lines have greatly expanded our understanding of the cellular pathogenesis of Shiga toxin, a key weakness has been that the established cell lines used are not appropriate target cells and therefore may be questionable models. With the development of methods to propagate human intestinal epithelial cells (14-18) has come the ability to study human enteropathogens and compare in vivo pathogenesis with an in vitro human cell culture model. In this study, the cytotoxicity of purified Shiga toxin for primary cultures of normal human intestinal epithelial cells was tested. Normal colon mucosa (NCM) epithelial cells were initiated from discard mucosa obtained from trauma patients without evidence of cancer or other diseases. The mucosal epithelium was scraped from the submucosa, rinsed eight times by centrifugation in autoclavable minimal essential *
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medium (Earle salts; M. A. Bioproducts) containing 4 mM glutamine and twice the normal concentrations of antibiotics used in complete culture medium. The complete culture medium was an L15-suspension minimal essential medium base medium containing antibiotics (penicillin, 100 U/ml; streptomycin, 50 ,ug/ml; gentamicin, 25 ,ug/ml; and amphotericin B [Fungizone; GIBCO Laboratories], 50 ,ug/ml) and supplemented with 2% (vol/vol) fetal bovine serum, 5% (vol/vol) L broth, 1% (vol/vol) stock pituitary extract, and other growth factors, as described by Moyer and Aust (17). The cells were seeded into 24-well plates at 8 x 105 cells (in 1.0 ml of complete culture medium) per well and incubated for several hours or overnight. The 96-well plates were seeded with 5 x 104 cells per well (in 0.1 ml). Replicate cultures were harvested to assess viability (by 0.5% trypan blue dye exclusion), DNA synthesis (by [3Hlthymidine incorporation), or protein synthesis (by incorporation of [3H]leucine). For rapid-screening assays of microtiter plates, protein assays (Bio-Rad Laboratories) were done as a measure of relative cell number. Purified Shiga toxin was maintained at -70°C until use. Dilutions from 10 pg/ml to 10 mg/ml were tested in cultures seeded into the multiwell plates. At various times, the cultures were harvested and assayed for viability or macromolecular syntheses as described above. Monospecific rabbit antiserum against Shiga toxin (2) was diluted (1:20) in complete medium and then premixed and incubated at 370C with an equal volume of the dilute Shiga toxin (500 ng/ml) for 30 min. To microcultures of the NCM cells (about 104 cells) in 0.1 ml were added 10 p.l of toxin, toxin plus antibody, or antibody only, with volumes adjusted to be equal and with the same amount of toxin or antibody in control and experimental cultures. After 18 h of incubation at 37°C, the cells were assayed for viability by trypan blue dye exclusion. In each experiment, two cultures and quadruplicate samples were studied. The toxin inhibited protein and DNA synthesis and depressed dye exclusion by NCM cells to about 50% at a concentration of 50 pg/ml (Fig. 1). Higher concentrations did not increase the level of cytotoxicity in these cultures. Differences between the mean values of the control cultures and all of the Shiga toxin-treated cultures were statistically significant (P 0.01) by analysis of variance determination, whereas differences were not significant among the toxin concentrations tested. Monospecific rabbit antiserum against Shiga toxin was able to completely neutralize the cytotoxic effect. -
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