The Afa/Dr adhesins of diffusely adhering Escherichia coli stimulate interleukin-8 secretion, activate mitogen-activated protein kinases, and promote polymorphonuclear transepithelial migration in T84 polarized epithelial cells

Abstract

Afa/Dr diffusely adhering Escherichia coli (Afa/Dr DAEC) strains cause symptomatic urinary tract and intestinal infections. The proinflammatory effects of Afa/Dr DAEC strains in vitro have been not investigated to date. In the present study, we used confluent polarized monolayers of intestinal cell line T84 to evaluate the consequences of epithelial infection by Afa/Dr DAEC strains in terms of proinflammatory response. Polymorphonuclear leukocyte (PMNL) migration across the epithelial barrier was induced after incubation of the T84 monolayers with the wild-type Afa/Dr DAEC strain C1845 harboring the fimbrial F1845 adhesin and strain IH11128 harboring the Dr hemagglutinin, and the E. coli laboratory strain HB101 was transformed with the pSSS1 plasmid, producing Afa/Dr F1845 adhesin. PMNL migrations were correlated with a basolateral secretion of interleukin-8 by T84 cells and were abolished after incubation of epithelial cells with an anti-decay accelerating factor (DAF) antibody that recognized the short consensus repeat 3 domain of DAF (monoclonal antibody 1H4). Moreover, Afa/Dr DAEC strains induced tyrosine phosphorylation of several T84 proteins and activated the mitogen-activated protein kinases (ERK1/2 mitogen-activated protein, P38, and Jun-C kinases). These data demonstrated for the first time that, in vitro, Afa/Dr DAEC strains exert a proinflammatory signal in intestinal epithelial cells.

FIG. 1.

Electron microscopy examination of Afa/Dr DAEC interaction with polarized T84 monolayers. (A) T84 cells infected with the nonadherent E. coli HB101 bacteria show a regular brush border. (B) Wild-type Afa/Dr DAEC C1845 bacteria are associated with the brush border and show a disappearance of the regular brush border. Bars, 0.5 μm.

FIG. 2.

Induction of physiologically directed transepithelial migration (basolateral to apical) of PMNL by apical colonization of T84 intestinal cells with wild-type Afa/Dr DAEC strain C1845. PMNL transepithelial migration in control cells was induced by the PMNL transmigrating inducing peptide fMLP (10⁻⁷ M). C1845 bacteria elicited >78% of the maximum transmigration response induced by fMLP, in contrast to the control HBSS or to E. coli HB101, a nonpathogen bacterium that induced no transepithelially directed chemotaxis of PMNL (✽, P < 0.005). Data represent the means and standard deviations of at least three experiments, each performed in triplicate.

FIG. 3.

Wild-type Afa/Dr DAEC strains C1845 and IH11128 induce tyrosine phosphorylation in T84 cells. T84 cells were lysed at various times after infection, and samples were then analyzed by immunoblotting with anti-phosphotyrosine antibody as described in Materials and Methods. The putative T84 cell-induced tyrosine-phosphorylated proteins are indicated by arrowheads. Preexposure of the T84 cell monolayers to MAb anti-DAF inhibited tyrosine-phosphorylated proteins induced by C1845. The positive control lane was obtained with cells treated with EGF (10 nM, 15 min). The negative control lane was obtained with cells infected with strain K12-HB101. The micrograph is representative of results obtained at least three times.

FIG. 4.

Activation of MAP kinases in Afa/Dr DAEC-infected epithelial intestinal T84 cells. Cells were lysed 60 min after infection by wild-type Afa/Dr DAEC strains C1845 and IH11128 and recombinant E. coli strain HB101/pSSS1 expressing F1845 adhesin. The positive controls were obtained from cells treated with EGF (10 nM, 15 min). The negative controls were obtained from cells infected with strain K12-HB101 and from uninfected control T84 cells. Samples were analyzed by immunoblotting with antiphosphorylated antibodies as described in Materials and Methods. (A) Activation of ERK1/2 in Afa/Dr DAEC-infected and EGF-stimulated cells. Note that control cells show a constitutive activation of ERK. (B) Activation of p38 MAP kinases in control, Afa/Dr DAEC-infected and EGF-stimulated cells. (C) Activation of JNK in control, Afa/Dr DAEC-infected, and EGF-stimulated cells. MAP kinase activation was not detectable in infected epithelial cells after preexposing T84 monolayers with MAb anti-DAF (1H4). Micrographs are representative of at least three experiments.

FIG. 5.

MAP kinase activation is required for the induction of the transepithelial migration of PMNL after apical colonization of T84 intestinal cells with wild-type Afa/Dr DAEC strain C1845. T84 monolayers were cultured alone (HBSS) or were infected with the C1845 bacteria with or without pretreatment with the p38 inhibitor SB203580 (10 μM) and/or the MEK1 inhibitor PD98059 (25 μM). Data represent the mean and standard deviation of at least three experiments, each performed in triplicate. One of three experiments performed. NS, not significantly different from control (HBSS).