Department of Pathology and Microbiology, University of Bristol, Bristol BS8 1TD, UK1
Author for correspondence: Timothy R. Hirst. Tel: +44 117 928 7538. Fax: +44 117 930 0543. e-mail: t.r.hirst{at}bristol.ac.uk
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ABSTRACT |
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Keywords: E. coli heat-labile enterotoxin, cholera toxin, cAMP, epithelial cells, cytokine expression
Abbreviations: Etx, E. coli heat-labile enterotoxin; Ctx, cholera toxin; dbcAMP, dibutyryl cAMP; PKA, cAMP-dependent protein kinase
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INTRODUCTION |
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Given that epithelial cells are the initial targets of the toxins, both during infection and following experimental mucosal immunization, it is conceivable that the apparent spectrum of adjuvanticity of the intact holotoxins, attenuated derivatives and B-subunits results from their differential effects on modulating epithelial cell cytokine production. McGee et al. (1993) showed that Ctx was able to strongly promote the production of IL-6 by rat IEC-6 epithelial cells, whilst non-recombinant preparations of CtxB had no effect. Similarly, Bromander et al. (1993)
found that Ctx-treatment of IEC-17 cells stimulated both IL-1 and IL-6 secretion. Here we show that recombinant preparations of fully intact and enzymically active Etx, but not an inactive mutant or EtxB, causes elevated cytokine secretion by human intestinal epithelial T84 cells. Such effects were found to dependent on activation of adenylate cyclase and protein kinase A. The nature of the cytokines activated, which included IL-10 and IL-1R
, is consistent with the toxins exerting an anti-inflammatory effect (Ryan et al., 2000
), which may contribute to the initial survival and growth of toxinogenic pathogens in vivo (Triadafilopoulos et al., 1989
). These findings provide a further insight into the enhanced immunomodulatory properties of the intact holotoxin as well as explaining how the toxin may alter the mucosal microenvironment in which immune responses occur.
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METHODS |
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T84 cell cultures.
The human epithelial cell line T84 was obtained from the European Centre for Applied Microbiology and Research (ECACC, Salisbury, UK) and routinely cultured in Dulbeccos modified minimal essential medium (D-MEM) supplemented with Hams F10, 10% (v/v) fetal bovine calf serum, glutamine, penicillin and streptomycin. Cells were passaged at 56 day intervals and were used between passages 3 and 12. T84 cell monolayers were grown by plating (5x105 cells per well) in 12-well tissue culture plates and cultured for 56 days until they were fully confluent and had the morphological appearance of polarized cells. Experiments were also performed using T84 cells grown for 3 weeks on 6-well polycarbonate plates (Costar) and with addition of fresh medium every 3 days. When cultures were treated with various agents the following concentrations were used unless otherwise stated: Etx 1 nM; Etx(E112D) 1 nM; EtxB 100 nM; V. cholerae lipopolysaccharide (LPS) 10 µg ml-1; forskolin 10 µM; dibutyryl cAMP (dbcAMP) 100 µM; H89 10 µM.
Immunofluorescence and confocal microscopy.
Immunofluorescence microscopy was performed on confluent adherent T84 cells grown for 56 days on collagen-coated glass coverslips as described by Casciola-Rosen et al. (1996) . In order to detect early apoptotic events in Etx-treated cells, we used Annexin-V staining of phosphatidylserine exposed on the plasma membrane. Control or Etx-treated cells were washed three times with ice-cold PBS and subsequently Annexin V-FITC was added for 20 min at 4 °C in the dark. Cover slips were then washed by dipping in PBS prior to fixation with 4% (w/v) paraformaldehyde and permeabilization with acetone. Cover slips were mounted on glass slides with Vectashield containing 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) (Vector Laboratories) before viewing on a Leica TCS-SP2 confocal Ar plus UV laser scanning microscope attached to a Leica DM IRBE inverted phase-contrast/epifluorescence microscope (Leica Microsystems). An oil-immersion objective lens 63x, NA 1·32, was used, and imaging parameters were selected to optimize resolution.
cAMP detection assay.
To measure intracellular cAMP in T84 cells the Biotrak cAMP scintillation proximity assay (SPA) kit from Amersham was used. Confluent T84 cells cultured in a 96-well plate were treated with cAMP-inducing agents for 1 h. Cells were then lysed and the level of cAMP determined. Briefly, the assay is based on the competition between intracellular unlabelled and a fixed quantity of 125I-labelled cAMP for a limited number of binding sites on cAMP-specific antibody. The antibody-bound cAMP is then reacted with the SPA reagent, which contains anti-rabbit secondary antibody bound to fluoromicrospheres. 125I-labelled cAMP that is bound to the primary rabbit antibody is immobilized on the fluoromicrospheres and the radiations emitted measured by a ß-scintillation counter.
Analysis of secreted cytokines.
Cytokine levels in T84 supernatants were determined by cytokine-specific ELISA. All cytokine standards, capture antibodies and detection antibodies were obtained from Pharmingen. Maxisorp immunoplates were coated overnight with monoclonal anti-human IL-2, IL-4, IL-6, IL-8, IL-10 and TGF-ß1. After blocking, samples and serial twofold dilutions of standards were added to duplicate wells and incubated overnight at 4 °C. The plates were then washed and incubated with the respective biotinylated antibody for 1 h at room temperature. After incubation, extravidin peroxidase conjugate was added and the reaction developed with tetramethylbenzidine and hydrogen peroxide.
Analysis of cytokine gene expression by multi-probe RNase protection assay.
RNase protection assays were performed according to the RiboQuant manual for human cytokines (hCK-2 template array, Pharmingen). Briefly, total RNA from T84 cells was prepared by the LS TRI-reagent protocol and quantified both spectrometrically and electrophoretically (TAE 1%, w/v, agarose gel). The Pharmingen multi-probe set contained a series of cDNA templates for IL-12, IL-1, IL-1ß, IL-1R
, IL-6, IFN-
and the housekeeping genes L32 and GAPDH to allow normalization of the samples. The multi-probe template set was transcribed as antisense RNA probes by T7 RNA polymerase in the presence of [
-32P]UTP (ICN). Labelled probes were hybridized overnight with 45 µg total RNA, after which free probe and other single-stranded RNA species were digested with RNase A and T1. The remaining RNase-protected probes were purified and resolved by urea denaturing PAGE. Mouse control RNA and yeast tRNA were also run. The expressed genes were detected by autoradiography and the level of gene expression determined using the Scion Image program (Scion Corporation). To accurately establish the identity of each protected fragment, the migration of each fragment was plotted against a standard curve of the migration distance for undigested probe versus nucleotide length on a logarithmic scale.
Statistical analysis.
Differences in cytokine production between treated and untreated T84 cells were analysed for statistical significance using unpaired Students t-test.
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RESULTS |
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Etx induces cytokine gene expression in T84 cells
It has been previously reported that Ctx triggers cytokine activation in epithelial cells (Bromander et al., 1993 ; McGee et al., 1993
), while no information is available on Etx-mediated effects. We assessed the multiple expression of cytokine mRNA transcripts in Etx-treated T84 cells using a highly sensitive and specific human cytokine multi-probe RNase protection assay system, which allows the simultaneous detection of expression of IL-12, IL-1
, IL-1ß, IL-1R
, IL-6 and IFN-
. Fig. 1(a)
shows that 1 nM Etx was able to induce the early expression of IL-1
, IL-1ß, IL-6 and IL-1R
. The data from three independent experiments were normalized against the mRNA levels of the housekeeping gene GAPDH and expressed as fold induction with respect to PBS control, and are shown in Fig. 1(cj
). We noted while the basal level of IL-1
and ß cytokine expression in the absence of treatment (PBS control) appeared to vary, the fold increase in expression of these cytokines following addition of Etx and other modulators of cAMP levels was remarkably consistent. The expression of IL-6, IL-1
, IL-1ß genes, which peaked at 6 h post-treatment (see Fig. 1c, g and i
, respectively), decreased at 24 h (see Fig. 1d, h and j
). By contrast, mRNA levels for IL-1R
remained elevated at 24 h post-Etx treatment (see Fig. 1e
and f
). Protected probes for IL-12 and IFN-
were barely detectable in samples derived from either PBS- or Etx-treated T84 cells, while their transcripts were present in a control human RNA sample.
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DISCUSSION |
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Ctx is known to elicit cytokine secretion by epithelial cells as well as by bone-marrow-derived macrophages (Bromander et al., 1991 , 1993
; Cong et al., 2001
; Elson & Cong, 1995
; Klimpel et al., 1995
; McGee et al., 1993
) and to inhibit the secretion of pro-inflammatory cytokines such as IL-12, TNF-
, RANTES and IL-1 by LPS-stimulated human dendritic cells (Braun et al., 1999
; Gagliardi et al., 2000
). Because of the similarity in structure and function of Ctx and Etx, it has been generally accepted that results obtained from studies on Ctx will also hold for Etx. Nonetheless, very few studies have been undertaken on Etx, and recent observations have highlighted some striking differences in the immunological properties of their respective B-subunit moieties (Millar & Hirst, 2001
). Thus, while CtxB appears to be a poor adjuvant, EtxB can trigger immune responses to co-administered antigens that approach levels seen using the full holotoxins of Ctx or Etx as adjuvants. In order to clarify the mechanism of action of such toxins, we investigated the effects of cholera-like enterotoxins on cytokine expression by epithelial cells and whether such modulation depended on (a) the identity of the toxins, (b) their ability to bind to GM1 receptors or (c) their intrinsic ADP-ribosylating activity. Interestingly, Ctx and Etx behaved identically with respect to their effects on cytokine expression by T84 epithelial cells. Moreover, these effects depended on the elevation of intracellular cAMP levels, since the non-toxic B-subunits alone or a mutant form of Etx, lacking enzymic activity, failed to increase any of the cytokines activated by Ctx/Etx or cAMP-elevating agents, such as forskolin and dbcAMP (Fig. 1
). By using the highly sensitive RNase protection assay, we were able to screen for transcriptional activation of a series of pro- and anti-inflammatory cytokines. Ctx and Etx, as well as forskolin and dbcAMP, triggered a large increase in IL-6 and IL-1R antagonist mRNA levels, while the expression of Th1-type cytokines like IL-12 was unaffected. The early activation of IL-1
and IL-1ß observed is in agreement with previous findings showing that Ctx enhances IL-1 production by macrophages (Bromander et al., 1991
) and epithelial cells (Bromander et al., 1993
). This potentiates antigen presentation by macrophages and modulates T-cell responses. The elevation in cAMP levels by cholera-like enterotoxins will lead to activation of cAMP-dependent protein kinase (PKA) and the translocation of PKA catalytic subunits to the nucleus (Zidek, 1999
). The subsequent phosphorylation of cAMP-responsive element binding proteins would be expected to activate transcription of specific genes carrying cAMP-regulatory elements in their promoter region (Seternes et al., 1999
), such as IL-6 (Krueger et al., 1991
) and IL-1ß genes (Chandra et al., 1995
). Our finding that H89, an inhibitor of PKA, abrogates the induction of IL-1
, IL-1ß, IL-6 and IL-1R
further corroborates the hypothesis of a direct effect of cAMP on Ctx/Etx-induced cytokine expression.
IL-1R is an important anti-inflammatory molecule that acts by blocking IL-1-induced inflammatory reactions (Dinarello, 1998
). Our finding that the expression of IL-1R antagonist is up-regulated in T84 cells by both Ctx and Etx provides evidence that these toxins might create an anti-inflammatory environment in the intestine by blocking IL-1 binding to its own receptor. Recently, Feng et al. (2000)
reported that Ctx and 8-bromo-cAMP up-regulated expression of IL-1R
, as well as IL-10, in LPS-stimulated macrophages.
IL-6 and IL-10 are believed to play an important role in modulating mucosal immune responses (Klimpel et al., 1995 ). Both Etx and Ctx up-regulated IL-6 levels in the supernatant of T84 human epithelial cells, while H89 completely blocked such effects (Fig. 2
). By contrast, CtxB, EtxB and the non-toxic mutant Etx(E112D) failed to induce the production of IL-6 in T84 cells. This is in agreement with previous findings indicating that Ctx triggers IL-6 production in rat epithelial cells by a mechanism involving cAMP (McGee et al., 1993
). While IL-6 is produced in response to certain inflammatory stimuli, it has been shown that this cytokine can exert anti-inflammatory effects in several systems by inhibiting Th1 responses (Barton, 1997
). In addition, IL-6 is known to act as a growth factor for activated T-cells, boosting the magnitude of the Th2 response, and promoting immunoglobulin production by B-cells, especially secretory IgA. The high-level production of IL-6 upon treatment of epithelial cells with Etx may contribute to both the polarization of the Th response and the apparent adjuvanticity of the toxin. IL-10 plays a pivotal role in determining the immunosuppressive environment resulting from the action of cholera-like toxins (Wiedermann et al., 1999
). This concept is strengthened by the fact that putative cAMP-responsive elements were localized within the promoter/enhancer region of the IL-10 gene (Platzer et al., 1999
). IL-10 levels were up-regulated by a cAMP-dependent pathway in the supernatants of Ctx/Etx-treated T84 cells. Whilst IL-10 secretion was clearly dependent on the ADP-ribosylating activity of Ctx/Etx, its delayed onset in production suggests that indirect autocrine mechanisms may be involved. In the intestinal mucosa IL-10 is released primarily by the Th2 subset of CD4+ T helper cells in response to common environmental antigens (Panja et al., 1995
). However, the fact that IL-10 is also produced by isolated epithelial cells suggests that it may play a role in the regulation of the intestinal cell function. Indeed, it has been shown that IL-10 enhances sodium and chloride absorption in rat small intestine, limits forskolin-induced chloride secretion and prevents disruption of monolayer barrier integrity in T84 epithelial cells (Madsen et al., 1997
).
In addition, Etx, as well as dbcAMP, slightly increased IL-8 levels and failed to induce expression of IL-12 in T84 cells. IL-8 is a potent neutrophil, T-cell and basophil chemoattractant involved in acute inflammatory responses (Harada et al., 1994 ) and it has been postulated that during intestinal invasion by luminal bacteria IL-8 can function as a signal for mucosal barrier penetration (Eckmann et al., 1993
). However, it has been shown that Ctx inhibits in vitro IL-8-induced lymphocyte migration (Bacon & Camp, 1990
) and that in a rabbit intestine model Ctx did not cause neutrophil infiltration in the lamina propria or necrosis of enterocytes (Triadafilopoulos et al., 1989
). Thus, while cAMP elevation by Etx or dbcAMP results in some IL-8 secretion by epithelial cells, the toxins appear to be able to couteract this, possibly by direct effects on leukocytes. Such findings are consistent with the clinical picture of disease in which diarrhoea ensues without any evident inflammatory response.
Taken together, these findings suggest that Etx and Ctx elevate expression of anti-inflammatory molecules such as IL-10 and IL-1R, and are unable to trigger the potent secretion IL-8 and IL-12. This is consistent with the view that activation of cAMP-mediated pathways in epithelial cells promotes an anti-inflammatory response, as well as a cytokine microenvironment that may favour induction of a polarized Th2 immune response.
The observation that neither an enzymically inactive mutant of Etx nor EtxB was able to induce cytokine secretion may in part explain why these molecules are not such potent mucosal adjuvants as their respective holotoxins. Moreover, the differential adjuvanticity being reported for EtxB, compared to CtxB, does not appear to be due to a differential induction of cytokine expression by epithelial cells, since both B-subunits behaved identically.
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ACKNOWLEDGEMENTS |
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Received 14 September 2001;
revised 26 October 2001;
accepted 7 November 2001.