Division of Food Sciences, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK1
Division of Gastroenterology, University Hospital Nottingham, Queens Medical Centre, Nottingham NG7 2UH, UK2
Author for correspondence: Kenneth H. Mellits. Tel: +44 115 95 16161. Fax: +44 115 95 16162. e-mail: ken.mellits{at}nottingham.ac.uk
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: transcription, innate immunity, interleukin-8, inflammation, food safety
Abbreviations: BCE, boiled-cell extract; EMS, electrophoretic mobility shift; IL, interleukin; LOS, lipo-oligosaccharide; TNF-; tumour necrosis factor-
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Patients suffering campylobacteriosis present a range of clinical symptoms, from mild watery diarrhoea to severe bloody diarrhoea accompanied with fever and abdominal cramps. A recent history of Campylobacter infection is also frequently associated with the neurological disorder GuillainBarré syndrome (Rees et al., 1993 ; Nachamkin et al., 1998
). GuillainBarré syndrome can result in paralysis and, occasionally, impaired respiratory function. The pathogenic mechanisms responsible for the acute intestinal infection of humans are poorly understood but are thought to involve the processes of colonization, adherence, cellular invasion and toxin production (Ketley, 1997
). There are clearly some variations in these processes, as not all clinical isolates of C. jejuni are demonstrably able to invade cultured human cells or produce defined toxins. However, a common feature of Campylobacter infectious enterocolitis is a localized acute inflammatory response that can lead to tissue damage and may be responsible for many of the clinical symptoms (Ketley, 1997
).
The NF-B/rel family of transcription factors comprises a structurally related series of DNA binding and transactivation proteins, which have been shown to play an early response role in a large number of cellular processes, including the host inflammatory response to microbial infection. NF-
B/rel members function as part of the innate immune response to microbial pathogens, acting to stimulate the transcription of the genes for cytokines and chemokines (Silverman & Maniatis, 2001
). The resulting secretion of cytokines/chemokines and other mediators leads to the activation of macrophages and the recruitment of polymorphonuclear leukocytes in the inflammatory response. Continued stimulation of these response mechanisms can result in chronic inflammatory states and fibrogenesis of the intestinal tract (Schmid & Adler, 2000
).
NF-B/rel members are composed of DNA-binding proteins (NF-
B1p50 and NF-
B2p52) in association with rel proteins (RelAp65, RelB and c-Rel) which bear the transactivation domain. NF-
B/rel complexes are held in the cytoplasm in non-induced cells by inhibitor proteins called I
B (I
B
, I
Bß, I
B
and I
B
). These proteins bind to NF-
B/rel and mask the nuclear localization domain in order to prevent nuclear translocation. Activation of NF-
B involves the phosphorylation and subsequent ubiquitin-mediated proteosomal degradation of I
B (Mellits et al., 1993
; Silverman & Maniatis, 2001
). I
B is phosphorylated by I
B kinase (IKK
, IKKß catalytic subunits and the regulatory subunit IKK
). IKK is subject to activation by the receptor pathway-dependent kinases MAP/ERK kinase kinase 1 (MEKK1) and NF-
B-inducing kinase (NIK) (Silverman & Maniatis, 2001
).
Here we show that C. jejuni, like other gastrointestinal pathogens (Salmonella typhimurium, Shigella flexneri, Helicobacter pylori, enterovirulent Escherichia coli and Yersinia enterocolitica), can activate NF-B in epithelial cells and thereby elicit a pro-inflammatory response. Moreover, we show that a cell-free, heat-stable extract of C. jejuni can activate NF-
B through the degradation of I
B
and the subsequent binding of NF-
B subunits to the NF-
B target DNA sequence. We also demonstrate that the NF-
B activation is co-ordinated with the production of the pro-inflammatory cytokine interleukin-8 (IL-8). As IL-8 is a chemotactic factor of immune-active cells and a mediator of local immune responses, these data therefore predict a mechanism by which C. jejuni can bring about the intestinal inflammation commonly associated with campylobacteriosis.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparation of Campylobacter extracts.
A 24 h culture of C. jejuni NCTC 11168T was used to inoculate 150 ml nutrient broth no. 2 (CM 67; Oxoid) dispensed in 250 ml conical flasks; the flasks were then shaken under microaerobic conditions for 24 h at 42 °C. The bacteria were collected by centrifugation at 10000 g for 15 min. The bacterial cell pellet was then resuspended in PBS and washed by centrifugation for a total of three times. The cell pellet was weighed then resuspended in PBS to 10% (w/v). This suspension was then boiled for 10 min and cooled on ice. The suspension was then centrifuged at 13000 g and the supernatant collected. This extract was then filtered through a 0·2 µm filter, to remove any residual bacteria, and stored at -20 °C until required.
The extract was fractionated by ultrafiltration using molecular mass cut-off filters of 30, 10, 5 and 3 kDa applied in PBS according to the manufacturers instructions (Millipore and Gelman Laboratories). The fractions were treated with proteinase K (100 µg ml-1) for 1 h at 55 °C and reboiled before use in reporter cell activation assays. Fractions pre- and post-proteinase K treatment were electrophoresed in 12·5% SDS-polyacrylamide gels and visualized with Coomassie blue and silver stain to ensure the correct functioning of the molecular mass cut-off filters and the complete digestion of the protein component of the extract.
Cell culture and induction.
HeLa 57A cervical epithelial cells (Rodriguez et al., 1999 ) and HCA-7 colonic epithelial cells (Kirkland, 1985
) were grown in monolayer cultures of approximately 5x106 in Dulbeccos Modified Eagles Medium supplemented with penicillin at 100 µg ml-1, streptomycin at 100 µg ml-1 and fetal calf serum at 10% (v/v). To select for the transcriptional markers present in HeLa 57A cells, these cultures were supplemented with G418 (Gibco) at 0·5 µg ml-1. Three hours prior to induction, cells were starved of serum, and inductions were carried out by adding tumour necrosis factor-
(TNF-
) at 50 ng ml-1 (obtained from the EU Programme EVA/MRC Centralized Facility for AIDS Reagents, NIBSC, UK; grant nos QLK2-CT-1999-00609 and GP828102) or C. jejuni extract at the concentration indicated. Live C. jejuni infections were performed using fresh overnight cultures of C. jejuni, which were allowed to equilibrate in Dulbeccos Modified Eagles Medium before introduction to tissue culture cells at an m.o.i. of 100.
Reporter cell assays.
HeLa 57A cells carry an NF-B-dependent promoter driving luc transcription, and an independent Rous sarcoma virus promoter driving the expression of lacZ (Rodriquez et al., 1999
). Replicate luciferase and ß-galactosidase reporter assays (four to six independent determinations) were performed with cytoplasmic protein extracts. Luciferase activity was measured using a Turner bioluminometer with luciferin as substrate, as recommended by the manufacturer (Promega). ß-Galactosidase activity was measured using a colorimetric assay with the substrate ONPG, as recommended by the manufacturer (Clontech). To calculate the degree of NF-
B induction, all luciferase activities were normalized against internal ß-galactosidase activities, so as to provide a constitutive control of the basal expression levels, and expressed as a multiple of the uninduced control (fold-activation).
Electrophoretic mobility shift (EMS) assay.
Nuclear and cytoplasmic protein extracts were prepared from tissue-culture cells as described by Mellits et al. (1993) , using 6 cm dishes (containing 5x105 cells). Protein concentrations were estimated by the Bradford assay (Bio-Rad). For EMS assays, nuclear protein extracts (10 µg) were incubated together with a 33P-labelled oligonucleotide probe containing the NF-
B binding site as characterized for the human ß-interferon promoter (positive regulatory domain II; Visvanathan & Goodbourn, 1989
) in binding buffer [10 mM HEPES/KOH (pH 7·9), 50 mM KCl, 5 mM DTT, 1 mM EDTA containing 2 µg poly(dI-dC)]. DNAprotein complexes were fractionated on 5% native polyacrylamide gels, dried and visualized using a phosphor-imager (Bio-Rad). Supershift assays were performed with polyclonal antibodies against the p50 and p65 subunits of NF-
B (gifts from Ronald T. Hay, University of St Andrews, UK).
Western blots.
Protein samples (10 µg) were heated in SDS loading buffer at (90 °C for 3 min) before fractionation on 12·5% SDS-polyacrylamide mini-gels and transfer to 0·45 µm PVDF membranes (Pierce). Membranes were blocked using milk protein and probed with primary antibodies directed against IB
(monoclonal 10B, a gift from Ronald T. Hay, University of St Andrews), I
Bß (polyclonal C-20, sc-945; Santa Cruz Biotech) and I
B
(polyclonal M-364, sc-7155; Santa Cruz Biotech) or as an independent control 14-3-3 protein (polyclonal K19, sc-629; Santa Cruz Biotech); bound antibodies were detected using species-specific secondary antibodies conjugated to alkaline phosphatase (Sigma) and visualized with nitroblue tetrazolium (Roche) and 5-bromo-4-chloro-3-indolyl phosphate (Roche).
IL-8 determination.
IL-8 released into cell-culture supernatants was determined for induced and mock-induced HeLa 57A and HCA-7 cells over a 9 h time-course. The induction factor for NF-B-dependent gene expression was determined in parallel for the HeLa 57A reporter cell line. IL-8 was measured using a sandwich ELISA. IL-8 was captured with murine anti-human IL-8 and detected with biotinylated goat anti-human IL-8 using streptavidin-coupled horseradish peroxidase according to the manufacturers instructions (R&D Systems).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
C. jejuni extract activates NF-B via I
B
degradation and DNA binding
NF-B is activated following the degradation of a cytoplasmic inhibitor protein, I
B. Degradation of I
B releases a heterodimer composed of NF-
B and rel proteins, which relocate to the nucleus to bind promoter DNA elements and exert transcriptional activation. To correlate the C. jejuni BCE-induced NF-
B-dependent gene expression with the degradation of a specific member of the I
B family, we performed a series of Western blots and probed these with antibodies against I
B proteins. Cytoplasmic protein extracts were prepared from HeLa 57A cells induced with either C. jejuni BCE or TNF-
. Western blots of cytoplasmic protein extracts harvested at 30 min intervals post-induction were probed with antibodies directed against I
B
and 14-3-3 proteins (Fig. 3
). The 14-3-3 protein levels are invariant with these treatments and act as a control of protein loading in these experiments. I
B
levels in 57A HeLa cells are reduced as compared with untreated cells at the 30 and 60 min time-points in either TNF-
- or C. jejuni BCE-treated cells (Fig. 3a
, b
; lanes 2 and 3). Later time-points show I
B
levels returning as a consequence of the resynthesis of I
B
(Fig. 3a
, b
; lanes 4 and 5).
|
The DNA-binding activities of the NF-B complexes from HeLa 57A and HCA-7 cells in response to treatments with either C. jejuni BCE or TNF-
were examined using an EMS assay. Nuclear protein extracts were prepared over a 4 h time-course post-treatment and incubated with a labelled oligonucleotide probe containing the NF-
B binding site derived from the positive regulatory domain II region of the human ß-interferon promoter. The probe detects NF-
B and a probe-specific complex marked B (Fig. 4
). The B complex provides a useful internal control of the formation of proteinDNA complexes in these assays, and is present in the untreated control. Treatment of HeLa 57A cells with either C. jejuni BCE or TNF-
produces an NF-
B-specific retarded band, after 30 min, which is absent with the untreated control extract (Fig. 4a
; lanes 1, 2 and 5). The NF-
BDNA complex produced by either treatment persists through to 240 min, but, by this time, the intensities of the corresponding bands are diminished (Fig. 4a
; lanes 3, 4, 6 and 7). To confirm the composition of the DNAprotein complexes observed in the EMS assay, supershift experiments were performed in which DNA-binding reactions were incubated with antibodies directed against the NF-
B component subunits p50 (NF-
B1) and p65 (RelA). Incubation with pre-immune serum does not further retard the treatment-specific band. However, incubation with either anti-p50 or anti-p65 produces a characteristic shift in the NF-
B proteinDNA complexes formed by either C. jejuni BCE or TNF-
treatment (Fig. 4a
; lanes 813). EMS assays and supershift experiments with HCA-7 cell nuclear extracts also indicate the formation of NF-
B proteinDNA complexes in response to either C. jejuni BCE or TNF-
treatment (Fig. 4b
). In corroboration of the I
B
degradation data, the DNA-binding activity observed for HCA-7 cells in response to TNF-
is delayed from 30 until 120 min post-treatment (Fig. 4b
, lanes 2 and 3) when compared with the response of HeLa 57A cells to TNF-
(Fig. 4a
, lane 3). In contrast, HCA-7 cells suffer no such delay in response to C. jejuni BCE treatment. C. jejuni BCE treatment leads to the formation of NF-
B proteinDNA complexes within 30 min, the levels of which remain unabated through to the 240 min time-point (Fig. 4b
, lanes 57). The NF-
B DNA-binding activity induced in HCA-7 cells by TNF-
, however, does not persist and is noticeably diminished by 240 min.
|
|
Fractionation and proteinase K sensitivity of the C. jejuni BCE
As a first step to identify the bioactive components present in C. jejuni BCE we fractionated the extract according to molecular mass by ultrafiltration. Soluble fractions containing molecules in the mass ranges >30, 1030, 310 and <3 kDa were assayed in HeLa 57A cells for their ability to induce NF-B-dependent gene expression. These fractions were electrophoresed in 12·5% SDS-polyacrylamide gels and visualized with Coomassie blue and silver stain to monitor the performance of the molecular mass cut-off filters. The majority of the activity was found in a low-molecular-mass <3 kDa fraction (70%); most of the remaining activity was retained in the >30 kDa fraction (20 %). Although the relative proportions of the total NF-
B stimulatory activities found in the <3 and >30 kDa fractions were observed to be fairly constant over several experiments, the luciferase induction values were never completely additive to that determined for the initial C. jejuni BCE. The extant activity was undetectable in the intermediate fractions.
Fig. 6 shows the effect of prior digestion with proteinase K on the abilities of the initial C. jejuni BCE and the <3 kDa fraction to stimulate NF-
B-dependent gene expression in HeLa 57A cells. Samples collected pre- and post-proteinase K digestion were electrophoresed in 12·5% SDS-polyacrylamide gels and visualized by Coomassie blue and silver stain to ensure the complete digestion of the protein components of the extract fractions. Proteinase K digestion will eliminate the TNF-
response but will only reduce the C. jejuni BCE response by 32%. The <3 kDa fraction is insensitive to proteinase K activity. These data imply that the majority of the NF-
B stimulatory activity is a low-molecular-mass non-protein component. However, it is probable that the initial C. jejuni BCE also contains a protein component with the ability to activate NF-
B, though constituting only a minor contribution.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
NF-B regulates the transcription of a series of pro-inflammatory proteins. In intestinal epithelial cells the responses to NF-
B activation include the production of cytokines and chemokines (IL-1ß, IL-6, IL-8, macrophage inflammatory protein-2, growth-related oncogenes
and ß), cell-surface receptors (IL-2R), adhesion molecules (ICAM-1), inflammatory enzymes (inducible nitric oxide synthase and cyclo-oxygenase-2), stress proteins (complement factors B, C3, C4) and immunoregulatory molecules (major histocompatibility complexes I and II) (Jobin & Sartor, 2000
). Amongst these, IL-8 is an important chemokine of epithelial cells: elaboration of IL-8 is associated with the recruitment of inflammatory cells such as polymorphonuclear leukocytes to sites of infection or tissue damage (Eckmann et al., 1993a
, b
, 1995
; Jung et al., 1995
). The promoter of the human IL-8 gene contains several consensus DNA-binding sites for transactivating proteins; these include
B sites which constitute the target DNA sequence of NF-
B (Mukaida et al., 1990
, 1994
; Kunsch & Rosen, 1993
). NF-
B has emerged as the critical element for transcription of the IL-8 gene, for although it may participate in cooperative activation with other transcription factors such as NF-IL-6 or AP-1, NF-
B remains a necessity (Mukaida et al., 1990
; Yasumoto et al., 1992
; Matsusaka et al., 1993
). Our experiments clearly correlate the elaboration of IL-8 with the activation of NF-
B by C. jejuni BCE. C. jejuni, in common with several enteric bacterial pathogens, has been shown to elicit IL-8 secretion from epithelial cells (Hickey et al., 1999
). Two mechanisms have been proposed by which C. jejuni can interact with intestinal epithelial cells and bring about the release of IL-8: the first requires the adherence and/or invasion of cells by C. jejuni (Hickey et al., 1999
); the second is through the direct action of the cytolethal distending toxin that is present in most strains of C. jejuni (Hickey et al., 2000
). Our initial data, in which we demonstrate that live C. jejuni cells can activate NF-
B and thereby provide the transactivation required to induce IL-8 gene expression, is consistent with the first of these proposals. We would suggest that the surface-active components associated with IL-8 production actually function by stimulating NF-
B, and that these components are represented in C. jejuni BCE. However, it is unclear whether the tripartite structure that constitutes the cytolethal distending toxin would survive the boiling process to form an active component of the C. jejuni BCE. If the cytolethal distending toxin were to be present in C. jejuni BCE then its contribution to the total NF-
B activation potential is likely to be minor, since the majority of the activity is proteinase K insensitive and falls into a low-molecular-mass fraction.
In these studies, we have used the cellular response to TNF- as an experimental control and benchmark of the ability of C. jejuni BCE to activate NF-
B. The signal transduction pathway by which cells respond to TNF-
has been the subject of considerable study, and probably represents one of the best examples of receptor-mediated induction of NF-
B (Jobin & Sartor, 2000
; Silverman & Maniatis, 2001
). TNF-
is a pro-inflammatory cytokine associated with inflammation and immune response. In these roles, TNF-
serves as an activator of NF-
B, and is itself subject to activation by NF-
B. In our experiments, C. jejuni BCE activates NF-
B within 2 h in a comparable time-frame to TNF-
. Given that TNF-
induces a rapid and orchestrated series of events, we suggest that the activation by C. jejuni BCE is also a direct response, independent of the production of any secondary inducers such as cytokines.
How C. jejuni BCE brings about this response is not clear at this time. However, we have demonstrated that the activation of NF-B by C. jejuni BCE, like TNF-
, is mediated through the transient degradation of I
B
. In cervical epithelial cells, the degradation of I
B
occurs within 30 min of induction by C. jejuni BCE but returns to untreated levels by 90 min. This occurs because newly synthesized I
B
is translocated to the nucleus, where it serves to sequester NF-
B and down-regulate the activation. It is quite noticeable that in human colonic epithelial cells the degradation of I
B
in response to TNF-
is delayed until 90 min. This was not the case when these cells were induced by C. jejuni BCE. This difference was also manifest in the DNA-binding assay, in which NF-
B-dependent gel retardation was not observed until 120 min after TNF-
treatment but was evident at 30 min after C. jejuni BCE treatment. These data are consistent with a previous report in which the kinetics of I
B
degradation in human colonic epithelial cells have been found to be delayed and incomplete in comparison with other cell types in response to cytokines such as TNF-
(Jobin et al., 1997
). Differences in the timing of the response of colonic epithelial cells between C. jejuni BCE and TNF-
probably reflect operational changes in the TNF-
signal pathway of these cells, since the C. jejuni BCE response is similar to that found for HeLa 57A cells. It would seem likely that the bioactive components of C. jejuni BCE operate through an alternative to the TNF-
signal pathway leading to I
B
degradation, and that this pathway is not subject to delay as observed for TNF-
-treated colonic epithelial cells.
In common with other gastric and enteric pathogens (H. pylori, Salmonella typhimurium, Shigella flexneri, Y. enterocolitica and enterovirulent E. coli), C. jejuni can activate NF-B. What the presentation of these pathogens to epithelial cells will have in common are elements of the surface carbohydrates, lipopolysaccharide (LPS) or lipo-oligosaccharide (LOS) linked to lipid A. LPS is a potent stimulator of NF-
B in endothelial cells, but epithelial cells are largely insensitive to LPS (Pugin et al., 1993
). Similarly, we have found that C. jejuni LOS preparations do not activate NF-
B (our unpublished data). In the interests of maintaining the homeostasis of intestinal epithelial cells in an environment awash with microbial flora, the threshold concentration at which the primary recognition of LPS/LOS might occur through a Toll-like receptor would have to be set high. It is therefore possible that at high concentrations of LPS, as experienced with invasive or intimately adhered bacteria, LPS recognition through an internal receptor may have a role to play. This possibility has been argued at least for Shigella flexneri on the basis that the microinjection of normally non-stimulatory LPS will activate NF-
B, and that LPS immunodepletion of bacteria-free supernatants will reduce their ability to activate NF-
B (Philpott et al., 2000
). The need to discriminate pathogens and their products from non-pathogens is exemplified by E. coli, which is normally a commensal in the human gut but is distinguishable by intestinal epithelial cells from enteropathogenic E. coli (Savkovic et al., 1997
). It is becoming evident that the mechanisms by which intestinal epithelial cells activate NF-
B in response to these pathogens are multifaceted. For example, the activation NF-
B to trigger IL-8 production in Y. enterocolitica has been correlated with the specific internalization of the carboxy-terminal region of the protein invasin (Schulte et al., 2000
). Invasion of Y. enterocolitica is dependent on the surface interaction of invasin with host ß1-integrins. By comparison, most strains of C. jejuni are capable of adhesion to host cells, but not all are observed to invade. Several cell-surface-associated proteins from C. jejuni have been reported to act as adhesins, including the major cell-binding factor (Fauchère et al., 1989
; Pei & Blaser, 1993
), which is also found to be a major antigenic component (Kervella et al., 1993
) and has been identified as a member of the ABC transporter family (Pei et al., 1998
). Disruption of the corresponding gene reduced, but did not abolish, epithelial cell adhesion, implying the presence of alternative adhesins. Similarly, disruption of the gene for the fibronectin-binding protein CadF reduced, but did not abolish, adhesion (Konkel et al., 1997
). Two proteins have been reported to bind the plasma membranes of epithelial cells, i.e. a 59 kDa outer-membrane protein and the 43 kDa major outer-membrane protein (Moser et al., 1997
; Schroder & Moser, 1997
). Finally, a surface-exposed lipoprotein, JlpA, has also been identified as functioning as an adhesion factor for C. jejuni (Jin et al., 2001
). All these various surface adhesion proteins are, at face value, candidates for the minor, but direct, proteinase-K-sensitive NF-
B stimulatory activity we find in C. jejuni BCE. However, the overall contribution of adhesin proteins to the stimulation of NF-
B by live C. jejuni may be more important, as they may be the basis for intimate cell binding that enables non-protein bacterial surface components to cross the host cell membrane. The nature of the non-protein low-molecular-mass molecules that constitute the bulk of the NF-
B stimulatory activity is not clear at this time. What can be said is that the low molecular mass of the active fraction would preclude intact versions of the obvious surface carbohydrates present in C. jejuni, i.e. the LOS and capsular polysaccharide (Karlyshev and Wren, 2001
), although it is implicit that fragments of these molecules might serve to stimulate NF-
B, whereas the complete molecules may not.
The activation of NF-B shows a positive correlation with several intestinal inflammatory diseases (Crohns disease, ulcerative colitis, self-limited colitis and inflammatory bowel disease); moreover, the degree of activation can be correlated with the severity of the mucosal inflammation (Barnes & Karin, 1997
; Jobin & Sartor, 2000
). Several anti-inflammatory drugs used in the treatment of inflammatory bowel disease act directly or indirectly to suppress NF-
B activation (Jobin et al., 1996
, 1999
; Ardite et al., 1998
; Egan et al., 1999
). It is therefore likely that inappropriate activation of NF-
B in gut tissues will lead to bouts of acute inflammation, and it is possible that repeated gratuitous activation could lead to chronic intestinal inflammatory conditions. The heat-dissociated components we find in C. jejuni BCE are likely to provoke such a response. The level of Campylobacter contamination entering the human food chain is high: this is evident from the fact that C. jejuni has, in recent times, become the most common form of bacterial food poisoning in the developed countries, but its impact may even be greater. Domestic poultry can carry up to 1000 million campylobacters in the gut, and is not abnormal for carcasses produced for retail to harbour up to 1 million campylobacters (Saleha et al., 1998
). Governments and retailers have, quite correctly, stressed the importance of cooking to prevent Campylobacter food poisoning, but the question arises as to whether, in so doing, we are in fact facilitating the extraction of potent, heat-stable, NF-
B-activating components from what could be substantial Campylobacter populations.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Baeuerle, P. A. & Henkle, T. (1994). Function and activation of NF-B in the immune system. Annu Rev Immunol 12, 141-179.[Medline]
Barnes, P. J. & Karin, M. (1997). Nuclear factor-B, a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 336, 1066-1071.
Eckmann, L., Jung, H. C., Schurer-Maly, C., Panja, A., Morzycka-Wroblewska, E. & Kagnoff, M. F. (1993a). Differential cytokine expression by human intestinal epithelial cell lines: regulated expression of interleukin-8. Gastroenterology 105, 1689-1697.[Medline]
Eckmann, L., Kagnoff, M. F. & Fierer, J. (1993b). Epithelial cells secrete the chemokine interleukin-8 in response to bacterial entry. Infect Immun 61, 4569-4574.[Abstract]
Eckmann, L., Kagnoff, M. F. & Fierer, J. (1995). Intestinal epithelial cells as watchdogs for the natural immune system. Trends Microbiol 3, 118-120.[Medline]
Egan, L. J., Mays, D. C., Huntoon, M., Bell, M., Pike, M. G., Sandborn, W. J., Lipsky, J. J. & McKean, D. J. (1999). Inhibition of interleukin-1 stimulated NF-B RelA/p65 phosphorylation by mesalamine is accompanied by decreased transcriptional activity. J Biol Chem 274, 26448-26453.
Fauchère, J. L., Kervella, M., Rosenau, A., Mohanna, K. & Véron, M. (1989). Adhesion to HeLa cells of Campylobacter jejuni and C. coli outer membrane components. Res Microbiol 140, 379-392.[Medline]
Hickey, T. E., Baqar, S., Bourgeois, A. L., Ewing, C. P. & Guerry, P. (1999). Campylobacter jejuni-stimulated secretion of interleukin-8 from INT-407 cells. Infect Immun 67, 88-93.
Hickey, T. E., McVeigh, A. L., Scott, D. A., Michielutti, R. E., Bixby, A., Carrol, S. A., Bourgeois, A. L. & Guerry, P. (2000). Campylobacter jejuni cytolethal distending toxin mediates release of interleukin-8 from intestinal epithelial cells. Infect Immun 68, 6535-6541.
Jin, S., Joe, A., Lynette, J., Hani, E. K., Sherman, P. & Chan, V. L. (2001). JplA, a novel surface-exposed lipoprotein specific to Campylobacter jejuni, mediates adherence to host epithelial cells. Mol Microbiol 39, 1225-1236.[Medline]
Jobin, C. & Sartor, R. B. (2000). The IB/NF-
B system: a key determinant of mucosal inflammation and protection. Am J Physiol Cell Physiol 278, C451-C462.
Jobin, C., Herfarth, H. H. & Sartor, R. B. (1996). Dexamethasone inhibits TNF- gene expression through an I
B/NF-
B pathway in intestinal IEC-6 cells. Gastroenterology 108, A-844.
Jobin, C., Haskill, S., Mayer, L., Panja, A. & Sartor, R. B. (1997). Evidence for altered regulation of IB
degradation in human colonic epithelial cells. J Immunol 158, 226-234.[Abstract]
Jobin, C., Bradham, C. A., Narula, A. S., Brenner, D. A. & Sartor, R. B. (1999). Curcumin blocks cytokine mediated NF-B activation & proinflammatory gene expression by inhibiting IKK activity. J Immunol 163, 3474-3483.
Jung, H. C., Eckmann, L., Yang, S.-K., Panja, A., Fierer, J., Morzycka-Wroblewska, E. & Kagnoff, M. F. (1995). A distinct array of proinflammatory cytokines is expressed in human colon epithelial cells in response to bacterial invasion. J Clin Invest 95, 55-65.[Medline]
Karlyshev, A. V. & Wren, B. W. (2001). Detection and initial characterization of novel capsular polysaccharide among diverse Campylobacter jejuni strains using alcian blue dye. J Clin Microbiol 39, 279-284.
Kervella, M., Pages, J. M., Pei, Z., Grollier, G., Blaser, M. J. & Fauchère, J. L. (1993). Isolation and characterization of two Campylobacter glycine-extracted proteins that bind to HeLa cell membranes. Infect Immun 61, 3440-3448.[Abstract]
Ketley, J. M. (1997). Pathogenesis of enteric infection by Campylobacter. Microbiology 143, 5-21.
Kirkland, S. C. (1985). Dome formation by a human colonic adenocarcinoma cell line (HCA-7). Cancer Res 45, 3790-3795.[Abstract]
Konkel, M. E., Garvis, S. G., Tipton, S. L., Anderson, D. E. & Cieplak, W. (1997). Identification and molecular cloning of a gene encoding a fibronectin-binding protein (CadF) from Campylobacter jejuni. Mol Microbiol 24, 953-963.[Medline]
Kunsch, C. & Rosen, C. A. (1993). NF-B subunit-specific regulation of the interleukin-8 promoter. Mol Cell Biol 13, 6137-6146.[Abstract]
Matsusaka, T., Fujikawa, K., Nishio, Y., Mukaida, N., Matsushima, K., Kishimoto, T. & Akira, S. (1993). Transcription factors NF-IL-6 and NF-B synergistically activate transcription of the inflammatory cytokines, interleukin-6 and interleukin-8. Proc Natl Acad Sci USA 90, 10193-10197.[Abstract]
Mellits, K. H., Hay, R. T. & Goodbourn, S. (1993). Proteolytic degradation of MAD3 (IB
) and enhanced processing of the NF-
B precursor p105 are obligatory steps in the activation of NF-
B. Nucleic Acids Res 21, 5059-5066.[Abstract]
Moser, I., Schroeder, W. & Salnikow, J. (1997). Campylobacter jejuni major outer membrane protein and a 59-kDa protein are involved in binding to fibronectin and INT 407 cell membranes. FEMS Microbiol Lett 157, 233-238.[Medline]
Mukaida, N., Mahe, Y. & Matsushima, K. (1990). Cooperative interaction of nuclear factor-B and cis-regulatory enhancer binding protein-like factor binding elements in activating the interleukin-8 gene by pro-inflammatory cytokines. J Biol Chem 265, 21128-21133.
Mukaida, N., Okamoto, S., Ishikawa, Y. & Matsushima, K. (1994). Molecular mechanism of interleukin-8 gene expression. J Leukoc Biol 56, 554-558.[Abstract]
Nachamkin, I., Allos, B. M. & Ho, T. (1998). Campylobacter species and Guillian-Barre syndrome. Clin Microbiol Rev 11, 555-567.
Parkhill, J., Wren, B. W., Mungall, K. & 18 other authors (2000). The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences. Nature 403, 665668.[Medline]
Pei, Z. & Blaser, M. J. (1993). PEB1, the major cell-binding factor of Campylobacter jejuni, is a homolog of the binding component in Gram-negative nutrient transport systems. J Biol Chem 268, 18717-18725.
Pei, Z., Burucoa, C., Grignon, B., Baqar, S., Huang, X., Kopecko, J., Bourgeois, A. L., Fauchere, J. L. & Blaser, M. J. (1998). Mutation in the peb1A locus of Campylobacter jejuni reduces interactions with epithelial cells and intestinal colonization of mice. Infect Immun 66, 938-943.
Philpott, D. J., Yamaoka, S., Israel, A. & Sansonetti, P. J. (2000). Invasive Shigella flexneri activates NF-B through a lipopolysaccharide-dependent innate intracellular response and leads to IL-8 expression in epithelial cells. J Immunol 165, 903-914.
Pugin, J., Schurer-Maly, C.-C., Leturcq, D., Moriarty, A., Ulevitch, R. J. & Tobias, P. S. (1993). Lipopolysaccharide activation of human endothelial and epithelial cells is mediated by lipopolysaccharide-binding protein and soluble CD14. Proc Natl Acad Sci USA 90, 2744-2748.[Abstract]
Rees, J. H., Gregson, N. A., Griffiths, P. L. & Hughes, R. A. C. (1993). Campylobacter jejuni and Guillain-Barre syndrome. Q J Medicine 86, 623-634.[Medline]
Rodriguez, M. S., Thompson, J., Hay, R. T. & Dargemont, C. (1999). Nuclear retention of IB
protects it from signal-induced degradation and inhibits nuclear factor
B transcriptional activation. J Biol Chem 274, 9108-9115.
Saleha, A. A., Mead, G. C. & Ibrahim, A. L. (1998). Campylobacter jejuni in poultry production and processing in relation to public health. World Poultry Sci J 54, 49-58.
Savkovic, S. D., Koutsouris, A. & Hecht, G. (1997). Activation of NF-B in intestinal epithelial cells by enteropathogenic Escherichia coli. Am J Physiol Cell Physiol 273, C1160-C1167.
Schmid, R. M. & Adler, G. (2000). NF-B/Rel/I
B: implications in gastrointestinal diseases. Gastroenterology 118, 1208-1228.[Medline]
Schroder, W. & Moser, I. (1997). Primary structure analysis and adhesion studies on the major outer membrane protein of Campylobacter jejuni. FEMS Microbiol Lett 150, 141-147.[Medline]
Schulte, R., Grassl, G. A., Preger, S., Fessele, S., Jacobi, C. A., Schaller, M., Nelson, P. J. & Autenrieth, I. B. (2000). Yersinia enterocolitica invasion protein triggers IL-8 production in epithelial cells via activation of Rel p65-p65 homodimers. FASEB J 14, 1471-1484.
Silverman, N. & Maniatis, T. (2001). NF-B signalling in mammalian and insect innate immunity. Genes Dev 15, 2321-2342.
Tauxe, R. V. (1992). Epidemiology of Campylobacter jejuni infections in the United States and other industrialized nations. In Campylobacter jejuni: Current Status and Future Trends. Edited by I. Nachamkin, M. J. Blaser & L. S. Tompkins. Washington, DC: American Society for Microbiology, pp. 919.
Visvanathan, K. V. & Goodbourn, S. (1989). Double-stranded RNA activates binding of NF-B to an inducible element in the human ß-interferon promoter. EMBO J 8, 1129-1138.[Abstract]
Yasumoto, K., Okamoto, S., Mukaida, N., Murakami, S., Mai, M. & Matsushima, K. (1992). Tumour necrosis factor and interferon
synergistically induce interleukin-8 production in a human gastric cancer cell line through activating concurrently on AP-1 and NF-
B-like binding sites of the interleukin-8 gene. J Biol Chem 267, 22506-22511.
Received 1 March 2002;
revised 1 May 2002;
accepted 15 May 2002.