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The Journal of Immunology, 2008, 181, 1438 -1445
Copyright © 2008 by The American Association of Immunologists, Inc.
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Selective Activation of Human Intestinal Mast Cells by Escherichia coli Hemolysin1

Sigrid Krämer2,*, Gernot Sellge2,{dagger}, Axel Lorentz*, Dagmar Krueger{ddagger}, Michael Schemann{ddagger}, Katharina Feilhauer§, Florian Gunzer and Stephan C. Bischoff3,*

* Department of Nutritional Medicine and Immunology, University of Hohenheim, Stuttgart, Germany; {dagger} Pathogénie Microbienne Moléculaire-Institut National de la Santé et de la Recherche Médicale Unité 786, Institut Pasteur, Paris, France; {ddagger} Department of Human Biology, Technical University Munich, Munich, Germany; § Clinic for Visceral Surgery, Katharinenhospital, Stuttgart, Germany; and Institute of Medical Microbiology and Hygiene, Faculty of Medicine Carl Gustav Carus, TU Dresden, Dresden, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Mast cells (MCs) are recognized to play an important role in bacterial host defense in the murine system. In this study, we studied the interaction of human MCs, isolated from the intestine and purified to homogeneity, with different Escherichia coli and Shigella flexneri strains. We show that {alpha}-hemolysin (Hly)-producing E. coli strains induce the release of histamine, leukotrienes, and proinflammatory cytokines in intestinal MCs. In contrast, MCs were virtually unresponsive to S. flexneri and several Hly-negative E. coli strains, including the isogenic Hly-deficient mutants of Hly+ strains. Hly+ E. coli but not Hly– E. coli caused an increase in intracellular Ca2+ levels. Blocking of extracellular Ca2+ and of the calmodulin/calcineurin pathway by cyclosporin A inhibited the response to Hly+ E. coli. Furthermore, inhibition of MAPKs p38 and ERK reduces activation of MCs by Hly+ E. coli. In addition, using an ex vivo system, we directly record the histamine release by MCs located in the lamina propria after infection with Hly+ E. coli. Our data indicate that human intestinal mast cells interact with selected Gram-negative bacteria, establish E. coli Hly as a factor regulating MC effector functions, and argue further for a role of human MCs in innate immunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
It has been presumed that mast cells (MCs),4 in addition to its well-established function in allergy play a relevant role in the innate immune response against pathogens (1, 2). Strong evidence that MCs protect the host against bacterial infections arose from studies using mouse models of Klebsiella pneumoniae-dependent lung infection and septic peritonitis. These studies showed that MC-derived TNF and leukotrienes mediate neutrophil influx and that MC deficiency impairs bacterial clearance and host survival (3, 4, 5). More recently, it has been shown that MCs can provide an important link between innate and adaptive immunity in bacterial infections (6, 7); however, the mechanisms of MC-pathogen interactions are not well known. Rodent MCs and human in vitro-derived MCs express "pattern recognition receptors" such as TLRs or CD48 known to interact with conserved microbiological structures. Moreover, bacterial components such as particular TLR agonists or the type I fimbrial protein FimH trigger MCs for the release of histamine, eicosanoids, and cytokines such as TNF, IL-1, and IL-6 (8, 9, 10, 11, 12, 13).

In this study, we investigated the interaction of human intestinal MCs with different Escherichia coli strains and Shigella flexneri, the causative agent of bacillary dysentery (14). S. flexneri and the majority of the E. coli strains including type 1 fimbriated E. coli known to activate murine and human cord blood-derived MCs had very little effect on intestinal MCs; however, we found that human intestinal MCs are highly sensitive target cells for an E. coli {alpha}-hemolysin (Hly) attack. Hly+ E. coli induced a biphasic response in human intestinal MCs accompanied by a Ca2+ influx and MAPK activation leading to the release of cytokines and inflammatory mediators followed by a phase of cell lysis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Bacteria

The human fecal isolate E. coli (O101:H-), the probiotic strain E. coli Nissle 1917 (EcN) (06:H1, Mutaflor; Ardeypharm), E. coli ATCC 25922 (Ec25) (O6:H1), E. coli ATCC 35218 (Ec35) (06:H31; both American Type Culture Collection), E. coli ORN103(pSH2) (expresses the pSH2 plasmid, encoding for the entire type 1 fimbrial gene cluster including FimH), E. coli ORN103(pUT2002) (isogenic FimH mutant of E. coli ORN103(pSH2), both described recently) (15), S. flexneri M90T (wild-type virulent strain, serotype 5a), and S. flexneri BS176 (nonvirulent, noninvasive derivative of M90T cured of the 220-kb virulence plasmid) (16) were used in this study. EcN was used as host strain for the introduction of plasmid pSF4000 (EcN Hly+) encoding for the Hly determinant, hlyCABD, and the control plasmid pACYC184 (EcN Hly–) (17). The isogenic, Hly-negative mutant of Ec25 (Ec25{Delta}Hly) and Ec35 (Ec35{Delta}Hly) was constructed using a suicide vector mutagenesis as described previously (18). Bacteria were grown overnight in Luria-Bertani medium at 37°C on a shaker and then subcultured and grown to late-log phase over 2–3 h before use. Chloramphenicol (40 µg/ml) was added for growing E. coli ORN103(pSH2), ORN103(pUT2002), EcN Hly+, and EcN Hly–.

Isolation, purification, and culture of human intestinal MCs

MCs were isolated from surgical tissue specimens (macroscopically normal tissue) derived from individuals who underwent bowel resection because of cancer. Permission to conduct the study was obtained from the local ethical committee of the Medical School of Hannover (Hannover, Germany). The methods of mechanical and enzymatic tissue dispersion yielding single-cell preparations have been described (19). After overnight culture of the cell suspension in culture medium (RPMI 1640 supplemented with 10% heat-inactivated FCS, 25 mM HEPES, 2 mM glutamine, 100 µg/ml streptomycin, 100 µg/ml gentamicin, 100 U/ml penicillin, and 0.5 µg/ml amphotericin; all from Invitrogen) c-kit-expressing MCs were enriched by positive selection using magnetic cell separation (MACS System; Miltenyi Biotec) and the anti-c-kit mAb YB5.B8 (5 ng/ml; BD Pharmingen) as described elsewhere (19). The fraction containing the c-kit-positive cells (MC purity, 50–90%) was cultured at a density of 1–2 x 105 MC/ml in the presence of recombinant stem cell factor (SCF, 50 ng/ml; Amgen) and IL-4 (2 ng/ml; Novartis). In some indicated experiments, MCs were cultured only in the presence of SCF. After 2–4 wk, MC purity increased to 98–100%. In some experiments in which MCs were not enriched by positive selection before culture, MC purity increased from 1 to 5 to 70- 99% after 3–4 wk of culture. Only cell preparations with a purity >98% were used for additional experiments. Once a week half of the culture medium was exchanged and cytokines were supplemented again.

Infection of MCs with bacteria

MCs (5–10 x 104) suspended in 200 µl of RPMI 1640 were incubated with bacteria at a 1:100 multiplicity of infection (MOI). Bacteria were centrifuged onto the cells at 400 x g for 5 min. For positive control, MCs were stimulated using the mAb 22E7 (100 ng/ml; Hoffmann-La Roche) directed against the high-affinity Fc{epsilon}RI {alpha}-chain. In some experiments, MCs were stimulated with culture supernatants derived from bacteria grown in late-log phase in RPMI 1640 (v/v 1/1). Cells and supernatants were harvested at indicated time points to analyze MC viability by trypan blue staining, mRNA expression, and release of cytokines (ELISA; BioSource International), histamine (RIA; Coulter-Immunotech), sulfido-leukotrienes (sLTs), and leukotriene B4 (LTB4) (ELISA; Biotrend). Apoptosis of MCs was determined using the Apo-ONE Homogeneous Caspase 3/7 Assay (Promega).

RNA preparation and RT-PCR

Total RNA was prepared from 5 to 10 x 104 MCs and RT-PCR was performed as described previously (20). The following primers were used for quantitative real-time RT-PCR: GDH (5'-TGG TCT CCT CTG ACT TCA AC-3'; 5'-CCT GTT GCT GTA GCC AAA TT-3' (133 bp; 56°C)), TNF-{alpha} (5'-CAA GCC TGT AGC CCA TGT TG-3'; 5'-AGA GGA CCT GGG AGT AGA TG-3' (151 bp; 56°C)), IL-5 (5'-ACT CTT GCA GGT AGT CTA GG-3'; 5'-GGA ATA GGC ACA CTG GAG AGT CAA-3' (157 bp; 56°C)), and CXCL8 (5'-CTG AGA GTG ATT GAG AGT GG-3'; 5'-ACA ACC CTC TGC ACC CAG TT-3' (112 bp; 56°C)). To quantify the mRNA expression, real-time PCR containing 1.5 µl of cDNA template (sample or standard), 10.5 µl of H2O, 12.5 µl of SYBR Green PCR Master Mix (Applied Biosystems), and 0.3 µl of 20 µM sense and antisense primers were performed in Optical tubes (Applied Biosystems). All reactions were performed in the Applied Biosystems PRISM 7700 Sequence Detector. Specificity of the reaction was controlled by creating a melt curve after each run. To calculate the relative transcription level of the genes of interest, the copy number for the mRNA of the gene of interest was calculated and divided by that of GAPDH mRNA.

Calcium mobilization

To measure changes in the cytosolic concentration of free calcium, MCs were labeled with Fluo-3 and Fura Red (4 µg/ml and 10 µg/ml, respectively; Molecular Probes) in the presence of probenicid (4 mM; Sigma-Aldrich) and pluronic F-127 (0.02%; Molecular Probes) for 30 min at 30°C in the dark, washed, and resuspended in 500 µl of RPMI 1640. MCs were stimulated with 500 µl of culture supernatants obtained from Ec25 or Ec25{Delta}Hly grown in a late-log phase in RPMI 1640. The changes in intracellular calcium were measured using excitation at 488 nm in a flow cytometer (FACSCanto; BD Biosciences). The relative ratios of fluorescence emitted at 530 and 670 nm vs time were recorded and displayed as a reflection of intracellular concentrations of calcium. Analyses were performed using the FlowJo software (Tree Star).

Western blot analysis

To obtain whole cell extracts for Western blot, 0.3–0.5 x 106 MCs were lysed in extraction buffer containing 25 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 0.5 mM EGTA, and 0.05% Triton X-100 supplemented with the protease inhibitor mixture Complete Mini (Roche Diagnostics). Cell extracts (10–20 µg of protein) were separated on a 12% SDS-polyacrylamide gel and proteins were transferred to Hybond T-P polyvinylidene difluoride membranes using a semidry electroblotter. The resulting blots were then probed with primary Abs against anti-ERK (MAPK) 2 and anti-phospho-ERK (MAPK) 1/2 mAbs (Alexis), respectively, and bands were visualized using a Super Signal Western Dura kit (Pierce).

Inhibitors and neutralizing Abs

In some experiments, MCs were treated with inhibitors 60 min before infection and Fc{epsilon}RI cross-linking. The following inhibitors and neutralizing Abs, respectively, were used in this study: cyclosporin A (1 µmol/L; Novartis), apigenin (20 µmol/L; Calbiochem), Gö 6976 (2 µmol/L; Calbiochem), wortmannin (100 nmol/L; Calbiochem), PD98059 (30 µM; BioSource International), PD169316 (10 µM; BioSource International), anti-TLR2 (clone TL2.1; eBioscience), and anti-TLR4 (clone HTA125; eBioscience).

Ussing chamber experiments

To test the effects of E. coli strains in intact submucosa/mucosa preparations of the human intestine, we used Ussing chamber techniques (Easy Mount Chambers; Physiologic Instruments). The area of the recording chamber was 0.5 cm2. Mucosal and serosal sides were bathed separately in 2 ml of Krebs solution. The bath was maintained at 37°C and continuously bubbled with 95% O2 and 5% CO2 (Carbogen). The transepithelial potential difference was measured by a pair of silver/AgCl electrodes connected to a voltage clamp apparatus (VCC 600; Physiologic Instruments) that compensated for the solution resistance between the electrodes. Chloride secretion was measured as short-circuit current (Isc) and expressed in µA cm–2. Positive Isc indicated a net anion current from the serosa to the lumen. Before starting the actual measurements, the tissues were allowed to equilibrate for at least 10 min. Ionomycin (10–9 M; Sigma-Aldrich) or bacteria were added to the mucosal side of the preparation. Spontaneous release of histamine was analyzed in the supernatant using a previously described method (21).

Statistics

Data are expressed as mean ± SEM. The Friedman test, followed by a post hoc Dunn’s test, was used for the determination of statistical significance among multiple groups. For parametric data, one-way ANOVA was performed. The Wilcoxon test was used to analyze differences between two groups. A value of p < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Infection of human intestinal MCs with selected E. coli strains induces cytokine mRNA expression, mediator release, and cell lysis

Infection of purified human intestinal MCs with the E. coli strains ATCC 25922 (Ec25) and ATCC 35218 (Ec35) caused significant induction of mRNA for IL-3, IL-5, IL-6, CXCL8, and TNF and pronounced release of histamine and sLTs# (Fig. 1, A–E and N). All effects were detectable after 90 min; however, the release of histamine was more pronounced after 3 and 6 h of infection (Fig. 1, F–K). Only marginal induction of cytokine mRNA expression and mediator release occurred after 90 min, 3 h, and 6 h of infection with the other bacteria strains tested including S. flexneri and E. coli ORN 103(pSH2)-expressing FimH (Fig. 1, A–K, and data not shown).


Figure 1
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  		FIGURE 1. Induction of cytokine mRNA expression and mediator release upon infection of human intestinal MCs with different E. coli and S. flexneri strains. MCs were incubated with indicated bacteria (MOI 1:100) or stimulated with mAb 22E7 inducing Fc{epsilon}RI cross-linking (positive control). TNF (A), CXCL8 (B), and IL-5 (C) mRNA expression after 6 h of incubation. D, sLT release after 90 min. E, Histamine release after 6 h. Time kinetics of mRNA expression for TNF (F), CXCL8 (G), and IL-5 (H) and of sLT (J) and histamine (K) release induced by mAb 22E7, EcN, Ec25, and Ec35.TNF (L) and CXCL8 (M) protein measured in the supernatants after 6 h of infection. A–M, Means ± SEM of five experiments are shown (D and J, n = 4). *, p < 0.05 in comparison to control. N, Induction of mRNA expression for TNF, IL-3, IL-5, IL-6, and CXCL8 after 6 h of infection with Ec25 and Ec35 analyzed by conventional RT-PCR. One representative experiment of three is shown.

 
In addition, we found that MCs produce TNF protein upon infection with Ec25 and Ec35, but not the other tested strains (Fig. 1L). CXCL8 protein release was induced by all bacterial strains; however, the production was more pronounced after challenge with Ec25 and Ec35 (Fig. 1M). The discrepancy between high induction of CXCL8 mRNA and a rather low protein production along with almost complete histamine liberation after 6 h of infection with Ec25 and Ec35 indicates MC death is preventing adequate mRNA translation (Fig. 2). S. flexneri M90T occasionally induced cell death upon 6 h of infection; however, this effect was significantly less pronounced than that of Ec25 and Ec35. No cell death was observed after infection of MCs with all other strains (Fig. 2, A, B, and D). Caspase 3/7 activity decreased after infection with Ec25 and Ec35 (Fig. 2C), suggesting that MC death was caused by cell necrosis rather than apoptosis.


Figure 2
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  		FIGURE 2. MC death upon infection with Ec25 and Ec35. A, Numbers of viable MCs after 6 h of incubation with the indicated bacteria strains (mean ± SEM, n = 5). *, p < 0.05 in comparison to control. B, Time dependency of cell death induction by Ec25 and Ec35 (mean ± SEM, n = 3). C, Caspase 3/7 activity in MCs after incubation with EcN, Ec25, or Ec35 for 90 min, 3 h, or 6 h (mean ± SEM, n = 3). D, Light microscopy of MCs after incubation with EcN and Ec35 for 6 h.

 
To exclude that the unresponsiveness of MCs to the other tested strains is not an artifact due to long-term IL-4 and SCF culture, we performed two experiments shortly after isolation and purification by magnetic cell separation (MACS) of human intestinal mast cells. The purity of these two mast cell isolates was 95 and 92% directly after MACS purification. Infection of MCs on day 3 after isolation with the other tested strains neither led to the release of histamine and sLT nor to the up-regulation of CXCL8 and TNF mRNA. In contrast, Ec25 induced the release of histamine (16.5 ± 2.5%) and sLT (2.2 ± 0.6 ng/106 MC) and caused induction of CXCL8 and TNF mRNA expression (1.8 ± 0.3 CXCL8/glycerol phosphate dehydrogenase and 0.17 ± 0.05 TNF/glycerol phosphate dehydrogenase, respectively). These experiments confirmed the results done with MCs after 3 or 4 wk of SCF and IL-4 culture. Since IL-4 changes the cytokine expression profile of intestinal MCs with enhanced production of IL-3, IL-5, and IL-13 and decreased production of IL-6 after Fc{epsilon}RI cross-linking (20), we compared the cytokine mRNA expression in MCs precultured with or without IL-4 after challenge with E. coli O101:H-, E. coli Nissle (EcN), Ec25, and Ec35. Only Ec25 and Ec35 up-regulated cytokine mRNA expression under both conditions. However, in MCs cultured without IL-4, Ec25 and Ec35 induced less IL-5 mRNA (53 ± 20% and 33 ± 26% reduction, respectively), the same amount of TNF-{alpha} mRNA, and more CXCL8 mRNA (86 ± 46% and 32 ± 19% increase, respectively; all mean ± SEM, n = 3, data not shown) compared with MCs cultured with IL-4 (condition used throughout the study).

E. coli Hly is essential for MC activation

Because Ec25 and Ec35, but not the other tested strains, produce Hly we hypothesized that MC activation and subsequent cell death induction by these strains might depend on E. coli Hly. To test this hypothesis, we transformed the Hly-negative strain EcN with the recombinant plasmid pSF4000 expressing the genes of the Hly operon. EcN (pSF4000) (EcN Hly+), but not EcN carrying the control plasmid pACYC184 (EcN Hly–), induced high amounts of mRNA for TNF, IL-5, and CXCL8, the release of sLT and histamine, as well as MC membrane damage (Fig. 3, A–G). In addition, we created mutant strains of Ec25 and Ec35 lacking the functional hlyA gene (Ec25{Delta}Hly and Ec35{Delta}Hly). These mutant strains had a greatly reduced capacity of inducing cytokine mRNA expression, histamine and sLT secretion, and cell death in MCs (Fig. 3, H–N). We also found production of LTB4 in response to Hly+, but not Hly– E. coli strains (Fig. 3, E and L).


Figure 3
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  		FIGURE 3. MC activation by E. coli depends on the production of Hly. A–G, MCs were infected with EcN Hly–, EcN Hly+, or were left untreated (Con). Expression of mRNA for TNF (A), CXCL8 (B), and IL-5 (C) after 3 h. Release of sLT (D), LTB4 (E), and histamine (F) after 3 h. G, MC viability after 3 and 6 h. H–M, MCs were infected with Ec25 (25 WT), Ec25{Delta}Hly (25 {Delta}Hly), Ec35 (35 WT), Ec35 {Delta}Hly (35 {Delta}Hly), or were left untreated (Con). CXCL8 (H) and IL-5 (J) mRNA expression and release of sLT (K), LTB4 (L), and histamine (M) after 3 h. N, MC viability after 3 and 6 h. Means ± SEM from nine (A–C), seven (F), five (H, J, and M), or three (D, E, G, K, L, and N) experiments are shown. *, p < 0.05 and **, p < 0.01 (in comparison to control if not indicated otherwise).

 
It is likely that MC lysis accounts for the majority of histamine released after 3 and 6 h of infection. The question remains whether MC activation by Hly can occur without subsequent cell death. Therefore, we challenged MCs with lower concentrations of Hly+ E. coli. Infection of MCs with Ec25 MOI 1:1 did not induce cell death up to 6 h postinfection. However, CXCL8 mRNA expression and histamine release was up-regulated ~3- or 2-fold, respectively, compared with nontreated controls (Fig. 4, A–C). Furthermore, culture supernatants of Ec25, but not the supernatant of the EC25{Delta}Hly strain, provoked cytokine mRNA expression and the release of histamine in MCs (Fig. 4, D and E). Supernatant of Ec25 did not induce cell death upon 6 h of challenge (Fig. 4F). This finding confirms that sublytic concentrations of Hly induce activation of MCs independent of cell death.


Figure 4
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  		FIGURE 4. MC activation upon infection with lower concentrations of Hly+ E. coli and bacterial supernatants. A–C, MCs were infected with Ec25 (MOI 1:100, 1:10, and 1:1) or medium as control (Con). Expression of mRNA for CXCL8 (A) and release of histamine (B) after 3 h of challenge. C, MC viability after 3 and 6 h. D–F, MCs were challenged with culture supernatants derived from Ec25 (25 WT), Ec25{Delta}Hly (25 {Delta}Hly), or medium as control (Con). Expression of mRNA for CXCL8 (D) and release of histamine (E) after 3 h of challenge. F, MC viability after 3 and 6 h. G, Histamine release at indicated time points after infection with Ec25 or Ec25{Delta}Hly. Means ± SEM from five experiments are shown. *, p < 0.05.

 
In addition, Ec25, but not Ec25{Delta}Hly, induced histamine release after only 15 min of infection. The amount of histamine released by MCs remained stable for up to 90 min after infection and only increased after 3 h when cell death started to occur (Fig. 4G). This observation further suggests that early histamine release is caused by degranulation rather than membrane damage.

Hly+ E. coli induces Ca2+flux

Binding of Hly to cell membranes and subsequent pore formation has been shown to cause flux of extracellular Ca2+ into the cells. We found that culture supernatants of Ec25, but not supernatants of Ec25{Delta}Hly, increased intracellular Ca2+ levels in MCs, the signal kinetics being different than those induced by Fc{epsilon}RI cross-linking (Fig. 5, A and B). It is tempting to speculate that Ca2+ influx was directly responsible for cell activation. To confirm this hypothesis, we conducted experiments with the Ca2+ chelator EDTA. MCs were incubated with Ec25 supernatants or control medium on ice in Ca2+-containing cell culture medium for 15 min. This allowed the toxin to bind in the absence of pore formation (22). Because Hly binding to membranes depends on Ca2+, neutralization of extracellular Ca2+ would block pore formation. Following two washes in ice-cold buffer without Ca2+, cells were resuspended in culture medium with or without EDTA at 37°C to allow pore formation of membrane-bound Hly. Expression of mRNA levels was analyzed after 3 h of culture. Under these modified conditions, Hly-containing Ec25 supernatant strongly up-regulated the expression of TNF (6.1 ± 2.7-fold), CXCL8 (9.0 ± 4.1-fold), and IL-5 (9.1 ± 5.0-fold, all mean ± SEM, n = 4) in MCs. As hypothesized, induction of cytokine mRNA expression in response to Hly-containing Ec25 was strongly inhibited through EDTA (Fig. 5C).


Figure 5
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  		FIGURE 5. Hly-induced Ca2+ signals. A and B, Ca2+ flux induced by stimulation of MCs with (A) culture supernatants derived from Ec25 (25 WT, black line) and Ec25{Delta}Hly (25 {Delta}Hly, gray line) and (B) mAb 22E7. Each graph shows one representative experiment of four. Arrow indicates the time point when stimulants were added. C, MCs were incubated with Ec25 supernatants or control medium on ice in Ca2+-containing cell culture medium and than cultured in medium with or without EDTA (1 mM). MCs stimulated with mAb 22E7 inducing Fc{epsilon}RI cross-linking in the presence or absence of EDTA (1 mM) served as positive control. Expression of mRNA for TNF, CXCL8, and IL-5 after 3 h is shown (expressed as percentage of specific induction in EDTA-treated cells in comparison to nontreated control cells (mean ± SEM, n = 4). MCs were treated with cyclosporin A (1 µM; D), Gö 6976 (2 µM; E), and wortmannin (100 nM; G) for 60 min before challenge with Ec25 and mAb 22E7 for 3 h. Expression of mRNA for TNF, CXCL8, and IL-5 and sLT release are shown (expressed as percentage of specific induction in nontreated control cells (mean ± SEM, n = 6; n = 3 for sLT). *, p < 0.05 (in comparison to control if not indicated otherwise).

 
To target Ca2+-dependent downstream signaling pathways, MCs were incubated with cyclosporin A inhibiting calcineurin, and Gö 6976 inhibiting Ca2+-dependent protein kinase C (PKC) {alpha}-isozyme and PKCβI. Cyclosporin A strongly decreased mRNA cytokine expression and sLT release in response to Ec25 and Fc{epsilon}RI cross-linking (Fig. 5D). Interestingly, Gö 6976 completely inhibited the response of MCs to cross-linking of the Fc{epsilon}RI, but only slightly blocked the Ec25-dependent effects (Fig. 5E). These results suggest that Ca2+ downstream signaling through the calmodulin/calcineurin pathway is required to establish the response to Hly+ E. coli, whereas PKC{alpha} and PKCβI are less important.

PI3K-dependent pathways regulate Fc{epsilon}RI-mediated calcium mobilization, degranulation, and cytokine production (23). The PI3K inhibitor wortmannin almost completely blocked the Fc{epsilon}RI-dependent response in intestinal MCs, whereas it had virtually no effects on MCs activated by Ec25 (Fig. 5F). This indicates that PI3K is not required to induce sLT release and TNF, CXCL8, and IL-5 mRNA expression in MCs after infection with Hly+ E. coli.

MAPK activation by Hly+ E. coli

The MAPK ERK has been shown to be necessary for transduction of signals derived from Fc{epsilon}RI aggregation and IL-4R activation in MCs (24, 25). We found that infection with Ec25 causes phosphorylation of ERK1/2 in MCs. ERK1/2 phosphorylation was also induced by Ec25{Delta}Hly, albeit a somewhat weaker response than by Ec25 (Fig. 6A). Furthermore, we observed enhanced mRNA transcription of c-fos, a downstream target of the MAPK ERK and p38, in MCs infected with Ec25. Ec25{Delta}Hly-dependent transcription of c-fos was less pronounced compared with Ec25-dependent c-fos activation. However, this difference was not statistically significant (Fig. 6B). The functional relevance of MAPK activation in the response of MCs to Ec25 is confirmed by the fact that the nonspecific MAPK inhibitor apigenin blocked ~80% of the cytokine mRNA expression and sLT secretion (Fig. 6C). In addition, we treated MCs with specific inhibitors for MEK, the upstream kinase of ERK, and p38. The MEK inhibitor PD98059 almost completely blocked Fc{epsilon}RI-dependent cytokine mRNA transcription and inhibited modestly Ec25-induced TNF and CXCL8 mRNA expression. However, PD98059 blocked effectively the release of sLT in response to Fc{epsilon}RI cross-linking and Ec25 (Fig. 6D). The p38 inhibitor PD169316 blocked 70–90% of cytokine mRNA expression and sLT secretion provoked by Ec25. PD169316 inhibited almost completely Fc{epsilon}RI-dependent TNF and CXCL8 mRNA expression and ~70% of Fc{epsilon}RI-induced sLT release, whereas it had only marginal effects on IL-5 mRNA expression induced by Fc{epsilon}RI cross-linking (Fig. 6E). These data indicate that the MAPK p38 and ERK are required for MC activation upon infection with EC25.


Figure 6
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  		FIGURE 6. MAPK is critical for the response of MCs to Hly+ E. coli. A, Activation of ERK1/2 in MCs challenged with Ec25 WT or Ec25{Delta}Hly was analyzed by Western blotting using Abs specific for the phosphorylated kinase. After stripping of the blot, reprobing with an Ab against total ERK1/2 served as equal loading control. One representative experiment of three is shown. B, c-fos mRNA expression after 30-min incubation with mAb 22E7, Ec25 (25 WT), or Ec25{Delta}Hly (25 {Delta}Hly). C–E, MCs were treated with apigenin (20 µM; C), PD98059 (30 µM; D), or PD169316 (10 µM; E) for 60 min before challenge with mAb 22E7 or Ec25. Expression of mRNA for TNF, CXCL8, and IL-5 and sLT release after 3 h is shown (expressed as percentage of specific induction in nontreated control cells). Means ± SEM from six (C; n = 3 for sLT), five (B), or three (D and E) experiments are shown. *, p < 0.05 (in comparison to control if not indicated otherwise).

 
TLR-independent activation of MCs by Hly+ E. coli

To determine the role of TLR in MC activation by Hly+ E. coli, MCs were incubated with neutralizing Ab against TLR2 or TLR4 as described previously (26, 27). The effectiveness of these neutralizing Abs was checked in control experiments with human peripheral blood monocytes. Neither anti-TLR2 nor anti-TLR4 Ab leads to a decrease of TNF and CXCL8 mRNA expression (Fig. 7, A and B). Taken together, these findings suggest that TLR2 and TLR4 are not involved in the Hly+ E. coli-dependent MC activation.


Figure 7
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  		FIGURE 7. TLR are not involved in the activation of MCs by Hly+ E. coli. MCs were treated with anti-TLR2 (20 µg/ml; A) and anti-TLR4 (20 µg/ml; B) for 60 min before challenge with Ec25 and mAb 22E7 for 3 h. As control, peripheral blood monocytes were stimulated with LPS (1 µg/ml) and LTA# (100 µg/ml), respectively, alone or along with anti-TLR4 or anti-TLR2 Ab. Expression of mRNA for TNF and CXCL8 is shown (expressed as percentage of specific induction in nontreated control cells (mean ± SEM, n = 4).

 
Hly+ E. coli induces histamine release in an ex vivo system

Hly might act as a soluble toxin released at the mucosal side. Since in our study bacteria (or culture supernatants) were in direct contact with the MCs, we were interested whether Hly released at the apical side of the mucosa might enter the lamina propria to activate MCs. Therefore, hemolytic and nonhemolytic bacteria, respectively, were added to the mucosal side of human intestinal tissue and histamine release was measured in the supernatant. During the experimental stage, the total tissue conductance was constant at 30–35 µA cm–2 in the control (Fig. 8A, upper trace). In contrast, the tissue conductance increased over time after challenge with ionomycin, Ec25, and Ec25{Delta}Hly. Both bacteria strains evoked a transient chloride secretion; however, the secretory response induced by Ec25{Delta}Hly was less pronounced compared with Ec25, suggesting that Hly enhances the E. coli-induced ion flux. To analyze whether the mucosal application of Hly+ and Hly- E. coli leads to MC degranulation, we measured the histamine content in the supernatants at the mucosal side of the Ussing chamber 2 h after ionomycin or the bacteria were added. Total amount of histamine content was 16.3 ± 4.8 ng/ml. Whereas ionomycin and Ec25 induced an enhanced secretion of histamine in the supernatant, the histamine content after challenge with Ec25{Delta}Hly did not change compared with nontreated controls (Fig. 8B). This indicates that the presence of Hly+ E. coli at the mucosal side can lead to an activation of MCs located in the lamina propria.


Figure 8
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  		FIGURE 8. Hly+ E. coli activate MCs in an ex vivo model. A and B, Ussing chamber experiments with human colonic tissue. Ionomycin (10–9 M), Ec25 WT or Ec25{Delta}Hly was added to the mucosal side. A, Chloride secretion was measured as Isc and expressed in µA cm–2. B, Histamine release in the supernatant was measured after 2 h. Means ± SEM from three patients. *, p < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Cytolytic pore-forming toxins (PFTs) comprise ~25% of all known bacterial protein toxins. PFTs contribute to and are sometimes essential for virulence (28). The pathogenic relevance of the pore-forming toxin Hly, a member of the RTX (repeats in toxin) toxin family, has been established in animal models and in humans (29, 30). E. coli Hly is produced by ~50% of E. coli isolates causing infections at extraintestinal locations such as the urinary tract. The role of Hly in intestinal pathologies is unclear at present. Most fecal isolates do not express Hly, while the percentage of hemolytic E. coli is increased in patients suffering from inflammatory bowel disease as well as in IgA-deficient individuals (31, 32, 33). In those patients, the progressive breakdown of the mucus barrier leads to translocation of bacteria in the subepithelium (34, 35, 36).

In this study, we show that E. coli Hly is a unique pathogen-derived factor causing activation of human intestinal MCs. Most of the tested Gram-negative bacterial strains, including S. flexneri and FimH-expressing E. coli, were ineffective in triggering human MCs for the production of inflammatory mediators. This finding was surprising because it was demonstrated that murine MCs and human in vitro-developed MCs respond to type I fimbriae protein, FimH, via binding to CD48 (8, 13) and to certain TLR ligands including LPS (1, 12). In contrast, the hemolytic E. coli strains Ec25 and Ec35 provoked cytokine mRNA expression and release of histamine, leukotrienes, and cytokines in intestinal MCs. Prolonged infection of MCs with hemolytic E. coli induced cell death after 3 h of incubation. In this study, we provide strong evidence that the bacterial exotoxin Hly is crucial for the response of intestinal MCs. Although nonhemolytic EcN does not activate MCs, we show that MCs strongly respond to infection with EcN transformed with a plasmid encoding for the Hly operon. In addition, Hly-deficient mutants of Ec25 and Ec35 failed to activate MCs. Thus, our data clearly indicate that bacterial stimulation and lysis of MCs was dependent on the production of Hly. However, cell lysis occurred subsequently and independently of MC activation. Intestinal MCs respond rapidly to Hly+ E. coli with histamine release being detected after 15 min and cell activation was also found in nonlytic conditions when MCs were challenged with bacterial cell culture supernatants.

The common view of Hly and other PFTs as virulence factors has long been ascribed to the cytolytic properties of the toxin. However, many mammalian cells do not lyse rapidly when treated with low concentrations of PFTs, but rather mount a cellular response which may be important for the innate immune defense against the pathogen. It has been shown that E. coli Hly mediates degranulation and production of eicosanoids, NO, respiratory burst, and different cytokines such as IL-1β, IL-6, and CXCL8. Various cells including human neutrophils, basophils, macrophages, and endothelial cells can be targeted by Hly (22, 37, 38, 39, 40, 41). Studies performed in the 1980s suggested that Hly+ E. coli provoke histamine release in rat MCs (38, 39).

Cellular responses induced by PFTs have been suggested to provoke a spectrum of events that depend on the target cell and type of PFT. Ion flux along their respective electrochemical gradients, e.g., Ca2+ influx and potassium efflux, are the earliest effects of pore formation (28). Sublytic doses of E. coli Hly have been reported to induce low-frequency Ca2+ oscillations in epithelial cells as a consequence of formation and rapid closure of Hly pores in cell membranes (42, 43). Ca2+ influx has been shown to be responsible for the production of IL-6 and CXCL8 in epithelial cells and oxidative burst production in granulocytes (22, 42). We show here that culture supernatants of Hly+ E. coli, but not supernatants, derived from the respective mutant induce a Ca2+ signal with a slow kinetic in MCs; however, we could not detect Ca2+ oscillations in human MCs with the methods used in this study. Ca2+ chelation and treatment with cyclosporin A almost abrogated the cellular responses of MCs, indicating that Ca2+ influx and downstream signaling through the calmodulin/calcineurin pathway is required to establish the response to Hly+ E. coli. Interestingly, pharmacological blockage of Ca2+-dependent PKC{alpha} and PKCβI or PI3K affected only slightly Hly+ E. coli-induced MC activation, whereas it strongly impaired Fc{epsilon}RI-mediated cell responses. These results clearly indicate that MC activation by Fc{epsilon}RI cross-linking and Hly involve different signal transduction pathways.

Our studies also show that MC activation by Hly+ E. coli is dependent on MAPK activation. Inhibition of MEK and p38 decreased MC cytokine mRNA expression and sLT release provoked by Hly+ E. coli. These results are supported by recent observations that p38 becomes phosphorylated in response to various PFTs such as staphylococcal {alpha}-toxin, streptococcal exotoxin streptolysin O (SLO), pneumolysin, and anthrolysin (26). Osmotic stress rather than signaling through possible toxin receptors seems to be involved in p38 activation (44). SLO has been shown to phosphorylate p38, resulting in TNF release in mouse bone marrow-derived MCs (45).

Although we show convincingly that Hly is necessary for E. coli to evoke MC activation, we cannot rule out the possibility that other bacterial factors are also required. Noteworthy is that Hly– E. coli induces ERK phosphorylation and up-regulation of c-fos mRNA in intestinal MCs, although the effect is less pronounced than with Hly+ E. coli. Possibly, Hly– E. coli are recognized by MCs but the signals are not sufficient to induce a functional response. Our unpublished results revealed that human intestinal MCs express several TLRs, including TLR2 and TLR4, as has been reported for MCs from other origins (1, 9, 10, 11, 12). According to these data, intestinal MCs should also be capable of recognizing all Gram-negative bacteria through their LPS. However, the functionality of TLRs on intestinal MCs remains elusive. Treatment of intestinal MCs with neutralizing Abs against TLR2 or TLR4 had no effect on the cellular response to Hly+ E. coli, suggesting that TLR2 and TLR4 do not participate in the activation of MCs by these bacteria. Whether TLR recognition is responsible for ERK phosphorylation and up-regulation of c-fos mRNA in MCs after challenge with Hly– E. coli requires further investigation.

Noteworthy, in the present study Ussing chamber experiments indicate that the presence of Hly+ E. coli at the mucosal side can lead to an activation of MCs located at the lamina propria most likely because Hly might pass the mucosal barrier.

In conclusion, we show that E. coli Hly is an important bacterial component regulating the production and secretion of proinflammatory cytokines, leukotrienes, and histamine in human intestinal MCs. Our study supports the concept that mucosal MCs have an important function in the innate immune response to bacteria. However, human intestinal MCs respond only to selected pathogens and are unresponsive to several well-established pathogen-associated molecular patterns of Gram-negative bacteria interacting with other immune cells, as well as murine MCs and human progenitor-derived MCs. Possibly, MCs of the intestine differ from MCs of other origins with respect to reactivity to bacterial products and thus represent a particular characteristic required to maintain homeostatic balance in the gut.


   Acknowledgments
 
We thank Lothar Gröbe and Nelson Gekara for support in intracellular calcium measurement. E. coli ORN103(pSH2) and E. coli ORN103(pUT2002) were provided by Soman Abraham; S. flexneri M90T and S. flexneri BS176 by Armelle Phalipon and Philippe Sansonetti, human recombinant SCF by L. Souza; human rIL-4 by E. Liehl; mAb 22E7 by R. Chizzonite; and pSF4000 by Herbert Schmidt.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by the Deutsche Forschungsgemeinschaft (SFB621-A8 to S.C.B.). Back

2 S.K. and G.S. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Stephan C. Bischoff, Department of Nutritional Medicine and Immunology, University of Hohenheim, Fruwirthstrasse 12, D-70593 Stuttgart, Germany. E-mail address: bischoff.stephan{at}uni-hohenheim.de Back

4 Abbreviations used in this paper: MC, mast cell; sLT, sulfido-leukotriene; Hly, hemolysin; PKC, protein kinase C; Ec, E. coli; SLO, streptococcal exotoxin streptolysin O; PFT, pore-forming toxin; SCF, stem cell factor; MOI, multiplicity of infection; Isc, short-circuit current; PKC, protein kinase C; PFT, pore-forming toxin; WT, wild type. Back

Received for publication June 27, 2006. Accepted for publication May 1, 2008.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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