Interleukin-10 and nerve growth factor have reciprocal upregulatory effects on intestinal epithelial cells

¹ Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5; and ² Unilever Health Institute, Vlaardingen 3133 AT, The Netherlands

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The intestinal mucosa is in a constant state of controlled inflammation, but the processes whereby this occurs are poorly understood. The aims of this study were to look at the role of IL-10 and nerve growth factor (NGF) in intestinal epithelial cell regulation. The human colon epithelial cell lines T84, HT-29, and CACO-2 were used. RT-PCR, flow cytometry analysis, and immunohistochemistry were applied to measure the cytokine changes in epithelial cells induced by recombinant cholera toxin and its B subunit, IL-10, and NGF. Cholera toxin B subunit caused selective dose-dependent increased mRNA for IL-10 in T84 cells and the protein in T84, HT-29, and CACO-2 cells. IL-10 dose dependently selectively increased NGF mRNA in T84 cells and intracellular protein synthesis in all three epithelial cell lines. The effect of NGF was reciprocal, selective, and dose dependent because it increased mRNA for IL-10 and IL-10 synthesis. Our results suggest that the epithelium may actively participate in downregulation through innate mechanisms involving IL-10 and NGF. The reciprocal interaction suggests for the first time that NGF may be involved in local downregulation by mucosal epithelium and thus may play a potent protective role in response to injury, by prevention of undue inflammation.

cholera toxin; cholera toxin B subunit

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

INTESTINAL MUCOSAL TISSUE is minimally inflamed and is in a constant state of downregulation under normal physiological circumstances, but the processes whereby this occurs are poorly understood. Recent work has suggested that nonpathogenic Salmonella organisms inhibit the synthesis of IL-8 by blocking a nuclear transcription degradation system (37). Little information exists as to other possible mechanisms of downregulation of local mucosal inflammation.

We have shown that a component of cholera toxin (CTX), cholera toxin B subunit (CTB), a potent factor that when conjugated to a variety of antigens promotes oral tolerance (8), upregulated and caused epithelial synthesis of IL-10, a significant immunodownregulatory molecule. This effect was selective, and no effects were seen on IL-6, IL-8, or transforming growth factor (TGF)-1. IL-10 dose dependently selectively upregulated synthesis of a neurotrophin, nerve growth factor (NGF), which in a variety of model systems appears to promote tissue repair and protection (28, 36, 41). Furthermore, we showed that NGF itself, which is found in relatively large amounts in normal saliva and other external secretions and is constitutively made in intestinal epithelial cells (31), selectively and reciprocally upregulated intestinal IL-10 synthesis. These findings raise the possibility that the epithelium can be induced to synthesize downregulatory molecules such as IL-10 and thus play a significant physiological role in mucosal homeostasis.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Reagents

Recombinant CTX and recombinant CTB were gifts from Prof. T. R. Hirst, Univ. of Bristol. The following reagents were obtained from the companies listed: DMEM, Eagle's minimum essential medium with Earle's salts, F12 nutrient mixture (Ham) medium, McCoy's medium, L-glutamine, nonessential amino acids, penicillin-streptomycin (GIBCO BRL, Grand Island, NY); IL-10, diethyl pyrocarbonate, Brefeldin A, saponin (Sigma, Oakville, ON); Caltag Fixation Medium (Reagent A) and Permeabilization Medium (Reagent B) (Caltag Laboratories, Burlingame, CA); phycoerythrin (PE)-conjugated rat anti-human IL-10 monoclonal antibody (mAb) and PE-conjugated R3-34 (rat IgG₁ isotype) (PharMingen, Mississauga, ON); affinity-purified FITC-conjugated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA); and rabbit IgG (DAKO Diagnostic Canada, Mississauga, ON). All primers (Table 1) were synthesized by the Institute for Molecular Biology and Biotechnology, McMaster University.

View this table: [in this window] [in a new window]   Table 1.   Oligonucleotide primers and PCR product sizes used in experiments

NGF was isolated from male mouse submandibular glands according to the methods of Petrides and Shooter (39). Rabbit anti-NGF was a generous gift from Dr. M. Coughlin, McMaster Univ. (7), and was affinity purified by passage over a CN-Br Sepharose 4B (Amersham Pharmacia Biotech, Piscataway, NJ) column coupled to NGF. Antibody was eluted with 0.5 M acetic acid, and the eluate was neutralized, concentrated, and tested for activity (48).

Cells

The T84, HT-29, and CACO-2 cell lines (human colon epithelial cells) were obtained from the American Type Culture Collection (Manassas, VA). T84 cells were grown in tissue culture flasks (Becton Dickinson Labware, Franklin Lake, NJ) with DMEM-Ham medium (1:1) supplemented with 10% FBS (GIBCO BRL), 2 mM L-glutamine, and 100 U/ml penicillin-100 µg/ml streptomycin. HT-29 cells were grown in McCoy's medium supplemented with 1.5 mM L-glutamine, 10% FBS. CACO-2 cells were grown in Eagle's minimum essential medium supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, and 20% FBS. All cells were maintained in 95% air-5% CO₂ at 37°C.

RNA Extraction and RT-PCR Analysis

T84 cells were cultured for 3 days in six-well plates (Becton Dickinson Labware) at an initial concentration of 1 × 10⁶/ml.

RNA extraction. RNA extraction was as per the vendor's instructions (GIBCO BRL).

RT-PCR. Total RNA (1 µg) was reverse transcribed at 37°C for 1 h by using 500 µM random hexamers [pd(N)6, Amersham Pharmacia Biotech], 500 µM deoxynucleotide-5'-triphosphate (dNTP, Amersham Pharmacia Biotech), 50 mM Tris · HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 10 mM DTT, and 400 U of moloney murine leukemia virus RT (GIBCO BRL) in a final volume of 40 µl. For each PCR, 2 µl of RT product was amplified with different primers designed to investigate the expression of target transcripts. PCR was performed in 1.5 mmol/l MgCl₂, 10 mM Tris · HCl (pH 8.3), 50 mM KCl, 500 µmol/l of each dNTP, 50 pmol of each primer, and 1 U of AmpliTag DNA polymerase (Roche Molecular Systems, Branchburg, NJ) in a final volume of 50 µl. A 5-min denaturation step at 94°C was followed by different cycles (30 cycles for IL-8 and TGF-1; 40 cycles for IL-6 and IL-10; 35 cycles for NGF). Each cycle consisted of 94°C for 1 min, 61°C for 1 min, 72°C for 1.5 min, and a final extension at 72°C for 8 min in an automated thermal cycler (Perkin Elmer Cetus, Norwalk, CT). GAPDH was used as an internal control, with a GAPDH/cytokine ratio of 1:20. Fifteen microliters of each reaction were run onto a 2% agarose gel, stained with ethidium bromide, and visualized by UV illumination. Lambda DNA-HindIII/X-174 DNA-HincII digest (Amersham Pharmacia Biotech) was run as molecular weight markers. Semiquantitative analysis of the DNA bands was performed by a Kodak Digital Science ID Electrophoresis Documentation and Analysis System. Data were expressed as the ratio of each specific expression to GAPDH calculated from the respective densities.

Flow Cytometry Analysis of Intracellular Cytokines

The methods used followed those described elsewhere (2), but with some changes. Based on RT-PCR results, optimal concentrations of CTB, NGF, or IL-10 were added, respectively, to the culture and incubated with T84 cells. After varying periods of incubation (see RESULTS for details), Brefeldin A (5 µg/ml) was added to each culture and incubated with the cells for 6 h. Cells were detached at 37°C by using trypsin (0.05%)-EDTA (0.53 mM) solution (GIBCO BRL), harvested, and centrifuged at 260 g for 5 min. The cells were resuspended at a final concentration of 1 × 10⁶/ml. One thousand microliters of cell suspension was placed into a florescence-activated cell sorting (FACS) tube (Becton Dickinson Labware). Cells were washed in ice-cold staining buffer (PBS containing 1% BSA, 0.02% EDTA, and 0.02% NaN₃). One hundred microliters of Caltag Reagent A [containing 4% paraformaldehyde (PFA)] was added to each tube, which was incubated for 20 min in the dark. Cells were then washed once. One hundred microliters of the staining solution (Caltag Reagent B containing 5% rat serum for anti-IL-10 staining, or 5% rabbit serum for anti-NGF staining) was added to each tube, and the suspensions were incubated for 15 min in the dark. For anti-IL-10 staining, 0.4 µg of PE-conjugated rat anti-human IL-10 mAb or PE-conjugated R3-34 was added to each tube. For anti-NGF staining, 1.5 µg of affinity-purified rabbit anti-NGF or rabbit IgG (isotype control) was added. All tubes were incubated for 30 min in the dark, followed by washing twice. For intracellular IL-10 staining, the cells were fixed with 500 µl of PBS containing 1% PFA (FACS buffer) and then analyzed with the FACScan (Becton Dickinson, San Jose, CA), the CELLQuest (Becton Dickinson), and WinMDI 2.8 analysis software (Scripps Research Institute, http://facs.scripps.edu). For NGF staining, 100 µl of 5% goat serum in Caltag Reagent B was added to each sample and incubated for 20 min, and then 0.2 µg of FITC-conjugated goat anti-rabbit IgG was added to each tube. After incubation for 30 min in the dark, the cells were washed, fixed, and analyzed as above.

Immunohistochemistry Staining of Intracellular IL-10 and NGF

To look at the effect of CTB, NGF, and IL-10 on all three cell lines, immunohistochemistry staining was also used to view intracellular IL-10 and NGF. Each of the cell lines was grown on coverslips in six-well plates for 3 days at a starting concentration of 10⁶/ml. Cells were incubated with CTB or NGF for 72 h or with IL-10 for 48 h, followed by incubation with Brefeldin A for 6 h. Coverslips were washed twice with PBS, fixed with 4% PFA for 10 min, and then washed twice with PBS containing 0.2% Tween 20 (PBST). For IL-10 staining, cells were blocked by 5% rat serum in permeabilization buffer (0.1% saponin, 1% FBS in PBS) for 15 min and washed three times in PBST. Cells were then incubated with 10 µg/ml of PE-conjugated rat anti-human IL-10 mAb or PE-conjugated R3-34 for 30 min, followed by wash with PBST (3 times), and mounted with glycerol-PBS. For NGF staining, cells were blocked by 5% rabbit serum in permeabilization buffer for 15 min and washed three times in PBST. Cells were incubated with 15 µg/ml of affinity-purified rabbit anti-NGF or rabbit IgG for 30 min and washed three times. Cells were blocked by using 5% goat serum in PBST for 15 min, followed by incubation with FITC-conjugated goat anti-rabbit IgG (1:8,000) for 30 min, washed three times, and mounted in glycerol-PBS. All the slides were viewed in a confocal microscope LSM510 (Zeiss, Oberkochen, Germany).

Statistics

Experimental results are expressed as means ± SE. Statistical analyses were performed with unpaired, two-tailed Student's t-tests, or one-way ANOVA followed by Newman-Keuls test for comparing all pairs of groups (GraphPad PRISM, version 2.0). P < 0.05 was considered statistically significant, and n represents the number of experiments performed.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

CTX and CTB Induce Differential Patterns of Cytokine mRNA Expressions

We determined by RT-PCR the effects of different concentrations (0.1, 1, 10, 100, and 1,000 ng/ml) for 2, 6, and 12 h of CTX and CTB on mRNA transcripts for IL-6, IL-8, IL-10, TGF-1, and NGF. CTX increased IL-8 and IL-6 mRNA in a dose-dependent manner. The optimal effect was found at 100 ng/ml (P < 0.01), and the maximal effects occurred at 6 h with both cytokines (data not shown). No significant effects were found on IL-10, TGF-1, and NGF mRNA transcripts with CTX at any concentration or time of incubation. CTB had no apparent effects on IL-6 (Fig. 1) or IL-8, TGF-1, or NGF (data not shown) but significantly increased the mRNA transcripts of IL-10 in a dose-dependent fashion (Fig. 2) at 6 h. These results showed differential cytokine induction by CTX and CTB and a selective induction of IL-10 by CTB.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Cholera toxin (CTX) and cholera toxin B subunit (CTB) effects on IL-6 mRNA expression in T84 cells. At a concentration of 100 ng/ml, CTX increased IL-6 mRNA expression significantly, but CTB had no effect on IL-6 mRNA compared with medium controls. Results were expressed as relative intensity of each IL-6 mRNA expression to GAPDH calculated for the respective densities. ** P < 0.01 compared with media control group, n = 3.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   CTB effect on IL-10 mRNA expression in T84 cells. Increasing concentrations of CTB (0.1-1,000 ng/ml) were added to T84 cells for 6 h. CTB dose dependently upregulated IL-10 mRNA expression. * P < 0.05, ** P < 0.01 compared with media control group, # P < 0.05 compared with CTB 10 ng/ml group, n = 3.

IL-10 Selectively Enhances NGF mRNA Expression

Because CTB differentially increased IL-10 mRNA expression, we wondered what the effect of IL-10 on cytokine induction would be on epithelial cells. Different concentrations (1, 10, and 100 ng/ml) of IL-10 were incubated with T84 cells for 0.5, 1, 2, and 6 h. IL-10 had no effect on IL-6, IL-8, TGF-1, and IL-10 (data not shown) at any time point. However, IL-10 significantly enhanced NGF mRNA expression in a dose-dependent manner (optimal dose was 10 ng/ml). Within 0.5 h incubation, NGF mRNA transcripts increased; the maximal effect occurred at 1 h (P < 0.05) and had returned to baseline by 6 h (Fig. 3).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Time course of IL-10 effect (10 ng/ml) on nerve growth factor (NGF) mRNA expression. NGF mRNA was constitutively expressed in T84 cells at low level at all time points. NGF mRNA was upregulated by IL-10 as early as 0.5 h of incubation but restored to medium control level after 6 h. The maximal effect of IL-10 on NGF mRNA expression occurred at 1 h. Results were expressed as percentage of NGF mRNA relative intensity of IL-10 treated to medium controls. * P < 0.05 compared with 0.5-h group, n = 3.

NGF Selectively Increases IL-10 mRNA Expressions

Because IL-10 selectively increased NGF mRNA transcripts, we examined whether NGF had any effect on cytokine induction. NGF had no effect at different concentrations (10, 100, and 1,000 ng/ml) for 1, 2, and 6 h on IL-6, IL-8, TGF-1, or NGF at any time point. However, NGF selectively and significantly increased IL-10 mRNA transcripts in a dose-dependent fashion (Fig. 4) after 1 and 2 h of incubation. The maximal response occurred at 1 h at an optimal concentration of 100 ng/ml. These results showed that NGF and IL-10 have a reciprocal relationship, and both effects occur at early stages of stimulation.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   NGF effect on IL-10 mRNA expression in T84 cells. Different concentrations (10-1,000 ng/ml) of NGF were added to the culture for 1 h. NGF increased IL-10 mRNA transcripts in a dose-dependent manner, and 100 ng/ml was optimal for the effect. * P < 0.05, ** P < 0.01 compared with media control group, # P < 0.05 compared with NGF 10 ng/ml group, n = 3.

Effects of CTB, IL-10, and NGF on Specific Protein Synthesis in T84, HT-29, and CACO-2 Cells

Because the above mRNA changes were found in T84 cells, we wondered if mRNA increases were accompanied by the relevant protein production as evidence of a translational effect in three human intestinal epithelial cell lines. Specific protein synthesis was measured by flow cytometry (Fig. 5) and viewed by confocal microscopy.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Flow cytometric analysis of intracellular IL-10 and NGF in T84, HT-29, and CACO-2 cells. T84 cells were incubated with either CTB (100 ng/ml) or NGF (100 ng/ml) for 72 h, and IL-10 (10 ng/ml) for 48 h, followed by 6-h incubation with Brefeldin A (5 µg/ml). Compared with medium controls (shaded), CTB increased intracellular IL-10 production (A). IL-10 enhanced intracellular NGF expression (B), and NGF upregulated intracellular IL-10 production (C). Similar results were shown in HT-29 (D-F, respectively) and CACO-2 (G-I, respectively) cells. Each graph was obtained from a single experiment and is representative of 3 experiments performed. See RESULTS.

CTB increases intracellular IL-10 production. The CTB effect on T84 intracellular IL-10 was examined by flow cytometry after incubation for 6, 12, 24, 48, and 72 h. Significant increases in intracellular IL-10 only occurred after 48 and 72 h of incubation (Fig. 5A). Similar results were also obtained in HT-29 (Fig. 5D) and CACO-2 (Fig. 5G) cells.

IL-10 increases intracellular NGF production. To show whether IL-10 also affected NGF protein production, T84 cells were examined for intracellular NGF by flow cytometry after incubation at 1 and 10 ng/ml for 6, 12, 24, and 48 h. As shown in Fig. 5B, intracellular NGF was significantly increased only after 48 h of incubation with IL-10 and only at 10 ng/ml. Examination of HT-29 (Fig. 5E) and CACO-2 (Fig. 5H) showed the same pattern of results.

NGF increases intracellular IL-10 production. Based on our RT-PCR results, the optimal concentration of NGF (100 ng/ml) was chosen and incubated with T84 cells for 6, 24, 48, and 72 h. Significant increases in intracellular IL-10 protein were seen by flow cytometry only after 48 and 72 h with a maximum effect at 72 h (Fig. 5C). Similar effects were also found in HT-29 (Fig. 5F) and CACO-2 (Fig. 5I) cell lines.

Immunohistochemical Evidence for Increase in Intracellular IL-10 and NGF by Confocal Microscopy

Increased intracellular IL-10 staining was seen in confocal microscopy in all three cell lines after incubation with CTB and NGF under exactly the same conditions as were used and represented above for the flow cytometry experiments. Similarly, when all three cell lines were incubated with IL-10, confocal microscopy revealed increased intracellular staining for NGF, which paralleled the observations already made by the cytometry. Because these experiments were confirmatory of results obtained by flow cytometry, examples of images obtained are not presented.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The intestinal mucosa is in a constant state of controlled inflammation. In recent years it has come to be recognized that the epithelium is an active participant in the maintenance of the integrity of the mucosal barrier, through a variety of physiological mechanisms mediated largely by molecules that it synthesizes and secretes (3, 13, 18, 21, 29). On interaction with pathogenic organisms, epithelial cells synthesize and secrete proinflammatory cytokines such as IL-6 and IL-8 (22, 45) and upregulate chemokine receptors (14) so as to promote the immigration of neutrophils and guide their orderly progression onto the cell surface (17).

The intestinal epithelium can synthesize a large number of cytokines, which include IL-1, TNF-, TGF-, granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemoattractant protein 1 (MCP1), IL-6, and IL-8 (3), but it has generally been assumed that IL-10 is not constitutively synthesized by T84 cells (15). In our results the cholera holotoxin caused upregulation of mRNA for IL-6 and IL-8 but had no effect on IL-10. It was therefore surprising that the recombinant CTB caused selective IL-10 upregulation with no evidence for increases in mRNA for TGF-1, IL-6, IL-8, or, for that matter, NGF.

IL-10 is a potent anti-inflammatory agent that can also suppress both Th1 and Th2 type inflammatory responses (44). It has been shown to be an important determinant factor in the development of colitis in experimental models, because transgenic IL-10 knockout mice spontaneously develop colitis if housed under conventional conditions (26). Feeding of Lactococcus lactis, engineered to secrete biologically active IL-10, to transgenic IL-10 knockout animals prevented the onset of colitis (43). Recently, IL-10 has been shown to cause synthesis of chemokine decoy receptors (10). This cytokine is generally thought to be an important but not essential molecule in the mediation of so-called oral tolerance. Indeed, the two molecules thought to be most important in this respect are TGF- and IL-10 (16). Because the most potent way of inducing oral tolerance appears to be via conjugation of protein and other antigens to the CTB (9), our observations that IL-10 is upregulated by CTB may be particularly significant especially because IL-10 promotes the growth and differentiation of a T cell regulatory subset that itself makes IL-10 (19). CTB causes its effect by binding to GM1 ganglioside on the cell surface, and it is highly relevant that other gangliosides have also been shown to induce IL-10 in human T-cells (23). It is not known whether the selective IL-10 induction shown in our experiments follows this or other pathways. However, it is known that ligation of CD1d, a nonclassical major histocompatibility complex (MHC) molecule expressed on the cell surface of intestinal epithelium, causes mRNA for IL-10 to increase in T84 cells (6). Furthermore, evidence for secretion of functionally bioactive IL-10 after CD1d ligation was also obtained. Receptors for IL-10 are expressed on epithelial cells (12), and through interaction with these receptors, IL-10 can block the effects of interferon- such as expression of MHC II and interruption of barrier integrity (32).

We were surprised to find that IL-10 in a dose-dependent fashion caused upregulation and synthesis of NGF. Neither holotoxin nor CTB caused this effect. The IL-10 effect itself was selective, because it did not cause upregulation of TGF-1, IL-6, IL-8, or IL-10 itself. Additionally, NGF caused selective upregulation of IL-10 with no effect on TGF-1, IL-6, IL-8, or NGF. It is pertinent, then, that astrocytes incubated with IL-10 upregulated the synthesis of NGF (4).

Very little information exists as to the production of NGF by intestinal epithelium (49). In our experiments, NGF was constitutively synthesized in low amounts by human T84 cells. NGF is essential for the growth and differentiation of many nerves, both in the peripheral and central nervous system (31). It is made by a variety of structural cell types such as keratinocytes, fibroblasts, and glial cells and also by a host of inflammatory and immune cells that include mast cells (30), eosinophils (42), dendritic and Langerhans cells (46), and T-cells (27), especially Th2. It is found in large amounts in external secretions, especially those of the submandibular glands. There is no information as to the extent of degradation of NGF that occurs in transit through the intestine.

NGF has both pro- and anti-inflammatory properties (11). It is a potent mast cell degranulator in association with lysophosphatidylserine (38), has a significant mastopoietic effect (34), promotes colony growth of basophils and eosinophils (35), and synergizes with IL-5 and GM-CSF (47) in this function. It has a potent anti-apoptotic effect on mast cells (25), eosinophils (20), and neutrophils (24). On the other hand, NGF has significant protective effects in a variety of situations and enhances repair processes. Thus it causes healing of human corneal ulceration (28), is a potent promoter of skin wound healing in both normal and diabetic animals (36), and has a highly anti-inflammatory effect in inhibiting the onset of experimental autoimmune encephalomyelitis (40). It has also been shown to prevent carrageenan-induced inflammation (1). Perhaps more importantly for the present study, it is involved in protection against the inflammation induced by trinitrobenzene sulfonic acid in a murine model of colitis (41). We have previously shown (33) that while NGF promotes the upregulation of synthesis of IL-6 by mast cells, it downregulates production of the proinflammatory cytokine TNF- through increase in synthesis and secretion of PGE₂. This may explain one of the mechanisms of its actions as an anti-inflammatory agent, the other being the upregulation of IL-10.

Bush et al. (5) using transgenic mice, which expressed the herpes simplex virus thymidine kinase gene coupled to a glial promoter, showed that administration of the antiviral drug ganciclovir caused selective enteroglial ablation in the jejunum and ileum. Consequently, there was patchy degeneration of neurons in this region, loss of integrity of the mucosa, and severe inflammatory bowel disease. Glial cells are a major local contributor of NGF to nerves and surrounding tissue. Thus there is evidence for the role of nerves in regulation of the integrity of the mucosa, and it is a high likelihood that neurotrophins are involved in that process.

In conclusion, while CTX did not affect NGF or IL-10 synthesis by epithelial cells, CTB selectively upregulated IL-10 (Fig. 6). NGF, a component of normal saliva, and itself synthesized by intestinal epithelial cells, selectively upregulated the synthesis of IL-10 and was in turn selectively upregulated by IL-10. Thus NGF may directly promote local downregulation of inflammation or act indirectly through upregulation of IL-10. This autacoid system may be worthy of further study with respect to innate mechanisms of homeostasis and defense.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Schematic diagram outlining results of experiments that show that CTB selectively upregulates IL-10, which in turn selectively upregulates NGF, which in an autacoid fashion upregulates NGF.

	ACKNOWLEDGEMENTS

The assistance of L. Builder and G. Goettsche are gratefully acknowledged, as are the contributions of Prof. M. Perdue and Dr. V. DiLeo for discussion and provision of recombinant human IL-10.

	FOOTNOTES

We acknowledge the Canadian Institutes for Health Research in support of this research.

Address for reprint requests and other correspondence: J. Bienenstock, Dept. of Pathology and Molecular Medicine, McMaster Univ., 1200 Main St. West, HSC 3N26, Hamilton, Ontario, Canada L8N 3Z5 (E-mail: bienens{at}mcmaster.ca).

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

First published January 23, 2003;10.1152/ajpregu.00756.2002

Received 16 December 2002; accepted in final form 15 January 2003.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

1.   Banks, BE, Vernon CA, and Warner JA. Nerve growth factor has anti-inflammatory activity in the rat hindpaw oedema test. Neurosci Lett 47: 41-45, 1984[Web of Science][Medline].

2.   Behan, DP, Maciejewski D, Chalmers D, and De Souza EB. Corticotropin releasing factor binding protein (CRF-BP) is expressed in neuronal and astrocytic cells. Brain Res 698: 259-264, 1995[Web of Science][Medline].

3.   Berin, MC, McKay DM, and Perdue MH. Immune-epithelial interactions in host defense. Am J Trop Med Hyg 60: 16-25, 1999[Abstract].

4.   Brodie, C. Differential effects of Th1 and Th2 derived cytokines on NGF synthesis by mouse astrocytes. FEBS Lett 394: 117-120, 1996[Web of Science][Medline].

5.   Bush, TG, Savidge TC, Freeman TC, Cox HJ, Campbell EA, Mucke L, Johnson MH, and Sofroniew MV. Fulminant jejuno-ileitis following ablation of enteric glia in adult transgenic mice. Cell 93: 189-201, 1998[Web of Science][Medline].

6.   Colgan, SP, Hershberg RM, Furuta GT, and Blumberg RS. Ligation of intestinal epithelial CD1d induces bioactive IL-10: critical role of the cytoplasmic tail in autocrine signaling. Proc Natl Acad Sci USA 96: 13938-13943, 1999[Abstract/Free Full Text].

7.   Coughlin, MD, and Kessler JA. Antiserum to a new neuronal growth factor: effects on neurite outgrowth. J Neurosci Res 8: 289-302, 1982[Web of Science][Medline].

8.   Czerkinsky, C, Anjuere F, McGhee JR, George-Chandy A, Holmgren J, Kieny MP, Fujiyashi K, Mestecky JF, Pierrefite-Carle V, Rask C, and Sun JB. Mucosal immunity and tolerance: relevance to vaccine development. Immunol Rev 170: 197-222, 1999[Web of Science][Medline].

9.   Czerkinsky, C, Sun JB, Lebens M, Li BL, Rask C, Lindblad M, and Holmgren J. Cholera toxin B subunit as transmucosal carrier-delivery and immunomodulating system for induction of anti-infectious and antipathological immunity. Ann NY Acad Sci 778: 185-193, 1996[Web of Science][Medline].

10.   D'Amico, G, Frascaroli G, Bianchi G, Transidico P, Doni A, Vecchi A, Sozzani S, Allavena P, and Mantovani A. Uncoupling of inflammatory chemokine receptors by IL-10 generation of functional decoys. Nature Immunology 1: 387-391, 2000[Web of Science][Medline].

11.   Dakin, R. Remarks of a cutaneous affection produced by certain poisonous vegetables. Am J Med Sci 4: 98-100, 1829.

12.   Denning, TL, Campbell NA, Song F, Garofalo RP, Klimpel GR, Reyes VE, and Ernst PB. Expression of IL-10 receptors on epithelial cells from the murine small and large intestine. Int Immunol 12: 133-139, 2000[Abstract/Free Full Text].

13.   Donnenberg, MS. Pathogenic strategies of enteric bacteria. Nature 406: 768-774, 2000[Medline].

14.   Dwinell, MB, Eckmann L, Leopard JD, Varki NM, and Kagnoff MF. Chemokine receptor expression by human intestinal epithelial cells. Gastroenterology 117: 359-367, 1999[Web of Science][Medline].

15.   Eckmann, L, Jung HC, Schurer-Maly C, Panja A, Morzycka-Wroblewska E, and Kagnoff MF. Differential cytokine expression by human intestinal epithelial cell lines: regulated expression of interleukin 8. Gastroenterology 105: 1689-1697, 1993[Web of Science][Medline].

16.   Faria, AM, and Weiner HL. Oral tolerance: mechanisms and therapeutic applications. Adv Immunol 73: 153-264, 1999[Web of Science][Medline].

17.   Godaly, G, Hang L, Frendeus B, and Svanborg C. Transepithelial neutrophil migration is CXCR1 dependent in vitro and is defective in IL-8 receptor knockout mice. J Immunol 165: 5287-5294, 2000[Abstract/Free Full Text].

18.   Gordon, JI, Hooper LV, McNevin MS, Wong M, and Bry L. Epithelial cell growth and differentiation. III. Promoting diversity in the intestine: conversations between the microflora, epithelium, and diffuse GALT. Am J Physiol Gastrointest Liver Physiol 273: G565-G570, 1997[Abstract/Free Full Text].

19.   Groux, H, O'Garra A, Bigler M, Rouleau M, Antonenko S, de VJ, and Roncarolo MG. A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 389: 737-742, 1997[Medline].

20.   Hamada, A, Watanabe N, Ohtomo H, and Matsuda H. Nerve growth factor enhances survival and cytotoxic activity of human eosinophils. Br J Haematol 93: 299-302, 1996[Web of Science][Medline].

21.   Hedlund, M, Wachtler C, Johansson E, Hang L, Somerville JE, Darveau RP, and Svanborg C. P. fimbriae-dependent, lipopolysaccharide-independent activation of epithelial cytokine responses. Mol Microbiol 33: 693-703, 1999[Web of Science][Medline].

22.   Kagnoff, MF, and Eckmann L. Epithelial cells as sensors for microbial infection. J Clin Invest 100: 6-10, 1997[Web of Science][Medline].

23.   Kanda, N. Gangliosides GD1a and GM3 induce interleukin-10 production by human T cells. Biochem Biophys Res Commun 256: 41-44, 1999[Web of Science][Medline].

24.   Kannan, Y, Usami K, Okada M, Shimizu S, and Matsuda H. Nerve growth factor suppresses apoptosis of murine neutrophils. Biochem Biophys Res Commun 186: 1050-1056, 1992[Web of Science][Medline].

25.   Kawamoto, K, Okada T, Kannan Y, Ushio H, Matsumoto M, and Matsuda H. Nerve growth factor prevents apoptosis of rat peritoneal mast cells through the trk proto-oncogene receptor. Blood 86: 4638-4644, 1995[Abstract/Free Full Text].

26.   Kuhn, R, Lohler J, Rennick D, Rajewsky K, and Muller W. Interleukin-10-deficient mice develop chronic enterocolitis. Cell 75: 263-274, 1993[Web of Science][Medline].

27.   Lambiase, A, Bracci-Laudiero L, Bonini S, Starace G, D'Elios MM, De CM, and Aloe L. Human CD4+ T cell clones produce and release nerve growth factor and express high-affinity nerve growth factor receptors. J Allergy Clin Immunol 100: 408-414, 1997[Web of Science][Medline].

28.   Lambiase, A, Rama P, Bonini S, Caprioglio G, and Aloe L. Topical treatment with nerve growth factor for corneal neurotrophic ulcers. N Engl J Med 338: 1174-1180, 1998[Abstract/Free Full Text].

29.   Lee, CA, Silva M, Siber AM, Kelly AJ, Galyov E, and McCormick BA. A secreted salmonella protein induces a proinflammatory response in epithelial cells, which promotes neutrophil migration. Proc Natl Acad Sci USA 97: 12283-12288, 2000[Abstract/Free Full Text].

30.   Leon, A, Buriani A, Dal TR, Fabris M, Romanello S, Aloe L, and Levi-Montalcini R. Mast cells synthesize, store, and release nerve growth factor. Proc Natl Acad Sci USA 91: 3739-3743, 1994[Abstract/Free Full Text].

31.   Levi-Montalcini, R, Skaper SD, Dal Toso R, Petrelli L, and Leon A. Nerve growth factor: from neurotrophin to neurokine. Trends Neurosci 19: 514-520, 1996[Web of Science][Medline].

32.   Madsen, KL, Lewis SA, Tavernini MM, Hibbard J, and Fedorak RN. Interleukin 10 prevents cytokine-induced disruption of T84 monolayer barrier integrity and limits chloride secretion. Gastroenterology 113: 151-159, 1997[Web of Science][Medline].

33.   Marshall, JS, Gomi K, Blennerhassett MG, and Bienenstock J. Nerve growth factor modifies the expression of inflammatory cytokines by mast cells via a prostanoid-dependent mechanism. J Immunol 162: 4271-4276, 1999[Abstract/Free Full Text].

34.   Marshall, JS, Stead RH, McSharry C, Nielsen L, and Bienenstock J. The role of mast cell degranulation products in mast cell hyperplasia. I. Mechanism of action of nerve growth factor. J Immunol 144: 1886-1892, 1990[Abstract].

35.   Matsuda, H, Coughlin MD, Bienenstock J, and Denburg JA. Nerve growth factor promotes human hemopoietic colony growth and differentiation. Proc Natl Acad Sci USA 85: 6508-6512, 1988[Abstract/Free Full Text].

36.   Matsuda, H, Koyama H, Sato H, Sawada J, Itakura A, Tanaka A, Matsumoto M, Konno K, Ushio H, and Matsuda K. Role of nerve growth factor in cutaneous wound healing: accelerating effects in normal and healing-impaired diabetic mice. J Exp Med 187: 297-306, 1998[Abstract/Free Full Text].

37.   Neish, AS, Gewirtz AT, Zeng H, Young AN, Hobert ME, Karmali V, Rao AS, and Madara JL. Prokaryotic regulation of epithelial responses by inhibition of IB- ubiquitination. Science 289: 1560-1563, 2000[Abstract/Free Full Text].

38.   Pearce, FL, and Thompson HL. Some characteristics of histamine secretion from rat peritoneal mast cells stimulated with nerve growth factor. J Physiol (Lond) 372: 379-393, 1986[Abstract/Free Full Text].

39.   Petrides, PE, and Shooter EM. Rapid isolation of the 7S-nerve growth factor complex and its subunits from murine submaxillary glands and saliva. J Neurochem 46: 721-725, 1986[Web of Science][Medline].

40.   Ransohoff, RM, and Trebst C. Surprising pleiotropy of nerve growth factor in the treatment of experimental autoimmune encephalomyelitis. J Exp Med 191: 1625-1630, 2000[Free Full Text].

41.   Reinshagen, M, Rohm H, Steinkamp M, Lieb K, Geerling I, Von Herbay A, Flamig G, Eysselein VE, and Adler G. Protective role of neurotrophins in experimental inflammation of the rat gut. Gastroenterology 119: 368-376, 2000[Web of Science][Medline].

42.   Solomon, A, Aloe L, Pe'er J, Frucht-Pery J, Bonini S, and Levi-Schaffer F. Nerve growth factor is preformed in and activates human peripheral blood eosinophils. J Allergy Clin Immunol 102: 454-460, 1998[Web of Science][Medline].

43.   Steidler, L, Hans W, Schotte L, Neirynck S, Obermeier F, Falk W, Fiers W, and Remaut E. Treatment of murine colitis by Lactococcus lactis secreting interleukin-10. Science 289: 1352-1355, 2000[Abstract/Free Full Text].

44.   Stordeur, P, and Goldman M. Interleukin-10 as a regulatory cytokine induced by cellular stress: molecular aspects. Int Rev Immunol 16: 501-522, 1998[Medline].

45.   Svanborg, C, Godaly G, and Hedlund M. Cytokine responses during mucosal infections: role in disease pathogenesis and host defence. Curr Opin Microbiol 2: 99-105, 1999[Web of Science][Medline].

46.   Torii, H, Yan Z, Hosoi J, and Granstein RD. Expression of neurotrophic factors and neuropeptide receptors by Langerhans cells and the Langerhans cell-like cell line XS52: further support for a functional relationship between Langerhans cells and epidermal nerves. J Invest Dermatol 109: 586-591, 1997[Web of Science][Medline].

47.   Tsuda, T, Wong D, Dolovich J, Bienenstock J, Marshall J, and Denburg JA. Synergistic effects of nerve growth factor and granulocyte-macrophage colony-stimulating factor on human basophilic cell differentiation. Blood 77: 971-979, 1991[Abstract/Free Full Text].

48.   Van der Zee, CE, Rashid K, Le K, Moore KA, Stanisz J, Diamond J, Racine RJ, and Fahnestock M. Intraventricular administration of antibodies to nerve growth factor retards kindling and blocks mossy fiber sprouting in adult rats. J Neurosci 15: 5316-5323, 1995[Abstract].

49.   Varilek, GW, Neil GA, Bishop WP, Lin J, and Pantazis NJ. Nerve growth factor synthesis by intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 269: G445-G452, 1995[Abstract/Free Full Text].