REPORT
PepT1-mediated fMLP transport induces intestinal inflammation in vivo

Marion Buyse1, Annick Tsocas2, Francine Walker2, Didier Merlin1, and Andre Bado2

1 Epithelial Pathology Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia 30322; and 2 Institut National de la Santé et de la Recherche Médicale Unité 410, Faculte de Medecine Xavier Bichat Paris, 75018 Paris, France


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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In the present study, the effect of H+/peptide transporter (PepT1)-mediated N-formylmethionyl-leucyl-phenylalanine (fMLP) transport on inflammation in vivo in the rat small intestine, which expresses high PepT1 levels, and in the rat colon, which does not express PepT1, were investigated using myeloperoxidase (MPO) activity and histological analysis. We found that 10 µM fMLP perfusion in the jejunum for 4 h significantly increased MPO activity and altered the architecture of jejunal villi. In contrast, 10 µM fMLP perfusion in the colon for 4 h did not induce any inflammation. In addition, we have shown that 50 mM Gly-Gly alone did not affect basal MPO activity but completely inhibited the MPO activity induced by 10 µM fMLP in the jejunum. Together, these experiments demonstrate that 1) the differential expression of PepT1 between the small intestine and the colon plays an important role in epithelial-neutrophil interactions and 2) the inhibition of fMLP uptake by jejunal epithelial cells (expressing PepT1) reduces the neutrophil ability to move across the epithelium, in agreement with our previously published in vitro study. This report constitutes the first in vivo study showing the implication of a membrane transporter (PepT1) in intestinal inflammation.

PepT1; N-formylmethionyl-leucyl-phenylalanine; myeloperoxidase; rat


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

CRYPT ABSCESSES represent a characteristic histological finding in acute intestinal inflammation and are thought to contribute to the epithelial dysfunction characterizing such disorders. The hallmark of this histological entity is the neutrophil adherence to and transmigration across the polarized intestinal epithelia. Inflammation is thought to arise from aberrant interaction of environmental and immunological factors. Recent studies suggest that the "normal" bacterial flora play a key role in the development of intestinal inflammation. For example, mice with a disrupted interleukin-2 gene (15) develop intestinal inflammation when raised in a conventional environment but not when raised in a germ-free environment. In addition, a spontaneous colitic mouse model, C3H/HeJBir, produces serum antibodies against normal enteric bacterial flora (2). Recognition of luminal pathogens by epithelial apical membranes results in polarized secretion of chemoattractants from epithelia that serve to orchestrate neutrophil movement through the underlying matrix as well as across the epithelial monolayer (8-10).

It has long been known that bacteria, such as Escherichia coli, release potent neutrophil chemotactic substance(s) (1). Partial characterization of culture filtrates from E. coli have established that at least a portion of this chemoattractant bioactivity relates to small heterogeneous peptides with blocked amino groups, so-called N-formyl peptides (7). For example, the tripeptide N-formylmethionyl-leucyl-phenylalanine (fMLP) has been demonstrated to be a major N-formylated peptide of the human colonic lumen (4, 7), and the total N-formyl peptide content of the human colon provides an fMLP equivalent concentration of ~10-7 M (4). This latter concentration is within the range of fMLP concentrations providing gradients that maximally influence polymorphonuclear neutrophil (PMN) migration when used in an in vitro system. It is likely that N-formyl peptide concentrations are substantially lower in the small intestine than in the colon, in parallel with the lower amounts of prokaryotes present in this former site in humans.

In a recent work, we demonstrated that the human peptide transporter hPepT1 transports bacteria-derived chemotactic peptides such as fMLP (13). A cDNA encoding the H+/peptide transporter (PepT1) derived from various mammalian species has been cloned. PEPT1 was identified as an integral membrane-spanning protein that is expressed predominantly in the small intestine and slightly in the kidney (6). Epithelial monolayers internalized apical fMLP in a fashion that was competitively inhibited by other hPepT1-recognized solutes such as Gly-Pro, Gly-Leu, and Gly-Gly but not by free amino acids such as glycine. Fluorescence analyses of intracellular pH (using BCECF-AM) revealed that fMLP uptake was accompanied by cytosolic acidification, consistent with the known function of hPepT1 as a peptide-H+ cotransporter (13). These results constituted the first demonstration that a major physiological chemotactic peptide released by bacteria, such as E. coli, is transported across the apical plasma membrane of intestinal cells by hPepT1. In our recent studies, we also demonstrated that PepT1 is absent from a colonic cell line and human colonic mucosa (12, 13). Furthermore, we have demonstrated that hPepT1-mediated uptake of N-formyl peptides has functional consequences on neutrophil-epithelial interactions in a cell line that expresses considerable high-hPepT1 protein (Caco2-BBE cells) (13). These results emphasize the importance of hPepT1 in mediating intestinal inflammation and provide the first evidence of a link between active transepithelial transport and neutrophil-epithelial interactions.

To confirm the validity of our in vitro findings, we examined the inflammatory effects of PepT1-mediated fMLP transport in vivo in the rat small intestine, which expresses a high level of PepT1, and in the rat colon, which does not express PepT1 (14).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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Animals. All studies were carried out in male Wistar rats (220-240 g; IFFA Credo, L'Abresle, France) that were deprived of food for 18 h with water available ad libitum. The animals were treated in accordance with European Community Standards concerning the care and use of laboratory animals (Institut National de la Santé et de la Recherche Médicale and Ministere de l'Agriculture et de la foret, France, Authorization no. 02249).

Intestinal perfusion technique. The rats were anesthetized with intramuscular ethylurethane (1.2 g/kg; Prolabo, Paris, France). After median laparotomy, the rats were equipped with a transpyloric duodenal inflow cannula made of Silastic tubing (1.65-mm outside diameter, 0.76-mm inside diameter, 10-cm length; Dow Corning, Midland, MI) and the outflow cannula in the jejunum was set up at ~15 cm below the ligament of Treitz. For the colon perfusion, the inflow cannula was inserted 1 cm below the caecum and the outflow cannula was set up at a distance of 1 cm above the rectum. The duodenojejunal or colonic segment was then flushed with saline solution prewarmed to 37°C to remove residual intestinal contents. Perfusion solutions were delivered with a peristaltic pump at a flow rate of 1 ml/15 min (Minipuls 2; Gilson, Paris, France) through an inlet tube water-jacketed at 37°C before its entry into the intestinal segment.

The solution used for intestinal perfusion was a Krebs-Ringer buffer (pH 7.5) containing 0.5 mM MgCl2, 4.5 mM KCl, 120 mM NaCl, 0.7 mM Na2HPO4, 1.2 mM CaCl2, 15 mM NaHCO3, and 10 mM glucose. After a 30-min stabilization period, fMLP (1-10 µM; Sigma Chemical, St. Louis, MO) or vehicle was added to the Krebs-Ringer buffer and perfused for at least 4 h. For the competition studies, Gly-Gly (50 mM; Sigma Chemical) was added to the buffer 1 h before addition of fMLP. Immediately after the rats were killed, the perfused intestinal segment was removed to assess the inflammation severity; the half used for histology was fixed in formol, and the second half was snap-frozen in liquid nitrogen and stored at -70°C for further measurement of myeloperoxidase (MPO) activity assay.

Histological examination. Each sample of rat jejunum (10 rats, jejunum perfusion with control solution; 10 rats, jejunum perfusion with 10 µM fMLP solution) or rat colon (7 rats, colonic perfusion with control solution; 7 rats, colonic perfusion with 10 µM fMLP solution) was fixed in buffered formalin for 24 h, dehydrated, and embedded in paraffin. Four-micrometer sections were stained with hematoxylin-eosin-safranin to reveal structural features and examined by light microscopy in a blinded manner.

MPO activity assay. The activity of intestinal MPO, an enzyme marker of the PMN primary granules, was assayed to monitor the degree of inflammation with the use of the method of Krawisz et al. with minor modification (5a). Briefly, intestinal tissue samples (~50-100 mg) were homogenized on ice by using a Polytron (13,500 rpm for 1 min) in a solution of 0.5% hexadecyltrimethylammonium bromide (Sigma Chemicals) in a 50 mM potassium phosphate buffer (pH 6.0, 1 ml/50 mg tissue). The resulting homogenate was subjected to three rapid freezing (-70°C) and thawing (immersion in warm water, 37°C) cycles. The samples were then centrifuged (4,000 rpm, 15 min, 4°C) to remove insoluble material. The MPO-containing supernatant (0.1 ml) was assayed spectrophotometrically after the addition of 2.88 ml of phosphate buffer (50 mM, pH 6.0) containing 0.167 mg/ml o-dianisidine hydrochloride (Sigma Chemicals) and 0.0005% hydrogen peroxide. The kinetics of absorbance changes at 470 nm was measured. One unit of MPO activity, defined as the quantity of enzyme able to convert 1 µmol of hydrogen peroxide to water in 1 min at room temperature, was expressed in units per gram of tissue.

Number of animals tested and statistical analysis. All values are reported as means ± SE. The number of animals used for the tissue-associated MPO activity in jejunum after luminal perfusion with or without fMLP for 4 h was 23 rats for jejunum perfusion with control solution, 10 rats for jejunum perfusion with 1 µM fMLP solution, and 19 rats for jejunum perfusion with 10 µM fMLP solution. The number of animals used for the tissue-associated MPO activity in the colon after luminal perfusion with or without fMLP for 4 h was 7 rats for colonic perfusion with control solution and 7 rats for colonic perfusion with 10 µM fMLP solution. To demonstrate that the inhibition of PepT1-mediated fMLP uptake decreases MPO activity in the jejunum, we used 20 rats for jejunum perfusion with control solution, 7 rats for jejunum perfusion with 50 mM Gly-Gly, 7 rats for jejunum perfusion with 10 µM fMLP, and 7 rats for jejunum perfusion with 10 µM fMLP in the presence of 50 mM Gly-Gly. An analysis of variance (ANOVA) was applied to the data followed by a multiple comparison Dunnett t-test. Statistical significance was accepted at a level of P < 0.05.

Electrophoretic mobility shift assay. Caco2-BBE cells were grown to confluency on 0.4-µm collagen-coated filters. Caco2-BBE monolayers were apically treated with 1 µM fMLP or vehicle for 30, 60, or 120 min. Nuclear extracts from Caco2-BBE monolayers were then isolated at these different time points (30, 60, and 120 min) as described previously (5). After nuclear extraction and protein determination, the nuclear proteins were used for electrophoretic mobility shift assays (EMSA). Double-stranded oligonucleotides containing the sequence of the binding site for transcription factor AP-1 (5'-CGCTTGATGAGTCAGCCGGAA-3'; Promega, Madison, WI) and nuclear factor (NF)-kappa B (5'-AGTTGAGGGGACTTTCCCAGCC-3'; Promega) were radiolabeled with 30 µCi of [gamma -32P]ATP by using a 5'-end labeling kit (Amersham Pharmacia Biotech, Piscataway, NJ). Nuclear proteins (6 µg) were incubated with 3,000 counts of labeled oligonucleotide in a buffer containing 1- mM HEPES, pH 7.5, 100 mM NaCl, 1 mM EDTA, 1.5 mM dithiothreitol, 5% glycerol, and 2 µg of poly(dI-dC) for 30 min at room temperature. The reaction mixture was loaded onto a 6% polyacrylamide gel buffered with 89 mM Tris, 89 mM boric acid, and 2 mM EDTA. After drying, the gel was placed in contact with X-ray film at -70°C. All radiographic images were reproduced by scanning, using Desk Scan software (Hewlett-Packard), and then exported into PowerPoint (Microsoft) for labeling.


    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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To investigate the effect of fMLP in the rat intestine, we perfused jejunum and colon with a solution with or without fMLP and monitored inflammation by using tissue-associated MPO activity as an index of neutrophil infiltration in the mucosa in these different intestinal segments.

In control rats, the mean MPO activity in the jejunum was three times higher than that detected in colon [jejunum vs. colon: 142 ± 12 (n = 23) vs. 39.2 ± 8.3 IU/g wet tissue (n = 7)]. These results indicate that the number of resident phagocytic cells in the lamina propria of the jejunum is more than that in the colon. As shown in Fig. 1A, 4 h after perfusion with 1 µM fMLP in the jejunum, the MPO activity did not significantly increase; after perfusion with 10 µM fMLP, the MPO activity increased by 40% (P < 0.01 vs. control). These results were supported by histological studies showing that perfusion of 10 µM fMLP for 4 h induced jejunal wall damage with epithelial exfoliation (Fig. 1C) compared with the control perfused jejunum segment (Fig. 1B). The jejunal villi were blunted and broadened with interstitial edema, and dilatation of vessels was associated with a general increase in inflammatory cells (lymphocytes and polynuclear cells) in the lamina propria (Fig. 1C). In contrast, 10 µM fMLP did not induce any inflammatory changes in the colon [39.2 ± 8.3 (n = 7) vs. 51.8 ± 16.4 IU/g wet tissue (n = 7)] (Fig. 2A). There was no macroscopic evidence of inflammation in any part of the colon except a slight edema in the submucosa that was similar in control (Fig. 2B) and fMLP-treated rats (Fig. 2C). Together, these results indicate that the mucosal inflammation induced by fMLP is confined to the jejunum, whereas the colonic mucosa is not affected by perfusion with fMLP. In previous studies it was suggested that fMLP has a better accessibility to phagocytic cells in the lamina propria in the small intestine compared with the colon (18, 19).


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Fig. 1.   N-formylmethionyl-leucyl-phenylalanine (fMLP) induces inflammation in the jejunum. A: tissue-associated myeloperoxidase (MPO) activity in jejunum before (control, Ctrl) and after luminal perfusion with fMLP for 4 h. Results are means ± SE of n = 23 rats for Ctrl, n = 10 rats for 1 µM fMLP, and n = 19 rats for 10 µM fMLP in the jejunum. ** P < 0.01 vs. Ctrl. Histological sections of rat jejunum are shown in untreated control rats (B), showing normal jejunal wall features, and 4 h after perfusion with 10 µM fMLP (C), showing jejunal wall damage. B and C: original magnification, ×10.



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Fig. 2.   fMLP does not induce inflammation in the colon. A: tissue-associated MPO activity in the colon before and after luminal perfusion with 10 µM fMLP for 4 h. Results are means ± SE of n = 7 rats for Ctrl and n = 7 rats for 10 µM fMLP. Histological sections of rat colon are shown in untreated control rats (B) and 4 h after perfusion with 10 µM fMLP (C). There is no macroscopic evidence of inflammation in the colon except a slight edema in the submucosa in both groups. B and C: original magnification, ×40.

There are two pathways available for passive diffusion of small molecules across epithelia: 1) the paracellular pathway (between cells) and 2) the transcellular pathway (across cells). The paracellular pathway consists of a circumferential sealing apical junction that is the rate-limiting barrier for movement through the paracellular space. fMLP diffuses passively across the tight junctions to establish an fMLP gradient. fMLP could have a better access to phagocytic cells in the small intestine than in the colon because of the segmental differences in paracellular permeability (higher paracellular permeability for small peptides such as fMLP in the small intestine than in colon) (19). In the small intestine, but not in the large intestine, di-tripeptides including fMLP can be transported by an apical membrane transporter, PepT1 (12, 13). The intestinal epithelial cells that express PepT1 could accumulate significant amounts of chemotactic peptide in the cytosol (13). It is not clear how the absorbed fMLP ultimately moves across the basolateral membrane to complete the transcellular transport of fMLP. As we have demonstrated previously, this differential expression of PepT1 between the small intestine and colon could play an important role in epithelial/neutrophil interactions (12, 13). Here we show that 50 or 10 µM Gly-Gly (data not shown) alone did not affect basal MPO activity compared with the control values (158 ± 8 vs. 142 ± 12 IU/g tissue). In contrast, 50 mM Gly-Gly completely inhibited the MPO activity induced by 10 µM fMLP in the jejunum [fMLP + 50 mM Gly-Gly vs. fMLP alone: 128 ± 10.9 vs. 201.4 ± 21 IU/g wet tissue (n = 23); P < 0.01] (Fig. 3). These experiments demonstrate 1) that the inhibition of fMLP uptake by jejunal epithelial cells (expressing PepT1) reduces the neutrophil ability to move across the epithelium, and they are in agreement with our previously published in vitro study (8) indicating 2) that the increase of MPO activity in the small intestine induced by fMLP is the result of PepT1-mediated fMLP uptake into the enterocytes but not of a paracellular diffusion of fMLP across the epithelium. The most logical interpretation of these observations is that PepT1 mediates an active transepithelial gradient of the chemoattractant, resulting in a more efficient movement of neutrophils across epithelial cells. Given the known sieving characteristics of tight junctions, small peptides such as fMLP should passively diffuse across the tight junctions in a limited amount so as to create an exceedingly steep transjunctional gradient of chemotactic peptide.


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Fig. 3.   Inhibition of H+/peptide transporter (PepT1)-mediated fMLP uptake decreases MPO activity in the jejunum. Tissue-associated MPO activity is shown in jejunum before and after luminal perfusion with control solution (n = 20 rats), with 50 mM Gly-Gly (n = 7 rats), and with 10 µM fMLP alone (n = 19 rats) or in the presence of 50 mM Gly-Gly (n = 7 rats) for 4 h. Results are means ± SE. ** P < 0.01 vs. Ctrl. ## P < 0.01 vs. 10 µM fMLP alone.

In the jejunum, but not in the colon, parallel transcellular transport of solutes such as fMLP can occur. Previous studies have shown the existence of a distinct peptide transporter in the basolateral membranes of Caco2-BBE (13) and in the rat small intestine (3) compared with the PepT1 cotransporter in their apical membranes. In addition, we have recently demonstrated that the transcellular fMLP transport is rate limited by its transport at the basolateral membrane, resulting in an intracellular accumulation of fMLP (13). Two potential possibilities exist: 1) fMLP can be translocated across the lateral membrane from tight junctions, and this would serve to lengthen and decrease the slope of the resulting paracellular gradient; or 2) intracellular uptake of N-formyl peptides may result in rapid signals that lead to modifications of the basolateral membranes with which the neutrophils interact during transepithelial migration. Recently, we demonstrated that hPepT1-mediated fMLP transport has a stimulatory effect on AP-1 and NF-kappa B activities. As shown in Fig. 4, we studied the DNA binding activity of the transcription factor AP-1 and NF-kappa B in Caco2-BBE monolayers with or without 1 µM fMLP added to the apical membranes. EMSA using 32P-labeled NF-kappa B (Fig. 4A) and 32P-labeled AP-1 (Fig. 4B) consensus sequences showed that nuclear extracts from monolayers apically treated with 1 µM fMLP evoked the appearance of one band, reflecting an enhanced association of nuclear protein with NF-kappa B (Fig. 4A) and AP-1 (Fig. 4B) that peaked within 60 min after fMLP treatment. The induction of NF- kappa B and AP-1 may lead to the activation of inflammatory responses by intestinal epithelial cells. However, further in vitro and in vivo studies need to be performed to elucidate these intracellular events induced by fMLP.


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Fig. 4.   fMLP induces activation of the nuclear factors NF-kappa B and AP-1 in Caco2-BBE monolayers. Caco2-BBE cells were grown to confluency on 0.4-µm collagen-coated filters. Caco2-BBE monolayers were apically treated with or without 1 µM fMLP for 30, 60, or 120 min. Nuclear extracts obtained from Caco2-BBE monolayers, either untreated (Ctrl) or treated for the indicated time (30, 60, or 120 min) with 1 µM fMLP, were then subjected to EMSA in the presence of NF-kappa B (A)- or AP-1 (B)-specific consensus sequence oligonucleotides (1). Competition assay was performed in the presence of 500-fold excess of unlabeled unspecific (2) and unlabeled specific (3) oligonucleotides.

This report constitutes the first in vivo study showing the implication of a membrane transporter (PepT1) in intestinal inflammation. fMLP has been demonstrated to be the major N-formylated peptide of the human colonic lumen (4, 7). In contrast, fMLP concentrations are less important in the small intestine, where bacterial populations are fewer compared with the colon. Under normal conditions, inflammation does not occur. However, and according to our model, PepT1-mediated transport of fMLP might play an important role in intestinal inflammation. In certain clinical syndromes, such as bacterial overgrowth in the small intestine, fMLP concentrations may become sufficient to induce PepT1-mediated inflammation. In addition, we observed that treatment of Caco2-BBE monolayer by luminal fMLP for 24 h increased hPepT1 mRNA and hPepT1-mediated dipeptide uptake (M. Buyse and D. Merlin, personal observation). These results corroborate the studies by other groups (16, 20). It is interesting that, under some conditions, another bacterial product (LPS) may reduce the PepT1 expression. However, in contrast to fMLP, its mechanism of action and its specificity to PepT1 was not elucidated (17). On the other hand, in chronic colitis, PepT1 expression occurs in the colon (13). Under these conditions, PepT1-mediated fMLP transport may participate to sustain inflammation in the colon. Such observations provide strong evidence for the relevance of modeling (in vitro) epithelial-neutrophil interactions as a first step to logically unravel the underlying molecular mechanism of such interactions.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-02831 (to D. Merlin) and Institut National de la Santé et de la Recherche Médicale APEX99 Grant 4X006E (to A. Bado). D. Merlin is a recipient of the Young Investigator Award from Crohn's and Colitis Foundation of America (2001).


    FOOTNOTES

Address for reprint requests and other correspondence: D. Merlin, Dept. of Pathology and Laboratory Medicine, Emory Univ., 615 Michael St., Atlanta, GA 30322 (E-mail: dmerlin{at}emory.edu).

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.

10.1152/ajpcell.00186.2002

Received 24 April 2002; accepted in final form 22 July 2002.


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ABSTRACT
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RESULTS AND DISCUSSION
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Am J Physiol Cell Physiol 283(6):C1795-C1800
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