Nippostrongylus brasiliensis: Infection Induces Upregulation of Acetylcholinesterase Activity on Rat Intestinal Epithelial Cells

Experimental Parasitology 96, 222–230 (2000) doi:10.1006/expr.2000.4565, available online at http://www.idealibrary.com on

Nippostrongylus brasiliensis: Infection Induces Upregulation of Acetylcholinesterase Activity on Rat Intestinal Epithelial Cells

Wayne S. Russell,1 Siaˆn M. Henson, Ayman S. Hussein, John R. Tippins, and Murray E. Selkirk2 Department of Biochemistry, Imperial College of Science, Technology and Medicine, London SW7 2AY, United Kingdom

Russell, W. S., Henson, S. M., Hussein, A. S., Tippins, J. R., and Selkirk, M. E. 2000. Nippostrongylus brasiliensis: Infection induces upregulation of acetylcholinesterase activity on rat intestinal epithelial cells. Experimental Parasitology 96, 222–230. Expression of cholinesterases and muscarinic acetylcholine receptors in the jejunal mucosa has been investigated during infection of rats with the nematode parasite Nippostrongylus brasiliensis. Selective expression of m3 receptors was observed on epithelial cells from uninfected rats and animals 7 days postinfection, and saturation binding with [3H]quinuclidinyl benzilate indicated that receptor expression on cell membranes was unaltered by infection. Butyrylcholinesterase was highly expressed in mucosal epithelia, but acetylcholinesterase was present at low levels in uninfected animals. In contrast, discrete foci of intense acetylcholinesterase activity were observed on the basement membrane of intestinal epithelial cells in animals infected with N. brasiliensis. This was demonstrated to be due to upregulation of expression of endogenous enzyme, which peaked at Day 10 postinfection and subsequently declined to preinfection levels. It is suggested that this occurs in response to hyper-activation of the enteric nervous system as a result of infection, and may benefit the host by limiting excessive fluid secretion due to cholinergic stimulation. 䉷 2000 Academic Press Index Descriptors and Abbreviations: Nippostrongylus brasiliensis; nematode; cholinesterase; muscarinic receptor. QNB, quinuclidinyl benzilate: AChE, acetylcholinesterase; BuChE, butyrylcholinesterase; BW284C51, 1,5,-bis(4 allyldimethylammoniumphenyl) pentan-3-one dibromide; iso-OMPA, tetraisopropylpyrophosphoramide.

INTRODUCTION

Infection of the intestinal tract by nematode parasites leads to alterations in the enteric nervous system. This is most evident in remodeling of nerves or changes in the levels of neurochemical transmitters, which ultimately result in effects on neuronal function and the regulation of intestinal motility and secretory responses (Collins 1996; McKay and Fairweather 1997). Alterations in the cholinergic component of the enteric nervous system, and in particular cholinergic stimulation of intestinal fluid secretion in rats infected with Nippostrongylus brasiliensis, have been demonstrated to be defective. Insensitivity of epithelial cells to ACh was discounted by the use of exogenous cholinergic agonists, which resulted in a secretory response comparable to that of uninfected animals (Masson et al. 1996). The enteric nervous system regulates several secretory processes of enterocytes. Nerve fibers in the mucosa terminate subjacent to the basement membrane of epithelial and enteroendocrine cells, on which muscarinic acetylcholine receptors (mAChRs) have been localized (Rimele et al. 1981). Muscarinic AChR agonists evoke chloride secretion from intestinal epithelial cells, and the electrogenic flux creates an osmotic gradient resulting in passive movement of water in the lumen (Cooke 1994). In addition, these agonists evoke mucus secretion from goblet cells (Bradbury et al. 1980) and exocytosis of Paneth cells, epithelial granulocytes located at the base of the crypts of Lieberku¨hn (Satoh et al. 1992).

1

Current address: Department of Cell and Molecular Biology, Lund University, PO Box 94, S-221 00 Lund, Sweden. 2 To whom correspondence should be addressed. Fax: (44) 207 225 0960. E-mail: [email protected].

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0014-4894/00 $35.00 Copyright 䉷 2000 by Academic Press All rights of reproduction in any form reserved.

CHOLINESTERASES IN PARASITISED INTESTINAL MUCOSA

One possibility which we have considered is that cholinergic stimulation of intestinal secretory responses, and fluid secretion in particular, might be influenced by acetylcholinesterases (AChEs) secreted by several species of parasitic nematode. This phenomenon is exhibited predominantly by nematodes that inhabit the gastrointestinal tract of their vertebrate hosts, and has provoked much discussion on the putative physiological role of these enzymes (Rhoads 1984; Lee 1996; Selkirk et al. 2001). We therefore sought to investigate this possibility by examining the expression of cholinesterases and acetylcholine receptors in the intestinal tract of rats infected with N. brasiliensis.

MATERIALS AND METHODS Isolation of intestinal epithelial cells. Sprague–Dawley rats were infected with 5000 infective larvae of N. brasiliensis, and intestinal epithelial cells isolated by an adaptation of a previously described procedure (Poussier and Julius 1997). The jejunum was removed and placed into a siliconized beaker containing sterile PBS at 37⬚C. It was flushed through several times with PBS, the Peyer’s patches were removed, and the jejunum was bisected longitudinally. The mucosal face was blotted to remove fecal material and mucus. The jejunum was cut into 1-cm pieces and incubated in Hanks balanced salt solution (HBSS) with 2 mM dithiothreitol (DTT), 5% foetal calf serum (FCS) for 15 min at 37⬚C with continuous stirring, and the supernatant removed and discarded. Tissue segments were then incubated in calcium and magnesium-free HBSS (HBSS-CMF) containing 2 mM DTT, 5% FCS, 1 mM EDTA at 37⬚C with continuous stirring. After 20 min the supernatant was removed, filtered, and centrifuged at 500g for 5 min at room temperature. Fresh medium was added to the tissue and the procedure repeated three times. The supernatants were discarded and the cell pellets recovered following resuspension in HBSSCMF containing 5% FCS and centrifugation at 500g for 5 min at room temperature. Radioligand binding. Cells were homogenized in membrane preparation buffer (0.25 M sucrose, 0.5 mM EDTA, 0.01 M triethanolamine HCl, pH 7.5) and centrifuged for 15 min at 30,000g at 4⬚C. The pellet was resuspended in membrane preparation buffer, centrifuged once more, and resuspended in 50 mM Tris–HCl, pH 7.4, and the protein content determined. Saturation binding was performed in 250 ␮l 50 mM Tris–HCl, pH 7.4, using concentrations of [3H]quinuclidinyl benzilate ([3H]QNB) ranging from 0.5 to 40 nM. Membranes were incubated with [3H]QNB at 37⬚C for 60 min, and nonspecific binding was determined in the presence of 100 ␮M atropine. Bound radioligand was separated from free ligand by filtration through Whatman GF/C filters and quantitated by scintillation counting. The dissociation constant (KD) and receptor density (Bmax) were determined from Scatchard plots of the binding data with the aid of Graphpad Prism. Binding experiments were determined for three animals per group, each performed in triplicate. Determination of mAChR expression by RT-PCR. Total RNA was isolated from epithelial cells using TRIzol reagent (GIBCO), treated for 25 min at 37⬚C with 5 U of RNase-free DNase I, 16.5 U of RNasin,

223 in 100 mM Tris–HCl, pH 7.4, 500 mM MgCl2, phenol/chloroform extracted, and precipitated. RT-PCR reactions were performed under standard conditions, with 30 cycles of 94⬚C for 1 min, 57⬚C for 1 min, and 72⬚C for 1.5 min using primers specific for rat mAChR subtypes m1-m5 as previously reported (Wei et al. 1994). Primers for rat ␤actin were used as positive controls. The products were resolved by electrophoresis in 1.2% agarose and stained with ethidium bromide. Competitive RT-PCR. Competitive RT-PCR was performed essentially as described by O’Connell et al. (1997). A heterologous competitor DNA fragment containing primer binding sites identical to the m3 amplicon was constructed by using looped oligonucleotide primers that amplified a fragment from the Echinococcus granulosus thioredoxin peroxidase cDNA (Salinas et al. 1998) of 327 bp which was easily distinguishable from the authentic m3 amplicon of 434 bp. RNA samples (10 ␮g) from epithelial cell samples were reverse-transcribed as described above. One-tenth of this test sample was used for each reaction along with serial dilutions of the cDNA competitor, ranging from 0.001 fg to 1 ng. The PCR reaction was carried out using m3 primers, the PCR products were separated on a 3% agarose gel, and the relative amounts of the amplified cDNAs corresponding to the 434 bp m3 test product and 327 bp competitive product were quantitated by densitometry of ethidium bromide-stained gels. Equivalence of PCR products occurs when target and standard templates are present in equal initial concentration, permitting quantitation of the target template. Immunocytochemistry. Segments of jejunum (1 cm) were dissected from sacrificed animals, flushed with ice-cold 4% paraformaldehyde and then ice-cold PBS. Segments were embedded with Tissue-Tek OCT compound (Miles, Inc.), mounted onto cork boards, and snapfrozen in isopentane on dry ice. Ten- to 12-nm-thick sections were cut on a cryostat and fixed in acetone at ⫺20⬚C. Endogenous peroxidase was blocked by incubating sections in hydrogen peroxide (3% in methanol) for 2 ⫻ 10-min incubations. Sections were overlaid with a monoclonal antibody to mammalian AChE (clone AE-2, Biogenesis) at 1:100 dilution overnight at 4⬚C, washed three times in PBS, and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (BioRad) at 1:200 dilution for 1 h at room temperature. The bound peroxidase was visualized in 0.05% 3,3⬘-diaminobenzidine/0.01% H2O2 in PBS, the reaction was stopped by three washes in dH2O, and sections were then counterstained with hematoxylin, dehydrated, mounted in DPX, and monitored by microscopy. Western blotting. Protein samples were resolved by 12% SDS– PAGE, transferred to nitrocellulose membranes, and overlaid with either AE-2 or rabbit anti-N. brasiliensis AChE B (Hussein et al. 1999). Binding was determined by standard procedures utilizing horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (Amersham RPN 2209). Histochemical determination of cholinesterase activity. Cholinesterase activity was detected by the method of Karnovsky and Roots (1964). Sections of intestine were fixed for 10 min in 4% paraformaldehyde at room temperature, and then washed three times in PBS. The sections were then incubated in substrate buffer for 1–2 h at room temperature in the dark. The buffer consisted of 5 mg acetylthiocholine iodide dissolved in 6.5 ml 0.1 M sodium phosphate buffer, pH 6.5, to which the following were added sequentially: 0.5 ml 0.1 M sodium citrate, 1 ml 30 mM copper sulfate, 1 ml H2O, and 1 ml 5 mM potassium ferricyanide. The specificity of enzyme activity was determined by preincubating the samples for 30 minutes in 10⫺4 M 1,5,-bis(4 allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284C51) or 10⫺4 M tetraisopropylpyrophosporamide (iso-OMPA), specific inhibitors of AChE and BuChE, respectively (Austin and Berry 1953). After

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incubation, sections were washed several times in water, dehydrated, and mounted.

RESULTS

Intestinal epithelial cells from both control and infected animals express m3 receptors. Given the documented defect in cholinergic stimulation of intestinal fluid secretion in rats infected with N. brasiliensis (Masson et al. 1996), we sought to determine whether muscarinic AChR expression on mucosal epithelial cells in the jejunum was altered. Saturation binding with [3H]QNB, a nonselective muscarinic receptor antagonist, determined that there was no significant difference in receptor expression on membranes from epithelial cells isolated from uninfected or 7-day-infected animals. A representative experiment is illustrated in Fig. 1. The KD for [3H]QNB binding was determined to be 22 ⫾ 6 nM for infected and 28 ⫾ 5 nM for uninfected rats. Receptor density was similarly unaffected, and the Bmax was calculated at 0.25 ⫾ 0.02 and 0.19 ⫾ 0.05 pmol/mg protein for membranes from uninfected and infected rats, respectively. These values were not significantly different (P ⬍ 0.05). We next utilized RT-PCR to determine the class of muscarinic receptor expressed on jejunal epithelial cells. Figure 2 illustrates the selective expression of m3 receptors in cells from both uninfected rats (A), and animals infected 7 days

FIG. 2. Expression of m3 receptors on jejunal epithelial cells. The presence of mRNAs for muscarinic receptor subtypes m1–m5 in epithelial cells of the rat jejunum was determined by RT-PCR, utilizing previously described primers (Wei et al. 1994). (A) Jejunal epithelial cells from an uninfected rat. (B) Jejunal epithelial cells from a rat infected 7 days previously with N. brasiliensis. (C) Control rat brain. Lane 1, ␤-actin primers; Lane 2, m1 primers; Lane 3, m2 primers; Lane 4, m3 primers; Lane 5, m4 primers; Lane 6, m5 primers; Lane 7, m2 primers with no cDNA; Lane 8, m2 primers with RNA. The position of markers is indicated in base pairs. FIG. 1. Muscarinic receptor expression on jejunal epithelial cell membranes. Binding experiments were performed with [3H]QNB on membrane preparations from epithelial cells isolated from rat jejunum as described under Materials and Methods. Assays were performed in triplicate, and on membranes from three infected and three uninfected animals. A representative binding curve and Scatchard plot for membranes from one animal is shown, with the bars representing standard deviations derived from triplicate samples.

previously with N. brasiliensis (B), whereas all the primer combinations gave products of the expected size when utilized with cDNA derived from total rat brain (C). Quantitative analysis of mRNA expression by competitive RT-PCR

CHOLINESTERASES IN PARASITISED INTESTINAL MUCOSA

showed no significant difference in the levels of expression of m3 mRNA in epithelial cells from infected or uninfected animals (Fig. 3). Cholinesterase expression in the jejunal mucosa. Segments of jejunum from normal and infected rats were therefore stained for ChE activity in conjunction with the AChEselective inhibitor BW284C51 and the BuChE-specific inhibitor iso-OMPA. In normal rats, high levels of BuChE activity were observed in the mucosal epithelium and the

225 external muscle layers (Figs. 4A, 4B). BuChE was distributed throughout the cytoplasm of the epithelial cells but was clearly absent from cells in the lamina propria. In contrast, AChE was expressed at very low levels in mucosal cells, but present in high concentration in the muscularis externa and submucosal nerve plexi (Figs. 4C, 4D). In animals infected 7 days previously with N. brasiliensis, the spatial distribution of BuChE expression in the jejunum appeared unaltered (Fig4. 4E, 4F), but a striking observation

FIG. 3. Quantitative analysis of m3 expression by competitive RT-PCR. (A, B) RNA isolated from epithelial cells of uninfected animals. (C, D) RNA isolated from epithelial cells of animals infected 7 days previously with N. brasiliensis. (A, C) m3 product at 434 bp and the competitor product at 327 bp, with 10-fold dilutions of the cDNA competitor from 1 ng (lane 2) to 0.001 fg (lane 11). DNA markers are shown in lane 1. (B, D) Densitometric analysis of (A) and (C), respectively: (●) competitor DNA; (⌬) m3 cDNA.

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CHOLINESTERASES IN PARASITISED INTESTINAL MUCOSA

was the presence of numerous discrete foci of intense AChE activity in the mucosa (Fig. 4G). Inspection at higher magnification revealed that this was localized to the basal lamina of epithelial cells (H). Infection induces upregulation of expression of endogenous AChE on mucosal epithelial cells. It was possible that the focal expression of AChE activity on epithelial cell basal lamina resulted either from translocation of enzyme secreted by individual parasites or from induction of expression of endogenous AChE at discrete sites. In order to discriminate between these possibilities and to determine the dynamics of enzyme expression, we isolated epithelial cells at different times postinfection and utilized extracts in Western blots with antibodies to mammalian AChE (AE-2, Biogenesis), or N. brasiliensis AChE B (Hussein et al. 1999). Only the former antibody bound to extracts of epithelial cells in Western blots. Figure 5 demonstrates a basal level of AChE on jejunal epithelial cells of uninfected rats, indicated by a band at approximately 65 kDa (lane 1). The level of expression increased progressively after Day 4 postinfection, reaching a peak at around Day 10 (lane 4), and subsequently declining to preinfection levels by Day 40 postinfection. Immunocytochemistry on tissue sections with AE-2 confirmed the localization of endogenous AChE on the basal lamina of jejunal epithelial cells from infected animals (Fig. 6B), with negligible binding to the same site in sections of jejuna from uninfected animals (Fig. 6A).

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FIG. 5. Dynamics of endogenous AChE expression on mucosal epithelial cells during infection. Expression of AChE determined by Western blotting of extracts of epithelial cells isolated from the jejunum of rats at different times postinfection with N. brasiliensis. Lane 1, uninfected; lanes 2–6, days 3, 7, 10, 17 and 40 postinfection, respectively. The blot was overlaid with a monoclonal antibody to mammalian AChE (clone AE-2, Biogenesis) and developed following incubation with a secondary horseradish peroxidase-conjugated antibody. The position of molecular mass markers is shown in kDa.

DISCUSSION

Use of subtype-specific antibodies has previously identified the presence of m1, m2, and m3 classes of mAChR in rat ileum, although these were not localized to different tissues within ileal segments (Wall et al. 1991). Studies based on the rank order of potency of selective antagonists,

however, suggested that ion and fluid secretion in rat intestine was mediated by m3 receptors on enterocytes (Przyborski and Levin 1997), consistent with previous pharmacological data obtained from pigs and guinea pigs (Kachur et al. 1990; Chandan et al. 1991). The use of RT-PCR in this investigation clearly indicates the selective expression of m3 receptors on mucosal epithelial cells, and the [3H]QNB

FIG. 4. Cholinesterase expression in the jejunum. (A–D) Uninfected animals; (E–H) infected animals. (A, B) BuChE activity in uninfected animals was localized by staining in the presence of BW284C51. High levels of activity can be seen in the muscularis externa (me) and the mucosa (m, A). At higher magnification, this is observed to be present in the cytoplasm of epithelial cells (e, B). Note the demarcation of goblet cells delineated by lack of BuChE activity in the secretory vesicles containing mucus. (C, D) AChE activity in uninfected animals was localized by staining in the presence of iso-OMPA. High levels of activity were observed in the muscularis externa (arrows) and submucosal nerve plexi (arrowhead, (C)). Very low levels were observed in the mucosa generally (m) and mucosal epithelial cells (e). (E, F) Spatial distribution of BuChE activity was unaltered in jejuna of infected animals. (G, H) In addition to intense staining in the muscularis externa, numerous discrete foci of AChE activity were observed in mucosal tissue ((G) arrows). Higher magnification demonstrates that this is localized to the basal lamina of epithelial cells (H). Bars: 100 ␮m (A, E, G); 25 ␮m (B, D, F, H); 65 ␮m (C).

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FIG. 6. Immunocytochemical localization of AChE in the jejunal mucosa. Localization of endogenous AChE by immunocytochemistry to the basal lamina of jejunal epithelial cells from infected animals (B). Uninfected animals show minimal binding of antibody at this location (A). Sections were stained with AE-2, binding was visualized following a secondary horseradish peroxidase-conjugated antibody and counterstaining with hematoxylin.Bars: 25 ␮m.

binding studies indicate that the expression of these receptors is not grossly perturbed by infection with N. brasiliensis. This result is consistent with data which indicate that epithelial ion secretion was equally stimulated by the muscarinic agonist bethanechol in control rats and those infected with N. brasiliensis, indicative of functionally intact receptors on epithelial cells (Masson et al. 1996). These authors demonstrated that ion secretion in response to electrical transmural stimulation of enteric nerves was significantly reduced in rats at 10 days postinfection, however, and showed no cholinergic component. Reponses to substance P were also reduced at this time point, although increased numbers of substance P-immunoreactive nerve fibers were detected in mucosal tissue (Masson et al. 1996). In the present study, high levels of BuChE activity were observed in epithelial cells and the intestinal lumen (the latter data not shown), which complements previous work demonstrating the presence of the major cholinesterase in rat intestinal mucosa as a dimeric amphiphilic (G2a) BuChE (Sine et al. 1992). In contrast, cytochemical staining revealed intense foci of AChE activity at the basal lamina of the mucosal epithelium in animals infected with N. brasiliensis, and this was demonstrated to be due to upregulation of AChE expression by epithelial cells rather than emanating from parasite secretions. Staining appeared to be more extensive at this site when examined by immunocytochemistry

(Fig. 6), although this is probably due to the higher sensitivity of this method, which will also detect AChE inactivated during tissue processing. This enzyme was not secreted into the gut lumen (data not shown), and is most likely membrane-bound. A reasonable interpretation of the data is that activation of the enteric nervous system by parasite infection leads to feedback inhibition of cholinergic stimulation of epithelial cells via upregulation of AChE expression on epithelial cells, and the focal expression of intense enzyme activity suggests that this may be maximally effected on cells in close proximity to nerve termini. This would be beneficial to the host in limiting continued fluid secretion, which could lead to debilitating diarrhea (Perdue and McKay 1994; Masson et al. 1996). Additional perturbation of the enteric nervous system has been documented during infection with parasitic nematodes, notably impaired ACh release from smooth muscle/myenteric plexus (SM/MP) preparations from the jejuna of rats infected with Trichinella spiralis (Collins et al. 1992). Choline uptake and choline acetyltransferase (ChAT) activity in SM/MP were both enhanced during infection, although ACh production was downregulated. Concomitant with these changes, AChE activity in SM/MP was decreased, although expression appeared to be minimally affected in mucosal tissue (Davis et al. 1998). These results differ significantly from our current data relating to N. brasiliensis infection,

CHOLINESTERASES IN PARASITISED INTESTINAL MUCOSA

in which AChE expression in the muscularis externa is unaffected, but upregulated at discrete sites on the mucosal epithelium. Although the reasons for these differences are not immediately apparent, they may derive from the distinct anatomical niches adopted by these two species of parasite, and the specific nature or extent of the ensuing immune response. N. brasiliensis is strictly a lumenal parasite, while T. spiralis invades and migrates through epithelial cells. Betamethasone treatment of rats infected with T. spiralis demonstrated that suppression of ACh release from SM/MP was dependent upon an inflammatory response. This defect was initially defined as T-cell-independent (Collins et al. 1992), and subsequently demonstrated to be dependent upon infiltration of the intestinal mucosa, muscle and myenteric plexus region with macrophages (Galeazzi et al. 2000). While providing insight into a potential mechanism for regulating excessive fluid secretion as a result of nematode infection, these data raise questions regarding the potential function of the parasite-secreted AChEs. They might operate in a redundant capacity to the same end or serve an alternative function, and we are therefore now working to resolve these possibilities.

ACKNOWLEDGMENTS This work was supported by the Wellcome Trust, and by the BBSRC and MRC through studentships to Wayne Russell and Siaˆn Henson, respectively.

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