Trichinella spiralis Secreted Enzymes Regulate Nucleotide-Induced Mast Cell Activation and Release of Mouse Mast Cell Protease 1

Abstract

Extracellular nucleotides are important triggers of innate immunity, acting on a wide variety of cells via signaling through purinergic receptors. Mucosal mast cells contribute to expulsion of a number of gastrointestinal nematode parasites, and mouse mast cell protease 1 has been shown to have a critical role in clearance of Trichinella spiralis from the intestinal tract. We show here that adenosine, ADP, ATP, UDP, and UTP all stimulate calcium mobilization in bone marrow-derived mast cells with a mucosal phenotype. Secreted proteins from T. spiralis infective larvae inhibit nucleotide-induced mast cell activation, and that induced by ADP and UDP is specifically blocked by parasite secretory 5′-nucleotidase. Release of mouse mast cell protease 1 is stimulated by ADP and ATP. Both parasite secreted products and the 5′-nucleotidase inhibit ADP-induced release of mast cell protease, whereas that stimulated by ATP is partially inhibited by secreted products alone. This indicates that the 5′-nucleotidase contributes to but is not solely responsible for inhibition of nucleotide-mediated effects on mast cell function. Secretion of nucleotide-metabolizing enzymes by parasitic nematodes most likely evolved as a strategy for suppression of innate immune responses and is discussed in this context.

INTRODUCTION

Extracellular nucleotides are important triggers of innate immunity and are released from cells either in a regulated manner or as a result of tissue damage (11). Signaling is effected through P1 and P2 purinergic receptors, which can directly activate or modulate many different cellular responses. The P1 family is composed of G-protein-coupled receptors, with 4 subtypes which respond differentially to adenosine (ADO) and inosine (INO) (10). The P2 family is comprised of G-protein-coupled P2Y receptors and ligand-gated ion channel P2X receptors. The eight human and seven murine P2Y receptors are differentially responsive to ADP, ATP, UDP, UTP, and UDP-glucose, with ligand affinities depending on the receptor subtype (48, 49), whereas the seven P2X receptors are all activated by ATP (37). In addition, the cysteinyl leukotriene receptors CysLT1 and -2 and GPR17 have been shown to mediate the actions of extracellular UDP (6, 32).

Mast cells from a variety of sources express a range of P1, P2Y, and P2X receptors, and extracellular nucleotides and nucleosides can directly activate an array of mast cell effector functions (5, 25). ATP was the first nucleotide described to act as an agonist of mast cell degranulation (7), but ADP also induces histamine and leukotriene release (42), and UDP-glucose was recently shown to act on P2Y14 receptors to mediate degranulation as indicated by beta-hexosaminidase release (12). The effects of adenosine are more complex, as low concentrations inhibit degranulation and release of inflammatory mediators, whereas adenosine at high concentrations acts as an agonist. These effects result from differential activation of P1 receptors, with low levels of adenosine preferentially acting on the high-affinity A2A receptors and high levels engaging A2B and A3 receptors (9, 40, 45). Mast cell activation contributes in turn to the pool of extracellular nucleotides, as secretory granules contain extremely high (up to 100 mM) concentrations of ATP (14). Although nucleotides do not appear to directly stimulate cytokine production by mast cells, they can modulate cytokine responses to other stimuli. For instance, UDP can induce production of interleukin-5 (IL-5), tumor necrosis factor alpha (TNF-α), and macrophage inflammatory protein 1β (MIP-1β) in human cord blood-derived mast cells primed with IL-4 (31).

Gastrointestinal nematode infection is accompanied by recruitment, differentiation, and activation of mucosal mast cells (MMCs), largely within epithelial tissues (33). Nematode-induced mast cell activation leads to release of a repertoire of mediators which result in leukocyte recruitment and activation, amplification of mucosal Th2 responses, smooth muscle contraction, and increased epithelial permeability (23, 26, 29, 33). Increased epithelial permeability is associated with the release of chymases (chymotrypsin-like serine proteases) (43), and the beta-chymase mouse mast cell protease 1 (mMCP-1) most likely effects this by degradation of the tight junction protein occludin (29). Subsequent efflux of water, solutes, and mediators such as antibodies into the intestinal lumen is thought to promote parasite expulsion, an idea that was formulated some 40 years ago and dubbed the leak lesion hypothesis (35). In support of this hypothesis, mice deficient in mMCP-1 exhibit delayed expulsion of the nematode parasite Trichinella spiralis (24), and this protease also appears to contribute to the intestinal pathology observed during infection (26). Activation of mucosal mast cells during nematode infection can occur independently of the high-affinity IgE receptor, as indicated by studies on mice with targeted disruption of the FcRγ chain. Although these mice display defects in mast cell-mediated allergic responses (46), mMCP-1 release appears unaffected in primary infections with T. spiralis (19) and Strongyloides stercoralis (38).

During the enteral phase of T. spiralis infection, infective larvae invade the mucosal epithelium of the small intestine, molt several times, mate, and release newborn larvae, which migrate through the lamina propria and enter local capillary vessels (50). This process causes severe damage to mucosal tissue, which must result in the release of high concentrations of nucleotides into the extracellular environment. Interestingly, T. spiralis secretes a range of nucleotide-metabolizing enzymes, specifically a nucleoside diphosphate kinase (NDPK), a 5′-nucleotidase (5′-NT), and an adenosine deaminase (15, 18). Secretion of these enzymes is anticipated to alter the amount and type of extracellular nucleotides available to bind host cell purinergic receptors, and we have postulated that these enzymes act to promote an anti-inflammatory environment that is more favorable for parasite survival (16). Given that mMCP-1 is released from mucosal mast cells during nematode infection in the absence of FcRεRI-mediated signaling and has a demonstrable role in expulsion of T. spiralis, we assessed whether protease release could be triggered by nucleotides and examined the effect of parasite nucleotide-metabolizing enzymes on this process.

MATERIALS AND METHODS

Parasites and parasite products.

Infective larvae of Trichinella spiralis were recovered from Sprague-Dawley rats 2 months after oral infection and maintained in vitro, and secreted proteins (SP) were collected as previously described (15). The secreted 5′-NT was expressed in Pichia pastoris and purified from culture medium, again as previously described (17). All procedures involving animals were approved by the Imperial College Ethical Review Committee and performed under license from the UK Home Office. Animal care and maintenance were performed by college staff as directed by the license and institutional guidelines.

mBMMC.

Mouse bone marrow-derived mast cells (mBMMC) were generated under conditions defined to maximize expression of the intestinal mucosal mast cell-specific chymase, mMCP-1 (34, 51). Bone marrow cell cultures were established from BALB/c mice by cell culture in Dulbecco's minimal essential medium (DMEM), 10% fetal calf serum (FCS), 2 mM l-glutamine, 100 U ml⁻¹ penicillin, 100 μg ml⁻¹ streptomycin, 2.5 μg ml⁻¹ amphotericin B (Fungizone), 1 mM sodium pyruvate, 15% WEHI-3B-conditioned medium as a source of IL-3, 50 ng ml⁻¹ recombinant rat stem cell factor (Amgen, Thousand Oaks, CA), 5 ng ml⁻¹ recombinant mouse IL-9 (R&D Systems), and 1 ng ml⁻¹ transforming growth factor β₁ (TGF-β₁; R&D Systems), grown for 21 days, and passaged at 7-day intervals. The mast cell phenotype was verified by staining cytospin preparations with toluidine blue and analyzing cell surface marker expression by flow cytometry. Expression of c-kit was determined by staining with 1 μg ml⁻¹ phycoerythrin (PE)-conjugated anti-c-kit or isotype control antibody (Caltag Labs). Expression of FcεRI was assayed by incubation with 10 μg ml⁻¹ anti-dinitrophenyl (DNP)-albumin IgE (clone SPE7) (Sigma), washing, and incubation with 1 μg ml⁻¹ fluorescein isothiocyanate (FITC)–rat anti-mouse IgE (Caltag). Cells were analyzed using a FACSCalibur flow cytometer (Becton, Dickinson and Co.) and Cell-Quest software.

RT-PCR.

RNA was isolated from mast cells by use of TRIzol (Invitrogen) according to the manufacturer's instructions, digested with DNase I, and reverse transcribed using Moloney murine leukemia virus reverse transcriptase primed with oligo(dT) (Promega). Expression of mRNAs for P1, P2Y, and P2X purinergic receptors and mMCP-1 was determined by reverse transcription-PCR (RT-PCR), using the primers and annealing temperatures listed in the supplemental material. Standard reaction conditions were utilized, with PCR cycle parameters of 95°C for 3 min followed by 35 cycles of 94°C for 30 s, annealing for 30 s, and 72°C for 1 min, with a final step of 72°C for 10 min.

Calcium mobilization assay.

Calcium flux assays were performed essentially as previously described (4). Cells were labeled with 5 μM Fura-2-AM in Ca²⁺ flux buffer (Hanks' balanced salt solution, 1 mM CaCl₂, 1 mM MgCl₂, 0.1% bovine serum albumin [BSA]) for 30 min at 37°C in the dark and then washed prior to resuspension in the same buffer at 0.5 × 10⁷ cells ml⁻¹. Subsequently, cells were added at 0.5 × 10⁶ cells per well to a 96-well black fluorescence plate (Nunc) prewarmed to 37°C. Changes in intracellular Ca²⁺ concentration induced by agonists were monitored using excitation at 340 nm (bound) and 380 nm (unbound) and emission at 510 nm in a fluorescence microplate spectrophotometer. In experiments with T. spiralis secreted proteins or recombinant 5′-NT, agonists were preincubated for 30 min with parasite proteins in 25 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM MgCl₂, and 2.5 mM dithiothreitol prior to addition to cells.

Release and assay of mouse mast cell protease 1.

Mast cells were added to 96-well plates containing the culture medium described above (DMEM plus additives) at 2 × 10⁵ cells per well and assayed for release of mMCP-1 following incubation with agonists for 2 h at 37°C. In experiments with T. spiralis secreted proteins or recombinant 5′-NT, agonists were preincubated for 30 min with parasite proteins in 25 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM MgCl₂, and 2.5 mM dithiothreitol prior to addition to cells. As a positive control for mMCP-1 release, mBMMC were passively sensitized with 10 μg ml⁻¹ anti-DNP-albumin IgE (clone SPE7; Sigma) for 2 h, washed, and stimulated with 30 ng ml⁻¹ DNP-albumin. Following centrifugation, cell supernatants were assayed for mMCP-1 by enzyme-linked immunosorbent assay (ELISA) as previously described (36), using 50 μl rat anti-mMCP-1 monoclonal antibody at 2 μg ml⁻¹ as the capture antibody and 50 μl of horseradish peroxidase (HRP)-conjugated rabbit anti-mMCP-1 antibody diluted 1:1,500 (Moredun Research Institute) for detection. The concentration of mMCP-1 in each sample was determined from a standard curve with recombinant enzyme (Moredun Research Institute).

Statistical analysis.

Data for mMCP-1 release are expressed as means ± standard deviations (SD) for triplicate cultures. Data were analyzed by unpaired Student's t test, and the significance of differences from controls is indicated in the figures (*, P < 0.05; **, P < 0.005; and ***, P < 0.001).

RESULTS

Expression of functional purinergic receptors by mouse bone marrow-derived mast cells.

mBMMC were cultured under conditions previously defined to create an in vitro analogue of intestinal mucosal mast cells (34, 51). From day 14 onwards, the population was relatively homogeneous, with over 90% c-kit⁺ FcεRI⁺ cells as assessed by flow cytometry. The granules showed a typical sulfated acid mucopolysaccharide content, as indicated by staining with toluidine blue, and RT-PCR confirmed that they expressed mMCP-1. Cells spontaneously secreted mMCP-1 at levels approximately 1,000 times greater than those in the absence of TGF-β₁, and mMCP-1 release was further stimulated (approximately 10×) by antigen stimulation through FcεRI, sensitization of cells with anti-DNP-albumin IgE, or stimulation with antigen (data not shown; see Materials and Methods). These data confirmed that the cells were of the intestinal mucosal phenotype, which is suitable for assaying release of mMCP-1, and all subsequent experiments were performed with cells which had been cultured under the conditions described for a minimum of 14 days.

Expression of mRNAs for purinergic receptors on mBMMC was examined by RT-PCR, using the primer pairs shown in the supplemental material. All primer pairs amplified the correct sequences in RT-PCRs using RNAs derived from the murine spleen, brain, or intestinal epithelia. Figure 1A shows that mast cells expressed transcripts for A1, A2, and A3 receptors, but a transcript for the A2A receptor was not observed. All of the P2Y receptors were represented, albeit at different levels, whereas only transcripts for P2X1 and P2X4 were observed consistently for the P2X receptors (Fig. 1A).

Fig 1.

Mucosal mast cells respond to nucleotide/nucleoside agonists. (A) Detection of mRNAs for purinergic receptors in mucosal mast cells by RT-PCR. Primers and annealing temperatures for amplification of P1 (lanes A₁ to A₃), P2Y (lanes Y₁ to Y₁₄), and P2X (lanes X₁ to X₇) receptors are given in the supplemental material. Lanes C, control PCRs for β-actin. Products were resolved by agarose gel electrophoresis and stained with ethidium bromide. No products were observed in the absence of reverse transcription. (B) Calcium mobilization in response to nucleotides. Cells were loaded with Fura-2-AM, and changes in intracellular Ca²⁺ concentration induced by agonists were monitored using a 340/380-nm fluorescence excitation ratio, with emission at 510 nm. The responses to agonists at concentrations between 10⁻³ M and 10⁻⁷ M are shown. Calcium fluxes are shown for single wells, and the experiments were performed 5 times.

In order to determine whether mast cells of a mucosal phenotype responded to stimulation with nucleotides and nucleosides, we assayed changes in cytosolic Ca²⁺ levels in cells labeled with the fluorescent indicator Fura-2-AM and challenged with agonists over a range of concentrations. The P1 agonists adenosine and inosine gave different results. Adenosine, which acts on all P1 receptor subtypes, elicited rapid Ca²⁺ flux in a dose-dependent manner at agonist concentrations as low as 100 nM, whereas inosine, which has been reported to act on the A3 receptor subtype (22, 47), did not elicit an increase in intracellular Ca²⁺ (Fig. 1B). AMP and UMP did not stimulate Ca²⁺ flux at concentrations of up to 1 mM, whereas ADP and ATP both triggered strong responses at concentrations down to 100 nM (Fig. 1B). P2Y receptor subtypes respond differentially to ADP and ATP (48), and the latter also signals through the P2X receptors (37). Mast cells were also responsive to UTP at concentrations down to 10 μM and to UDP at 100 μM (Fig. 1B).

Inhibition of nucleotide-induced mast cell activation by T. spiralis secreted proteins.

We next assayed whether T. spiralis infective larval SP affected nucleotide-induced activation of mast cells. SP inhibited calcium flux induced by adenosine at concentrations up to 10 μM (Fig. 2A), as well as that induced by 100 μM UDP (Fig. 2B). Inhibition of ADP-induced activation was evident with 1 μM agonist but not with 10 μM ADP (Fig. 2C), and calcium flux induced by 1 μM ATP appeared unaffected by SP (Fig. 2D). Mast cell activation by UDP and ADP could also be blocked by purified recombinant 5′-NT, but as with SP, the effects on ADP were less potent (Fig. 3A and B). Given that the parasite 5′-NT hydrolyzes ADP to AMP and AMP to adenosine, we hypothesized that the reason for partial inhibition could be that the reaction product adenosine was stimulating calcium flux, and this was confirmed by the use of receptor antagonists. ADP appeared to be acting predominantly through the P2Y1 receptor, as this was blocked by the P2Y1-specific antagonist MRS 2179 (39), whereas the effect of adenosine was blocked by the A3-specific receptor antagonist MRS 1523 (13) (Fig. 3C). Repetition of the experiment with SP substituting for 5′-NT gave a similar result, with a blockade of calcium flux effected by inclusion of both receptor antagonists, implying that the 5′-NT is responsible for a significant component of the overall effects of parasite SP (Fig. 3D). Neither parasite SP nor the 5′-NT affected calcium flux in cells when applied on its own (data not shown).

Fig 2.

T. spiralis secreted proteins inhibit nucleotide-induced calcium mobilization in mast cells. (A) SP inhibit adenosine-induced flux (1 μM and 10 μM adenosine, with or without 10 μg ml⁻¹ SP). (B) SP inhibit UDP-induced flux (100 μM UDP, with or without 10 μg ml⁻¹ SP). (C) SP partially inhibit ADP-induced flux (1 μM and 10 μM ADP, with or without 10 μg ml⁻¹ SP). (D) SP do not inhibit ATP-induced flux (1 μM ATP, with or without 10 μg ml⁻¹ SP). Calcium fluxes are shown for single wells, and the experiments were performed 3 times.

Fig 3.

T. spiralis 5′-nucleotidase inhibits nucleotide-induced calcium mobilization. (A) 5′-NT inhibits UDP-induced flux (100 μM UDP, with or without 5′-NT). (B) 5′-NT partially inhibits ADP-induced flux (1 μM and 10 μM ADP, with or without 5′-NT). (C) ADP-induced flux occurs via P2Y1 receptor activation (10 μM and 100 μM ADP, with or without 50 ng ml⁻¹ 5′-NT). Where indicated, cells were preincubated for 10 min with the receptor antagonists MRS 2179 and/or MRS 1523 at 10 μM. (D) The experiment shown in panel C was performed in the same manner, but replacing 5′-NT with 10 μg ml⁻¹ SP. Calcium fluxes are shown for single wells, and the experiments were performed 3 times.

Release of mouse mast cell protease 1 is inhibited by the parasite 5′-nucleotidase.

To determine whether nucleotides could induce the release of mMCP-1, mBMMC were stimulated with a range of concentrations of inosine, adenosine, ADP, ATP, UDP, and UTP. The background level of mMCP-1 release was 0.27 μg ml⁻¹, and cross-linking of FcεRI with anti-DNP IgE plus DNP resulted in a 9.6× increase in mMCP-1 release (Fig. 4).

Fig 4.

Release of mMCP-1 is induced by specific nucleotides and inhibited by parasite secreted proteins. Mast cells were assayed for release of mMCP-1 following incubation with agonists at concentrations between 1 and 100 μM for 2 h at 37°C (see Materials and Methods). Sensitization with anti-DNP-albumin IgE plus antigen challenge was used as a positive control in each experiment (IgE+Ag). (A) Adenosine does not induce mMCP-1 release. (B) UDP does not induce mMCP-1 release. (C) UTP does not induce mMCP-1 release. (D) Release of mMCP-1 by ADP. (E) Release of mMCP-1 by ATP. (F) ADP-induced release of mMCP-1 is inhibited by T. spiralis SP (10 μg ml⁻¹). (G) ADP-induced release of mMCP-1 is inhibited by T. spiralis 5′-NT (100 ng ml⁻¹). (H) ATP-induced release of mMCP-1 is partially inhibited by T. spiralis SP (10 μg ml⁻¹). (I) ATP-induced release of mMCP-1 is not inhibited by T. spiralis 5′-NT (100 ng ml⁻¹). Filled bars indicate the effect of SP (F and H) or 5′-NT (G and I) in comparison to controls (open bars). Data are presented as means ± SD for triplicate cultures. In panels A to E, stimulation of mMCP-1 release by nucleotides is indicated as follows: **, P < 0.005; and ***, P < 0.001. In panels F to I, suppression of nucleotide-induced mMCP-1 release by SP or 5′-NT is indicated as follows: *, P < 0.05; **, P < 0.005; and ***, P < 0.001.

Adenosine, UDP, and UTP, in addition to inosine (data not shown), could not induce mMCP-1 secretion at concentrations up to 100 μM (Fig. 4A to C). However, ADP and ATP both induced a dose-dependent increase in release (Fig. 4D and E). The slowly hydrolyzable nucleotide analogues ADP-βS and ATPγS had the same effect, confirming that these nucleotides, rather than hydrolyzed products, induced mMCP-1 release, and MRS 2179 inhibited the response to ADP, confirming that ADP does indeed signal via the P2Y1 receptor to stimulate release of mMCP-1 (data not shown). A summary of the effects of nucleotides on calcium mobilization and mMCP-1 release is provided in Table 1.

Table 1.

Summary of effects of nucleotides and nucleosides on mucosal mast cell function^a

Nucleotide or nucleoside	Calcium flux	mMCP-1 release
Inosine	−	−
Adenosine	+++	−
AMP	−	−
ADP	+++	+++
ATP	+++	+++
UMP	−	−
UDP	+	−
UTP	++	−

^aEffects on calcium flux and mMCP-1 release are indicated, with an indication of the potency determined from dose-response experiments shown with arbitrary units.

Larval parasite SP inhibited release of mMCP-1 from mBMMC stimulated with ADP (Fig. 4F), and replacement of SP with 5′-NT demonstrated that this enzyme was the active inhibitory constituent (Fig. 4G). SP significantly reduced 10 μM ATP-induced mMCP-1 secretion but had no effect on that induced by higher concentrations of agonist (Fig. 4H). Replacement of SP by 5′-NT, however, failed to inhibit secretion of mMCP-1, even at the lowest concentration of ATP (Fig. 4I), indicating that additional components of SP were responsible for the effect on this agonist.

Previous studies have shown that mMCP-1 is stored in secretory granules, which act as a repository for regulated release, but that constitutive secretion also occurs, which requires continuous protein synthesis and transport via the Golgi complex (3, 8). Consistent with these data, we observed that ADP and ATP induced a substantial release of mMCP-1 in the presence of cycloheximide, which blocks protein synthesis, or brefeldin A, which blocks membrane vesicular traffic out of the endoplasmic reticulum. This indicates that adenine nucleotides induce release of preformed mMCP-1 from secretory granules distal to the Golgi complex (Fig. 5). In these experiments, cells were preincubated with cycloheximide or brefeldin A for 24 h, which also reduced constitutive secretion of mMCP-1.

Fig 5.

Adenine nucleotides induce release of preformed mMCP-1 via degranulation. Mast cells were incubated for 24 h with methanol vehicle control, cycloheximide (120 ng ml⁻¹), or brefeldin A (120 ng ml⁻¹) and then stimulated with 10 μM ADP (filled bars) or 10 μM ATP (striped bars) or left unstimulated (open bars). Culture supernatants were collected 2 h later and assayed for mMCP-1. Data are presented as means ± SD for triplicate cultures. Values which differ significantly from unstimulated controls are indicated (**, P < 0.005).

DISCUSSION

Mast cells of various types express a variety of functional P1, P2Y, and P2X receptor subtypes that can be activated in both an autocrine and a paracrine manner (5, 52). In this study, we showed that bone marrow-derived mucosal mast cells expressed transcripts for A1, A2B, and A3 receptors, all of the P2Y receptors, P2X1, and P2X4. Changes in cytosolic calcium levels were monitored, and cells were observed to respond to stimulation with adenosine, ADP, ATP, UDP, or UTP but not inosine, AMP, or UMP. Inosine has been observed to signal through P1 receptors in a variety of cell types, such as macrophages (21) and mast cells (22). In contrast, it was long thought that there was no receptor for AMP or UMP, although recent data suggest that AMP can activate the human A1 receptor (41).

Maximal stimulation of calcium flux in the current study was observed with relatively high concentrations of adenosine, suggesting a predominant involvement of the low-affinity A2B and A3 receptors (10), and this was largely blocked by the A3-specific antagonist MRS 1523 (13). ADP is known to signal via murine P2Y1, P2Y12, and P2Y13 receptors (48). ADP-induced calcium mobilization was blocked by the P2Y1-selective antagonist MRS 2179, suggesting a predominant involvement of this receptor subtype (2, 39).

UDP and UTP were observed to stimulate calcium flux, although this did not result in release of mMCP-1. UDP can signal via the P2Y6 receptor (27) as well as the CysLT1 (32) and GPR17 (6) receptors. A high concentration (100 μM) of UDP was needed to mobilize calcium in our hands, suggestive of low-affinity receptor expression in murine mucosal mast cells. Lower concentrations of UTP, known to signal predominantly through P2Y2 and P2Y4 receptors (27), elicited a response.

T. spiralis SP inhibited activation of mast cells by adenosine at concentrations of 10 μM or less. This can most likely be attributed to an adenosine deaminase which we have shown to be secreted by the parasite (15). Increasing the concentration of adenosine to 100 μM overcame the inhibition (data not shown), which would be limited by the concentration and specific activity of the enzyme in SP. The deamination product of adenosine (inosine) might be expected to act on A3 receptors in some cell types, but as recorded here, inosine did not elicit calcium mobilization in mucosal mast cells.

Activation of mast cells by UDP and ADP was also blocked by SP and, as predicted, by purified recombinant 5′-NT. The 5′-NT is unusual in that it can catalyze the hydrolysis of nucleoside 5′-diphosphates and 5′-monophosphates at the same active site (17). It is monospecific for AMP among the nucleoside 5′-monophosphates and catalyzes the hydrolysis of nucleoside 5′-diphosphates in the rank order of UDP ≫ ADP. The end product of hydrolysis of UDP would therefore be UMP, and it is consistent that inactivation of UDP, which appears to bind its receptor with low affinity, is very efficient. Inactivation of ADP-induced calcium mobilization is less efficient, as the reaction product of the 5′-NT, i.e., adenosine, may then act in turn on P1 receptors. Use of specific receptor antagonists confirmed that this is the most likely scenario.

Despite the wide range of agonists which stimulated calcium flux, only ADP and ATP induced mMCP-1 secretion. Inhibition of ADP-induced secretion was effected by the 5′-NT, which had no effect on ATP-induced release. This was expected, as the T. spiralis 5′-NT is highly active against ADP but shows no activity against ATP or any other nucleoside 5′-triphosphate (17). It was therefore interesting that parasite SP significantly reduced release of mMCP-1 induced by 10 μM ATP, and there are several possible reasons for this. First, the NDPK secreted by infective larvae can convert ATP to ADP, or UTP to UDP, in the presence of nucleoside diphosphate acceptors (18), and it could work in conjunction with the 5′-NT to reduce the concentration of extracellular nucleoside triphosphates. Second, T. spiralis secretes serine/threonine protein kinases, which consume ATP in the presence of suitable substrates, some of which are also present in SP (1). Although ATP-induced calcium flux appeared unaffected by SP, a critical threshold and/or additional signaling pathways may be necessary to stimulate degranulation, and these may be more susceptible to inhibition. Infective larvae also express ecto-ATPases on their surface (44), and although these could contribute to depletion of extracellular ATP in vivo, they would not be present in the SP utilized here.

In conclusion, we have shown that mucosal mast cells respond to stimulation with nucleosides/nucleotides by secreting the chymase mMCP-1, an important determinant of immunity to the parasitic nematode T. spiralis, and demonstrated in turn that secreted enzymes of the parasite inhibit this response. In addition to prolonging residence in the intestinal tract, it is possible that these enzymes could aid larvae in skeletal muscle by inhibiting the effects of nucleotides on a variety of cells, including connective tissue mast cells. Mice deficient in mMCP-1 have significantly larger numbers of muscle larvae, although this most likely results from an increased output of L1 from adult worms during the enteral phase (24). Parasitic nematodes have evolved other mechanisms to inhibit mast cell function. The filarial parasite secreted protein ES-62 inhibits FcεRΙ-mediated degranulation of human mast cells by blocking calcium mobilization and NF-κB activation via binding to Toll-like receptor 4 and subsequent sequestration of protein kinase C-α (30). Given the recent interest in using parasitic helminths to treat inflammatory disorders (20, 28), we can expect further dissection of the mechanisms used by these organisms to suppress immune responses.