Mast cell-independent impairment of host defense and muscle contraction in T. spiralis-infected W/WV mice

Bruce A. Vallance, Patricia A. Blennerhassett, Jan D. Huizinga, and Stephen M. Collins

Intestinal Diseases Research Program, Department of Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In response to nematode infection, the host presumably attempts to create an unfavorable environment to prevent larval penetration of the host and to expedite parasite expulsion from the gut. In this study, we have used W/WV mice with or without mast cells after bone marrow reconstitution (BMR-W/WV) to examine the role of mast cells in the host response. W/WV, BMR-W/WV, and wild-type (+/+) mice were infected with Trichinella spiralis. Infected W/WV mice exhibited less tissue damage and experienced a delay in worm expulsion and a greater degree of larval penetration of the gut leading to encystment in skeletal muscle. Tissue injury was greater and worm expulsion was normalized in BMR-W/WV mice, but larval penetration remained unchanged. Spontaneous contractile activity of jejunal muscle was disrupted in W/WV mice, as was the contractile response to carbachol. These abnormalities were also present in BMR-W/WV mice. These results indicate that mast cells mediate tissue damage and contribute to the timely expulsion of nematodes from the gut during primary infection.

interstitial cell; nematode; intestine; primary infection


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TISSUE MAST CELL HYPERPLASIA is characteristic of the mammalian host response to nematode infections (17, 21, 42, 51). Activation of mast cells and their release of mediators has been implicated not only in the eventual clearance of these infections (17, 21) but also in the development of tissue pathology and altered physiology seen during infection (19, 26, 36, 37). The implication of mast cells in these processes is based largely on studies (1, 12, 22, 33, 34, 35) on the c-kit-defective W/WV mice that are mast cell deficient. These mice exhibit impaired host defense and delayed expulsion after infection with several species of nematode and helminthic parasites (1, 12, 22, 33, 34, 35). In some cases, the delayed parasite expulsion was reversible through bone marrow reconstitution (22, 34), indicating a role for mast cells, whereas with other parasites, restoration of mast cells had no effect (11, 24). A possible explanation for these inconsistencies was the recent demonstration that W/WV mice also lack a population of c-kit-dependent interstitial cells of Cajal (ICC) (23). These cells regulate myoelectrical slow wave activity and are important in the control of the propulsive motor activity of the small bowel (14, 23).

Nematode infections are accompanied by increases in intestinal muscle contractility and propulsive activity (2, 16, 44, 45) as well as accelerated intestinal transit (8). Our laboratory and others (10, 17, 43) have postulated that, during such infections, the intestinal motor apparatus acts as an extension of the immune system, aiding in the expulsion of pathogens through increased propulsive activity. Because W/WV mice lack an important population of small bowel ICC, leading to abnormal motility and delayed intestinal transit (23), their inability to clear nematode infections efficiently could reflect impaired intestinal propulsion rather than mast cell deficiency. The almost complete loss of function of the tyrosine kinase receptor c-kit results in a broad array of hematopoietic cellular deficiencies in W/WV mice. These deficiencies include mast cells, erythrocytes, and a subpopulation of intraepithelial lymphocytes as well as nonhematopoietic cells such as melanocytes, germ cells, and ICC; however, only mast cells and ICC have so far been implicated in the regulation of intestinal muscle function.

To evaluate the relative contributions of mast cells and other non-bone marrow-derived c-kit dependent cells such as ICC, we investigated Trichinella spiralis-infected W/WV mice with or without bone marrow reconstitution. Bone marrow grafts from congenic wild-type mice restore mast cells as well as other hematopoietic cell populations to W/WV mice but do not correct deficiencies in nonhematopoietic cell types such as ICC. As expected, W/WV mice exhibited delayed expulsion of adult worms from the gut and more larvae gained access to the skeletal muscle of the host. Bone marrow grafting partially corrected the rate of worm expulsion but did not alter the penetration to skeletal muscle by T. spiralis larvae. W/WV mice also exhibited changes in spontaneous contractile activity of intestinal muscle and an attenuation of infection-induced muscle hypercontractility after carbachol stimulation. These findings were also evident in bone marrow-reconstituted W/WV mice. Together, these results indicate that non-bone marrow-derived cells such as ICC, in cooperation with mast cells, play separate but integrated roles in the host's defense against an enteric nematode infection.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mice. WBB6F1-KitW/KitW-v (W/WV ) mice and congenic wild-type WBB6F1-+/+ (+/+) mice have been well described previously (18) and were obtained from Jackson Laboratories (Bar Harbor, ME). Mice were kept in sterilized filter-topped cages, handled in tissue culture hoods, and fed autoclaved food under specific pathogen-free conditions at our animal facilities. Sentinel animals were routinely tested for common pathogens. The protocols used were in direct accordance with guidelines drafted by the McMaster University Animal Care Committee and the Canadian Council on the Use of Laboratory Animals.

Bone marrow reconstitution. The method for reconstituting mast cell populations in W/WV mice by injecting bone marrow cells of congenic +/+ mice was described previously (36). Briefly, donor +/+ mice were euthanized by cervical dislocation, and bone marrow was flushed from their femurs using Hanks' balanced salt solution. The bone marrow cells were then pooled, washed, and counted using trypan blue exclusion to assess viability. Recipient W/WV mice were then reconstituted by tail vein injection of 20,000,000 viable bone marrow cells suspended in sterile PBS. Bone marrow-reconstituted W/WV mice (designated BMR-W/WV) were studied 8-12 wk later. Successful reconstitution was confirmed by testing the hematocrit of sample mice that revealed the correction of the anemia suffered by W/WV mice.

Trichinella infection. The T. spiralis parasites used in this study originated in the Department of Zoology at the University of Toronto, and the colony was maintained through serial infections alternating between male Sprague-Dawley rats and male CD1 mice. The larvae were obtained from infected rodents 60-90 days after infection, using a modification of the technique described by Castro and Fairbairn (9). Mice were infected by administration of 0.1 ml of PBS containing 375 T. spiralis by gavage. To minimize differences between infections, all three groups of mice were infected with the same T. spiralis preparations.

Body weight measurement. To evaluate the systemic effects of infection and inflammation, changes in body weight were assessed over the infection, with mice weighed just before infection as well as at 4-day intervals during the course of the T. spiralis infection until day 16. Data are from one representative experiment and are presented as the mean of the percent start weight from five mice at each time point.

Histology and mast cell counts. Full-thickness jejunal tissues were fixed in Carnoy's fixative. Sections (3 µm) were cut and stained with hematoxylin and eosin or Alcian blue and lightly counterstained with safranin for visualization of mast cells. The number of mucosal mast cells per villus crypt unit in the jejunal mucosa was determined for a minimum of 20 villus crypt units, and data were expressed per villus crypt unit. The number of mast cells identified in a full cross section in the muscle layers and myenteric plexus region, as well as subserosal mast cells, was counted separately and expressed as mast cells per cross-section. Additional mast cell counts were performed and expressed as cells per square millimeter of muscle tissue. Photomicrographs were taken using a Zeiss camera.

Adult worm counts. The entire length of the small intestine was removed and opened longitudinally, and all adult worms within the small intestine were then counted using a modification of the method described by Castro and Fairbairn (9). Briefly, the mucosa was separated from the underlying muscularis by scraping with a glass microscope slide and mixed with 1 ml of PBS. The worms were then counted using a scored petri dish and an inverted microscope. In accordance with established practices, worm rejection was considered complete when at least 98% of the infective dose had been expelled from the gut (48).

Skeletal muscle larvae counts. Skeletal muscle larvae numbers were determined by homogenizing skinned, eviscerated mice by using a mixture of PBS, hydrochloric acid, and pepsin in a kitchen blender, again by using a modification of the method described by Castro and Fairbairn (9). The homogenate was transferred to 1,000-ml flasks and bubbled with 95% O2-5% CO2 for 2 h. The digestate was then strained through several layers of gauze, and the worms were allowed to settle at unit gravity. After tissue digestion, the supernatant was removed and the worms were resuspended in PBS, pH 7.4. The larvae were then washed, collected, and counted under an inverted dissecting microscope.

Preparation of antigen. Antigen solutions were made fresh each day from frozen stock solutions. Antigen was prepared from frozen T. spiralis skeletal muscle larvae using the method of Russell and Castro (39). Larvae were kept on ice in PBS, homogenized, and centrifuged for 30 min at 104 g. Antigen was diluted to ~40,000 larvae/ml.

Muscle function. The preparation of the sections of jejunal longitudinal muscle for muscle contractility analysis and the analysis of the length-tension relationships were described previously (3, 44). Briefly, the jejunum was removed and placed in oxygenated (95% O2-5% CO2) Krebs solution, and 1-cm sections of whole gut were cut from the jejunum, beginning at the ligament of Treitz and proceeding distally. The lumen of each segment was flushed with Krebs buffer before the insertion of short (2-3 mm) lengths of Silastic tubing (0.065-in. OD, 0.030-in. ID) (Dow Corning, Midland, MI) into the open ends of the gut segments. Tubing was then tied in place with surgical silk. The insertion of the tubing was found to maintain patency of the gut segments over the course of the experiments. Segments were then hung in the longitudinal axis and attached at one end to a Grass FT03C force transducer (Quincy, MA), and responses were recorded on a Grass 7D polygraph. Tissues were equilibrated for 30 min at 37°C in Krebs solution oxygenated with 95% O2-5% CO2 before starting the experiment. Experiments were then conducted to examine the length-tension characteristics of the muscle before and after infection. Segments were stretched by applying tension equivalent to 0-1,250 mg of weight, and contraction was assessed after stimulation with 1 µM carbachol (Sigma Chemical, St. Louis, MO). Initial experiments indicated that this tension range was sufficient to determine the maximal responsiveness of both control and inflamed tissues. After each application of tension, the length of the tissue and the contractile response were recorded. Once the optimal applied tension was reached, the frequency of spontaneous activity was assessed by counting the number of contractions generated within a 1-min period, with at least four periods examined per tissue, each at least 5 min apart from the others. At least four tissues were examined per mouse, with five mice examined per group. At the end of the experiment, the tissue was removed, blotted, and weighed. The tissue weight and length found to generate the maximum contractile response (optimal tension) were used to calculate the cross-sectional area of the tissue (44).

Antigen challenge. In some experiments, the tissue response to Trichinella antigen was also assessed. In these experiments, tissues were stretched to their optimal applied tension and allowed to equilibrate, the tissue length was recorded, and then the tissue was stimulated by 1 µM carbachol. After application of carbachol, the contractile response was recorded, the tissue baths were washed out, the tissues were allowed to reequilibrate, and then the contractile response to 100 µl of the Trichinella antigen (equivalent to 4,000 larvae) was determined and compared with the carbachol response. The mast cell stabilizer disodium cromoglycate (1 mM; Fisons Laboratories) or the calcium ionophore compound 48/80 (1 µg/ml; Sigma Chemical) was used in some experiments.

Data presentation and statistical analysis. Responses to carbachol were recorded from tracings, and then the contractile activity was calculated and expressed as milligrams of tension per cross-sectional area as described previously (3, 44). For each mouse, the mean tension was calculated from at least three segments. All the results are expressed as means ± SE; n refers to the number of mice tested. Statistical significance was calculated using the Student's t-test for comparison of two means or a one-way ANOVA for comparison of three or more means. Multiple comparisons were performed using the Newman-Keuls multiple-comparison test. P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

T. spiralis infection: macroscopic appearance and body weight. W/WV and +/+ mice developed similar changes in behavior characteristic of T. spiralis infection, including the adoption of a hunched appearance, reduced movement, and piloerection by days 4-6 of infection. Weight loss was gradual for both strains and was maximal on postinfection day 12. However, +/+ mice lost significantly more weight (10-15%) compared with W/WV mice (6-8%) (see Fig. 1). BMR-W/WV mice also lost significantly more weight than the W/WV mice. However, unlike both the +/+ and W/WV mice, which showed a rebound in body weight by postinfection day 16, the weight of the mast cell reconstituted mice fell even further, to ~80% of their initial weight by postinfection day 16.


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Fig. 1.   Weight changes of +/+ (), W/WV (open circle ), and bone marrow-reconstituted (BMR)-W/WV mice () over the first 16 days of a Trichinella spiralis infection. Weight data from 1 representative experiment is shown. Each point represents average weight data pooled from 5 mice and is expressed as %initial body weight. * P < 0.05 vs. W/WV at same time point.

Histology, mast cell counts, and distribution. During the course of the infection, an inflammatory response and intestinal tissue damage characteristic of T. spiralis infection were observed to occur in the jejunal mucosa of both +/+ and W/WV mice. Corresponding with the time of greatest weight loss, the observed tissue damage was also maximal at postinfection day 12. However, tissue damage and villus blunting were greater in +/+ mice compared with W/WV mice. After bone marrow grafting, inflammation and tissue damage were increased (see Fig. 2, A and B).


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Fig. 2.   Histology showing representative tissue damage seen at postinfection (PI) day 12 in W/WV (A) and BMR-W/WV mice (B). Note the mild blunting and maintenance of normal villus architecture of the W/WV mice and the more severe tissue damage and blunting of the jejunal villi in the mast cell-reconstituted mice. C: 2 connective tissue mast cells within the muscle layers of a +/+ mouse infected 16 days prior with Trichinella. In general, few mast cells were seen in the muscle layers of the +/+ mice, and those identified were scattered throughout the muscularis externa. D: the muscle layer of a BMR-W/WV mouse infected and killed at the same time as the mouse in C. Note the numerous mast cells within the field of view, many found within the myenteric plexus and serosa. Original magnification: A and B, ×100; C and D, ×400.

The number of mast cells identified microscopically in the jejunum of W/WV, +/+, and BMR-W/WV mice were evaluated per villus crypt unit both before and during a primary T. spiralis infection (see Table 1). Before infection, as expected, W/WV mice were essentially devoid of intestinal mast cells (0.1), whereas mast cells were present but of similar low numbers in both the +/+ and BMR-W/WV mice (0.8 ± 0.2 vs. 1.1 ± 0.2, respectively), confirming the success of the bone marrow grafting. During infection, mucosal mast cell numbers were seen to rise in all groups, even in W/WV mice, with numbers peaking at postinfection day 16. However, the number of mucosal mast cells increased more rapidly and peaked at much higher levels in the +/+ and BMR-W/WV mice compared with the W/WV mice, with similar numbers of mast cells seen in these two groups throughout the infection.

                              
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Table 1.   Mucosal mast cell numbers per villus crypt unit

Few, if any, connective tissue mast cells were seen in the muscle layers of the W/WV mice during the course of the infection (see Table 2). In the +/+ mice, occasional Alcian blue-positive mast cells were seen in the longitudinal muscle or subserosally (see Fig. 2C), with numbers increasing over the course of the infection to peak at day 21. Staining of tissues from BMR-W/WV mice led to the discovery of large numbers of mast cells within the muscle layers, myenteric plexus, and subserosa. These unexpectedly large numbers of cells were evident by postinfection day 8, with numbers peaking on day 21, at levels increased at least 25-fold over that seen in the +/+ mice (see Fig. 2D).

                              
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Table 2.   Mast cell numbers in muscle layers and serosa

Worm counts. Evaluation of the parasite load in the small intestine of T. spiralis-infected W/WV and +/+ mice revealed a similar time-dependent decrease in worm numbers from the small bowel of both mouse strains over the first 12 days of a primary infection (Fig. 3). However, although expulsion of the infection was complete by day 16 in the +/+ mice, a significant number of worms (32 ± 7) could still be found in the W/WV mice until at least postinfection day 21. By day 28, this number had dwindled to <10 worms per animal. Mast cell reconstitution of the W/WV mice restored worm expulsion to levels similar to those found in the +/+ mice, with expulsion complete by postinfection day 16 (see Fig. 3).


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Fig. 3.   The number of adult T. spiralis worms recovered from the small bowel of infected +/+ (solid bar), W/WV (open bar), and BMR-W/WV mice (hatched bar) on days 8, 12, 16, and 21 PI, after a primary infection with 375 T. spiralis larvae by gavage. Results shown represent means ± SE of groups of 5 mice. Note that the T. spiralis worms had been completely expelled from the small bowel of both +/+ and BMR-W/WV mice by day 21 PI. * P < 0.05, recovery of significantly fewer worms compared with W/WV mice at the same time point.

Skeletal muscle larvae counts. During infection, T. spiralis larvae are released by adult worms and migrate from the intestine via the lymphatics to the skeletal muscle. There the larvae invade muscle cells, and by 40-60 days after infection the larvae are completely encysted within the skeletal muscle. To assess the fecundity of the adult worms, the bodies of previously infected mice were digested, and the number of recovered larvae was counted. Approximately 50,000 muscle larvae were recovered per W/WV mouse, significantly more than the 28,000 recovered from the +/+ mice. Mast cell-reconstituted W/WV mice were found to have on average 53,000 larvae, again significantly more than the +/+ mice and similar to the recovery from unreconstituted W/WV mice (Fig. 4).


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Fig. 4.   The number of T. spiralis skeletal muscle larvae recovered after digestion of the body of +/+ (solid bar), W/WV (open bar), and BMR-W/WV mice (hatched bar) infected 40-80 days previously with 375 T. spiralis worms. Results are means ± SE of the number of larvae recovered per body for groups of 5 animals. * P < 0.05 vs. +/+ mice.

Spontaneous muscle activity. One of the characteristics of the loss of pacemaker activity in the W/WV mouse is an alteration of spontaneous activity with a reduction in the frequency of spontaneous contractions and abnormal contractile patterns. These changes become even more exaggerated during T. spiralis infection. As shown in Fig. 5, tissues from postinfection day 8 +/+ mice show a regular pattern of spontaneous activity, with higher frequency (37.3 ± 0.7 contractions/min) than that seen in the W/WV mice (22 ± 2 contractions/min). Note that the pattern and frequency (23.2 ± 2 contractions/min) of spontaneous activity of BMR-W/WV mice, also at postinfection day 8, were similar to that generated by an unreconstituted W/WV mouse during infection. This indicates that bone marrow reconstitution did not restore normal spontaneous activity and presumably did not affect the loss of pacemaker activity in the W/WV mouse.


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Fig. 5.   Representative tracing of the contraction pattern seen in +/+, W/WV, and BMR-W/WV mice at day 8 PI. Note the regular contraction pattern and higher frequency in the +/+ mice compared with the irregular and slower pattern in the W/WV. Note that the pattern does not change after BMR.

Muscle response to carbachol. The contractile response by jejunal longitudinal muscle to 10-6 M carbachol was similar among all mouse groups before infection. As can be seen in Fig. 6, 8 days after T. spiralis infection, there was a substantial and significant (P < 0.01) increase in the tension generated by longitudinal muscle from both W/WV and +/+ mice. However, the increase in muscle tension by W/WV mouse tissue was significantly reduced compared with +/+ mice. Despite their mast cell reconstitution, tissues from BMR-W/WV produced tension at levels similar to those of W/WV mice and were also significantly reduced compared with the +/+ mice. Although tissues from +/+ mice still generated significantly more tension than tissues from controls, the contractile response generated by tissues from W/WV and BMR-W/WV on postinfection day 16 had decreased to levels similar to those seen before infection. In contrast, the significant elevation in muscle tension in the +/+ mice was maintained at postinfection day 16.


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Fig. 6.   The tension generated in response to 10-6 M carbachol in jejunal longitudinal muscle from uninfected +/+ (solid bar), W/WV (open bar), and BMR-W/WV mice (hatched bar) as well as mice infected 8 and 16 days earlier. Results shown represent means ± SE of groups of 4 mice. * P < 0.05 vs. W/WV mice at the same time point.

Muscle response to antigen. Because the accumulation of mast cells in the muscle layers was so large and rapid in the reconstituted mice, we examined whether these mast cells were functionally active and could therefore play a role in host defense by stimulating muscle contraction in response to Trichinella antigen. We tested tissues at postinfection day 16, because this was the first infection time point at which the presence of mast cells was seen to have an effect on worm expulsion (see Fig. 3). As expected, tissues from W/WV mice, which lack mast cells, did not respond to Trichinella antigen (see Table 3). As for the +/+ mice, the response was variable. A small contractile response was seen in some tissues, whereas in other mouse tissues antigen exposure caused no contraction. This poor response was not unexpected, because in previous infection studies (31, 47) in the rat, connective tissue mast cells and their ability to cause muscle contraction in response to antigen have been shown to be maximal long after the primary infection was resolved. However, tissues removed from BMR-W/WV mice strongly contracted in response to the antigen. Surprisingly, the contraction could not be blocked by cromoglycate. Furthermore, the contractile response could not be mimicked by administration of compound 48/80, suggesting a response uncharacteristic of a normal connective tissue mast cell response. Whether this reflects the immaturity or bone marrow-derived nature of the mast cells or indicates a non-mast cell-mediated mechanism is at present unknown.

                              
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Table 3.   Muscle contraction in response to antigen


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Enteric infections elicit a highly integrated response by the host aimed toward the neutralization and expulsion of the pathogen (7, 17). During nematode infections, this involves the activation of the immune system along several pathways, including the proliferation of putative effector cell types, such as mast cells, as well as the immune-mediated development of changes in enteric physiology (7, 10, 17, 37). Although a functionally intact immune system is necessary for the expulsion of these infections (38, 42), it remains unclear whether this reflects the direct actions of the immune system on the infectious organism or indirect actions through alterations in gut physiology. The c-kit-deficient W/WV mouse offers a unique opportunity to distinguish between these two possibilities. The inability of W/WV mice to rapidly expel a number of nematode species (1, 12, 22, 33, 35) has been attributed to their relative lack of tissue mast cells. However, findings (23) indicate that the c-kit deficiency seen in these mice also results in the loss of normal intestinal motility due to a deficiency in small bowel ICC (23). Although W/WV mice do suffer from other cellular deficiencies as mentioned above, mast cells and ICC are the most likely candidates that influence intestinal muscle function and worm expulsion. However, any potential role for the ICC in host defense is, as of yet, incompletely understood.

As expected, a significant mast cell hyperplasia was seen during Trichinella infection in the c-kit-expressing +/+ mice. There was a prominent increase in mast cell numbers in the mucosa that peaked at postinfection day 16. We also observed a small increase in the number of mast cells found within the muscularis externa and serosa at the later stages of the infection, consistent with previous studies (31). The maximum number of mast cells seen in W/WV mice after infection was reduced by ~95% compared with findings in +/+ mice. Grafting bone marrow from +/+ mice to W/WV mice restored the mastocytosis seen during infection. However, although similar numbers of mucosal mast cells were seen in the BMR-W/WV and +/+ mice, the maximum number of mast cells seen in the muscularis externa and serosa was at least 25-fold greater than that seen in the tissues of +/+ mice after infection. This unexpected distribution of mast cells in infected BMR-W/WV mice may reflect previously described (18) differences in the phenotype of mast cells derived directly from bone marrow and those found in other tissues. In addition, it is known that the microenvironment influences mast cell phenotype (28). It is likely that infection-induced changes in the muscularis externa influence mast cell chemotaxis. For example, substance P-containing nerves have a close affinity for mast cells (40), and there is a substantial increase in the substance P content of the muscularis externa during nematode infection (41). It is therefore possible that neural remodeling in the muscle layers may result in a stronger chemoattraction for mast cells. Thus differences in environment between the mucosal and muscle compartments may contribute to the altered distribution of mast cells in BMR-W/WV mice.

The results of this study identified those components of the host response that are mast cell mediated. Body weight loss was attenuated in W/WV mice but was apparent in infected BMR-W/WV to a degree that exceeded that in +/+ mice. This could reflect the release of mast cell-derived mediators such as interleukin-6 (5, 27) and tumor necrosis factor-alpha (4, 20, 30) that have been associated with anorexia and cachexia. The injury to host mucosal tissues seen during T. spiralis infection is likely caused by the local effects of mast cell products, as damage was attenuated in infected W/WV mice but prominent in infected BMR-W/WV mice. The efficient expulsion of worms from the gut required the presence of mast cells or other c-kit-dependent hematopoietic cells, because 10-15% of the worm burden remained in the gut at postinfection day 16 and stayed for almost 2 wk beyond this point in W/WV mice but not in infected BMR-W/WV mice.

Our implication of mast cells in accelerating expulsion of worms during the intestinal phase of the infection addresses a controversial issue. Some studies (1, 12, 22, 33, 34, 35) support such a role, whereas others (11, 24, 26) reject a critical role for mast cells in the expulsion of nematode parasites. The mechanisms involved in such a role have also been unclear. Although an important role for mast cells in regulating innate immunity and host defense through the recruitment of other inflammatory cells has been demonstrated (15, 30, 32) during bacterial infections, this role has yet to be investigated in nematode infections. Mast cells have been well characterized during nematode infections as releasing a number of potent inflammatory mediators. Although such mediators damage the parasite, they also damage host tissues as well as induce changes in enteric physiology. Mediators released by mast cells have been implicated in increased fluid secretion as well as mucin release (6, 7, 37) within the intestine. Mast cell-derived mediators have also been shown to contract smooth muscle (31, 47). Although it is not clear that the response by BMR-W/WV muscle to Trichinella antigen was mediated by mast cells, muscle contraction in response to parasite antigen was only reproducibly seen in the BMR-W/WV mice, which are the only mice to possess large numbers of mast cells in their jejunal muscle layers. Such a muscle response is usually observed only after repeated sensitization to parasites (31, 47) or other antigens and has not been previously described during a primary infection. In combination with other changes in enteric physiology, stimulation of muscle contraction by parasite antigens could perhaps explain the more rapid worm expulsion seen in the BMR-W/WV mice compared with that seen in unreconstituted W/WV mice.

Our results indicate that other aspects of the host response to the parasite are mast cell independent. It is well documented that T. spiralis larvae gain access to the host via the intestinal epithelium and migrate to skeletal muscle, where they encyst. A major increase in the number of encysted larvae as found in the W/WV mice is biologically significant, because it influences the viability of the host and improves the chances of the parasite being passed on to a new host. The increased numbers of encysted larvae recovered from the skeletal muscle of W/WV compared with their wild-type littermates in this study indicate that host defense against this parasite is impaired in c-kit-deficient W/WV mice. Because an even greater number of larvae were recovered from infected BMR-W/WV mice, this impairment of host response is mast cell independent. This does not conflict with our demonstration of slow worm expulsion in the W/WV mouse and its normalization in the BMR-W/WV mice, because previous studies (25) have shown that the release of larvae from adult worms within the intestine begins by day 5, peaks at days 6-7, and ends by day 10. At days 8 and 12 in our study, there were no differences in intestinal adult worm burdens between +/+, W/WV, and BMR-W/WV mice. The factors that control parasite fecundity must come into play within the first 10 days of the infection and must involve non-bone marrow-derived cell types lacking in the W/WV mice, such as ICC.

This study also confirms that BMR does not normalize the irregular spontaneous contractile activity seen in WW/V mice and invokes a role for ICC. In the +/+ mice, electrical slow wave activity determines the appearance of action potentials, which occur superimposed on the electrical slow waves. Consequently, contractions occur in a regular pattern, matching the slow wave frequency. With the loss of ICC in W/WV and BMR-W/WV mice, action potential generation is not restricted to slow waves and irregular patterns of contraction result (14). Interestingly, ICC are among the first cells within the intestinal neuromuscular layers to develop ultrastructural changes and alterations in function during T. spiralis infection (13) as well as other forms of intestinal inflammation (29). Although this leads to early disruption of motor patterns, these changes are transient, and previous studies (2, 8) have shown that T. spiralis infection is associated at later time points with increased propulsive activity and more rapid intestinal transit. This may involve the restoration of ICC function, in concert with the previously demonstrated immune-mediated increases in longitudinal muscle contraction that together may contribute to the altered transit. Although T cells are known to be involved in mediating these changes (46), other cell types such as mast cells have also been hypothesized to play a role (19, 43). However, after restoration of the mastocytosis seen during infection, BMR-W/WV mice exhibited impaired generation of muscle hypercontractility, suggesting that non-bone marrow-derived cells are responsible for the defective response and that the immune system may need to act through ICC to alter longitudinal muscle function. Although the role of ICC or other nonhematopoietic cell types in the motility changes that occur during infection still needs to be defined, their absence appears to place the host at a considerable disadvantage in host defense, as measured by the increased larval burden recovered from mice lacking ICC.

The present study is the first to demonstrate a role for non-bone marrow-derived c-kit-dependent cells in enteric host defense against T. spiralis. A previous study (24) examining Nippostrongylus brasiliensis infection in W/WV mice reported that mast cell reconstitution did not alter the delayed expulsion seen at the intestinal phase of the infection, although it did reduce parasite numbers in the migratory phase through the lungs. Ishikawa et al. (24) suggested that a non-mast cell, c-kit-dependent cell type was involved in the host defense. In both the previous (24) and the current study, the ICC is the cell type most likely responsible. In the Nippostrongylus study (24), it was the intestinal phase of the infection that was unaffected by bone marrow grafting, and this probably reflects differences in the intestinal niche occupied by the parasite. Because adult Nippostrongylus worms dwell within the bowel lumen, they can presumably evade much of the immune response of the host and are less likely to suffer from the effects of bone marrow reconstitution. However, they are likely to be more susceptible to factors altering propulsive activity. In contrast, adult Trichinella worms that invade the intestinal epithelium are probably less vulnerable to propulsive forces designed to evict them. These results suggest that, although the end result may be pathogen specific, certain generalized defense mechanisms exist against nematode infections. Although mast cells are major effector cells of the immune response, intestinal motor activity is another component of the integrated host response. Together, these observations suggest that the loss of propulsive forces may retard eviction of the parasite from the gut, as seen with N. brasiliensis (24), and may provide invasive parasites such as T. spiralis a greater opportunity to gain access to the host.


    ACKNOWLEDGEMENTS

We thank Ryan Barrett for technical expertise.


    FOOTNOTES

This study was funded by the Medical Research Council of Canada.

Address for reprint requests and other correspondence: S. M. Collins, Rm. 4W8, McMaster Univ. Medical Center, Hamilton, Ontario, Canada L8N 3Z5 (E-mail: scollins{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.

Received 22 February 2000; accepted in final form 14 November 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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