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* Division of Rheumatology, Allergy and Immunology, Departments of Medicine and
Pathology, Brigham and Women’s Hospital and Harvard Medical School, and
Department of Pediatrics, Beth Israel Hospital, Children’s Hospital, and Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The normal immune response to Trichinella spiralis (Ts) in mice depends on both MC (19, 20, 21) and Th2 cells (21, 22). In the absence of either, mice cannot reject adult worms from the intestine and exhibit increased larval burdens. All MC development in vivo requires stem cell factor and its receptor c-kit, and the local mucosal mastocytosis that occurs during helminth infection requires the additional comitogenic function of Th2-derived cytokines (19, 20, 21). The intestinal immune response to Ts is also accompanied by eosinophilia, but the eosinophil is not thought to be essential for the elimination of adult worms after a primary infection (19, 21, 23, 24, 25, 26). Moreover, although both human and mouse eosinophils kill Ts larvae in vitro (27, 28, 29), their importance in clearance of larvae that are released by adult worms in vivo remains controversial based on the results of studies in mice (23, 24, 25, 26). Depletion of eosinophils in Ts-infected mice with a rabbit anti-eosinophil serum resulted in increased larval burdens (23), whereas eosinophil depletion by an mAb that blocked the actions of IL-5, a cytokine essential for normal eosinophil responses, had no effect on larval burden (24). Recently, studies with transgenic mice overexpressing IL-5 (25) and with IL-5-null mice (26) did not find evidence that eosinophils play a significant role in the rejection of adult worms after a primary infection or in larval killing. Thus, whereas the importance of MC and Th2 cells in host defense to a primary Ts infection is well established, the role of eosinophils remains incompletely defined. Therefore, we investigated the response by eosinophils and MC in the intestine of Ts-infected mice with a targeted disruption of the CCR3 gene to clarify the role of CCR3 in the trafficking of these cell types and to delineate their respective contributions to the primary anti-helminth response as defined by clearance of the adult worms from the intestines and limitation of larval encystment in skeletal muscle.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All animals were 10–20 wk of age when infected with Ts. CCR3-/- mice were generated as described, and the strain was maintained at the Children’s Hospital Institutional Animal Care and Use Committee-approved facility (Boston, MA) (15). Male mice from the N2 and N3 generations of (BALB/c x 129) x BALB/c were used in all experiments. CCR3+/+ and CCR3-/- animals were age matched within 2 wk of each other. All experiments were conducted with the approval of the Dana-Farber Cancer Institute Animal Care and Use Committee in accordance with Public Health Service Policy and provisions of the Animal Welfare Act.
Infection and enumeration of Ts
Stage 3 infectious larvae were isolated from the skeletal muscle
of mice previously infected as described (30, 31).
Briefly, the skeletal muscle was obtained by dissection, sliced into
small pieces, and digested in 1% HCl/1% pepsin (Sigma-Aldrich, St.
Louis, MO) for 1–2 h at 37°C with stirring. The larvae were isolated
and washed by low speed centrifugation (50 x g for 5
min), counted, and diluted with distilled water to a concentration of
2000 larvae/ml. Mice were infected with 400 larvae each by
gavage. To minimize variability in infection rates, we constantly
alternated between +/+ and -/- mice. Then, at each time point, mice
from different cages were selected for analysis. Three mice from each
group were selected for histological analysis and six mice for
determination of the intestinal worm burden at each time point.
Adult Ts were enumerated by removing the small intestine, opening it, and sectioning it into 1- to 2-cm pieces in a 100-mm petri dish containing 25 ml of HBSS. The dish was sealed and rocked slowly at 37°C for 2–3 h (31). The adult worms were then counted with an inverted microscope.
Histology
Animals were killed at the indicated time points, and tissue samples were obtained and immediately fixed in 4% paraformaldehyde as described (30). The tissues were embedded in JB4 glycolmethacrylate, sectioned at 2-µm thickness, and placed on glass slides. Eosinophils were counted after the tissues were stained with Congo Red, and MC were counted after the tissues were stained for choloroacetate esterase (32, 33, 34). At least 20 high power fields (hpf; x50 objective) were counted for each mouse. Each data point represents the average of six mice from two separate experiments. Blood eosinophil counts and tissue eosinophil peroxidase content were obtained as described (35, 36). Larval cysts and degenerate cysts containing necrotic larvae were assessed histologically in sections of the tongue after staining with DiffQuik (Dade-Behring, Newark, DE). At least 10 low power fields (lpf; x10 objective) were counted for each animal, and a mean number of cysts/lpf was calculated. Cysts with necrotic larvae were counted simultaneously and were expressed as the percentage of cysts with necrotic larvae in the tongue for each animal. Values are the mean (± SEM) from 15 animals in two experiments with three animals per group and 2–3 time points per experiment (days 28, 35, and 56).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We and others have previously characterized the kinetics of the
intestinal eosinophilia (which peaks at about day 11) and MC
hyperplasia (which peaks at about day 14) that occur during the
induction of a primary Ts infection in BALB/c mice
(19, 20, 21, 22, 23, 24, 25, 26, 30, 32, 33). In the present study, contrasting
the response to Ts infection in
CCR3-/- mice and +/+ littermates, the
CCR3-/- mice failed to accumulate appreciable
numbers of eosinophils in the jejunum, whereas the +/+ mice exhibited
jejunal eosinophilia 4 days after infection, with progression to a
sharp peak at day 11 before declining to a nadir at day 20 (Fig. 1
a). A second increase in
jejunal eosinophils in the +/+ mice followed at day 30, and a plateau
was maintained through day 56. The jejunal MC hyperplasia peaked at 14
days and was similar in magnitude in both -/- and +/+ mice (Fig. 1b). In contrast to the dramatic difference in
jejunal-localized eosinophils, eosinophil counts in the blood of
CCR3-/- mice were higher than in the +/+
littermates on days 4–35 (Fig. 1c). The peripheral blood
eosinophilia in the -/- mice increased up to day 21 and then remained
fairly constant through day 56, when the experiment was terminated. The
eosinophilia in the +/+ mice peaked at day 21, declined, and rose again
at the end of the experiment at day 56.
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, a and
b). No differences in the number or localization of the
jejunal MCs were observed in the -/- and +/+ mice (Fig. 2, c and d). In the caecum of the
CCR3-/- mice, some eosinophils did permeate
7–14 days after infection (Fig. 3, a and c), with the peak value at day 14 being
<1/4 of the peak value for the same anatomic region of the +/+
mice at day 11 (Fig. 3c). In the
CCR3-/- mice, similar to the jejunum, most of
the caecal eosinophils appeared within the venous and lymphatic
vasculature (Fig. 3a), whereas in the +/+ mice most of the
eosinophils were in the connective tissue of the lamina propria (Fig. 3b). Eosinophils did not intercalate into the intestinal
epithelium of the jejunum or the caecum in either the
CCR3-/- or the +/+ mice (Figs. 2 and 3). The
caecum also demonstrated a mastocytosis from day 7 to the end of the
experiment on day 56 that was similar in both
CCR3-/- and +/+ mice (data not shown). With
resolution of the worm burden, the MCs that remained in the caecum were
all intraepithelial in location. In both the
CCR3-/- and +/+ mice, MC accumulated in the
spleen concomitantly with the resolution phase of the MC hyperplasia
(data not shown) as we have previously described (33).
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e). In addition, the cells
are in a different location, being primarily intermixed in the lymphoid
follicles in -/- mice (Fig. 4a), and in the peripheral
sinusoids of the lymph nodes of +/+ mice (Fig. 4b). These
eosinophil locations suggest migration from the blood in the -/- mice
and migration from the intestine via the lymph in the +/+ littermates.
None of the nondraining lymph nodes exhibited any eosinophilia at any
time (Fig. 4, c and d). In both sets of animals,
the eosinophils progressed more deeply into the lymphoid follicular
areas with time, commonly becoming apoptotic and ingested by resident
macrophages (data not shown). The CCR3-/- mice
also had more eosinophils in the spleen than the +/+ mice before
infection and at all time points examined after infection (data not
shown).
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The CCR3-/- and +/+ mice both had abundant
numbers of adult worms in their intestines on day 4 after infection
(Fig. 5). The numbers of worms declined
progressively thereafter, and worm expulsion was essentially complete
by day 14 in both groups of mice. There was a statistically significant
delay in worm expulsion in the CCR3-/- mice at
day 11 (p < 0.05), but this delay did not
alter the final worm expulsion kinetics.
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). Inspection revealed no quantitative
or kinetic differences in encystment among the intercostal,
paravertebral, diaphragmatic, abdominal wall, and tongue skeletal
muscles (data not shown); and thus, tongue was used as the reference
organ for monitoring larval encystment. Few larvae were identified in
muscles at day 21 postinfection. By day 28, encysted larvae were
plentiful in the tongues of both CCR3-/- and
+/+ mice (Fig. 6). Numerous eosinophils permeated the tissue and
congregated around the encysting larvae in the +/+ mice (Fig. 6, d and e), but almost no eosinophils appeared at
these sites in the CCR3-/- mice (Fig. 6, a and c). In many instances in the +/+ mice,
eosinophils breached the matrix and appeared to abut the encysted
larvae (Fig. 6, d and e), where many exocytosed
their granules. Some of the larvae where eosinophils had congregated
and made contact were necrotic (Fig. 6d). In striking
contrast, the skeletal muscle in CCR3-/- mice
lacked eosinophils, and virtually all of their encysted larvae were
surrounded by intact cyst walls (Fig. 6, a and
c). Furthermore, the number of cysts in the
CCR3-/- mice was increased by 2-fold over their
controls (Fig. 7a,
p < 0.05), and the number of degenerate cysts
containing necrotic larvae in the null mice was negligible. Almost no
degenerate cysts were observed in the CCR3-/-
mice (<1%), whereas 11% were observed in the +/+ controls (Fig. 7b).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4 integrins), and the eosinophilopoietic
cytokine IL-5 (1, 2, 3, 4, 5, 12, 13, 14, 15, 36, 37, 38, 39). IL-5 induces
eosinophil maturation and emigration from bone marrow and
cytoprotection of the cells in peripheral tissues, while locally
produced chemokines such as CCL11 facilitate their activated adhesion
to endothelial cells, and direct their movement into the tissues. At
the same time, other Th2-derived cytokines provide the comitogenic
stimulus for stem cell factor-dependent expansion and maturation of the
MC progenitors already localized in peripheral tissues, most notably
the jejunum (21, 40, 41, 42, 43, 44, 45). CCR3 is found on both MC and
eosinophils, effector cells of the Th2 cell-dependent inflammatory
response to intestinal nematode parasites. Thus, we compared
CCR3-/- and congenic BALB/c mice for the
appearance and distribution of these cells within select tissues and
importantly, for their role in host defense against the intestinal
adult worm and skeletal muscle larvae of Ts over the course
of 8 wk.
CCR3 deficiency did not affect the capacity of mice to mount a
peripheral blood eosinophilia in response to Ts (Fig. 1
c), indicating that CCR3 is not essential to up-regulate
the number of eosinophils produced in the bone marrow or to their exit
from this tissue. In contrast, the lack of eosinophils in the jejunal
submucosa in the infected CCR3-/- mice (Figs. 1a and 2a) highlights the critical nature of the
CCR3 interaction with its ligands at the vascular interface for
adhesion, transendothelial migration, and/or retention of eosinophils
in this organ. These data are consistent with the marked effects of
CCL11 deficiency in both basal and inflammation-based recruitment of
eosinophils to the small intestine (12, 14). The
recruitment of eosinophils to the caecum was partly preserved (Fig. 3),
suggesting a secondary specificity for another chemokine-mediated
recruitment step, e.g., CCR1, which is expressed and active in
eosinophils (46, 47). Eosinophils were found in regional,
but not distant, lymph nodes of CCR3-/- animals
(Fig. 4), and their location suggests migration out of the vasculature
as opposed to the sinusoidal location of eosinophils in the nodes
of +/+ mice, indicative of transit via the lymphatics. Therefore,
movement of the eosinophils into the draining nodes of the
CCR3-/- mice likely reflects a local change in
vasculature permeability and adhesion characteristics rather than a
transient gastrointestinal residence. These data indicate that
eosinophil recruitment into different inflammatory sites is
regulated in a tissue-specific manner and that CCR3 is critical for
movement into the small intestine, less so for the large intestine, and
not at all for regional lymph nodes.
Despite the profound deficit in jejunal eosinophil recruitment in the
CCR3-/- mice, the expulsion of the adult worms
was affected only transiently on day 11 (Fig. 5), before the
establishment of MC hyperplasia was complete (Fig. 1b). This
observation is consistent with earlier studies that failed to
demonstrate a requirement for eosinophils in the elimination of the
adult parasites after a primary infection (19, 21, 23, 24, 25, 26, 48, 49). IL-5-/- mice, on a C57BL/6
background, also showed a statistically significant increase in
intestinal worm burdens at a single time point, day 16 postinfection,
but all the mice had rejected the worms on day 21 (26).
The absence of any delay in the overall rejection kinetics for the
adult worm (Fig. 5) substantiates the mast cell dependence of the
integrated response and indicates that the eosinophil does not have a
meaningful role in this function.
The CCR3-/- mice also failed to recruit
eosinophils to the skeletal muscle in response to encystment of larvae
(Fig. 6), indicating that eosinophil movement into this tissue also is
strictly dependent on CCR3. In the +/+ mice, necrotic larvae were
always surrounded by eosinophils, whereas both eosinophils and necrosis
of encysted larvae were lacking in the CCR3-/-
mice (Fig. 6). This finding was accompanied by a 2-fold increase in the
numbers of larval cysts in the muscle of
CCR3-/- mice, as compared with their BALB/c
controls (Fig. 7). Although several studies indicated that eosinophils
could kill Ts larvae in vitro (27, 28, 29), in vivo
studies have generated conflicting results on this issue. Grove et al.
(23) found that suppression of the eosinophil response in
CF1 outbred mice with an anti-eosinophil serum resulted in
increased numbers of Ts larvae, counted after digestion of
the host tissues. In contrast, Herndon and Kayes (24),
using the same mouse strain, prevented eosinophilia by treating the
mice with anti-IL-5 and found no difference in adult worm rejection
from the intestines or in the number of larvae isolated from the
infected animals. Also, C3H/HeN mice overexpressing IL-5
(25) and C57BL/6 IL-5-null mice (26) were
similar to their wild-type controls in the number of larvae recovered
from host tissues and in their overall ability to reject the adult
worms from the small intestine after a primary infection. Our findings
of an increased larval burden, defined by a quantitative increase in
skeletal muscle cysts, and a marked decrease in the fraction of
encysted larvae undergoing necrosis in CCR3-/-
mice lacking infiltration of skeletal muscle with eosinophils supports
the in vitro evidence that human and mouse eosinophils are cytotoxic
for Ts larvae and the in vivo finding obtained with a
polyclonal anti-eosinophil serum (23, 27, 28, 29). Some of
the discrepancy regarding the role of the eosinophils in the primary
infection may be due to the different mouse strains used in these
different analyses and/or to subtle differences in the biology
resulting from the different interventions or procedures. Also, because
of the appreciable constitutive differences between rodent and human
eosinophils, the experimental findings for the mouse do not necessarily
apply to clinical disease in humans.
CCR3 is expressed on both committed MC progenitors and their mature
counterparts in humans (9, 10), and more recently, has
been shown to be expressed and active on the immature MC generated in
vitro from mouse bone marrow cells (6). Although mature MC
are sparse in the normal mouse intestine and are largely confined to
the submucosal connective tissues under baseline conditions, mouse
intestines are rich in committed MC progenitors (40, 41, 42, 43, 44, 45);
and inflammation, such as that which occurs after a helminth infection,
causes increased numbers of these cells to appear in the intestine
(43, 44, 45). The CCR3-/- mice
exhibited typical MC hyperplasia (Figs. 1b and 2,
c and d) and normal worm rejection from the small
intestine (Fig. 5) after infection with Ts. Because the MC
progenitors represent a pool of cells that constitutively home to the
intestine and then respond to cytokines, particularly IL-3, IL-4, and
IL-9, which are provided by Th2 cells, it is evident that the homing of
MC progenitors is not dependent on signaling via CCR3. This conclusion
contrasts with the evidence that the 4
7
integrin is required for basal homing of MC progenitors to the small
intestine (42), thereby contributing to the delayed
intestinal mastocytosis in 7 integrin-deficient mice
after infection with Ts (50). Furthermore,
because MC responses were normal in number and protective function, it
is apparent that the effect of CCR3 deficiency on eosinophil responses
is a direct effect on eosinophils rather than through a MC effect, as
has been implicated in eosinophil migration into the peritoneal cavity
and skin pouches of OVA-sensitized mice (16, 17, 18).
The helminth-induced MC hyperplasia is dependent on an intact T cell
response (21, 22). The abundant increase in mucosal MC in
the CCR3-/- mice (Figs. 1b and
2c) indicates that the function of Th2 lymphocytes also is
not appreciably altered by CCR3 deficiency. Further support for this
interpretation is the observation that the infected CCR3-deficient mice
have a blood eosinophilia and increased numbers of eosinophils in the
draining lymph nodes. Because the increase in blood-borne eosinophils
is due to the production of IL-5, a Th2-derived cytokine (3, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39), ample production of this cytokine is occurring. Also,
although other investigators have implicated CCR3 in the Th2 response
in the lung (7), it appeared most critical in the early
stages. In an ongoing response to intestinal helminths that requires
>1 wk to develop, any early role of CCR3 in the Th2 response may be
compensated by other pathways, such as the CCR4/monocyte chemotactic
protein that is important in Th2 responses in the lung
(7). Thus, although CCR3 is expressed on both adaptive and
innate immunocytes involved in the anti-Ts response,
loss of this receptor does not dramatically impair this coordinated
effort between the different components.
This study confirms the essential role of CCR3 and its ligands for eosinophil recruitment to the intestines and skeletal muscle in a helminth infection. It also suggests clear distinctions for the roles of MC and eosinophils in the elimination of adult helminths and their larvae, with each cell type playing a direct role in controlling the numbers of the respective life stages of the parasite. Moreover, our study establishes that CCR3 is required neither for MC recruitment to the small intestine nor for the upstream functions of Th2 cells, as evidenced by the MC hyperplasia and adult worm rejection kinetics.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michael F. Gurish, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Smith Research Building Room 624, One Jimmy Fund Way, Boston, MA 02115. E-mail address: mgurish{at}rics.bwh.harvard.edu
3 Abbreviations used in this paper: MC, mast cell; Ts, Trichinella spiralis; hpf, high power field; lpf, low power field; CCL, CC chemokine ligand.
Received for publication February 5, 2002. Accepted for publication April 1, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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7 (47 integrin) for tissue-specific homing. J. Exp. Med. 194:1243.7 integrin-deficient mice: delayed leukocyte recruitment and attenuated protective immunity in the small intestine during enteric helminth infection. Eur. J. Immunol. 30:1656.[Medline]This article has been cited by other articles:
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D. P. Beiting, L. F. Gagliardo, M. Hesse, S. K. Bliss, D. Meskill, and J. A. Appleton Coordinated Control of Immunity to Muscle Stage Trichinella spiralis by IL-10, Regulatory T Cells, and TGF-beta J. Immunol., January 15, 2007; 178(2): 1039 - 1047. [Abstract] [Full Text] [PDF]
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 A. M. Galioto, J. A. Hess, T. J. Nolan, G. A. Schad, J. J. Lee, and D. Abraham Role of Eosinophils and Neutrophils in Innate and Adaptive Protective Immunity to Larval Strongyloides stercoralis in Mice. Infect. Immun., October 1, 2006; 74(10): 5730 - 5738. [Abstract] [Full Text] [PDF]
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