of June 13, 2013.
This information is current as
Immunity in the Chronic Phase of Trichinella
Critical Link between Adaptive and Innate
Mouse Mast Cell Tryptase mMCP-6 Is a
M.Friend, Alan D. Pemberton, Michael F. Gurish and David
Kichul Shin, Gerald F. M. Watts, Hans C. Oettgen, Daniel S.
2008; 180:4885-4891; ;
, 28 of which you can access for free at:
cites 53 articles
is online at:
The Journal of Immunology
Information about subscribing to
Submit copyright permission requests at:
Receive free email-alerts when new articles cite this article. Sign up at:
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Immunologists All rights reserved.
Copyright © 2008 by The American Association of
9650 Rockville Pike, Bethesda, MD 20814-3994.
The American Association of Immunologists, Inc.,
is published twice each month by
The Journal of Immunology
by guest on June 13, 2013
Mouse Mast Cell Tryptase mMCP-6 Is a Critical Link
between Adaptive and Innate Immunity in the Chronic
Phase of Trichinella spiralis Infection1
Kichul Shin,* Gerald F. M. Watts,* Hans C. Oettgen,†Daniel S. Friend,* Alan D. Pemberton,‡
Michael F. Gurish,* and David M. Lee2*
Although the innate immune function of mast cells in the acute phase of parasitic and bacterial infections is well established,
their participation in chronic immune responses to indolent infection remains incompletely understood. In parasitic infection
with Trichinella spiralis, the immune response incorporates both lymphocyte and mast cell-dependent effector functions for
pathogen eradication. Among the mechanistic insights still unresolved in the reaction to T. spiralis are the means by which
mast cells respond to parasites and the mast cell effector functions that contribute to the immunologic response to this
pathogen. We hypothesized that mast cell elaboration of tryptase may comprise an important effector component in this
response. Indeed, we find that mice deficient in the tryptase mouse mast cell protease-6 (mMCP-6) display a significant
difference in their response to T. spiralis larvae in chronically infected skeletal muscle tissue. Mechanistically, this is asso-
ciated with a profound inability to recruit eosinophils to larvae in mMCP-6-deficient mice. Analysis of IgE-deficient mice
demonstrates an identical defect in eosinophil recruitment. These findings establish that mast cell secretion of the tryptase
mMCP-6, a function directed by the activity of the adaptive immune system, contributes to eosinophil recruitment to the site
of larval infection, thereby comprising an integral link in the chronic immune response to parasitic infection.
of Immunology, 2008, 180: 4885–4891.
of stimuli and organisms via recognition by pathogen-associated
molecular pattern receptors, including Toll-like receptors, manno-
syl receptors, and others (reviewed in Ref. 3). In this context, mast
cells participate in rapid mobilization of the innate immune re-
sponse by elaborating leukocyte chemoattractants and can partic-
ipate in stimulating initial adaptive immune responses either by
direct Ag presentation or by inducing the migration of dendritic
cells or Langerhans cells to draining lymph nodes (4–6).
What remains less clear is the mast cell participation in chronic
responses to pathogens whose infections are long term and whose
clearance requires orchestration by the adaptive immune system.
Are mast cells members of the “orchestra” directed by the adaptive
immune response to chronic infection? Mast cells are theoretically
capable of responding to signals from the adaptive immune system
through stimulation of cytokine receptors (IL-1R, IL-10R, IL-12R,
he role of mast cells as sentinels in innate immune re-
sponses acting acutely against infectious pathogens is
well established (1, 2). They respond rapidly to a variety
IFN-?R) (reviewed in Ref. 3) or by activation via Ig receptors such
as Fc?RI or Fc?RIII (7). Indeed, the clearest examples of mast cell
responses directed by adaptive immunity are their involvement in
allergic disease via stimulation by IgE (reviewed in Ref. 8) and
their participation in autoantibody-driven autoimmune diseases (9,
10). Aside from these pathologic states, very little else is known
about the use of mast cells in the adaptive immune system directed
toward clearance of chronic infections.
Chronic parasitic infections elicit robust humoral responses
from the adaptive immune system, and this suggests a role for mast
cells in the coordinated immune response required for pathogen
clearance. A well-established model of parasitic infection is that of
the intestinal nematode Trichinella spiralis (11–13). Acute infec-
tion by T. spiralis proceeds from ingestion of larvae, which mature
and move into the small intestine and burrow into the intestinal
mucosa. There, they produce larvae that penetrate the intestine and
disseminate to muscle. In this chronic phase of infection, larvae
infect muscle tissue, forming nurse cells (14). Active muscles,
such as diaphragm and tongue, typically display the highest levels
of chronic parasite burden. Within 2 wk of infection, high titers of
serum IgE are present and a T cell-dependent intestinal mastocy-
tosis is prominent (15–18). Previous studies have demonstrated a
functional contribution from both lymphocytes and mast cells in
the clearance of adult parasites from the intestine. Mast cell-defi-
cient mice or wild-type mice whose mast cells have been depleted
by anti-c-kit Abs display profoundly delayed expulsion of the par-
asite (19–24). Similarly, mice lacking lymphocytes also exhibit a
significant inability to resolve the primary gastrointestinal infec-
tion by T. spiralis (24). Furthermore, the exogenous addition of the
mast cell growth factor IL-3 or the Th2 cytokine IL-4 increases the
rate of intestinal pathogen clearance (24, 25).
The mast cell population present within the infected intestinal
tissues is not homogenous. Rather, mast cells from both the
*Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hos-
pital and†Division of Immunology, Children’s Hospital, Harvard Medical School,
Boston, MA 02115; and‡Division of Veterinary Clinical Studies, University of Ed-
inburgh, Easter Bush Veterinary Centre, Roslin, Midlothian, United Kingdom
Received for publication April 13, 2007. Accepted for publication January 21, 2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This study was supported by National Institutes of Health Grants AI059746-01,
HL036110, AI031599, and AI05447, an Arthritis Foundation postdoctoral fellowship
(100972), and the Cogan Family Foundation. A.D.P. was funded by the Veterinary
Training Research Initiative (VT0102).
2Address correspondence and reprint requests to Dr. David M. Lee, 1 Jimmy Fund
Way, Smith Building Room 552B, Boston, MA 02115. E-mail address:
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
The Journal of Immunology
by guest on June 13, 2013
mucosal mast cell (MMC)3subpopulation (that express mouse
mast cell protease (mMCP)-1 and mMCP-2)) and the connec-
tive tissue-type mast cell (CTMC) subpopulation (that express
mMCP-4, mMCP-5, mMCP-6, and mMCP-7) are present. The
MMC subset undergoes massive hyperplasia in the infected in-
testinal mucosa, while the CTMC population remains sparse
and located predominantly in the intestinal serosal region.
Mechanistically, mast cells have been shown to participate in
intestinal expulsion of Trichinella by elaboration of the chy-
mase mMCP-1 (26, 27). This observation was consistent with
the functional importance of the expanded MMC subpopulation
in this tissue in the initial responses to this early stage of T.
Unlike the intestinal phase of T. spiralis infection, little is
known about the mechanisms of host immune response in the
chronic, skeletal muscle stage of disease. Histologically, clear-
ance of the larvae is signified by patchy necrotic lesions char-
acterized by infiltration of eosinophils, lymphocytes, and mac-
rophages, which later turn into calcified deposits (28, 29).
Eosinophils have been implicated in newborn larval cytotoxic-
ity by both in vitro and in vivo studies (30–32). This has been
further supported by more recent in vivo studies. In chemokine
receptor CCR3-deficient mice, there is little eosinophil influx
around the larvae infecting skeletal muscle, unlike in wild-type
mice, and there is decreased immune response to these larvae
(28). IgE also contributes to the immune response to larval pres-
ence through a mechanism not fully understood, although acti-
vation of mast cells found in the skeletal muscle has been hy-
pothesized (29). Mouse eosinophils are not thought to express
the high-affinity IgE receptor; however, eosinophil activation
via antibody-dependent cytotoxicity is not excluded and has
been suggested by in vitro studies (31).
Skeletal muscle is populated exclusively with the CTMC sub-
population (33) that expresses both the chymotryptic-like serine
proteases called chymases and the tryptic-like serine proteases
called tryptases. Previous in vivo analyses with recombinant
tryptases mMCP-6 and mMCP-7 have demonstrated potent leuko-
cyte chemoattractant properties for these neutral proteases (34,
35). Knowing that degranulation and release of granule proteases
are among the effector functions elicited by IgE stimulation, we
hypothesized that IgE-stimulated release of mast cell tryptase com-
prises an important mediator in the immunologic response to par-
asites in skeletal muscle. Herein, using a newly generated strain of
mMCP-6-deficient mice, we demonstrate that this tryptase func-
tions to direct the immune response to T. spiralis-infected skeletal
muscle via the recruitment of eosinophils. Furthermore, we dem-
onstrate the same functional immunologic deficit in mice lacking
IgE. Taken together, these observations demonstrate that mast cell
activation, directed by the adaptive immune system, comprises an
important facet of immune system function in response to chronic
Materials and Methods
Generation of mMCP-6?/?mice
The mMCP-6 gene locus was knocked out using a transgenic construct
transfected to 129/Sv strain embryonic stem cells (Fig. 1). Sequence of the
mMCP-6 gene was obtained by probing both National Institutes of Health
and Celera mouse genome sequences. The 5? and 3? arms of the targeting
construct were obtained by using a PCR cloning strategy. These genomic
sequences were cloned into recipient plasmids (pCR-TOPO, Invitrogen)
and subcloned into pBlueScript (Stratagene) along with a loxP-flanked
neomycin selection marker from pLNTK. The construct was cloned into a
diphtheria toxin expressing polylinker 915 vector. 129/Sv embryonic stem
3Abbreviations used in this paper: MMC, mucosal mast cell; CTMC, connective
tissue-type mast cell; mMCP, mouse mast cell protease; LPF, low power field.
through the proximal portion of exon 3. Stop codons in all frames are introduced after exon 2 into the mMCP-6 locus. Founder mice were mated with the
Cre-expressing strain B6.FVB-Tg(EIIa-cre)C5379Lmgd/J (36) to delete the PGK-neorselecting element before backcrossing (Bg indicates BglII; Bm,
BamHI; K, KpnI; N, NheI).
Map of mMCP-6 gene and targeting construct. After homologous recombination, the final product deletes exon 2 after the initial fMet codon
4886TRYPTASE mMCP-6 PARTICIPATION IN ADAPTIVE IMMUNITY
by guest on June 13, 2013
cells were electroporated with 10 ?g of vector and screened for homolo-
gous recombination with Southern blot hybridization. Positive clones were
injected into C57BL/6 blastocysts, and chimeric males were mated to
C57BL/6 females. Germline transmission was assayed by Southern blot of
tissue from the F1offspring. Heterozygous mice were first mated to the
Cre-expressing strain B6.FVB-Tg(EIIa-cre)C5379Lmgd/J (36), and dele-
tion of the loxP-flanked PGK-neorwas confirmed by PCR. These offspring
were further backcrossed to C57BL/6J mice for five generations (N5), and
then intercrossed to generate N5 homozygous mMCP-6?/?and control
Mice infected with T. spiralis
In addition to mMCP-6?/?mice, we used IgE-deficient mice backcrossed
through 10 generations onto the BALB/cAnTac (Taconic Farms) back-
ground (29, 37). BALB/cAnTac mice were used as controls. All mice were
9–12 wk of age when infected with T. spiralis. All experiments were
conducted with the approval of the Dana-Farber Cancer Institute Animal
Care and Use Committee.
T. spiralis infection and worm burden analysis
Mice were infected orally with freshly isolated T. spiralis larvae, as pre-
viously described (38). With a gastric gavage, ?450 larvae were injected
to each mouse. Five mice per group were selected for determination of the
intestinal worm burden at each time point. The small intestine was sec-
tioned into 1–2-cm pieces in a 100-mm petri dish containing 20 ml of PBS.
After rocking the dish slowly for 3 h, adult worms were counted under an
inverted microscope (Nikon). To evaluate worm burdens histomorpho-
metrically, 9–10 mice per group per experiment were infected with T.
spiralis larvae and euthanized at 5 wk. Tongue tissues were obtained and
worm burdens were histomorphometrically quantified as previously de-
scribed (28, 29). To quantify skeletal worm burdens via isolation of par-
asites from tissue, tongues from experimental mice were weighed and then
digested with 1% pespsin, 0.1 N HCl for 4 h, and the total numbers of
larvae per digested tongue were enumerated via inverted microscopy (29).
Organs including ear skin, stomach, lung, spleen, tongue, and a small piece
(?2 cm) of jejunum of both healthy and T. spiralis-infected mice were
fixed in 4% paraformaldehyde in PBS for 24 h, and then embedded in
paraffin. Tissue was cut into 3-?m sections. Slides were deparaffinized in
xylene for 5 min twice and then hydrated in graded ethanol. For toluidine
blue staining, a working solution of 0.1% toluidine blue O (Sigma-Aldrich)
in water was prepared. Tissue sections were stained for 30 s, rinsed in
distilled water, next dehydrated in graded alcohol, and mounted with Cy-
toseal 60 (Richard-Allan Scientific). H&E staining and Congo red staining
were performed as previously described (28).
Immunohistochemistry of mast cell proteases mMCP-1, mMCP-4,
mMCP-5, mMCP-6, and mMCP-7 was performed with Vectastain ABC-
alkaline phosphatase kit (Vector Laboratories) as previously described
(39). After a final series of washes in PBS, 200 ?l of Fast Red (1 mg/ml)
(Sigma-Aldrich) substrate was applied for 25 min. Tissue was subsequently
counterstained with Gill II hematoxylin and mounted with Crystal/Mount
(Biomeda). Immunohistochemistry of IgE was done with Vectastain ABC-
peroxidase kit (Vector Laboratories) using anti-IgE (Novus Biologicals) as
previously described (29).
Mast cell density in tongue or intestine was determined by the average
number of toluidine blue-positive cells in five low power fields (LPF) un-
der a light microscope (Leica Microsystems). Necrotic larvae in the tongue
were defined by lesions with heavy infiltration of inflammatory cells in-
vading the nurse cell around the parasite as previously described (28, 29).
To increase our sampling accuracy, sections were obtained at three depths
separated by at least 100 ?m for each tongue. The percentage of necrotic
larvae was enumerated at each depth in each tongue section. The number
of live larvae in five LPF was also calculated in all sections. For the eo-
sinophil density around the larva, an eyepiece reticule (Leica) was used to
define a square unit of 0.04 mm2encompassing individual larva. Congo
red-positive eosinophils inside the unit area were enumerated in 40 larvae
per experimental group. Separately, serial sections within a single depth
were evaluated to confirm consistency of enumeration for individual lar-
vae. Other cell lineages were enumerated as described (40). Observers
were blinded to the experimental condition and genotype in all analyses.
Western blot analysis
Bone marrow-derived mast cells were cultured from both N5 mMCP-6?/?
and wild-type mice. Briefly, cells obtained from femur flushes with DMEM
(Life Technologies) were cultured in media with 10 ng/ml stem cell factor
(PeproTech) and 10 ng/ml recombinant IL-3 (Pierce) at 10% CO2, 37°C for
5 wk. Media was changed weekly and added with fresh cytokines. Cells
were lysed with Triton X-100-based buffer supplemented with a protease
inhibitor cocktail (Sigma-Aldrich). Proteins were blotted onto a PolyScreen
membrane (0.45 ?m pore size, PerkinElmer) and then the membrane was
blocked with 1% milk in TBS containing 0.05% Tween 20 (Sigma-Al-
drich) for 2 h. After incubation with mMCP-6 or mMCP-7 Ab diluted
1/10,000 in TBS/Tween 20, membrane was washed and then incubated
with HRP-conjugated donkey anti-rabbit Ab (Jackson ImmunoResearch)
diluted 1/10,000 with TBS/Tween 20. Western Lighting (PerkinElmer) was
used as the substrate for development.
Serum IgE levels were determined as previously described (29). Briefly,
96-well plates were coated with purified anti-IgE (BD Pharmingen) at 1
?g/ml overnight at 4°C. The next day, plates were washed twice with PBS
containing 0.05% Tween 20. Serum was added at dilutions ranging from
1/500 to 1/1500. Standards and sera were diluted in PBS/1% BSA (Sigma-
Aldrich) and left on plates for 2 h. Plates were then washed and incubated
for 45 min with biotinylated anti-IgE at 1 ?g/ml. After washes, plates were
given avidin conjugated to HRP (Zymed Laboratories) and incubated for
30 min. Plates were washed before adding substrate, 2,2?-azino-bis(3-ethyl-
benzthiazoline-6-sulfonic acid) (Zymed Laboratories). OD405was measured
10–20 min after addition of substrate. Specific IgG against T. spiralis was
the manufacturer’s protocol.
Data were analyzed using Prism software v4.0 (GraphPad Software). All
mean values are presented as the means ? SE. For comparison of the mean
values, Student’s t test (two-tailed) was performed. p values ?0.01 were
We generated tryptase mMCP-6?/?mice by disrupting exons 1 and
2 via homologous recombination in 129/Sv ES cells (Fig. 1). mMCP-
6?/?mice are viable, fertile, devoid of any opportunistic infection in
specific pathogen-free housing conditions, and lack detectable
mMCP-6 protein in tissue mast cells and in bone marrow-derived
cultured mast cells (Fig. 2A). We backcrossed the 129/Sv strain
founder mice for five generations onto the C57BL/6 strain for these
studies. To exclude aberrant mast cell protease expression in vivo, we
stained tongue CTMC with protease-specific Abs and found expres-
sion of the chymases mMCP-4 and mMCP-5 intact in mMCP-6?/?
mice (Fig. 2B). Since the mMCP-7 gene locus resides immediately
adjacent to the mMCP-6 locus, and since 129/Sv strain mice have
intact mMCP-7 while C57BL/6 mice contain a null mMCP-7 gene
(41), we also examined mMCP-7 expression in tongue CTMC. As
expected, mMCP-7 was expressed in mMCP-6?/?CTMCs, in con-
trast to wild-type littermates (Fig. 2). Our data indicate that mMCP-6
is not essential for the expression of these CTMC proteases.
This finding contrasts with some secretory granule proteases
whose absence perturbs expression of other constituents of the
secretory granule (35, 42).
mMCP-6 is not required for intestinal T. spiralis expulsion
To explore a role for tryptase mMCP-6 in the response to parasitic
infection, we first investigated the contribution of mMCP-6 in the
acute stage of T. spiralis infection. Previous studies identified a
role for the intestinal mucosal cell subpopulation through their
elaboration of mMCP-1, a population that does not express
mMCP-6 to any significant degree (26, 27). Herein we find no
difference in the worm expulsion kinetics between mMCP-6?/?
and wild-type mice (Fig. 3). Worm expulsion in both groups was
4887The Journal of Immunology
by guest on June 13, 2013
complete by day 18, and similar numbers of adult worms were
found in the intestine of the two strains at various earlier time
points. Thus, although mMCP-6-positive CTMCs are present in
the jejunal lamina propria after T. spiralis infection (18), mMCP-6
is dispensable for the intestinal expulsion of the parasite.
mMCP-6 enhances the immune response to T. spiralis
We next examined whether there may be a role for mMCP-6 in the
immune response during the chronic stages of T. spiralis infection.
After 5 wk of infection, we confirmed large numbers of larvae in
the diaphragm, quadriceps, and tongue (28, 29). Tongue mast cell
density at baseline was comparable between the two groups (wild-
type vs mMCP-6?/?, 29.0 ? 3.1/LPF vs 26.0 ? 3.5/LPF; p ?
0.54). Moreover, the tissue mastocytosis after larval invasion was
indistinguishable in the absence of mMCP-6 (48.9 ? 4.1/LPF vs
57.7 ? 2.6/LPF, respectively; p ? 0.21). Analysis of tongue tissue
shows degranulating mast cells located around larvae in both wild-
type and mMCP-6?/?mice (Fig. 4, A and B). Despite the simi-
larity in tongue mast cell numbers, examination of necrosis of
larvae revealed significant decreases in mMCP-6?/?mice (Fig. 4,
C and D). More specifically, there was a significant decrease in the
percentage of necrotic larvae in the tongue of mMCP-6?/?mice in
comparison to wild-type mice infected in parallel with a concom-
itant increase in intact larvae (Fig. 4, E and F, p ? 0.001). We
further quantified muscle larvae burden at 5 wk after infection.
Although we found a trend toward higher average parasite burden
in the mMCP-6?/?mice, the differences between the two strains
does not achieve statistical significance (3433 ? 428/g vs 2953 ?
348/g, p ? 0.3872).
Tissue eosinophil recruitment by mMCP-6
Since leukocyte recruitment is thought to comprise one of the pri-
mary functions of tryptase (34, 43), we hypothesized that a defect
mice. A, Western blots of wild-type and mMCP-6?/?bone marrow-derived
mast cell (BMMC) lysates demonstrate lack of detectable mMCP-6 pro-
tein. Note that in contrast to C57BL/6 wild-type BMMCs, mMCP-6?/?
BMMCs express mMCP-7. B, Immunohistochemistry of tongue connec-
tive tissue mast cells from wild-type and mMCP-6?/?mice demonstrate
lack of mMCP-6 expression in vivo as well as intact mMCP-4 and
mMCP-5 expression. Note mMCP-7 expression in the wild-type strain (a,
isotype control; b, anti-mMCP-1; c, anti-mMCP-4; d, anti-mMCP-5; e,
anti-mMCP-6; f, anti-mMCP-7; magnification of ?200).
Absence of mMCP-6 protein expression in mMCP-6?/?
acutely infected with T. spiralis were analyzed for intestinal worm burden.
Each time point shows the mean number of worms (with SE) recovered per
mouse from two independent experiments with 5 mice per group in each
experiment (p ? NS).
Kinetics of T. spiralis expulsion from small intestine. Mice
cle. A and B, Mast cells (arrowheads) locate at the periphery of larvae in
both wild-type and mMCP-6?/?mice. Insets, Degranulating mast cells in
higher magnification (?630). C and D, H&E stain of tongue skeletal mus-
cle from chronically infected wild-type (C) and mMCP-6?/?(D) mice (NL
indicates necrotic larva; arrow, live larva). E and F, Histomorphometric
quantification of necrotic and live larvae in tongue skeletal muscle tissue
sections from 4 mice per group pooled from two independent experiments.
Values are means ? SE.
Chronic phase elimination of T. spiralis from skeletal mus-
4888TRYPTASE mMCP-6 PARTICIPATION IN ADAPTIVE IMMUNITY
by guest on June 13, 2013
in leukocyte recruitment may be evident in the immune response to
chronic T. spiralis infection of mMCP-6?/?skeletal muscle. We
therefore quantified leukocyte populations around T. spiralis lar-
vae (Fig. 5C). We found that eosinophils, a major infiltrate in the
leukocytic population surrounding infecting parasites (28), were
markedly decreased in the mMCP-6?/?mice (Fig. 5, p ? 0.0091).
We did not note decreases in the fraction of other leukocyte pop-
ulations infiltrating skeletal muscle larvae.
Since we had previously noted a role for IgE in the immune
response to chronic T. spiralis infection (29), it was possible that
mMCP-6?/?mice displayed a lesion in their capacity to mount an
effective adaptive immune response, especially production of IgE.
We measured serum IgE levels 2 wk post infection and found no
defect in IgE production by mMCP-6?/?mice (mMCP-6?/?vs
wild-type mice, 260.6 ? 30.9 vs 134.7 ? 14.5 ?g/ml). We further
assessed IgE binding to the infecting parasite in vivo via immu-
nohistochemistry (29) and found indistinguishable, robust IgE
coating of T. spiralis larvae in both strains of mice (Fig. 6). Ad-
ditionally, we also quantified the anti-T. spiralis humoral response
via ELISA (44) and found no difference in IgG levels between
mMCP-6?/?and wild-type mice (OD 0.085 ? 0.01 vs 0.082 ?
0.01, n ? 10 mice/group, p ? 0.7304). These data provide evi-
dence that the mast cell tryptase mMCP-6 contributes to the eosi-
nophil-rich immune response to T. spiralis in skeletal muscle in the
context of an intact adaptive immune response to parasite.
IgE-deficient mice demonstrate defective skeletal muscle tissue
Previous analyses in IgE-deficient mice also exhibited an altered
immune response to skeletal muscle T. spiralis infection, high-
lighting the importance of the adaptive immune system response in
chronic T. spiralis infection and implicating mast cells in this pro-
cess (29). Having observed the identical phenotype in mMCP-
6?/?mice, we hypothesized that the phenotype in IgE?/?mice
resulted from their inability to stimulate mast cell mMCP-6 re-
lease. This led us to predict that eosinophil infiltration of T. spiralis
larvae would also be decreased in IgE?/?mice. Indeed, histomor-
phometric quantification of leukocyte populations infiltrating lar-
val structures in skeletal muscle revealed markedly depressed eo-
sinophil numbers in infected IgE-deficient mice compared with
infected wild-type mice (Fig. 7). Thus, IgE?/?and mMCP-6?/?
mice both demonstrate prominent defects in their immune re-
sponse to skeletal muscle T. spiralis infection via a decrease in the
eosinophil-rich leukocytic infiltrate.
As a mast cell-, lymphocye-, and IgE-dependent infection model
with distinct acute (intestinal) and chronic (skeletal muscle) stages
of infection, T. spiralis is an ideal pathogen to investigate immu-
nologic mechanisms of parasite elimination. Infection of both hu-
mans and rodents has long been known to elicit prominent IgE
responses and tissue mast cell hyperplasia (45, 46). Although pre-
vious studies in mast cell-deficient mice and in mMCP-1-deficient
mice have provided insight into the role of mast cells in the intes-
tinal phase of infection, little was known about the role of mast
cells in the chronic response to this pathogen in skeletal muscle
(29). In this study we demonstrate that mast cells participate in the
immune response to chronic T. spiralis infection via elaboration of
the tryptase mMCP-6. Furthermore, we confirm that leukocyte re-
cruitment, particularly eosinophil recruitment, is an important
physiologic role for mast cells and mMCP-6 in vivo.
More broadly, these studies highlight that the effector function
of the mast cell is an integral aspect of the responses orchestrated
by the adaptive immune system in reaction to an important class of
and B, Representative tongue skeletal muscle sections containing larva
from wild-type (A) and mMCP-6?/?(B) mice stained with Congo red to
identify eosinophils (arrows point to examples). C and D, Histomorphometric
quantification of (C) different cell lineages and (D) eosinophil density in the
perilarval space. Values are means ? SE.
Eosinophil recruitment to larvae in mMCP-6?/?mice. A
Wild-type mouse stained with isotype control. Representative skeletal mus-
cle sections containing larva from wild-type (B) and mMCP-6?/?(C) mice
were stained with anti-IgE Ab (arrows point to IgE-positive mast cells)
(magnification of ?400).
IgE immunostaining of larvae in mMCP-6?/?mice. A,
Representative tongue skeletal muscle sections containing larva from wild-
type (A) and IgE?/?(B) mice stained with Congo red to identify eosino-
phils (arrows point to examples). C, Histomorphometric quantification of
perilarval eosinophil density in tongue tissue from 4 mice per group pooled
from two independent experiments. Values are means ? SE.
Eosinophil recruitment to larvae in IgE?/?mice. A and B,
4889 The Journal of Immunology
by guest on June 13, 2013
infectious pathogens. Previous analyses in IgE-deficient mice and
in IgE-depleted rats demonstrated the importance of the adaptive
immune response in clearance of T. spiralis from skeletal muscle
(29, 47). Because the mouse eosinophil does not express the high-
affinity IgE receptor, this dependence implicated either mast
cells in this process based on their expression of the high-af-
finity IgE receptor (although no direct assessment of mast cells
around the lesion was performed) or antibody-dependent cyto-
toxicity by the eosinophil based on killing of newborn larvae in
vitro (31). We herein extend those studies and show that mast cells
are recruited to the site of larval infection, and that mMCP-6?/?
mice, like IgE?/?mice, exhibit profound decreases in eosinophil
recruitment to T. spiralis larvae despite an intact humoral IgE re-
sponse. These observations, combined with previous studies dem-
onstrating IgE binding to skeletal muscle larvae (29) and the well-
documented ability of IgE to cause mast cell degranulation,
suggest a chain of events by which the adaptive immune system
directs mast cell and eosinophil participation in chronic T. spiralis
infection. Initially, the adaptive immune response produces Ag-
specific IgE that binds to T. spiralis larvae in skeletal muscle; the
T. spiralis Ag-binding IgE activates CTMC expressing mMCP-6,
causing release of this tryptase, which leads to eosinophil
Although the significant decrease in necrotic larvae and increase
in infected muscle cells are consistent with less efficient killing of
T. spiralis larvae in mMCP-6?/?mice, our studies do not directly
demonstrate increased parasitic cytotoxicity or increased parasite
burden. Previous studies have demonstrated that eosinophils are
capable of killing T. spiralis newborn larvae (29–32); however,
definitive studies demonstrating this activity on mature larvae,
such as those present in chronically infected muscle tissue, are
lacking. It is thus possible that mast cells contribute to the immu-
nologic response to dead larvae in these studies via elaboration of
Our results provide further evidence for distinct functional ac-
tivities of discrete mast cell subpopulations in T. spiralis infection.
Numerous previous studies have identified a role for the intestinal
MMC subpopulation, in part through their elaboration of
mMCP-1, to direct rejection of the adult worms from the small
intestine (26, 27). The defects in mMCP-6-deficient mice now pro-
vide evidence for participation of the CTMC subpopulation in a
temporally and anatomically distinct phase of the immunologic
reaction to this parasite from that of the MMC population. As such,
we think that our data provide the first definitive example of a
primary immunologic contribution from the connective tissue mast
cell population in a chronic parasitic infection.
Previous studies on the role of mast cells in tissue eosinophilia
have focused primarily on cytokine production. Mast cell elabo-
ration of cytokines and chemokines such as IL-3, IL-5, GM-CSF,
and eotaxin (48–50) are thought to recruit eosinophils and may
potentiate eosinophil survival and function (51). The observations
in this study add the mast cell tryptase mMCP-6 to this list of
molecules recruiting eosinophils and extend previous observations
regarding the roles of the mast cell tryptases mMCP-6 and
mMCP-7 in leukocyte recruitment.
More specifically, experiments using recombinant mMCP-6
demonstrated a neutrophil influx after intraperitoneal injection,
whereas recombinant mMCP-7 induced eosinophil infiltration in-
side the peritoneum (34, 43, 52). Herein, we find a profound de-
crease in eosinophil recruitment in mMCP-6-deficient, mMCP-7-
sufficient mice while eosinophil recruitment remains intact in
mMCP-7-deficient C57BL/6 (41) control mice. Thus, our results
suggest that in the skeletal muscle, unlike in the peritoneum,
mMCP-6 rather than mMCP-7 is critical to eosinophil recruitment.
Furthermore, our findings underscore the importance of mast cell
tryptases in leukocyte recruitment and provide evidence that mast
cell protease function may be anatomically or stimulation context-
specific. These observations have a broader context in that mast
cell neutral protease function may be of importance in chronic
inflammation in other tissues such as the asthmatic lung, where
eosinophilic recruitment is implicated in pathobiology (53); the
molecular basis for these distinctions awaits further experimental
In summary, we have delineated that the tryptase mMCP-6, pro-
duced by CTMCs, modulates the immunologic response to the
parasite T. spiralis, specifically in the chronic, skeletal muscle col-
onization phase. Moreover, tryptase-dependent eosinophil recruit-
ment is a plausible effector mechanism by which CTMC contrib-
utes to adaptive immunity-driven (IgE-dependent) elimination of
this parasite. This demonstrates the first example of a contribution
by tryptase in the immunologic response to a chronic parasitic
pathogen. Furthermore, our study highlights the discrete functional
contributions of mast cell subsets as well as the functional diver-
sity of the responses, driven by either adaptive immunity or by
innate stimuli, of the mast cell lineage.
We thank Teresa Bowman for expert histotechnology input.
The authors have no financial conflicts of interest.
1. Echtenacher, B., D. N. Mannel, and L. Hultner. 1996. Critical protective role of
mast cells in a model of acute septic peritonitis. Nature 381: 75–77.
2. Maurer, M., B. Echtenacher, L. Hultner, G. Kollias, D. N. Mannel, K. E. Langley,
and S. J. Galli. 1998. The c-kit ligand, stem cell factor, can enhance innate
immunity through effects on mast cells. J. Exp. Med. 188: 2343–2348.
3. Marshall, J. S. 2004. Mast-cell responses to pathogens. Nat. Rev. Immunol. 4:
4. Malaviya, R., N. J. Twesten, E. A. Ross, S. N. Abraham, and J. D. Pfeifer. 1996.
Mast cells process bacterial Ags through a phagocytic route for class I MHC
presentation to T cells. J. Immunol. 156: 1490–1496.
5. Bryce, P. J., M. L. Miller, I. Miyajima, M. Tsai, S. J. Galli, and H. C. Oettgen.
2004. Immune sensitization in the skin is enhanced by antigen-independent ef-
fects of IgE. Immunity 20: 381–392.
6. Jawdat, D. M., E. J. Albert, G. Rowden, I. D. Haidl, and J. S. Marshall. 2004.
IgE-mediated mast cell activation induces Langerhans cell migration in vivo.
J. Immunol. 173: 5275–5282.
7. Nigrovic, P. A., and D. M. Lee. 2005. Mast cells in inflammatory arthritis. Ar-
thritis Res. Ther. 7: 1–11.
8. Oettgen, H. C., and R. S. Geha. 2001. IgE regulation and roles in asthma patho-
genesis. J. Allergy Clin. Immunol. 107: 429–440.
9. Chen, R., G. Ning, M. L. Zhao, M. G. Fleming, L. A. Diaz, Z. Werb, and Z. Liu.
2001. Mast cells play a key role in neutrophil recruitment in experimental bullous
pemphigoid. J. Clin. Invest. 108: 1151–1158.
10. Nigrovic, P. A., B. A. Binstadt, P. A. Monach, A. Johnsen, M. Gurish,
Y. Iwakura, C. Benoist, D. Mathis, and D. M. Lee. 2007. Mast cells contribute to
initiation of autoantibody-mediated arthritis via IL-1. Proc. Natl. Acad. Sci. USA
11. Ruitenberg, E. J., and A. Elgersma. 1976. Absence of intestinal mast cell re-
sponse in congenitally athymic mice during Trichinella spiralis infection. Nature
12. Briggs, N. T. 1963. Immunological injury of mast cells in mice actively and
passively sensitized to antigens from Trichinella spiralis. J. Infect. Dis. 113:
13. Keller, R., H. Cottier, and M. W. Hess. 1974. Mast cell responses in mesenteric
lymph nodes to infection of rats with the nematode, Nippostrongylus brasiliensis.
Immunology 27: 1039–1044.
14. Ko, R. C., L. Fan, D. L. Lee, and H. Compton. 1994. Changes in host muscles
induced by excretory/secretory products of larval Trichinella spiralis and
Trichinella pseudospiralis. Parasitology 108: 195–205.
15. Ghildyal, N., H. P. McNeil, S. Stechschulte, K. F. Austen, D. Silberstein,
M. F. Gurish, L. L. Somerville, and R. L. Stevens. 1992. IL-10 induces tran-
scription of the gene for mouse mast cell protease-1, a serine protease preferen-
tially expressed in mucosal mast cells of Trichinella spiralis-infected mice. J. Im-
munol. 149: 2123–2129.
16. Lantz, C. S., J. Boesiger, C. H. Song, N. Mach, T. Kobayashi, R. C. Mulligan,
Y. Nawa, G. Dranoff, and S. J. Galli. 1998. Role for interleukin-3 in mast-cell and
basophil development and in immunity to parasites. Nature 392: 90–93.
4890 TRYPTASE mMCP-6 PARTICIPATION IN ADAPTIVE IMMUNITY
by guest on June 13, 2013
17. Madden, K. B., J. F. Urban, Jr., H. J. Ziltener, J. W. Schrader, F. D. Finkelman,
and I. M. Katona. 1991. Antibodies to IL-3 and IL-4 suppress helminth-induced
intestinal mastocytosis. J. Immunol. 147: 1387–1391.
18. Friend, D. S., N. Ghildyal, M. F. Gurish, J. Hunt, X. Hu, K. F. Austen, and
R. L. Stevens. 1998. Reversible expression of tryptases and chymases in the
jejunal mast cells of mice infected with Trichinella spiralis. J. Immunol. 160:
19. Ha, T. Y., N. D. Reed, and P. K. Crowle. 1983. Delayed expulsion of adult
Trichinella spiralis by mast cell-deficient W/Wv mice. Infect. Immun. 41:
20. Donaldson, L. E., E. Schmitt, J. F. Huntley, G. F. Newlands, and R. K. Grencis.
1996. A critical role for stem cell factor and c-kit in host protective immunity to
an intestinal helminth. Int. Immunol. 8: 559–567.
21. Newlands, G. F., H. R. Miller, A. MacKellar, and S. J. Galli. 1995. Stem cell
factor contributes to intestinal mucosal mast cell hyperplasia in rats infected with
Nippostrongylus brasiliensis or Trichinella spiralis, but anti-stem cell factor
treatment decreases parasite egg production during N. brasiliensis infection.
Blood 86: 1968–1976.
22. Grencis, R. K., K. J. Else, J. F. Huntley, and S. I. Nishikawa. 1993. The in vivo
role of stem cell factor (c-kit ligand) on mastocytosis and host protective immu-
nity to the intestinal nematode Trichinella spiralis in mice. Parasite Immunol. 15:
23. Alizadeh, H., and K. D. Murrell. 1984. The intestinal mast cell response to
Trichinella spiralis infection in mast cell-deficient w/wv mice. J. Parasitol. 70:
24. Urban, J. F., Jr., L. Schopf, S. C. Morris, T. Orekhova, K. B. Madden, C. J. Betts,
H. R. Gamble, C. Byrd, D. Donaldson, K. Else, and F. D. Finkelman. 2000. Stat6
signaling promotes protective immunity against Trichinella spiralis through a
mast cell- and T cell-dependent mechanism. J. Immunol. 164: 2046–2052.
25. Korenaga, M., T. Abe, and Y. Hashiguchi. 1996. Injection of recombinant inter-
leukin 3 hastens worm expulsion in mice infected with Trichinella spiralis. Para-
sitol. Res. 82: 108–113.
26. Lawrence, C. E., Y. Y. Paterson, S. H. Wright, P. A. Knight, and H. R. Miller.
2004. Mouse mast cell protease-1 is required for the enteropathy induced by
gastrointestinal helminth infection in the mouse. Gastroenterology 127: 155–165.
27. Knight, P. A., S. H. Wright, C. E. Lawrence, Y. Y. Paterson, and H. R. Miller.
2000. Delayed expulsion of the nematode Trichinella spiralis in mice lacking the
mucosal mast cell-specific granule chymase, mouse mast cell protease-1. J. Exp.
Med. 192: 1849–1856.
28. Gurish, M. F., A. Humbles, H. Tao, S. Finkelstein, J. A. Boyce, C. Gerard,
D. S. Friend, and K. F. Austen. 2002. CCR3 is required for tissue eosinophilia
and larval cytotoxicity after infection with Trichinella spiralis. J. Immunol. 168:
29. Gurish, M. F., P. J. Bryce, H. Tao, A. B. Kisselgof, E. M. Thornton, H. R. Miller,
D. S. Friend, and H. C. Oettgen. 2004. IgE enhances parasite clearance and
regulates mast cell responses in mice infected with Trichinella spiralis. J. Im-
munol. 172: 1139–1145.
30. Bell, R. G., and C. H. Wang. 1987. The Trichinella spiralis newborn larvae:
production, migration, and immunity in vivo. Wiad. Parazytol. 33: 453–478.
31. Gansmuller, A., A. Anteunis, S. M. Venturiello, F. Bruschi, and R. A. Binaghi.
1987. Antibody-dependent in-vitro cytotoxicity of newborn Trichinella spiralis
larvae: nature of the cells involved. Parasite Immunol. 9: 281–292.
32. Grove, D. I., A. A. Mahmoud, and K. S. Warren. 1977. Eosinophils and resis-
tance to Trichinella spiralis. J. Exp. Med. 145: 755–759.
33. Gurish, M. F., and K. F. Austen. 2001. The diverse roles of mast cells. J. Exp.
Med. 194: F1–F5.
34. Huang, C., G. T. De Sanctis, P. J. O’Brien, J. P. Mizgerd, D. S. Friend,
J. M. Drazen, L. F. Brass, and R. L. Stevens. 2001. Evaluation of the substrate
specificity of human mast cell tryptase ?I and demonstration of its importance in
bacterial infections of the lung. J. Biol. Chem. 276: 26276–26284.
35. Huang, C., A. Sali, and R. L. Stevens. 1998. Regulation and function of mast cell
proteases in inflammation. J. Clin. Immunol. 18: 169–183.
36. Lakso, M., J. G. Pichel, J. R. Gorman, B. Sauer, Y. Okamoto, E. Lee, F. W. Alt,
and H. Westphal. 1996. Efficient in vivo manipulation of mouse genomic se-
quences at the zygote stage. Proc. Natl. Acad. Sci. USA 93: 5860–5865.
37. Oettgen, H. C., T. R. Martin, A. Wynshaw-Boris, C. Deng, J. M. Drazen, and
P. Leder. 1994. Active anaphylaxis in IgE-deficient mice. Nature 370: 367–370.
38. Friend, D. S., N. Ghildyal, K. F. Austen, M. F. Gurish, R. Matsumoto, and
R. L. Stevens. 1996. Mast cells that reside at different locations in the jejunum of
mice infected with Trichinella spiralis exhibit sequential changes in their granule
ultrastructure and chymase phenotype. J. Cell Biol. 135: 279–290.
39. Shin, K., M. F. Gurish, D. S. Friend, A. D. Pemberton, E. M. Thornton,
H. R. Miller, and D. M. Lee. 2006. Lymphocyte-independent connective tissue
mast cells populate murine synovium. Arthritis Rheum. 54: 2863–2871.
40. Friend, D. S., M. F. Gurish, K. F. Austen, J. Hunt, and R. L. Stevens. 2000.
Senescent jejunal mast cells and eosinophils in the mouse preferentially translo-
cate to the spleen and draining lymph node, respectively, during the recovery
phase of helminth infection. J. Immunol. 165: 344–352.
41. Hunt, J. E., R. L. Stevens, K. F. Austen, J. Zhang, Z. Xia, and N. Ghildyal. 1996.
Natural disruption of the mouse mast cell protease 7 gene in the C57BL/6 mouse.
J. Biol. Chem. 271: 2851–2855.
42. Feyerabend, T. B., H. Hausser, A. Tietz, C. Blum, L. Hellman, A. H. Straus,
H. K. Takahashi, E. S. Morgan, A. M. Dvorak, H. J. Fehling, and
H. R. Rodewald. 2005. Loss of histochemical identity in mast cells lacking car-
boxypeptidase A. Mol. Cell. Biol. 25: 6199–6210.
43. Huang, C., D. S. Friend, W. T. Qiu, G. W. Wong, G. Morales, J. Hunt, and
R. L. Stevens. 1998. Induction of a selective and persistent extravasation of
neutrophils into the peritoneal cavity by tryptase mouse mast cell protease 6.
J. Immunol. 160: 1910–1919.
44. Marva, E., A. Markovics, M. Gdalevich, N. Asor, C. Sadik, and A. Leventhal.
2005. Trichinellosis outbreak. Emerg. Infect. Dis. 11: 1979–1981.
45. Morakote, N.,K. Sukhavat,C.
Suphawitayanukul, and W. Thamasonthi. 1992. Persistence of IgG, IgM, and IgE
antibodies in human trichinosis. Trop. Med. Parasitol. 43: 167–169.
46. Alizadeh, H., J. F. Urban, Jr., I. M. Katona, and F. D. Finkelman. 1986. Cells
containing IgE in the intestinal mucosa of mice infected with the nematode par-
asite Trichinella spiralis are predominantly of a mast cell lineage. J. Immunol.
47. Dessein, A. J., W. L. Parker, S. L. James, and J. R. David. 1981. IgE antibody and
resistance to infection: I. Selective suppression of the IgE antibody response in
rats diminishes the resistance and the eosinophil response to Trichinella spiralis
infection. J. Exp. Med. 153: 423–436.
48. Bressler, R. B., J. Lesko, M. L. Jones, M. Wasserman, R. R. Dickason,
M. M. Huston, S. W. Cook, and D. P. Huston. 1997. Production of IL-5 and
granulocyte-macrophage colony-stimulating factor by naive human mast cells
activated by high-affinity IgE receptor ligation. J. Allergy Clin. Immunol. 99:
49. Hogaboam, C., S. L. Kunkel, R. M. Strieter, D. D. Taub, P. Lincoln,
T. J. Standiford, and N. W. Lukacs. 1998. Novel role of transmembrane SCF for
mast cell activation and eotaxin production in mast cell-fibroblast interactions.
J. Immunol. 160: 6166–6171.
50. Wallaert, B., P. Desreumaux, M. C. Copin, I. Tillie, A. Benard, J. F. Colombel,
B. Gosselin, A. B. Tonnel, and A. Janin. 1995. Immunoreactivity for interleukin
3 and 5 and granulocyte/macrophage colony-stimulating factor of intestinal mu-
cosa in bronchial asthma. J. Exp. Med. 182: 1897–1904.
51. Levi-Schaffer, F., and A. M. Piliponsky. 2003. Tryptase, a novel link between
allergic inflammation and fibrosis. Trends Immunol. 24: 158–161.
52. Hallgren, J., U. Karlson, M. Poorafshar, L. Hellman, and G. Pejler. 2000. Mech-
anism for activation of mouse mast cell tryptase: dependence on heparin and
acidic pH for formation of active tetramers of mouse mast cell protease 6. Bio-
chemistry 39: 13068–13077.
53. Bousquet, J., P. Chanez, J. Y. Lacoste, G. Barneon, N. Ghavanian, I. Enander,
P. Venge, S. Ahlstedt, J. Simony-Lafontaine, P. Godard, et al. 1990. Eosinophilic
inflammation in asthma. N. Engl. J. Med. 323: 1033–1039.
4891The Journal of Immunology
by guest on June 13, 2013