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