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Infection and Immunity, December 2001, p. 7743-7752, Vol. 69, No. 12
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.12.7743-7752.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Interleukin-4 Receptor-Stat6 Signaling in Murine
Infections with a Tissue-Dwelling Nematode Parasite
L.
Spencer,1
L.
Shultz,2 and
T. V.
Rajan1,*
Department of Pathology, University of
Connecticut Health Center, Farmington,
Connecticut,1 and Jackson
Laboratories, Bar Harbor, Maine2
Received 2 May 2001/Returned for modification 2 July 2001/Accepted 4 September 2001
 |
ABSTRACT |
Interleukin-4 (IL-4) has been shown to be crucial in parasite
expulsion in several gastrointestinal nematode infection models. Data
from both epidemiological studies with humans and experimental infections in animals imply a critical role for the type II helper response, dominated by IL-4, in host protection. Here we utilized inbred mice on two distinct backgrounds to document the involvement of
IL-4 in the clearance of a primary infection of Brugia
from the murine host. Our data from infections of IL-4
receptor
/ and Stat6/ mice further
indicate that IL-4 exerts its effects by activating the Stat6 molecule
in host target cells, a finding which links clearance requirements of a
gastrointestinal tract-dwelling nematode with those of a
tissue-dwelling nematode. Additionally, we show that the requirements
for IL-4 receptor binding and Stat6 activation extend to accelerated
clearance of a secondary infection as well. The data shown here,
including analysis of cell populations at the site of infection and
infection of immunoglobulin E (IgE)/ mice, lead us to
suggest that deficiencies in eosinophil recruitment and isotype
switching to IgE production may be at least partially responsible for
slower parasite clearance in the absence of IL-4.
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INTRODUCTION |
In rodent models of helminth
infections, a Th2-like response is generally associated with parasite
clearance, while a Th1-dominated response leads to a state of chronic
infection (11). This protective role for Th2 host
responses has been documented most extensively in the gastrointestinal
nematode models, in which an absolute requirement for the prototypical
Th2 cytokine interleukin-4 (IL-4), the IL-4 receptor (IL-4R), or Stat6
signaling has been shown for the clearance of Heligmosomoides
polygyrus, Trichuris muris, Trichinella spiralis, and Nippostrongylus brasiliensis from the
murine host (4, 7, 8, 38-40). In contrast, gamma
interferon mediates exacerbation of infection, at least in N. brasiliensis infections (36).
The human-infecting parasite Brugia malayi and its
feline-infecting counterpart Brugia pahangi are
tissue-dwelling nematodes against which a Th2-dominated response also
seems to be rapidly induced and beneficial in a rodent host. The murine
model has been extensively utilized in studies of Brugia
infection. Immunocompetent mice are able to clear an infection with
Brugia before patency, thereby providing a mammalian model
of filarial infection in which host immune responses are successful in
clearance of the parasite. Antigen-presenting cells responding to a
Brugia infection in wild-type (WT) mice induce Th2
differentiation of naive T cells (17). Analysis of
draining lymph node cells as well as splenocytes following intraperitoneal (i.p.), subcutaneous, or hind-footpad injections with
live Brugia L3 infective-stage larvae (hereafter referred to
as L3) shows an induction of Th2-like cytokines and an impairment in
concanavalin A-driven Th1 cell proliferation and IL-2 and gamma interferon production (15, 27, 28). If the L3 are
irradiated prior to subcutaneous injection, a strong Th2 response is
still elicited (3). Osborne and Devaney have demonstrated
that increased levels of mRNA encoding IL-4 can be detected in draining
lymph nodes as early as 24 h postinfection with B. pahangi L3, from T-cell receptor 
+
CD4 CD8 cells
(27). A critical requirement for IL-4 in this system has
been described (2), although studies with IL-4-deficient mice have shown variable results, seemingly dependent upon the background strains used (11, 15).
Although the necessity for IL-4 signaling has been well established in
the clearance of several gastrointestinal nematodes, the precise
role(s) of IL-4 has been more difficult to determine. Targets of IL-4
signaling in general include B cells, T cells, macrophages, stromal
cells, hematopoietic precursors, intestinal epithelial cells,
fibroblasts, and muscle cells (18, 26, 29, 30). Effects of
IL-4 include inhibition of superoxide anion production by macrophages
(34), upregulation of isotype switching to immunoglobulin
G1 (IgG1) and IgE and major histocompatibility complex class II and
CD23 expression on B cells (9, 33, 41), enhancement of
eosinophil extravasation and recruitment (7, 21, 22, 32),
and induction of Th2 cell differentiation in T cells (23).
Finkelman et al. (10) and Urban et al.
(37-39) have shown a dependence on both T cells
and mast cells for the function of Stat6 signaling in clearance of
Trichinella spiralis, while the Stat6 requirement in a model
of N. brasiliensis infection is independent of B cells, T
cells, or mast cells. It has been hypothesized that the target cell
within the latter system is the intestinal epithelial cell (10,
37-39). Despite the target cells involved, the clearance
mechanisms of these models involve expulsion of the live parasite from
the gastrointestinal tract of the host.
A tissue-dwelling nematode such as Brugia differs in this
respect from its gastrointestinal relatives in that elimination of the
parasite load can be accomplished only by killing the worm while it is
still inside the host. It is therefore very probable that the final
effector mechanisms resulting in parasite clearance will be very
different in the two nematode groups. Nonetheless, it is tempting to
speculate that successful clearance of both gastrointestinal tract- and
tissue-dwelling nematodes from the murine host shares the
characteristic of inducing the IL-4R chain-Stat6 signaling pathway
within a host target cell(s).
In this study we utilized inbred mice on two distinct backgrounds to
make a stronger case for the necessity of IL-4 in clearance of a
primary infection of Brugia from the murine host. Further, through infection of IL-4R chain/
(hereafter IL-4R/) and
Stat6/ mice, we show that IL-4-induced Stat6
signaling within a host target cell(s) does in fact play a role in the
killing of a tissue-dwelling parasite. We have begun to further
elucidate the mechanisms by which IL-4 may aid in clearance of
Brugia from the murine host through cellular analysis of
intact and IL-4-deficient animals at the site of infection, in addition
to infections of mutant mice with deficiencies downstream of Stat6 signaling.
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MATERIALS AND METHODS |
Mice.
BALB/c-Pkrdcscid/Pkrdcscid,
BALB/c Stat6/,
BALB/cByJ+/+, C57BL/6+/+,
C57BL/6-Il4tm1Nnt
(IL-4/), and
BALB/c-Il4tm2Nnt
(IL-4/) animals were obtained from the
Jackson Laboratory (Bar Harbor, Maine).
BALB/c-Il4ratm1Sz
(IL-4R/) mice were initially obtained from
the Jackson Laboratory and were subsequently bred and housed under
specific-pathogen-free conditions in microisolator cages in the
American Association for the Accreditation of Laboratory Animal
Care-accredited vivarium of the University of Connecticut Health
Center. BALB/c IgE/ mice were originally
obtained as a gift from M. Oettgen (Harvard University School of
Medicine) and subsequently bred at our facility. All mice used were
males between 6 and 12 weeks of age. Confirmation of the genotype of
the IL-4R/ animals was carried out by random
PCR analysis of tail DNA using primers specific for the neomycin
resistance gene as well as specific segments of the intact IL-4R gene
as described previously (25). Leakiness of the SCID
phenotype was ruled out through serum Ouchterloney tests. The IgE
deficiency of IgE/ animals was periodically
confirmed using an IgE-specific enzyme-linked immunosorbent assay
(ELISA) of serum.
Parasite.
B. malayi was harvested at the
insectarium of Thomas Klei (Louisiana State University, Baton Rouge)
from infected Aedes aegypti mosquitoes and shipped overnight
in RPMI medium containing antibiotics and fluconazole. B. pahangi was harvested from infected mosquitoes at the University
of Georgia and shipped in a similar manner.
Experimental infection.
Mice were inoculated with 50 to 80 B. pahangi or B. malayi L3 i.p. using a 25 5/8-gauge needle for a primary infection. For challenge infections, 50 L3 of the same species were injected i.p. into mice previously
sensitized with 30 to 50 L3 4 to 6 weeks earlier.
Worm recovery.
Mice were sacrificed at various time points
postinfection and subjected to a cardiac bleed for retrieval of serum.
Peritoneal lavages were performed using RPMI medium supplemented with 5 U of heparin per ml. At time points of 4 weeks and later, lavage was
extracted from the peritoneal cavity using a soft plastic pipette to
prevent shearing of the adult worms. Following lavage, intestines were
removed and soaked in phosphate-buffered saline (PBS). Testes were cut,
and carcasses were placed in PBS for further soaking. Carcasses were
then rinsed several times with PBS. Viable worms were counted from
peritoneal lavage, intestinal washes, and carcass soaks under a
dissecting microscope.
Preparation of inflammatory nodules.
Inflammatory nodules
recovered from peritoneal lavages were transferred to petri dishes
containing RPMI with 5 U of heparin per ml as a washing step. Nodules
were then transferred to a 4% glutaraldehyde fixative and stored at
4°C prior to embedding.
IgE ELISA.
Total serum IgE-specific sandwich ELISA was
performed following standard BD Pharmingen (San Diego, Calif.)
protocol. Purified anti-mouse IgE R35-72 (Pharmingen catalog no.
02111D) was used as a capture antibody, and alkaline
phosphatase-conjugated anti-mouse IgE (Pharmingen catalog no. 02133E)
was used as a detecting antibody. Total concentrations in serum were
determined by comparison to a purified mouse IgE standard (Pharmingen
catalog no. 03121D).
Statistical analysis.
Statistical significance was analyzed
by parametric as well as nonparametric methods. The Student
t test (Stt) was used in the parametric analysis, and the
Mann-Whitney test (MWt) was used in nonparametric determinations.
P values of less than 0.05 were considered statistically significant.
Fluorescence-activated cell sorting.
Peritoneal lavage cells
were collected and passed through nylon mesh to remove debris.
Conjugated monoclonal antibodies (CD19-phycoerythrin, CD3-fluorescein
isothiocyanate, Ly6G-fluorescein isothiocyanate, CD43-biotin,
CD11b-biotin, and CD138-phycoerythrin) were obtained from Pharmingen
and used at a dilution of 1:100 unless otherwise stated.
Streptavidin-CyChrome (Pharmingen catalog no. 554062) was used at a
dilution of 1:400 as a secondary antibody with biotin-conjugated antibodies. Stained cells were fixed with paraformaldehyde, and data
were acquired through the CellQuest software using the FACSCalibur (Becton Dickinson). Data were subsequently analyzed using WinMDI software. Cell sorts were performed using the FACSVANTAGE SE.
Cytospins.
Cells (104) were taken from
peritoneal lavages prior to fluorescence-activated cell sorter staining
or following sorting and resuspended in 1% bovine serum albumin in
PBS. Using a Cytospin 2, these cells were transferred to slides.
Staining of slides was carried out with the Platinum Line Quick-Dip
three-part staining system (Mercedes Medical, Inc.).
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RESULTS |
IL-4 is required for clearance of Brugia from
C57BL/6 mice.
Our previous studies showed that BALB/c mice
deficient in IL-4 were significantly more permissive to infection with
B. malayi than WT mice at 6, 10, and 12 weeks postinfection.
In addition, microfilariae (Mf) were found in the
IL-4/ mice at the 10- and 12-week time points
(2). These results were in conflict with those obtained by
Lawrence et al. (15) using IL-4/
mice on the segregating (C57BL/6J × 129) background, suggesting that a requirement for IL-4 in host protection may be a phenomenon depending on the background strain of the murine host. In order to
determine if the requirement for IL-4 for resistance to infection was
unique to the BALB/c strain or a more universal requirement, we
examined C57BL/6 mice. Although both BALB/c and C57BL/6 mice have been
shown to be "resistant" to patent infection, our laboratory has
consistently found that clearance of an i.p. injection of L3 from a
C57BL/6 mouse occurs significantly more rapidly than clearance from a
BALB/c mouse (unpublished data).
Figure 1 compares the clearance kinetics
of C57BL/6 WT and IL-4-deficient mice. As early as 2 weeks following an
i.p. injection of L3, a significantly higher percentage of live worms
were recoverable from the C57BL/6 IL-4-deficient cohort. In this
experiment (which is representative of four independent experiments),
WT animals supported survival of 8% ± 8% of injected worms at 2 weeks, while their IL-4-deficient counterparts harbored 28% ± 9%
(Stt, P = 0.005; MWt, P = 0.021). This
difference in live worm recoveries remained significant until 4 weeks
postinfection, at which point 2% ± 3% of worms remained in the WT
animals, while the IL-4-deficient cohort still maintained 12% ± 3%
(Stt, P = 0.001; MWt, P = 0.008). By 6 weeks postinfection both WT and IL-4-deficient mice supported less than
10% of injected worms. Unlike the case for BALB/c
IL-4/ mice, we have not detected Mf in B6
IL-4/ animals. Similar results were obtained
upon infection with B. pahangi (data not shown).
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FIG. 1.
C57BL/6 mice are delayed in their ability to clear a
primary infection of Brugia. Groups of 5 to 10 C57BL/6+/+ or IL-4/ mice were injected i.p.
with 50 L3 of B. malayi and necropsied at indicated time
points postinfection. The data represent the percentage of the initial
inoculation recovered as live worms. *, statistical significance
(P < 0.05 by both parametric and nonparametric
analyses) between percentages of worm recoveries from the WT and
IL-4/ groups. Error bars indicate standard
deviations.
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IL-4-induced parasite clearance requires signaling through IL-4R
and activation of Stat6 within host cells.
To differentiate
between an activity of IL-4 directly against the worm and a function
requiring signaling through an intermediary host cell, we utilized
BALB/c IL-4R/ mice. As shown in Fig.
2A, at 7 weeks postinfection 23% ± 18% of injected worms were recovered from IL-4R/
mice, while cohorts of intact mice at this time point harbored only 4% ± 6% of injected worms. At 12 weeks postinfection
IL-4R/ mice continued to harbor significantly
more worms than their WT counterparts, with
IL-4R/ mice supporting 17% ± 12% of
injected worms, while fewer than 3% of worms could be recovered from
the WT cohort (Stt, P = 0.02; MWt, P = 0.013). In addition, live worm recoveries from
IL-4/ cohorts in the same experiment were
nearly identical to those from the IL-4R/
animals. This experiment is representative of eight independent experiments analyzing various time points from 4 to 12 weeks
postinfection.
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FIG. 2.
IL-4 exerts its effects by signaling a host target
cell(s) through IL-4R and subsequent Stat6 activation. (A) Groups of 5 to 10 BALB/c+/+ or BALB/c IL-4R/ mice were
injected i.p. with 50 B. malayi L3 and necropsied at 7 or 12 weeks following infection. The data represent the average
percentage of the original inoculation recovered as live worms.
P values at weeks 7 and 12 between WT and
IL-4R/ are 0.06 and 0.02, respectively, by Stt and
0.098 and 0.013, respectively, by MWt analysis. Error bars indicate
standard deviations. (B) Groups of five BALB/c+/+,
IL-4/, or Stat6/ mice were injected
i.p. with 50 B. malayi L3 and necropsied at 6 weeks
postinfection. P values between WT and
Stat6/ mice are 0.0006 by Stt and 0.007 by MWt.
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To ascertain whether signaling of IL-4 through IL-4R involved
activation of the Stat6 pathway, we utilized BALB/c mice in
which the
Stat6 gene had been disrupted. Figure
2B shows the results
of one
representative experiment of three, which highlights significantly
higher recoveries from Stat6
/ mice than from
WT mice. At 6 weeks postinfection fewer than 1%
of injected worms were
recovered from WT mice, while IL-4
/ and
Stat6
/ mice still harbored 9% ± 5% and
17% ± 7%, respectively, of the
initial
inoculum.
Effects of IL-4 defect compared to severe combined
immunodeficiency.
Immunodeficient SCID mice, which lack T and B
cells, have been utilized as an example of a completely susceptible
host for Brugia infection (24). At 10 to 12 weeks postinfection with L3, SCID mice become patent for Mf. Although
IL-4R/ mice on the BALB/c background
maintained significant worm burdens at late time points and became
patent for Mf, live worm recoveries from these animals were
consistently lower than those from completely immunodeficient SCID mice
(data not shown).
A requirement for IL-4 signaling extends to a challenge
infection.
Immunocompetent BALB/c mice previously exposed to live
Brugia L3 exhibit accelerated clearance of a second
infection with L3 given 4 to 6 weeks later. Worms remaining from the
primary infection are easily distinguishable from those remaining from the challenge infection due to their much larger size. In an effort to
establish a role for IL-4 signaling in this rapid secondary clearance,
BALB/c IL-4R/ animals were subjected to the
same challenge protocol. As shown in Fig.
3, worm recoveries from naive WT mice
averaged 35%, while previously exposed WT mice harbored 4% of the
challenge injection (Stt, P = 0.004; MWt,
P = 0.008). In contrast, naive
IL-4R/ mice harbored 58% of injected worms,
while previously exposed IL-4R/ mice
supported 40% of the challenge injection at this same time point (Stt,
P = 0.129; MWt, P = 0.1).
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FIG. 3.
IL-4 signaling is required for accelerated clearance of
a challenge infection with Brugia. Five previously
exposed or naive BALB/c+/+ or IL-4R/ mice
were challenged with 50 B. malayi L3 i.p. and necropsied
at 14 days postchallenge. The data represent the average percentage of
the challenge inoculation recovered as live worms. *, statistical
significance (P < 0.5) between naive and
previously exposed cohorts by both parametric and nonparametric
analyses. Error bars indicate standard deviations.
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Cellular analysis of the site of infection.
In search of
mechanisms underlying the requirement for IL-4 signaling in our system,
we analyzed the cellular compartment responding to the worms at the
site of infection at several time points throughout infection.
Peritoneal exudate cells (PEC) recovered from a peritoneal lavage were
analyzed cytologically as well as by flow cytometry.
Figure
4A shows representative forward-
and side-scatter plots generated from PEC isolated from C57BL/6 WT and
IL-4
/ mice at 2 and 5 weeks postinfection. In
these plots the
y axis
is a measurement of the relative
complexity, or granularity, of
the cells (side scatter) and the
x axis represents the relative
sizes of the cells (forward
scatter). Thus, the cells in the lowest,
leftmost regions are the
smallest, least granular cells, while
cells found at the rightmost,
higher region of the plot represent
the largest, most complex
populations. Staining with fluorochrome-conjugated
monoclonal
antibodies has allowed us to identify the discreet
populations which
appear on this plot, as illustrated in Fig.
4B. Thus, the region
arbitrarily designated R1 consists predominantly
of T and B cells
(CD3
+ and CD19
+ staining,
respectively), while R2 appears to be predominantly
larger B1 cells
with some B2 cells (CD19
+
CD43
+ and CD19
+
CD43
, respectively). The region designated R3
presumably represents
a predominantly macrophage population, as
suggested by the high
degree of nonspecific staining and
autofluorescence seen in this
population and a strong reaction with
CD11b (Mac-1) antibody (data
not shown), as well as the large size and
complexity implied by
its location on the scatter plot (Fig.
4B).
Positive staining
for CD138 has also been observed in this region,
indicating the
presence of plasma cells (data not shown). The R4 region
has been
difficult to identify by cell surface staining techniques,
although
intermediate Ly6G (Gr-1) staining is consistently measured.
These
cells appear to be eosinophils, based on the observation that
upon sorting of this region a population enriched for eosinophils
is
recovered (Fig.
4B). Further support for this identification
comes from
findings that this population is significantly reduced
in
IL-5
/ mice, as well as in WT mice treated
with a monoclonal antibody
against CCR3 (unpublished data).
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FIG. 4.
Infected C57BL/6 IL-4/ mice display an
increased percentage of small lymphocytes and an impairment in their
ability to recruit eosinophils to the site of infection. (A) PEC were
recovered from C57BL/6+/+ or IL-4/ mice at
2 or 5 weeks postinfection and analyzed by flow cytometry using the
FACSCalibur. Results are shown as a forward-scatter (FSC)-side-scatter
(SSC) plot. (B) Discrete cell populations of
forward-scatter-side-scatter plots were gated and analyzed for
expression of specific fluorochrome-conjugated antibodies. The R4 gate
was sorted using the FACSVANTAGE SE and reanalyzed with the FACSCalibur
to ensure purity of the sorted population. Sorted cells were then
transferred to slides with a Cytospin 2 and stained.
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The cellular compositions of the peritoneal cavities of WT and
IL-4
/ mice at 2 and 5 weeks postinfection are
calculated in Table
1 from a
representative experiment involving five mice in each group.
Amounts of
different cell types are expressed as percentages of
total
cells, to account for any variability in lavage efficiency
or mouse
size. However, as Table
2 illustrates, a
comparison
of total cell numbers yields the same results. At 2 weeks
postinfection,
a significant difference was observed between the
percentages
of worms recovered from WT and
IL-4
/ mice, with WT mice harboring 8% ± 8%
of the injected worms, while
IL-4
/ mice
supported 28% ± 9%. Comparison of the cellular compositions
of these
animals at this time point indicates that the two cohorts
differed
significantly in the numbers of small lymphocytes (comprising
45% ± 6% of the IL-4
/ PEC and 25% ± 0.6% of the
WT PEC [Stt,
P = 0.00009]) and eosinophils
(comprising 4% ± 1% of the IL-4
/ PEC and
14% ± 2% of the WT PEC [Stt,
P = 0.0001]). By 5 weeks
postinfection the IL-4
/ cohort had
begun to decrease its worm burden, as 12% ± 4% of
the injected worms
were recovered, while the WT mice harbored
1% ± 2%. Analysis of the
cellular compartments at this time point,
as shown in Table
1,
indicates no significant differences in
the compositions of PEC between
the two groups. As seen in Table
2, a comparison of total cell numbers
yields similar results,
with a fivefold decrease in the absolute number
of eosinophils
seen in IL-4
/ mice in
comparison to their WT counterparts at 2 weeks postinfection
and no
significant discrepancies in any cell population at 5 weeks
postinfection.
Analysis of PEC from IL-4R
/ and
Stat6
/ mice in comparison to WT mice
highlights a similar increase in small lymphocytes and
decrease in
eosinophils seen in the mutant cohorts, as shown in
Fig.
5 and Table
3. At 2 weeks postinfection, an
average of 11%
± 16% of injected worms were recovered from five
BALB/c WT mice,
while five each of
IL-4R
/ and
Stat6
/ mice harbored averages of 75% ± 19%
and 43% ± 23%, respectively.
At this time point 38% ± 13% of PEC
from IL-4R
/ mice compared to only 23% ± 3%
of PEC from WT mice are small
lymphocytes (Stt,
P = 0.04). Similar to the case for IL-4R
/ mice,
small lymphocytes represent 37% ± 3% of PEC in
Stat6
/ mice (Stt,
P = 0.00004 between Stat6
/ and WT mice). The eosinophil
deficiency is seen in both the IL-4R
/ and
Stat6
/ mice as well, as eosinophils comprise
1% ± 0.8% of PEC from IL-4R
/ mice, 1% ± 0.9% of PEC from Stat6
/ mice, and 11% ± 2% of PEC from WT mice (Stt,
P = 0.00003 between
IL-4R
/ and WT mice and
P = 0.00003 between Stat6
/ and WT mice). Unlike
the comparison between the C57BL/6 WT and
IL-4
/ cohorts, the percentage of total PEC
made up of large, complex
cells was also significantly deficient in
IL-4R
/ and Stat6
/
mice.
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FIG. 5.
Infected BALB/c IL-4R/ and
Stat6/ mice display an increased percentage of small
lymphocytes and an impairment in their ability to recruit eosinophils
to the site of infection. PEC were recovered from
BALB/c+/+, IL-4R/, or
Stat6/ mice at 2 weeks postinfection. Cells were
analyzed by flow cytometry as in Fig. 4 and are shown as
forward-scatter (FSC)-side-scatter (SSC) plots.
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TABLE 3.
Percentages of different cell types in PEC after
infection of WT, IL-4R/, and Stat6/
BALB/c mice with B. malayia
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|
Examination of cytospins obtained from IL-4
/,
IL-4R
/, Stat6
/, and
WT PEC confirms the flow cytometric results (data not
shown).
Analysis of PEC during challenge infection.
Table
4 lists the breakdown of the PEC
compartment from WT or IL-4R/ mice 2 weeks
following a challenge infection. Again, an increase in small
lymphocytes is observed in IL-4R/ (21% ± 3%) compared to WT (8% ± 3%) animals, as is a deficit in the
percentage of eosinophils found within the peritoneal cavity upon
challenge infection of IL-4R/ mice (13% ± 6%) compared to WT controls (28% ± 5%). In addition, a
significantly higher percentage of the PEC are comprised of large,
granular cells and a significantly lower percentage of large
lymphocytes are found within the WT cohort compared with the mutant
animals.
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TABLE 4.
Percentages of different cell types in PEC after
challenge infection of previously exposed WT and IL-4R/
BALB/c mice
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Importance of eosinophils in worm clearance.
The inability of
IL-4/, IL-4R/, and
Stat6/ mice to recruit eosinophils to the
infection site in a timely manner is not surprising, as a role for IL-4
in eosinophil recruitment has been well documented (7, 21, 22,
32). A role for eosinophils in clearance of filarial infections
has not been formally documented. However, evidence exists for their
inclusion in host cell nodules encompassing worms within a murine host
(12, 13, 20, 35). These "coated worms" (Fig.
6A) are found within the peritoneal
cavity during phases of maximum worm clearance and presumably represent
a final step before worm death, as illustrated by the necrotic
appearance of portions of the encased worms when observed by electron
microscopy. Analysis of the cellular composition of these nodules by
light microscopy and transmission electron microscopy indicates that the predominant host cell types are eosinophils and macrophages. Figure
6B illustrates the high level of eosinophil involvement in host cell
nodules. Further analysis of this nodule revealed eosinophil
involvement ranging from 10 to 80% in every field examined. This
dramatic variation in eosinophil numbers makes a precise quantitation
of eosinophils nearly impossible.
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FIG. 6.
Worms recovered from peritoneal cavities encased in host
cell nodules contain a high percentage of eosinophils. (A) L4 stage
worms encased in host cells recovered from a WT mouse at 2 weeks
postinfection with B. malayi. Measuring marks represent
1-mm increments. (B) Transmission electron micrograph of a section of a
host cell nodule recovered from a WT mouse at 2 weeks postinfection
with B. malayi. Magnification, ×900.
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Isotype switching to IgE as a possible target of IL-4.
Another
IL-4-mediated effect which has been attributed to Stat6 activation is
the induction of isotype switching to IgE production upon costimulation
of B cells through CD40 (5, 6, 42). IL-4/ mice have been shown to produce
significantly less parasite-specific IgE than WT mice in response to
B. malayi (2). Similarly, we find little to no
IgE in sera from IL-4R/ and
Stat6/ mice following a primary
infection with Brugia (Table
5).
In order to ascertain whether the decrease in IgE production in these
animals may be responsible for the impairment in worm
clearance
abilities, we infected BALB/c IgE
/ mice. A
comparison of worm clearance kinetics among WT,
IL-4
/, and IgE
/
mice is shown in Fig.
7. By 4 weeks
postinfection, IL-4
/ and
IgE
/ mice harbored 44% ± 13% and 54% ± 7% of injected worms, respectively.
In contrast, the WT animals had
decreased their worm burdens to
12% ± 5% at this time point. This
increase in worm recoveries
from IgE
/
compared to WT mice is maintained at least until week 6 postinfection,
at which point 8% ± 9% of injected worms were recovered in the
WT
mice, while 27% ± 12% were found in the
IgE
/ group. It is important to note, however,
that worm recoveries
from IgE
/ mice at 6 weeks postinfection were significantly lower than those
seen at 4 weeks
postinfection (Stt,
P = 0.003; MWt,
P = 0.014).
Additionally, the recoveries at 6 weeks were significantly
lower
in IgE
/ animals than in
IL-4
/ mice (Stt,
P = 0.034;
MWt,
P = 0.019).
View larger version (23K):
[in this window]
[in a new window]
|
FIG. 7.
IgE is involved in clearance of a primary infection with
B. malayi. Ten BALB/c+/+, 10 IL-4/, and 10 IgE/ mice were injected
with 35 B. malayi L3. Five mice from each of these
cohorts were necropsied at each time point following injection. The
data represent the average percentage of the initial inoculation
recovered as live worms. Error bars indicate standard deviations.
|
|
Using our challenge infection protocol, we analyzed the levels of
circulating serum IgE in BALB/c
+/+ and
IL-4R
/ mice. In agreement with other
published reports, previously exposed
WT mice displayed an increased
level of circulating IgE at 2 weeks
after a challenge infection. As
measured by sandwich ELISA, serum
IgE levels in WT mice were 5,058 ± 3,672 ng/ml. In contrast, no
serum IgE could be detected in any of
the IL-4R
/ mice previously exposed to L3.
Thus, the presence of circulating
IgE appears to correlate with the
ability of the murine host to
clear a challenge injection of L3 in an
accelerated
manner.
| |
DISCUSSION |
We chose to utilize BALB/c and C57BL/6 mice to address the
question of a role for IL-4 in murine immunity to Brugia for
two reasons: (i) the two strains differ in major histocompatibility complex haplotype and follow slightly different kinetics of worm clearance, and (ii) both strains are "resistant" to infection, as
they are eventually able to reduce parasite numbers to zero or near
zero, and Mf are not found, even at very late time points. The data
shown here therefore indicate that IL-4 does in fact play a role in
otherwise resistant strains, irrespective of haplotype or worm
clearance kinetics. The lack of IL-4 signaling significantly delays the
worm clearance kinetics of mice on the C57BL/6 as well as the BALB/c
background. Due to the slower killing kinetics seen in BALB/c mice
infected with Brugia, the further delay caused by a
deficiency in IL-4 leads to patency for Mf. This effect of IL-4
deficiency was seen following infection with B. pahangi as well as B. malayi.
We hypothesized that IL-4 was acting through a host target cell(s)
rather than through a direct cytotoxic effect on the worm itself. This
hypothesis was based on the observations that although SCID mice lack T
cells as a source of IL-4, other IL-4-producing cells such as NK cells,
mast cells, and eosinophils are present, and yet these animals are
unable to eliminate a substantial number of worms (24).
Additionally, in vitro studies with worms recovered from WT or
IL-4/ hosts showed identical survival times
in culture at all time points analyzed, suggesting that the mere
presence of IL-4 within the host does not predispose these worms to
earlier destruction (data not shown). Two receptors have been described
for IL-4 (type I and type II) (14, 16, 31). The type I
receptor is composed of the IL-4R alpha chain complexed to the common
gamma chain, while the type II receptor is made up of the IL-4R alpha
chain and the IL-13R alpha chain. IL-13 has been shown to bind to the type II form of the receptor (44). In order to more
convincingly differentiate between a direct or indirect effect of IL-4,
we infected mice in which the gene encoding the alpha chain of IL-4R had been disrupted, thus disabling both type I and type II receptors. These IL-4R/ mice exhibit an impairment in
worm clearance capabilities comparable to that of the
IL-4/ animals, indicating that IL-4 is indeed
exerting its effects by signaling through an intermediary host cell. In
addition, these data suggest that IL-13 is not able to compensate for
the absence of IL-4 in a primary-infection model.
This dependence upon IL-4R signaling is reminiscent of several
gastrointestinal nematode models, including T. muris,
H. polygyrus, T. spiralis, and N. brasiliensis. In these models the crucial IL-4 signaling has been
shown to work through a Stat6-dependent pathway, to induce mastocytosis
or act on the intestinal epithelial cell to induce expulsion of the
parasites. Our data indicate a similar requirement for Stat6 activation
downstream of IL-4R in clearance of B. malayi and B. pahangi from the murine host, a novel concept in that in this
system the action of IL-4 must result in destruction of the parasite
while it is still within the host, rather than induction of a mechanism
for expulsion of the live worms.
Having established a role for IL-4R-Stat6 signaling in a primary
infection, we questioned whether there existed a similar requirement
for accelerated worm clearance in a challenge infection model. As shown
in Fig. 3, IL-4R-Stat6 signaling is necessary for accelerated
clearance of a challenge infection as well, at least at the time points
analyzed. It should be noted that in the challenge infection protocol,
adult worms remaining from the primary infection are seldom if ever
found within WT animals but are nearly always present in the
IL-4R/ and Stat6/
animals. The argument may be made that the inability of the
IL-4R/ and Stat6/
mice to clear the challenge L3 may therefore be attributable to the
existence of proliferative suppressive capabilities of the PEC induced
by the adult worms remaining from the primary injection. The concept of
lymphocytic proliferative suppression has been described for murine
B. malayi infections. In studies where adult B. malayi organisms were implanted into the peritoneal cavities of
mice, adherent peritoneal cells removed from these animals suppressed
lymphocytic proliferation ex vivo, while appearing to leave cytokine
production unhampered (1). In later studies a similar
suppressive ability in response to L3 was described as well
(19). Work by MacDonald et al. highlights an indirect requirement for IL-4 in the induction of this suppressive PEC population, arguing against the ability of adult worms to elicit such a
response in the IL-4R/ animals
(19).
Although these data have identified an essential role for Stat6
activation in both a primary and a challenge infection of B. malayi, the crucial effector mechanism(s) downstream of this Stat6
signaling remains elusive. Analysis of the cellular components responding to the site of infection by both cytological and flow cytometric analyses highlights a significant defect in the ability of
IL-4-, IL-4R-, and Stat6-deficient mice to recruit eosinophils at time
points when a significant difference in live worm recoveries is
observed. The importance of IL-4 signaling in eosinophil recruitment is
not a new concept but has been well established in models such as the
cellular response to schistosomal egg antigens in rodents and allergy
in humans (7, 21, 22, 32). Human studies have shown that
IL-4R is expressed constitutively on the surface of eosinophils
(7). While a directly cytotoxic role for eosinophils in
the host response to Brugia has not yet been established,
eosinophil involvement in host cell nodules encasing the worms is shown
here (Fig. 6B) and has been extensively described in the literature (12, 13, 20, 35). It is therefore likely that one factor contributing to the impairment of the worm clearance capabilities of a
mouse deficient in IL-4 signaling will be the inability to recruit
eosinophils to the site of worm invasion.
An increase in the percentage of small lymphocytes was observed in all
groups of mutant animals, and a significant decrease in percentages of
large, granular cells responding to infection was observed in
IL-4R/ and Stat6/
animals compared with WT controls. The significance of these cellular
abnormalities has yet to be determined. The high level of involvement
of macrophages in inflammatory nodules in conjunction with the many
known effects of IL-4 signaling on these cells suggests that this
population may also contribute to the involvement of IL-4 signaling in
worm clearance.
In addition to these deficiencies in cellular recruitment, it is
tempting to speculate that another important player may be the
induction of IgE production from B cells. B cells are induced to
undergo class switching to the IgE isotype following signaling through
IL-4R and CD40. Analyses of antibody isotypes responding to a primary
infection with Brugia illustrate a severe defect in IgE
production from IL-4/,
IL-4R/, and Stat6/
mice (2) (Table 5). A further increase in IgE levels has been observed in the challenge infection in WT mice (43).
IL-4R/ and Stat6/
mice continue to exhibit an inability to mount a substantial IgE
response in the challenge infection protocol as well; thus, the absence
of IgE appears to correlate with a deficiency in worm clearance.
Further support for the importance of IgE in parasite clearance is
provided by the decreased ability of IgE/
mice to clear a primary infection in comparison to WT mice. In contrast
to our findings, Watanabe et al. have found no difference in worm
clearance abilities between control mice and mice treated from birth
with an antiepsilon monoclonal antibody (43). The discrepancies between our results and those of Watanabe et al. may
reflect an incomplete depletion of IgE in their protocol of neonatal
antibody neutralization. Our system utilized mice deficient in IgE due
to a targeted gene disruption. This strategy is also subject to
misleading results due to the ability of organisms to compensate for a
missing gene when forced to develop in its absence. In general,
compensation would result in a normal phenotype. Since our data suggest
that the absence of IgE alters the phenotype compared to WT controls,
it is unlikely that compensation is playing a major role.
Although the data generated here suggest roles for both eosinophil
recruitment and IgE production in the clearance of tissue-dwelling filarial worms, we have no data to support any commonalities between these mechanisms except for a mutual dependence upon IL-4R signaling for their implementation. Unlike their human counterparts, murine eosinophils do not express the high-affinity IgE receptor. In light of
this, it may be interesting to look at the contribution, if any, of the
low-affinity IgE receptor in parasite killing.
IL-4 signaling and subsequent activation of Stat6 have been shown to be
a key component of a successful host response to gastrointestinal nematodes. Despite the vast differences in mechanisms of parasite clearance, the data shown here indicate that a similar signaling pattern is utilized for the elimination of Brugia, a
tissue-dwelling nematode, from the rodent host. Identification of the
crucial IL-4-mediated mechanism(s) has yet to be achieved; however, it is likely that at least two of these mechanisms may be the inability of
the murine host, in the absence of IL-4, to produce sufficient levels
of IgE and to recruit eosinophils to the site of infection.
| |
ACKNOWLEDGMENTS |
This work was made possible by grants AI 39705 and AI 42362 to
T.V.R.
We thank Lynn Puddington and Steve Wikel for critical review of the
manuscript, and we thank Sharon Coleman and Thomas Klei for some of the
infectious larvae used in this study.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Pathology, University of Connecticut Health Center, 263 Farmington
Ave., Farmington, CT 06030-3105. Phone: (860) 679-3221. Fax: (860)
679-2936. E-mail: rajan{at}neuron.uchc.edu.
Editor:
J. M. Mansfield
| |
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Infection and Immunity, December 2001, p. 7743-7752, Vol. 69, No. 12
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.12.7743-7752.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
