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*
Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and
Biomedical Research Institute, Rockville, MD 20852
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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- and IL-4-secreting
cells, elevated perivascular/peribronchial inflammatory responses in
the lung, and greater in vitro schistosomulacidal capacity of parasite
Ag-elicited cells. These results suggest that optimal vaccine-induced
immunity against schistosomes is linked not to the development of a
highly polarized response, but, rather, to the induction of both type
1- and type 2-associated immune responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although it is generally agreed that a vaccine will have at its core the generation of an Ag-specific CD4+ T cell response (7), it is still unclear which subset of Th cells, Th1 or Th2 (8, 9, 10), should be induced in a schistosome vaccine. Because of the potent counter-regulatory activity exhibited between these two subsets of Th cells (11), it is generally believed that it may be difficult to develop a vaccine that effectively exploits both arms of the immune response (12).
Induction of resistance in mice by vaccination with
radiation-attenuated cercariae is an important model used in the study
of schistosome immunity. Mice receiving one exposure to attenuated
cercarial infection (singly vaccinated) develop partial immunity to a
challenge infection with viable parasites that is dependent upon the
production of IFN- from CD4+ T cells; this
immunity is reduced after CD4+ T cell
(13, 14, 15) or IFN- (3, 16) depletion.
Furthermore, numerous studies in this model identified a dominant role,
not only for IFN-, but also for IL-12 in resistance (3, 16, 17, 18). Indeed, extremely high levels of protection (exceeding
90%) were reported in mice vaccinated in the presence of the
IFN--inducing cytokine, IL-12 (18). Inducible NO
(6) and B cells (19) also participate in the
protective response. Therefore, type 1-associated immune responses
generally are thought to be responsible for the partial immunity
observed in mice receiving one exposure of the irradiated cercariae
vaccine. However, when mice receive booster exposures to the irradiated
cercariae vaccine (multiply vaccinated), they demonstrate increased
expression of type 2 cytokines (20) and develop
parasite-specific Abs that can effectively transfer resistance to naive
animals (21).
To further clarify which immune mechanisms correlate with optimal schistosome immunity, we have compared vaccine-induced immunity in cytokine-deficient animals that have been engineered to develop highly polarized type 1 (IL-10/IL-4 double-deficient mice) or type 2 (IL-10/IL-12 double-deficient mice) immune responses (22). Surprisingly, the findings of this study suggest that polarizing the immune response in the type 1 or type 2 direction provides no significant advantage for the development of optimal schistosome immunity. In fact, high levels of protection were consistently observed in IL-10-deficient animals that exhibited a completely nonpolarized or mixed Th0-like phenotype. These findings demonstrate that simultaneous expression of both type 1/type 2-associated responses can contribute to anti-schistosome immunity and suggest a novel strategy by which IL-10 antagonism might be exploited in the development of a vaccine that induces highly effective humoral and cell-mediated immunity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-10-deficient mice were generated from IL-10-deficient mice on a 129J background backcrossed 10 times to the C57BL/10 strain and obtained from Taconic Farms (Germantown, PA). Single IL-4-/--deficient (C57BL/6) and double IL-4/IL-10-deficient (IL-10, C57BL/6) mice were generated by the crossing of IL-4-/-/IL-10+/- parents and screening for IL-4-/-/IL-10+/+ or IL-4-/-/IL-10-/- offspring, respectively (22). The IL-12 knockout (KO)2 mice used to generate the IL-10/IL-12 double-deficient animals were derived from IL-12-deficient mice on a 129/sv background backcrossed five times to the C57BL/6 strain (23). Double-deficient IL-10/IL-12 mice were generated by crossing IL-10-/- males (C57BL/6) to IL-12-/- females, and the IL-12/IL-10+/- progeny were again crossed to generate IL-12-/-/IL-10+/- offspring. The IL-12-/-/IL-10+/- animals were mated to generate the double-deficient animals used. Single IL-12-/--deficient siblings generated in this crossing were also used. Male and female littermates (IL-4-/-/IL-10-/-, IL-4-/-, IL-12-/-, IL-10-/-/IL-12-/-) were used equally throughout this study, with no immunological difference observed between the sexes. C57BL/10 mice controls were from Charles River Laboratories (Raleigh, NC) or Taconic Farms. All mice were housed in a NIH American Association for the Accreditation of Laboratory Animal Care-approved animal facility in sterile filter-top cages and were maintained on water containing antibiotics until studied (Bactrin, Hoffmann-La Roche, Nutley, NJ). Cercariae of a Puerto Rican strain of Schistosoma mansoni (NMRI) were obtained from infected Biomphalaria glabrata snails (Biomedical Research Institute, Rockville, MD). Soluble worm (SWAP) Ag preparation was derived from homogenized adult parasites as previously described (24).
Immunizations and infections
Vaccination of mice with irradiated S. mansoni cercariae was performed as previously described (17). Briefly, cercariae were attenuated with 40 krad of gamma irradiation from a 137Cs source. Mice were vaccinated by immersion of their tails in water containing approximately 500 attenuated parasites for 40 min. This procedure was repeated 4 wk after the primary vaccination in those mice receiving two immunizations (twice vaccinated). In protection studies, 4 wk following the initial vaccination (once vaccinated) or the last vaccination (twice vaccinated), mice were challenged with 120 cercariae percutaneously through the tail. Six weeks following challenge infection, worms were collected by perfusion from the hepatic portal system. Worms were counted using a dissecting microscope. Control mouse groups were unvaccinated, genotype-matched siblings.
For cytokine measurements, singly vaccinated mice were challenged with 500 attenuated parasites, and on day 18 postchallenge the animals were sacrificed. Additional immunological studies with vaccinated IL-10-deficient and C57BL/10 mice involved challenging mice with 500 attenuated parasites and sacrificing animals on days 0, 12, and 21 postchallenge.
Lymphocyte culture and cytokine assays
For in vitro cytokine measurements, spleens, gluteal lymph nodes
draining the tail (TALN), and lung-associated lymph nodes (LALN;
thoracic) were removed aseptically on day 18 after challenge, and
single-cell suspensions were prepared. Spleens, TALN, or LALN were
pooled from five animals per group, and cells were plated in 24-well
tissue culture plates at a final concentration of 4 x
106 cells/ml (spleens) or 3 x
106 cells/ml (TALN or LALN) in RPMI supplemented
with 2 mM glutamine, 25 mM HEPES, 10% FCS, 50 µM 2-ME, penicillin,
and streptomycin. Cultures were incubated at 37°C in an atmosphere of
5% CO2. Cells were stimulated with Con A (5
µg/ml), SWAP (50 µg/ml), or medium alone. Blocking of the APC/T
cell interaction was achieved by incubating cultured cells in the
presence of an anti-CD4 mAb (50 µg/ml; Clone GK1.5). Supernatant
fluids were harvested at 72 h and assayed for cytokine activity.
IFN-, IL-5, and IL-10 were measured by specific two-site ELISA as
previously described (24). IL-4 levels were determined by
proliferation of CT4S cells. Cytokine levels were calculated using
standard curves constructed using recombinant murine cytokines.
Measurement of SWAP-specific Ab responses
For assessment of serum Ig during protection experiments, sera were collected at 3 wk after secondary immunization and 3 wk after challenge infection. Immulon 4 (Dynatech, Chantilly, VA) microtiter plates were coated overnight at 4°C with SWAP (2 µg in 50 µl/well) diluted in PBS. Plates were blocked with 200 µl of 5% nonfat dry milk/PBS for 2 h at 37°C. The blocking solution was aspirated, and the wells were washed six times with PBS/0.05% Tween-20 (Sigma). Individual mouse serum was serially diluted 1/100 to 1/102,500 in 1% BSA/PBS, and 50 µl was added to appropriate wells. Plates were incubated at 37°C for 90 min and then washed six times with PBS/0.05% Tween-20. Fifty microliters of isotype specific HRP-conjugated rabbit anti-mouse Abs in 1% BSA/PBS diluted at 1/1,000 (measurement of IgG1 and IgG2b; Zymed, San Francisco, CA) were added to the wells and incubated at 37°C for 2 h. Wells were again washed six times with PBS/0.05% Tween-20, 100 µl of 2,2'-azino-di(3-ethyl-benzthiazoline sulfonate) (ABTS:H2O2 substrate, Kirkegaard & Perry, Gaithersburg, MD) was added, and the reactions were developed in the dark at room temperature for 20–30 min. Absorbance at 405 nm was determined using a Vmax Kinetic Microplate Reader (Molecular Devices, Palo Alto, CA). Specific SWAP isotype titers were calculated by the product of absorbance and the reciprocal of the sera dilution from an average of two points in the linear portion of the dilution curve.
Measurement of total SWAP-specific IgG/A/M was performed from the sera of vaccinated IL-10-deficient and C57BL/10 mice on days 0, 12, and 21 postchallenge. This ELISA was performed in essentially the same way as the isotype-specific Ab ELISAs described above using a Zymed HRP-conjugated rabbit anti-mouse IgG/A/M (diluted 1/1000 in 1% BSA/PBS).
Total serum IgE Abs were quantitated by ELISA using a protocol provided by PharMingen (San Diego, CA). Briefly, plates were coated with anti-mouse IgE capture mAb from clone R35-72 in 0.1 M NaHCO3 pH 8.2, overnight at 4°C. The secondary mAb was a biotinylated anti-mouse IgE from clone R35-92, and the streptavidin-peroxidase reagent was diluted 1/1000 in 1% BSA/PBS. A purified mouse IgE from clone IgE-2 (PharMingen) was used as the control standard.
Ag-specific cellular proliferation
TALN and LALN cells from vaccinated IL-10-deficient and C57BL/10 mice were aseptically removed on days 0, 12, and 21 after challenge. Single-cell suspensions were made, cultured in 96-well tissue culture (Costar, Cambridge, MA) plates (5 x 105 cells/well), and stimulated to proliferate by addition of 50 µg/ml SWAP or 1 µg/ml Con A. One microcurie of [3H]thymidine was added to each well at 48 h, and the cells were incubated for an additional 18–24 h before harvesting. Between 66 and 72 h the cells were harvested, and the amount of Ag-specific proliferation was assessed by incorporated thymidine. Each sample was assayed in duplicate.
Frequency of cytokine-producing cells
The frequency of IFN-- and IL-4-producing cells was
determined by ELISPOT analysis as previously described
(25). Briefly, Immulon 4 microtiter plates (Dynatech) were
coated with rat anti-mouse IL-4 (Endogen, Boston, MA) or rat
anti-mouse IFN- (BioSource, Camarillo, CA) in 50 µl of PBS
overnight at 4°C. The plates were subsequently blocked by addition of
200 µl of 5% BSA/PBS overnight at 4°C. The blocking solution was
aspirated, and the wells were washed six times with PBS/0.05%
Tween-20. TALN and LALN cells from vaccinated IL-10-deficient and
C57BL/10 mice were plated at 2 x 105
cells/well and at 2-fold dilutions up to 5 x
104 cells/well. Cultures were stimulated by
addition of 50 µg/ml SWAP. After a 24-h incubation at 37°C in 5%
CO2, the plates were washed and incubated for
2 h at room temperature with 50 µl/well of biotinylated
secondary Abs (rat anti-mouse IL-4, no. 18042D; rat anti-mouse
IFN-, no. 18112D; PharMingen) diluted 1/1000 in 5% FCS/PBS/0.05%
Tween-20. After secondary Ab incubation, the plates were again washed,
and incubated at room temperature for 1 h with 50 µl of a 1/5000
dilution (5% FCS/PBS/0.05% Tween-20) of streptavidin-alkaline
phosphatase (PharMingen). Finally, the plates were washed, and 175 µl
of a 4/1 solution of 5-bromo-4-chloro-3-indolyl-phosphate (BCIP)
phosphatase substrate (; Sigma, St. Louis. MO) and low EEO agarose
(Sigma) was added at 56°C. The blue spots were allowed to develop
overnight, and individual cytokine-secreting cells were counted using a
x10 objective on a Zeiss inverted microscope (Zeiss, New York,
NY).
Histopathology
The degree of cellular inflammation associated with vascular and bronchial structures of the lung was examined in Giemsa-stained tissue sections. Essentially, two lung lobes were inflated with and fixed in Bouin-Hollande solution, and histologic sections were processed and stained with Giemsa (Histo-Path of America, Clinton, MD). The degree of inflammation was estimated using an arbitrary scale, in which 1 represents minimal inflammation, and 6 represents maximal inflammation.
Peritoneal lavage and in vitro larvicidal assay
Peritoneal lavage cells were isolated in DMEM from immunized IL-10-deficient and C57BL/10 mice injected 18–20 h previously with 250 µg of SWAP in 0.5 ml of PBS. Three-hour mechanically transformed schistosomula were prepared as previously described (20). Lavage cells were incubated at 37°C with schistosomula at a cell:target ratio of 104:1 in DMEM containing 4.5 mg/ml glucose, 10% FCS, and antibiotics. Schistosomula viability was assessed at 40 h by the criteria of mobility and internal substructure granularity (20). NO production by lavage cells was assayed by the Greiss reaction from 100 µl of supernatant collected after 40 h of culture.
Statistics
Values for worm burdens, secreted cytokine proteins, serum ELISA data, perivascular inflammation, proliferation assays, and ELISPOTs were compared using Student’s two-tailed t test. p < 0.05 was regarded as significant. A minimum of two separate experiments was performed for all data.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To test the hypothesis that polarization of the immune response
toward the type 1 or type 2 direction may lead to differences in
vaccine-induced immunity against S. mansoni, mouse strains
displaying unique cytokine-producing profiles were vaccinated twice
with 500 attenuated cercariae. To confirm that immune polarization
occurred, vaccinated mice were analyzed for their in vitro parasite
Ag-stimulated (SWAP) cytokine responses on day 18 after the second
immunization (Fig. 1
, A and
B). Measurement of their SWAP-specific isotype profiles
before and after challenge with unattenuated parasites was also
performed to confirm immune polarization (Fig. 2, A and B). As
described previously (22), vaccinated wild-type (wt) mice
exhibited a mixed Th0-like profile of cytokine production in their
gluteal (TALN; Fig. 1A) and LALN (Fig. 1B),
although the IL-4 and IFN- responses were of greater magnitude in
the TALN cultures. IL-10-deficient animals displayed a similar mixed
IFN-/IL-4 SWAP-specific profile, although the response in general,
particularly for IFN-, was significantly increased compared with
that in wt animals. IL-4 production was more variable in the
IL-10-deficient animals, showing an average increased level in the LALN
cultures (Fig. 1B) and a similar or slightly decreased
response in TALN cultures (Fig. 1A). Nevertheless, the
overall mixed cytokine response in vaccinated IL-10-deficient mice was
identical with the pattern previously reported in the spleens and
mesenteric lymph nodes of 8-wk-infected IL-10 KO mice
(26). IL-12-deficient animals showed very little change in
their type 2-like cytokine profile compared with wt animals, while
IFN- production was decreased in both the TALN (Fig. 1A)
and LALN (Fig. 1B) cultures. IL-4-deficient mice, by
contrast, showed an increased IFN- response. Nevertheless, the
greatest increase in Ag-specific IFN- production was consistently
observed in double IL-4/IL-10-deficient animals (Fig. 1, A
and B), which when compared with wt, IL-10-deficient, or
IL-4-deficient animals also showed the greatest defect in Th2-like
cytokine production. Indeed, RT-PCR analysis confirmed no detectable
IL-5 mRNA response in the lungs of IL-4/IL-10-deficient animals while
wt, IL-10-deficient, and, to a lesser degree, IL-4-deficient animals,
all developed significant IL-5 mRNA responses (wt-137, IL-10 KO-90,
IL-4 KO-15, IL-10/IL-4 KO-0 arbitrary densitometric units; data not
shown). The double IL-12/IL-10-deficient animals compared with wt,
IL-10-deficient, or IL-12-deficient mice showed a similar and
significant increase in their IL-4 responses (Fig. 1, A and
B). Similar cytokine profiles were detected in splenocyte
cultures from these animals, and RT-PCR analysis of cytokine mRNA
expression in the lungs further confirmed that mice deficient in both
IL-4 and IL-10 showed the greatest increase in IFN- mRNA expression
(and a low Th2-type profile), while IL-12/IL-10-deficient animals
developed a maximal IL-4/IL-5 mRNA response (and low IFN- message;
data not shown). Furthermore, inclusion of anti-CD4 mAbs in
SWAP-stimulated cultures confirmed that cytokine expression in vitro
was largely attributable to a CD4+ T cell
response (Fig. 1, A and B, far right
panels).
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) in large part reflected the
cytokine-producing phenotype of the various cytokine-deficient animals.
Thus, wt animals, as reported previously (20), produced
Abs associated with type 1-associated (IgG2b) as well as type
2-associated (IgG1) immune responses. In accordance with their elevated
and mixed cytokine profiles, the IL-10-deficient animals displayed
elevated IgG2b and IgG1 titers at the postchallenge time point (Fig. 2B) and elevated IgG1 and IgE titers at the prechallenge
time point (Fig. 2A). In some cases, these differences were
not statistically different from those of wt mice. Not surprisingly,
the double IL-12/IL-10-deficient animals developed a decreased IgG2b
but similar IgE/IgG1 response compared with the IL-10-deficient mice
(Fig. 2B), confirming that a more polarized type 2 immune
response had developed in these animals. Single-IL-12-deficient animals
showed a similarly skewed profile. The IL-4/IL-10 double-deficient
animals, in contrast, routinely showed the highest IgG2b titers both
before (Fig. 2A) and after (Fig. 2B) challenge.
These mice also displayed little or no IgG1 or IgE Abs, confirming that
a highly polarized type 1/IFN--dominant cytokine response was
driving Ab production. IL-4-deficient animals displayed an Ab phenotype
similar to but of lesser magnitude than the IL-4/IL-10 double-deficient
animals, particularly at the prechallenge time point. Indeed, compared
with the other mice in this study, the highest ratio of SWAP-specific
IFN-/IL-4 and IgG2b/IgG1 was observed in mice deficient in both IL-4
and IL-10. In contrast, the IL-12/IL-10 double-deficient mice exhibited
the highest ratio between their Ag-specific IL-4/IFN- and IgG1/IgG2b
responses. Interestingly, the cytokine phenotypes were also extremely
stable among the various cytokine-deficient groups. This was determined
at the termination of the vaccine experiments (all challenged mice were
perfused at 6 wk postchallenge), when the cytokine-producing profile of
SWAP-stimulated spleen and LALN cell cultures reflected the profiles at
earlier time points (data not shown). Regardless of their cytokine/Ab production profile, mice vaccinated twice with irradiated cercariae develop highly significant, but similar, levels of immunity to a challenge infection
When the twice vaccinated mice were challenged with S.
mansoni parasites and perfused 6 wk later, no significant
difference in worm burdens or overall percentage of protection was
observed between the type 1 polarized (IL-4/IL-10-deficient, 83.2%),
type 2 polarized (IL-12/IL-10-deficient, 75.9%), or wt (84.2%) mice
(Fig. 3). Indeed, all groups of
vaccinated wt and cytokine-deficient animals showed highly significant
levels of immunity compared with their respective nonvaccinated
control group. Worm burdens were reduced between 75.9–91.7% in the
various cytokine-deficient animals, and only the IL-10-deficient group
showed a significantly increased level of immunity compared with the
vaccinated wt animals (p < 0.05). The results
obtained with IL-4-deficient animals confirmed the results reported by
King et al., who also reported no difference between multiply
vaccinated IL-4-deficient and wt mice (27). More
importantly, however, they extend these observations and demonstrate
that a similar level of protection can develop in multiply vaccinated
animals regardless of the nature of the type 1 and/or type 2 cytokine
or the Ab phenotype that dominates the response.
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The preceding results were surprising, since earlier
reports have clearly shown a requirement for IFN- (3, 16) and IL-12 (28) in the protective response
induced by vaccination with attenuated parasites. Nevertheless, these
previously published studies only examined immunity in mice vaccinated
a single time with attenuated parasites in which cellular mechanisms
are generally thought to dominate the protective response. Therefore,
in subsequent experiments we examined the once vaccinated model to
again determine whether mice exhibiting type 1 or type 2 skewed immune
responses would display different patterns of immunity. In these
experiments, mice were vaccinated with 500 attenuated cercariae and
then challenged with 120 unattenuated parasites 4–5 wk later. At 6 wk
postinfection the mice were perfused to assess their worm burdens, and
spleens were examined for their Ag-specific cytokine production
profiles. As expected, all groups again showed the predicted cytokine
response (data not shown). Nevertheless, in contrast to the twice
vaccinated studies, significant differences in protection were observed
between wt animals and several of the cytokine-deficient groups.
Indeed, all vaccinated groups except the IL-10-deficient mice showed
statistically significant differences in the number of recovered adult
worms compared with the wt animals (Fig. 4). IL-10/IL-12-, IL-12-, IL-4-, and
IL-10/IL-4-deficient mice all demonstrated slightly reduced resistance
compared with wt vaccinated mice, whereas IL-10-deficient mice again
demonstrated a slightly increased protective response (not
significant). Regardless of these differences, however, all groups
displayed a relatively high immunity, varying between 53.6–83.9%.
These findings in part agree with the recent results of Anderson
et al., who also showed that once vaccinated IL-12-deficient mice were
partially, but not completely, defective in their protective response
(28).
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Because of the surprising findings with the IL-10-deficient mice,
in subsequent studies we examined their immune phenotype in more detail
to determine the mechanisms of their protective response. In the
following experiments, wt and IL-10-deficient mice were vaccinated with
radiation-attenuated cercariae and then exposed 4 wk later to a second
dose of attenuated parasites. This approach mimicked the procedures
used in the two-vaccine experiments described above and assured that
the immune response generated against the parasites in both groups of
mice was not influenced by differences in Ag exposure, which can occur
when mice are challenged with unattenuated parasites. At all time
points (Fig. 5) IL-10-deficient mice
exhibited significantly higher titers of SWAP-specific Abs than the wt
control group.
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or IL-4
ELISPOT studies were performed to determine whether the altered
IFN- and IL-4 responses previously observed in IL-10-deficient mice
(Fig. 1) were due to increased cytokine production or were the result
of changes in the frequencies of cytokine-producing cells. TALN and
LALN cells from twice vaccinated mice were used in ELISPOT assays to
determine the frequencies of IFN-- and IL-4-producing cells on days
0, 12, and 21 after the second exposure. As shown in Fig. 6, TALN and LALN cell cultures from
IL-10-deficient mice consistently displayed a marked increase in
IFN--secreting cells compared with wt mice. This was evident
at all three time points examined. Interestingly, a higher
frequency of IL-4-secreting cells was also observed in both TALN and
LALN cultures from IL-10-deficient mice on days 0 and 21. IL-4
frequencies were not significantly different on day 12, and in
agreement with the IFN- results, day 12 appeared to be the peak time
point.
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- and IL-4-producing
cells were adjusted to take into account differences in total cell
number, the frequencies of IFN-- and IL-4-producing cells per
lymphoid organ were significantly increased at all time points in the
IL-10-deficient mice (Fig. 7).
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A) and tail (TALN, Fig. 8B), cells from IL-10-deficient vs wt mice displayed greater
proliferation in response to SWAP. Similar results were obtained with
cultures stimulated with mitogen (Con A; data not shown). The
proliferative capacities of TALN and LALN cultures in general appeared
similar in magnitude at most time points; however, the response on day
21 was stronger in the LALN and remained elevated in the
IL-10-deficient vs wt mice. This pattern might be expected, since day
21 corresponds to the time when most of the attenuated schistosomula
have reached the lung and are beginning to die (29). These
results may explain why the number of cytokine-producing cells was
increased in the vaccinated IL-10-deficient animals. In addition, an
augmented effector T cell response may, in turn, provide a mechanistic
explanation for why serum Ab titers were also elevated in the KO mice.
Interestingly, no noticeable difference was observed in the
expression of several cell surface markers between IL-10-deficient and
wt mice when FACS analysis was employed (data not shown) on cells
collected from the TALN and LALN. Cell surface markers examined
included Mac-1, CD40, CD40 ligand, CD28, B7.2, MHC II, B220, and
CD11c.
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Because it is believed that the primary site of parasite
elimination in vaccinated mice is in the lungs (30), by
mechanisms still incompletely understood, we examined the morphological
changes in the lungs of vaccinated wt and IL-10-deficient mice
following a challenge infection with attenuated parasites. At the three
time points examined, there was significantly greater
peribronchial/perivascular inflammation in the lungs of IL-10-deficient
vs wt mice (Fig. 9A). The
inflammatory reactions were characterized by a larger accumulation of
cells surrounding bronchial and vascular structures (Fig. 9B). There were also fewer infiltrating eosinophils
associated with the lung tissue of IL-10-deficient compared with wt
mice (data not shown), which may be explained by the increased IFN-
response in the KO animals (31). Since it has been
proposed that immature parasites may become trapped in pulmonary
inflammatory foci and thus are prevented from completing their onward
migration and maturation (32), the accentuated
peribronchial inflammatory reactions may provide an additional
mechanistic explanation for the excellent protective response of the
IL-10-deficient animals.
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To determine whether cells from vaccinated IL-10-deficient mice
could kill immature schistosomula, we performed an in vitro
schistosomulacidal assay. In these experiments once vaccinated wt and
IL-10-deficient mice were injected with SWAP i.p., and 18 h later
parasite Ag-elicited cells were harvested and placed in culture with
mechanically transformed schistosomula (Fig. 10). Surprisingly, SWAP-elicited cells
from IL-10-deficient mice killed schistosomula ex vivo without the need
for additional activation by IFN- (Fig. 9A). Indeed, the
degree of killing by nonstimulated (medium) IL-10-deficient cells
mirrored the levels seen with IFN--activated wt cells. The
schistosomulacidal capacity of cells from both wt and IL-10-deficient
mice was almost completely inhibited by
NG-monomethyl-L-arginine
monoacetate (L-NMMA), suggesting that NO was the
primary toxic mediator in both situations. Production of NO was
confirmed by measuring NO2 levels in the culture
supernatants (Fig. 9B). As might be predicted from the
parasite killing data, SWAP-elicited cells from IL-10-deficient mice
were producing significant levels of NO directly ex vivo, while wt
cells required the addition of IFN- to attain comparable levels.
IFN- failed to further enhance the level of killing with
IL-10-deficient cells, suggesting that the macrophages were already
fully activated. Together, these data demonstrate that SWAP-elicited
cells from vaccinated IL-10-deficient mice have an increased intrinsic
capacity to kill schistosomula in vitro, through a NO-mediated
pathway.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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According to the classic Th1/Th2 paradigm, Th1-type cytokines activate
macrophages and induce strong cell-mediated immune responses, while
Th2-type responses preferentially elicit nonphagocytic humoral defense
mechanisms (9). Because the relative contributions of
cellular and humoral anti-parasite effector mechanisms have been
under debate in schistosomiasis, the double cytokine-deficient mice
described here provided an excellent system to formally compare these
two pathways in mice that are otherwise genetically matched. In
agreement with previous pulmonary egg granuloma studies
(22), mice vaccinated with attenuated parasites developed
Ag-specific cytokine production profiles predicted from their cytokine
deficiencies (Fig. 1). Thus, IL-4/IL-10-deficient animals produced
abundant IFN- and no IL-4, while IL-12/IL-10 double-deficient
mice produced the highest amount of IL-4 but little or no IFN-.
These contrasting cytokine patterns were accompanied by markedly
different Ab isotype profiles (Fig. 2), consistent with the accepted
Th1/Th2 paradigm where IFN- triggers high IgG2 and low IgG1/IgE, and
IL-4 induces high IgG1/IgE and low IgG2 Ab titers (37).
Nevertheless, despite highly divergent cytokine and Ab profiles, the KO
mice showed no significant difference in vaccine-induced immunity,
particularly in the two-vaccination experiments in which a high level
of immunity was achieved in all groups of mice. These findings suggest
that while distinct anti-parasite effector mechanisms may be
operating in type 1 vs type 2-polarized mice, both mechanisms are
equally capable of evoking extremely high levels of protection.
Alternatively, a similar protective mechanism may function on both
poles of the immune response that is not significantly influenced by
the phenotype of the responding CD4+ T cell
population or the relative contribution of cellular vs humoral effector
mechanisms.
These findings were somewhat surprising, since numerous studies
suggested a central role for CD4+ T cells
(13, 15), IFN- (3, 16), and IL-12
(28) in the protection elicited by attenuated cercariae.
Nevertheless, these conclusions were based almost entirely on results
obtained with the one-vaccine model, where, in contrast to the
two-vaccine model, cellular, not humoral, mechanisms are believed to
play a major role in the protective response. Indeed, it has been
suggested that polarizing the immune response in favor of Th1-dependent
cell-based mechanisms might prove an effective strategy to achieve
maximal levels of immunity in mice receiving one vaccine (38, 39). Th2-type cytokines are induced by one vaccine in mice
(39) and have been hypothesized to antagonize the
Th1-dependent effector mechanisms (40). In support of
these conclusions, we observed a reduced level of protection in the
once vaccinated IL-10/IL-12- and IL-12-deficient mice (Fig. 4).
Nevertheless, in agreement with the recent published results of
Anderson et al., we still observed quite high levels of protection in
the IL-12-deficient animals (exceeding 50%) (28). Perhaps
more importantly, however, by the second immunization, there appeared
to be no significant requirement for IL-12 in the development of the
protective response (Fig. 3). Indeed, mice that developed the most type
1 polarized phenotype (IL-10/IL-4 deficient) failed to show increased
protection and, in fact, displayed reduced levels compared with wt or
single IL-10-deficient mice (Fig. 4). This result along with the
excellent protection data observed in the IL-10/IL-12-deficient mice
(type 2 polarized) suggest that IFN--mediated effector devices are
not the sole immunological mechanisms driving immunity in the
one-vaccine model. These findings suggest that the humoral arm of the
immune response may play a more significant role in protection than was
previously hypothesized. Indeed, recent studies in B cell-deficient
mice confirmed an important role for Abs, even in the one-vaccine model
(19).
Based on our findings from the KO mice (Fig. 2), we argue that the
major Ab isotype contributing to protection is probably of the IgG
class. This is in agreement with serum transfer experiments of Mangold
and Dean, who showed that the IgG class is the Ab isotype responsible
for protection in immune mice (21). IgE is believed to
play little or no role in immunity in murine schistosomiasis (2, 3), which is consistent with our observations, since highly
divergent levels of IgE were detected between the two double
cytokine-deficient groups (Fig. 2), yet they displayed similar levels
of protection (Fig. 3). Our data suggest that as long as a significant
anti-parasite IgG response is generated, the specific IgG subclass
may not be an important determinant in generating high levels of
immunity. While we have no data to support whether the IgG Abs on the
type 1 pole (IgG2) vs those on the type 2 pole (IgG1) would be more or
less protective, purified IgG1 Abs have been used to successfully
transfer protection to naive recipients (41). We are
currently planning passive transfer experiments to address this
question. The double cytokine-deficient mouse provide an ideal model to
perform such studies.
More importantly, however, these data suggest that polarizing the
immune response in the context of an anti-schistosome vaccine may
be a less important goal than was previously thought (12).
In fact, our data demonstrate that generating an immune response
intermediate between the Th1/Th2 poles may be even more
advantageous. This was most clearly observed in the vaccinated
IL-10-deficient mice that developed the most nonpolarized
cytokine-producing phenotype. These mice developed very high levels of
protection and displayed increases in both type 1 and type 2-associated
cytokine responses. Because wt mice display a similar phenotype
(Figs. 1 and 2), but of a lesser magnitude, a mixed or nonpolarized
type immune response may be a more realistic goal for an
anti-schistosome vaccine, as has recently been hypothesized
(42).
Interestingly, the IL-10-deficient mice displayed several
characteristics that would be considered ideal for a vaccine. Perhaps
most importantly, the IL-10-deficient animals responded to the
attenuated parasites with a more vigorous and rapid Ab response (Fig. 5). In addition, there was a highly significant expansion of the
Ag-specific cytokine-producing effector cell population in the
IL-10-deficient mice (Figs. 6 and 7). This expansion was characterized
by an increased frequency of both type 1 and type 2 cytokine-producing
cells, which probably explains the presence of both IgG2b and IgG1 Ab
isotypes in these animals. The cellular expansion was also accompanied
by an increased proliferative response to parasite Ags (Fig. 8),
complementing the work of Sher et al. and King et al., who showed that
IL-10 inhibits lymphocyte proliferation in murine and human
schistosomiasis (43, 44). The enhanced proliferative
response probably explains the markedly enhanced lymph node sizes of
the vaccinated IL-10-deficient animals. These observations are
potentially important for vaccine research, because they suggest a
mechanism in which IL-10 antagonism might be used to significantly
expand the CD4+ T cell memory population.
The increased perivascular inflammatory response observed in
IL-10-deficient mice might also contribute to the high levels of
protection in these animals (Fig. 9). Similar findings were also
recently reported in 8-wk-infected mice, where increased egg-induced
granuloma formation was observed in IL-10-deficient animals
(26). It has been suggested that the inflammatory
reactions that surround the lung stage schistosomula might contribute
to the trapping of parasites and thus prevent their onward migration
and maturation (32, 45). Indeed, Th1-like responses were
hypothesized to promote parasite attrition in vaccinated mice by
allowing the formation of tight inflammatory foci, which effectively
traps the lung stage parasites (46, 47). These tight
inflammatory foci potentially increase the parasite’s exposure to the
schistosomulacidal mediator NO, which is produced at maximal levels by
IFN--activated macrophages and endothelial cells (48).
Our data show that parasite Ag–elicited cells from IL-10-deficient
mice have an increased capacity to kill parasites, ex vivo, through an
NO-dependent mechanism (Fig. 10). However, it is important to note that
the inflammatory reactions that we observed in the IL-10-deficient mice
were peribronchial and perivascular and were not in all cases directly
associated with the lung stage parasites themselves. Nevertheless,
these findings support the results of Gazinnelli et al., who showed
that IL-10 could inhibit the killing of schistosomula by parasite
Ag-elicited cells (49).
Previously, our laboratory demonstrated that cellular and humoral
immune responses were increased in vaccinated mice exposed to
attenuated parasites in the presence of exogenous rIL-12
(18). Interestingly, we observed a similar situation in
the vaccinated IL-10-deficient mice, since both humoral and
cellular-dependent mechanisms were increased. However, the humoral
response in IL-12-vaccinated mice was dominated by IgG2 Abs, an Ab
isotype typically associated with polarized type 1 immune responses,
while both type 1 and type 2 Abs were observed in the IL-10-deficient
animals. This observation was surprising, since we have previously
hypothesized that the presence of type 2-associated effector mechanisms
might inhibit the protective mechanisms that operate on the type 1 pole
(39, 50). The current data show that type 1 effector
mechanisms are indeed functional in IL-10-deficient mice (Fig. 10),
which supports the conclusions of Caulada-Benedetti et al., who also
demonstrated highly effective cell-mediated anti-schistosome
effector mechanisms in the midst of a significant type 2 response
(20).
Our results suggest that a major consequence of schistosome-induced IL-10 production is to down-regulate the host’s immune response against the parasite. We recently demonstrated that IL-10 also plays an important role in down-regulating hepatic granuloma formation during the acute stage of infection (26). Thus, the induction of IL-10 by the parasite may be an important factor ensuring the survival of the parasite as well as the host, since it reduces several key anti-parasite effector mechanisms and simultaneously limits the extent of tissue pathology. Both effects would help insure a favorable host-parasite relationship. Thus, as has been suggested by Muraille and Leo (51), IL-10 appears to be a central dampening cytokine of the immune response. Moreover, these studies support the recent findings of Pearce et al., who have also suggested that generating a balance between Th1- and Th2-type cytokine expression may be a more beneficial goal for preventing severe disease in schistosomiasis (52). Additionally, results obtained from humans suggest that IL-10, in fact, may be a commonly used protective cytokine induced by many helminth parasites (44, 53, 54). Finally, these data revealed a potential strategy whereby short term IL-10 antagonism might be exploited to boost cellular and humoral protective mechanisms induced by vaccination. Nevertheless, whether IL-10 neutralization at the time of vaccination alone can duplicate the results obtained from the IL-10-deficient animals has yet to be determined. Studies with schistosome vaccine candidates have often yielded disappointing results (36); targeting IL-10 might prove an effective approach to increase the efficacy of some of the defined vaccine Ags and contribute to a highly effective vaccine for schistosomiasis.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: KO, knockout; SWAP, soluble worm Ag preparation; TALN, tail lymph nodes; LALN, lung-associated lymph nodes; ELISPOT, enzyme-linked immunospot; wt, wild type.
Received for publication February 19, 1999. Accepted for publication May 4, 1999.
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