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Previous Article | Next Article 
Infection and Immunity, December 2003, p. 6835-6843, Vol. 71, No. 12
0019-9567/03/$08.00+0 DOI: 10.1128/IAI.71.12.6835-6843.2003
Copyright © 2003, American
Society for
Microbiology. All Rights Reserved.
Specificity and Mechanism of Immunoglobulin M (IgM)- and IgG-Dependent Protective Immunity to Larval Strongyloides stercoralis in Mice
Jessica A. Ligas,1 Laura A. Kerepesi,1 Ann Marie Galioto,1 Sara Lustigman,2 Thomas J. Nolan,3 Gerhard A. Schad,3 and David Abraham1*
Department
of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson
University, Philadelphia, Pennsylvania
19107,1
The Lindsley F. Kimball
Research Institute, New York Blood Center, New York, New York
10021,2
Department of Pathobiology,
School of Veterinary Medicine, University of Pennsylvania,
Philadelphia, Pennsylvania
191043
Received 25 June 2003/
Returned for modification 17 August 2003/
Accepted 18 September 2003
 |
ABSTRACT
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|---|
Protective
immunity in mice to the infective third-stage larvae (L3) of
Strongyloides stercoralis was shown to be dependent on
immunoglobulin M (IgM), complement activation, and granulocytes. The
objectives of the present study were to determine whether IgG was also
a protective antibody isotype and to define the specificity and the
mechanism by which IgG functions. Purified IgG recovered from mice 3
weeks after a booster immunization with live L3 was shown to transfer
high levels of protective immunity to naïve mice. IgG
transferred into mice treated to block complement activation or to
eliminate granulocytes failed to kill the challenge larvae. Transfer of
immune IgG into IL-5 knockout (KO) mice, which are deficient in
eosinophils, resulted in larval attrition, while transfer into
FcR
KO mice did not result in larval killing. These findings
suggest that IgG from mice immunized with live L3 requires complement
activation and neutrophils for killing of L3 through an
antibody-dependent cellular cytotoxicity (ADCC) mechanism.
This is in contrast to the results of investigations using IgM from
mice immunized with live L3 and IgG from mice immunized with larval
antigens soluble in deoxycholate in which protective immunity was shown
to be ADCC independent. Western blot analyses with immune IgM and IgG
identified few antigens recognized by all protective antibody isotypes.
Results from immunoelectron microscopy demonstrated that the protective
antibodies bound to different regions in the L3. It was therefore
concluded that while IgM and IgG antibodies are both protective against
larval S. stercoralis, they recognize different antigens and
utilize different killing
mechanisms.
|
INTRODUCTION
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Large multicellular organisms like Strongyloides stercoralis
present a significant challenge to the immune system, and the
mechanisms by which this parasite is controlled and eliminated by the
immune response remain poorly defined. In humans with severe
strongyloidiasis, a significant decrease in the immunoglobulin M (IgM)
and IgG levels was observed compared to the levels found in people with
asymptomatic and mildly symptomatic infections. Eosinophil levels were
also lower in people with severe strongyloidiasis than in individuals
with asymptomatic or symptomatic infections. These findings suggest
that antibody and eosinophils play a role in protective immunity to
larval S. stercoralis in humans
(5). In Erythrocebus
patas monkeys and dogs infected with S. stercoralis,
elevated anti-larval IgG titers were observed; however, it was not
determined whether the elevated antibody titers were related to
decreases in parasite survival
(9,
12). Jirds can also
support the complete S. stercoralis life cycle. It was
determined that after a single primary infection, jirds eliminated the
challenge infection within 24 h via a mechanism dependent on
cell contact and a factor found in serum
(27).
To gain an
understanding of how the immune response eliminates the larval stages
of S. stercoralis, a mouse model was developed. In this model
it was demonstrated that immunization of mice with live infective
third-stage larvae (L3) induced protective immunity which resulted in
the elimination of approximately 90% of the challenge larvae
within 24 h (1).
Serum collected from mice 1 week after a booster immunization could
passively transfer immunity into naïve mice
(4). Parasite-specific
IgG1, IgM, and IgA titers were elevated in the immunized mice
(1); however, it was shown
that IgM was the only antibody isotype capable of participating in
parasite elimination at that time. IgM-mediated killing of the L3 was
dependent on cell contact, the presence of granulocytes, and complement
activation (4).
Furthermore, it was shown that IgM from immunized mice could passively
transfer immunity to naïve IL-5 knockout (KO) mice, which are
deficient in eosinophils
(20), thus suggesting
that neutrophils were the required granulocyte for the IgM-dependent
killing mechanism
(15).
IgG and not
IgM was protective against larval S. stercoralis in mice
immunized with L3 antigens solubilized in deoxycholate (DOC)
(16). This observation
suggested that IgG could function in the killing of S.
stercoralis L3 and that the development of a protective IgG
response was dependent on how the mice were immunized. IgG has been
shown to be responsible for immunity-dependent protection in other
nematode infections, including Strongyloides ratti
(25), Nematospiroides
dubius (41),
Trichinella spiralis
(3), and Brugia
pahangi (24). The
goal of the present study was therefore to determine whether and when
IgG from mice immunized with live L3 functioned as a component of the
larval killing process. In addition, comparisons were made between the
mechanisms used by IgM and IgG to kill the larvae. Finally, the
molecular and morphological targets used by IgM and IgG were
identified.
|
MATERIALS AND
METHODS
|
|---|
Animals and parasites.
BALB/cByJ mice and C57BL/6J mice 6 to
8 weeks of age were purchased from Jackson Laboratories (Bar Harbor,
Maine). IL-5 KO and FcR KO mice (both on a C57BL/6 background)
were produced in our breeding colony in the Laboratory Animal Sciences
facility at Thomas Jefferson University (Philadelphia, Penn.). IL-5 KO
mice were a generous gift from Manfred Kopf of the Basel Institute for
Immunology (20), and
FcR KO mice were a generous gift from Jeffery Ravetch of
Rockefeller University
(37). All mice were
housed in microisolator boxes (Lab Products Inc., Maywood, N.J.) and
kept in a climate-controlled environment.
S. stercoralis
L3 were harvested from the feces of an infected laboratory dog as
previously described
(33). L3 were washed five
times with a 1:1 mixture of NCTC-135 and IMDM supplemented with 100 U
of penicillin (Gibco, Grand Island, N.Y.)/ml, 0.1 mg of streptomycin
(Gibco)/ml, and 0.1 mg of gentamicin
(Gibco)/ml.
Live immunization and
challenge.
Mice were
immunized with 5,000 live L3 on day 0 and received a similar booster
immunization on day 14. Immunized mice received challenge infections of
50 S. stercoralis L3 (contained in diffusion chambers) at
various time points after the booster immunization. Diffusion chambers
were made from 14-mm-diameter Lucite rings covered with
2.0-µm-pore-size polycarbonate Isopore membranes (Millipore,
Bedford, Ma.). The rings are glued together with a 1:1 mixture of
1,2-dichloroethane (Sigma, St. Louis, Mo.) and acryloid resin (Rohm and
Haas Co., Philadelphia, Pa.). The membranes are attached to the rings
with cyanoacrylate (Super Glue, Hollis, N.Y.). The completed diffusion
chambers were then sterilized by exposure to 100% ethylene oxide
gas followed by 12 h of aeration. Diffusion chambers
containing the challenge L3 were placed in a subcutaneous pocket on the
rear flank of the mice. At 24 h later, the diffusion chambers
were recovered and larval survival was determined on the basis of
morphology and mobility of the worms. Host cells found in the diffusion
chambers were centrifuged onto slides through the use of a Cytospin 3
apparatus (Shandon, Pittsburgh, Pa.) and then stained for differential
counts with DiffQuik (Baxter Healthcare, Miami,
Fla.).
Preparation and immunization with
soluble antigen.
Mice were
immunized with larval antigens solubilized in DOC as previously
described (17). Briefly,
S. stercoralis L3 were incubated in a petri dish for 20 min
with a 1% solution of low-melt agarose (Type I-A Low EEO;
Sigma). Phosphate-buffered saline (PBS) supplemented with antibiotics
(100 U of penicillin/ml, 0.1 mg of streptomycin/ml, and 0.1 mg of
gentamicin/ml) was added, and larvae that migrated into the PBS were
harvested and stored at -80°C in 50%
glycerol-PBS. L3 were homogenized on ice in PBS supplemented
with a protease inhibitor cocktail (Sigma) and 50 mM EDTA and then
sonicated. DOC-soluble L3 antigens were obtained first by extraction
from the L3 with PBS and then by extraction of the remaining pellet
with 20 mM Tris-HCl-0.5% DOC in the presence of protease
inhibitors; the extracted antigens were then dialyzed overnight in PBS.
DOC antigens (DOC Ag) were then passed through a
0.2-µm-pore-size syringe filter, and protein concentrations
were determined with a Micro BCA protein assay (Pierce, Rockford,
Ill.).
Mice were immunized with soluble antigens through the use
of the following protocol: 2% aluminum hydroxide low-viscosity
Rehydragel (alum) (Reheis, Inc., Berkeley Heights, N.J.) was diluted
1:10 with PBS containing 25 µg of DOC Ag. Mice were injected
with 200 µl of the solution in the nape of the neck on days 0
and 14 which was followed on day 28 by a challenge infection consisting
of L3 contained within a diffusion chamber (which was recovered 4 days
later).
Serum transfers and antibody
purification.
Serum used in
transfers was obtained from mice which were immunized and challenged as
described above. The serum was collected at either 3 or 5 weeks after a
booster immunization from mice immunized with live L3 and 18 days after
a booster immunization from mice immunized with the DOC Ag. Serum
(recovered from blood after exsanguination of mice anesthetized with
isoflurane) was filter sterilized with a 0.2-µm-pore-size
syringe filter and stored at -80°C.
Serum was
passed over a protein G-Sepharose column (Pharmacia, Peapack, N.J.) to
purify the IgG fraction. The flowthrough volume containing IgA, IgE,
and IgM was then passed over IgA and IgE affinity columns, leaving only
IgM in the flowthrough solution. Antibody fractions were eluted from
the column with 0.5 M acetic acid (pH 3.0), neutralized with saturated
Tris-HCl (pH 9.8), dialyzed overnight in PBS, and concentrated with an
Ultrafree-15 centrifugal filter device (Millipore). Total IgM and IgG
concentrations in the serum were determined by enzyme-linked
immunosorbent assay (as described below) before and after
purification.
Passive transfer of the purified antibody and whole
immune sera was done by injection either directly into the diffusion
chamber or into the subcutaneous pocket around the implanted diffusion
chamber at the time of challenge. No differences were seen between the
results obtained with each method. The amount of purified antibody
transferred into the mice was equal to the amount of antibody found in
100 µl of either unfractionated naive or immune serum. The
antibody was administered in 200 µl of PBS. All serum transfer
experiments were terminated at 24 h
postchallenge.
Cell and complement
depletion.
Depletion of
granulocytes was accomplished using the monoclonal antibody (MAb)
RB6-8C5 (18). Mice were
given an intraperitoneal injection of 0.5 mg of antibody 3 days before
the challenge as well as at the time of challenge. An isotype control
antibody was not included in these studies, because multiple previous
experiments have shown that these antibodies have no effect on parasite
viability (31),
(32). Granulocyte
depletion was verified with blood smears taken at the time of recovery
and from analysis of the fluid in the diffusion chambers.
Cobra
venom factor (CVF) (Sigma) (used for depletion of complement) was
injected (400 µg per kg of body weight) intraperitoneally into
mice at 24 h prior to and at the time of challenge
(14).
Enzyme-linked
immunosorbent assay.
Maxisorp 96-well plates (Nunc Inc.,
Naperville, Il.) were coated with 2 µg of rat anti-mouse IgG
(Sigma)/ml or 5 µg of rat anti-mouse IgM (Pharmingen, San
Diego, Calif.)/ml overnight at 4°C. Plates were then blocked
with borate buffer solution (0.17 M boric acid, 0.12 M NaCl,
0.05% Tween 20, 0.025% bovine serum albumin, 1 mM EDTA,
pH 8.2) at room temperature (RT) for 30 min. The plates were washed
with distilled water, and the samples and appropriate standards were
serially diluted in borate buffer solution and then incubated for
2 h at 37°C. The plates were washed with distilled
water and then incubated with biotinylated goat anti-mouse IgG or IgM
(Pharmingen) for 1 h at RT. The plates were
washed, and ExtrAvidin peroxidase (Sigma) was added for 30 min at RT.
After the final washing, the peroxidase substrate ABTS
[2,2'-azino-di-(3-ethylbenzthiazoline-6-sulfonate)]
(Kirkegaard & Perry Laboratories, Gaithersburg, Md.) was added.
ABTS color reactions were measured at 410 nm on an MR5000 microplate
reader (Dynatech Laboratories, Chantilly,
Va.).
Western blotting.
DOC-soluble L3 antigens (100
µg) were separated in a sodium dodecyl sulfate-8 to
16% polyacrylamide electrophoresis slab gel on a
Mini-PROTEAN 3 apparatus (Bio-Rad Corporation, Hercules, Calif.).
Electrophoresis was run at 30 mA for 2 h, after which
proteins were transferred to a nitrocellulose membrane (Bio-Rad)
through the use of a Trans-Blot apparatus (Bio-Rad) at 100 V for
1 h at 4°C. The nitrocellulose membranes were then
blocked with 5% Carnation brand nonfat dry instant milk
(Nestlé Food, Glendale, Calif.). The membranes were washed in
PBS-0.05% Tween 20, cut into strips, and then incubated
in a 1:1000 dilution of whole serum for 1 h at RT. The
membrane strips were washed in PBS-0.05% Tween 20 and
then treated with anti-mouse IgG horseradish peroxidase-conjugated
antibody (Pharmingen) or anti-mouse IgM F(ab')2
horseradish peroxidase-conjugated antibody (ICN/Cappell, Aurora, Ohio)
for 45 min at RT and washed. Western blots were processed using
enhanced chemiluminescence (NEN Life Sciences Products Inc., Boston,
Mass.) and then developed with Biomax ML film (Eastman Kodak,
Rochester, N.Y.).
Electron
microscopy.
L3 were fixed
for 30 min in 0.25% glutaraldehyde in 0.1 M phosphate buffer (pH
7.4) containing 1% sucrose, embedded, and then processed for
immunoelectron microscopy. Thin sections of embedded worms were
incubated with purified IgM (diluted 1:5) or IgG (diluted 1:100) from
control and immunized mice followed by incubation with a suspension of
15-nm-diameter gold particles coated with protein A, as previously
described (19,
21,
22).
Statistics.
All experiments consisted of at least
five mice per group; experiments were performed at least twice, and
consistent results were obtained from the repeated experiments. Data
presented are from all of the performed experiments. Statistical
analysis was determined by multifactorial analysis of
variance; probability values of less than 0.05 were considered
significant.
|
RESULTS
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Passive
transfer of immunity to mice with serum and antibody.
Serum and antibody transfers were
performed to determine when after immunization with live L3, IgG became
protective against larval S. stercoralis in mice. IgG and IgM
antibody fractions were purified from whole serum at 3 and 5 weeks
after the booster immunization. Transfer of IgG, IgM, and whole serum
was by subcutaneous injection of antibody around the implanted
diffusion chamber. Serum collected at 3 weeks after the booster
immunization caused a more than 80% reduction in the number of
live larvae recovered. Larval killing at that time was mediated by
whole immune serum, purified IgM, and IgG (Fig.
1A). Transfer of whole serum collected at 5 weeks after the booster
immunization caused a significant reduction in the survival of the
challenge infection, as did transfer of purified IgG and IgM (Fig.
1B). As the level of
protection attributed to IgG was higher at 3 weeks after the booster
immunization than at 5 weeks, IgG collected 3 weeks after the booster
immunization was used in the subsequent
studies.
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FIG. 1. Mean
percentage of live larval recovery from mice that received the transfer
of whole immune sera, purified IgM, and purified IgG recovered 3 weeks
(A) and 5 weeks (B) after booster immunization with
live L3. The data represent the means and standard deviations of the
results for 10 animals per group. The asterisks indicate statistical
differences (P  0.05) from control
values.
|
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The mechanism by which IgG from
mice immunized with live L3 elicits larval killing.
To test whether IgG requires complement
activation to kill the larvae, CVF was used to block the complement
cascade by the depletion of the C3 component. The ability of immune IgG
to kill the parasites was significantly diminished when complement
activation was blocked (Fig.
2). Cell recruitment into diffusion chambers did not alter either in the
number of cells or in the types of cells after CVF treatment.
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FIG. 2. Effect
of CVF treatment of mice that have received transfer of naïve
and immune IgG on the survival of challenge larvae. The data represent
the means and standard deviations of the results for 10 animals per
group. The asterisks indicate statistical differences (P
0.05) from control
values.
|
|
The
role of cells in the IgG killing process was initially studied by using
the MAb RB6-8C5 to deplete both eosinophils and neutrophils. Treatment
of mice with RB6-8C5 inhibited larval killing after passive transfer of
immune IgG (Fig.
3). Differential analysis of the cells in the diffusion chambers in the
RB6-8C5-treated mice showed significantly decreased levels of
neutrophils. Equally low levels of eosinophils were found in both the
MAb-treated mice and nontreated controls (data not shown). Immune IgG
was then transferred into IL-5 KO mice, which are deficient
in eosinophils. Purified immune IgG transferred protective immunity to
the IL-5 KO mice (Fig.
4), thus suggesting that eosinophils are not required for larval killing by
IgG but rather that neutrophils are the required
cells.
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FIG. 3. Effect
of granulocyte depletion with MAb RB6-8C5 on immunity transferred with
purified immune IgG. The data represent the means and standard
deviations of the results for 10 animals per group. The asterisks
indicate statistical differences (P 0.05) from
control
values.
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FIG. 4. Transfer
of naïve and immune IgG into wild-type C57BL/6J mice and IL-5
KO mice to determine whether eosinophils are required for protective
immunity. The data represent the means and standard deviations of the
results for 10 animals per group. The asterisks indicate statistical
differences (P 0.05) from control
values.
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|
FcR KO mice are missing the -chain
required for FcRI, FcRIII, and Fc
R receptor
surface expression (37).
Cells in these mice are therefore unable to bind to IgG and IgE.
FcR KO mice were immunized with L3 to determine whether
protective immunity would be different from that seen with wild-type
mice. Immunization of FcR KO mice with challenge either 1 week
after the booster immunization (when IgM was the functioning isotype)
(4) or 3 weeks after the
booster immunization (when IgM and IgG were the functioning isotypes)
resulted in protective immunity (Fig.
5). However, transfer of purified IgG (recovered 3 weeks after booster
immunization with live L3) into FcR KO mice failed to transfer
immunity (Fig.
6A). This was in marked contrast to the results seen with IgG recovered from
mice immunized with DOC Ag, which could transfer immunity to the
FcR KO mice (Fig.
6B). Diffusion chambers
implanted in wild-type and FcR KO mice that received either no
IgG, control IgG, or IgG from animals immunized with live or
solubilized L3 all contained equal numbers of infiltrating neutrophils.
The numbers of infiltrating neutrophils ranged from 4 x
106 to 8 x 106, with no statistical
differences between groups.
Morphological
and molecular targets of protective IgM and IgG.
Western blotting was performed to
determine what antigens the protective IgM and IgG recognized in
DOC-solubilized S. stercoralis larvae. Analysis of the Western
blots showed that both the IgM and the IgG recognized approximately 20
antigens. Of these 20 antigens, only 8 were recognized by both IgM and
IgG (Fig.
7). IgM and IgG from naïve mice did not bind to any of the
solubilized S. stercoralis larval antigens (data not
shown).
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FIG. 7. Antigen
recognition in Western blot analysis of protective IgM and
IgG (recovered from mice 3 weeks after immunization with live L3) from
solubilized S. stercoralis larval antigens. The bars represent
bands recognized by both IgM and
IgG.
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Immunoelectron microscopy was performed to determine
where protective IgM (recovered 1 week after booster immunization) and
IgG (recovered 3 weeks after booster immunization) bound to the L3. It
was determined that IgM bound to the surface of the cuticle, basal
cuticle-hypodermis, coelomic cavity, and glandular
esophagus (Fig.
8), whereas the IgG bound only to the basal cuticle-hypodermis and the
coelomic cavity (Fig.
9).
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FIG. 8. Immunoelectron
microscopy performed to determine where protective IgM recovered 1 week
after booster immunization bound to the larvae of S.
stercoralis. Cu, cuticle; g, glandular esophagus granules. The
thin arrows indicate labeling in the basal cuticle-hypodermis area, the
thick arrows indicate labeling on the surface of the cuticle, and the
arrowheads indicate labeling in the coelomic cavity. Bar, 250
nm.
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FIG. 9. Immunoelectron
microscopy performed to determine where protective IgG, recovered at 3
weeks after booster immunization, bound to the larvae of S.
stercoralis. Cu, cuticle; g, glandular esophagus granules. The
thin arrows indicate labeling in the basal cuticle-hypodermis area; the
arrowheads indicate labeling in the coelomic cavity. Bar, 250
nm.
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DISCUSSION
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The first objective of this
study was to determine whether IgG from mice immunized with live L3 had
the ability to transfer protection to challenge infections in
naïve mice. Previous studies have shown that IgM was the only
protective antibody isotype in sera obtained 1 week after booster
immunization (4). IgG was
purified from the sera of mice immunized with live worms at 3 or 5
weeks after booster immunization to determine whether allowing the
immune response to mature would result in a protective IgG response. It
was determined that both IgM and IgG were protective at 3 and 5 weeks
after booster immunization and that the levels of protection produced
by the two antibody isotypes were equal at both time points. Purified
IgM and IgG from 3 weeks after booster immunization could individually
transfer immunity at the same high level as the whole serum. However,
this result was not seen at 5 weeks after the booster immunization,
whole serum being more protective than IgM or IgG alone. It is possible
that at 5 weeks after booster immunization, effective protective
immunity requires both IgM and IgG; alternatively, another isotype or
serum component may be required. IgG recovered from mice 3 weeks after
booster immunization with live L3 was capable of contributing to an
effective protective immune response against larval S.
stercoralis and was studied in the ensuing experiments.
Mice
were treated with CVF to eliminate complement activation and thereby
determine whether activation of the complement cascade is required for
IgG to function in killing the larvae. It was determined that IgG could
not transfer immunity when the recipient mice were treated to block
complement activation. Both the alternative and the classical pathways
of complement activation have been shown to occur on the surface S.
stercoralis L3 in vitro
(7), and it was previously
shown that complement activation was also required for IgM to effect
larval killing in mice
(4). In vitro studies have
also shown a requirement for complement activation in killing the
helminths T. spiralis
(38), Loa loa
(30), Angiostrongylus
cantonensis (34),
Nippostrongylus brasiliensis
(8), and Schistosoma
mansoni (2) and in
the control of S. mansoni in vivo
(10,
39).
In previous
studies it was shown that contact between host cells and the parasites
was required for IgM to kill the larvae of S. stercoralis.
Furthermore, depletion of granulocytes with the MAb RB6-8C5 also
eliminated IgM-dependent immunity
(4). Granulocytes have
also been shown to be important in controlling S. ratti
infections (40). Evidence
that neutrophils were the effector cells against larval S.
stercoralis was derived from experiments in which IgM was
transferred into IL-5 KO mice. These mice had virtually no eosinophil
recruitment into the parasite's microenvironment and yet the
larvae were still effectively killed, thus suggesting that neutrophils
can function as the effector cells in IgM-dependent immunity
(15). Results from the
present study regarding the mechanism of IgG-dependent immunity
mirrored the results obtained for IgM. Specifically, granulocytes were
required, and judging on the basis of the results obtained in studies
with IgG transferred into IL-5 KO mice, it appears that neutrophils are
the effector cells in IgG-dependent immunity as well. A role for
neutrophils in killing N. dubius larvae in vivo in
collaboration with IgG has been previously reported
(29); in vitro results
with A. cantonensis also confirm the function of IgG-dependent
and complement-dependent neutrophil cytotoxicity to the infective
larvae
(34).
FcR
KO mice were immunized with S. stercoralis L3 to investigate
whether the IgM and IgG protection functioned by an antibody-dependent
cellular cytotoxicity (ADCC) mechanism. These animals lack Fc
receptors; consequently, cells cannot bind to IgG, thereby impairing
cell-mediated ADCC (37).
Fc receptor-mediated mechanisms have been shown to be crucial in
controlling primary Strongyloides venezuelensis infections
(28) and resistance to
the microfilariae of Brugia malayi in mice
(11). Immunized
FcR KO mice that received challenge infections either 1 or 3
weeks after a booster immunization developed protective immunity. These
results can be interpreted to mean either that ADCC dependent on the
presence of IgG is not required for protective immunity or that the
protective IgM found in the serum at both time periods was sufficient
for protective immunity in the absence of a functional IgG
response.
To test the second hypothesis, IgG from mice
immunized with live L3 was transferred into FcR KO mice; it
was shown that IgG could not transfer immunity to the FcR KO
mice. FcR KO mice have also been shown to have a deficit in
neutrophil recruitment into sites of inflammation
(6,
13,
14). In the present
study, neutrophils were recruited in equal numbers in the wild-type and
FcR KO mice receiving transferred IgG. Therefore, the deficit
in protective immunity after passive transfer of IgG from mice
immunized with live L3 was not caused by an absence of effector cells
but rather suggests a dependency of IgG on ADCC as the mechanism of
larval killing. It is of interest that IgG recovered from mice
immunized with DOC Ag could transfer immunity to the FcR KO
mice. The difference in the modes of action for the two protective IgGs
may be explained by their different antigenic binding sites, with IgG
from mice immunized with live L3 binding to the cuticle while IgG from
mice immunized with DOC Ag bound only to internal tissues
(17). These experiments
therefore show that IgM from mice immunized with live L3 and IgG from
mice immunized with DOC Ag kill the L3 in an ADCC-independent
mechanism. This result is in contrast to that seen with IgG from mice
immunized with live L3, which appears to function through a classical
ADCC mechanism.
Western blot analyses performed with the
protective IgM and IgG from mice immunized with live L3 showed that IgM
and IgG each recognized approximately 20 antigens. DOC Ag was used in
these analyses, since it was previously demonstrated that lymphocytes
from mice immunized with live L3 proliferated and produced IL-5 in
response to antigens found in DOC Ag and not to antigens soluble in PBS
(17). Of the 20
recognized antigens, only 8 were recognized by both IgM and IgG.
Immunoelectron microscopy was performed to determine where the
protective IgM and IgG bound to the larvae. It was determined that IgM
bound to the surface of the cuticle, the basal cuticle-hypodermis, the
coelomic cavity, and the glandular esophagus, whereas IgG bound only to
the basal cuticle-hypodermis and the coelomic cavity. Previous studies
using fixed intact L3 in indirect fluorescent antibody assays have
shown that IgM was the only antibody isotype found to bind to the
surface of the larvae (4),
thus corroborating the results of the present study.
The antigens
jointly recognized by IgM and IgG may be in the basal
cuticle-hypodermis and the coelomic cavity, while the antigens uniquely
recognized by IgM may be on the surface of the cuticle and in the
glandular esophagus. The IgM response which temporally occurs first may
be directed at the antigens initially seen by the host, surface
antigens, and antigens secreted by the glandular esophagus during the
larval migration process. In previous studies it was shown that the
protective IgG from mice immunized with DOC Ag recognized seven
antigens (17), four of
which were also apparently recognized (as determined on the basis of
their molecular weights) by the protective IgG from mice immunized with
live L3. IgG from mice immunized with DOC Ag was found to bind to the
muscles and nerve cord
(17). The antigens
recognized by IgG from mice immunized with DOC Ag may represent
proteins which are normally not seen in the immune response. These
antigens may become evident only after the larvae are disrupted and
solubilized. Antigens normally concealed from the host were shown to be
effective immunogens against the nematodes Haemonchus
contortus and Dirofilaria immitis
(23,
35,
36). Therefore, it
appears that IgG from mice immunized with DOC Ag and IgG from mice
immunized with live L3 do not bind to the cuticle of the larvae and
differ in the internal organs of the L3 to which they bind. Although
they share reactivity with many antigens, the mechanisms by which they
kill the larvae are different.
As the immune response to larval
S. stercoralis matures in mice, a protective IgG response
develops which requires complement activation, neutrophils, and
FcR to function. It appears that killing occurs via an ADCC
mechanism whereby IgG binds to the larvae and the neutrophils then bind
to the Fc portion of the antibody. To kill the larvae, the neutrophils
may then release one of the many toxic molecules which they contain
(26). The hypothesis that
IgG killing of the larvae functions through a classical ADCC mechanism
is difficult to reconcile with the antibody localization studies, since
IgG was found to bind to various internal organs and not to the surface
of the L3. It is possible that the surface antigens to which IgG binds
are not seen in the immunoelectron micrographs because they are
secreted antigens that are loosely bound to the larval surface or
because they represent antigens stripped during the fixation
process.
It is clear from these studies that in the killing of
larval S. stercoralis, IgM and IgG from mice immunized with
live L3 and IgG from mice immunized with DOC Ag function by using
different target antigens and different mechanisms. This finding
confirms the observation made with other pathogens that the immune
response is pluripotent in its ability to kill pathogens. It is
therefore proposed that highly effective vaccines against S.
stercoralis infections could be developed that are composed of
multiple antigens inducing different types of protective immune
responses. The synergy of the different types of protective immune
responses would result in a vaccine that could efficiently kill the
pathogen regardless of the parasite strain and host genetic
background.
|
ACKNOWLEDGMENTS
|
|---|
We acknowledge support from
NIH grants AI 47189 and AI 22662.
We also thank Ofra Leon and
Shalom Leon for expert technical assistance. We give special thanks to
Yelena Oskov for assistance with the electron
microscopy.
|
FOOTNOTES
|
|---|
* Corresponding
author. Mailing address: Department of Microbiology and Immunology,
Thomas Jefferson University, 233 South 10th St., Philadelphia, PA
19107. Phone: (215) 503-8917. Fax: (215) 923-9248. E-mail:
David.Abraham{at}jefferson.edu. 
Editor:
W. A. Petri, Jr.
|
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Infection and Immunity, December 2003, p. 6835-6843, Vol. 71, No. 12
0019-9567/03/$08.00+0 DOI: 10.1128/IAI.71.12.6835-6843.2003
Copyright © 2003, American
Society for
Microbiology. All Rights Reserved.
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Top
Abstract
Introduction
