Parasite Immunology, 1997: 19: 29–39
IL-12 eliminates the Th-2 dependent protective immune response of
mice to larval Strongyloides stercoralis
H.L.ROTMAN 1 , S.SCHNYDER-CANDRIAN 1 , P.SCOTT 2 , T.J.NOLAN 2 , G.A.SCHAD 2 & D.ABRAHAM 1
1
Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, PA 19107, USA
2
Department of Pathobiology, University of Pennsylvania, Philadelphia, PA 19104, USA
SUMMARY
The goal of the present study was to determine if immune-
mediated killing of S. stercoralis L3 in mice could be
modulated by shifting from a Th-2 to a Th-1 type immune
response. L3 killing in immunized mice was ablated in
CD4þ T cell-depleted animals, but not in CD8þ T cell-
depleted or b2-microglobulin-deficient mice. Treatment of
immunized mice with IL-4 or IL-5 neutralizing MoAb sig-
nificantly reduced the protective effects of vaccination
against S. stercoralis, while protective immunity was unim-
paired in IFN-g knockout mice. Recombinant IL-12 was
administered to infected mice to switch the immune response
from a Th-2 to a Th-1 type response. Protective immunity
was ablated in immunized mice that received IL-12 therapy.
Eosinophil numbers, eosinophil peroxidase levels, and
parasite-specific IgG1 levels were lowered in IL-12 treated
immunized animals, and parasite-specific IgG2a levels were
increased in these animals. The data indicate that eosino-
phils are important as mediators of larval killing, and that
the establishment of Th-2 type immunity results in killing of
infective S. stercoralis L3, while a shift to Th-1 type
immunity abrogates protective responses.
Keywords IL-12, Strongyloides stercoralis, nematode,
Th-2 immunity
Correspondence: D.Abraham
Received: 28 August 1996
Accepted for publication: 17 October 1996
q 1997 Blackwell Science Ltd
INTRODUCTION
Strongyloidiasis, a major worldwide human intestinal infec-
tion, is caused by the nematode Strongyloides stercoralis.
Natural hosts for this organism include humans, nonhuman
primates, and dogs (Schad 1989). Chronic infections are
found in many patients and are thought to be maintained by
an autoinfection process. Massive and uncontrolled auto-
infection may result in hyperinfection, which can be fatal in
immunocompromised individuals (Genta 1992).
Mice have been used as a means for studying the
systematic phase of S. stercoralis infection, as infective
larvae migrate to the tissues but do not develop further
(Grove, Northern & Heenan 1986). Larvae are found in the
muscles for nine days in S. stercoralis-infected mice before
disappearing spontaneously. This disappearance is asso-
ciated with inflammatory reactions around dying larvae,
predominantly eosinophilic and histiocytic (Grove, Northern
& Heenan 1986). Using live third-stage larvae (L3) for
immunization, protective immunity to L3 has been demon-
strated in BALB/cByJ mice (Abraham et al. 1995). Diffusion
chambers have been used to assess larval survival in immune
mice, and the only cell type significantly increased in
immunized mice in diffusion chamber fluid and peripheral
blood was the eosinophil (Abraham et al. 1995). In mice
treated with MoAb to eliminate eosinophils, protective
immunity to L3 contained within diffusion chambers is
significantly reduced (Rotman et al. 1996). Human eosinophil
granule products are toxic for host-adapted L3, indicating that
larvae are susceptible to eosinophil-mediated destruction
(Rotman et al. 1996). Humoral immune components also
play an essential role in S. stercoralis larval killing. IgM is
necessary for protection against L3, in that IgM, and no other
isotype, purified from immune sera mediates larval killing in
naive mice (Brigandi et al., 1996). Only IgM binds to the
surface of S. stercoralis L3 in immune serum, while no
antibody isotypes from control serum bind significantly to
the surface of L3 (Abraham et al. 1995). Complement was
also found to be essential in L3 killing as shown in cobra
29
H.L.Rotman et al.
venom factor-treated mice, where no larval killing took place
(Brigandi et al. 1996).
The importance of Th-2 cytokines such as IL-4 and IL-5
in protective immunity to challenge infection and resistance
to primary infection has been demonstrated in several
nematode infections, including Strongyloides venezuelensis
(Korenaga et al. 1991), Angiostrongylus cantonensis
(Sasaki et al. 1993), Onchocerca volvulus (Lange et al.
1994), Heligmosomoides polygyrus (Urban et al. 1991), and
Trichuris muris (Else & Grencis 1991). IL-5 is essential for
eosinophil generation and development (Coffman et al.
1989), and IL-4 plays a role in several processes (Tepper
1994), including isotype switching to IgG1 and IgE (Snap-
per, Finkelman & Paul 1988), the adhesion and migration of
eosinophils across vascular membranes (Moser, Fehr &
Bruijnzeel 1992), as well as many other effects on B and
T cells, macrophages, and mast cells (Howard et al. 1982,
Snapper & Paul 1987, Trenn et al. 1988, Fernandez-Botran
et al. 1988, Mosmann et al. 1986, McInnes & Rennick 1988,
Crawford et al. 1987). IL-12, a heterodimeric cytokine
produced primarily by macrophages and other accessory
cell types, can modulate Th-2 responses, favouring devel-
opment and activation of Th-1 cells (Manetti et al. 1993,
Hsieh et al. 1993, Afonso et al. 1994).
The objective of the present study was to determine how
the immune response to S. stercoralis infection in mice
could be regulated. Immunized mice depleted of CD4þ T
cells were unable to generate protective immunity to L3,
while depletion of CD8þ T cells had no negative effect on
L3 killing. Inactivation of the Th-2 cytokines IL-4 or IL-5
resulted in significant reductions in immunity to third-stage
larvae, while depletion of the Th-1 cytokine IFN-g had no
effect on protective immunity. Mice treated with recombi-
nant IL-12 to shift the immune response to a Th-1-type
response lost their ability to kill challenge L3. Taken
together, these findings indicate that Th-2-type responses
are essential for the immune-mediated killing of S. stercor-
alis L3 to take place.
MATERIALS AND METHODS
Mice
Male BALB/cByJ, C57BL/6J, b2 -microglobulin-deficient
(C57BL/6 background) mice, and IFN-g knockout mice
(BALB/c background) were purchased from Jackson
Laboratories, Bar Harbor, ME, USA. The mice ranged
from six to 12 weeks of age at the beginning of all
experiments. Animals were housed in Micro-Isolator
boxes (Lab Products Inc., Maywood, NJ, USA) and were
fed Autoclaveable Laboratory Rodent Chow (Ralston
Purina, St. Louis, MO, USA), and sterilized acid water
30
Parasite Immunology
(pH 2.7) ad libitum. The animal housing room was tempera-
ture, humidity, and light cycle controlled.
Parasites
S. stercoralis L3 were harvested from the fresh stools of a
laboratory dog-infected with the parasite according to
methods previously described (Schad, Hellman & Muncey
1984). In brief, faeces were mixed with a small amount of
water and charcoal and placed in a humidified petri dish at
278C for 5–7 days. The L3 were cleaned of faecal debris by
mixing with a liquid low-gelling-temperature agar solution
(with a final concentration of 1%) (Sigma Chemical Co., St.
Louis, MO, USA) and allowing the L3 to migrate to the
surface of the solidified mixture. The larvae were washed
five times by centrifugation and resuspended in a 1 : 1
mixture of Iscove’s Modified Dulbecco’s Medium
(IMDM) and NCTC-135 (Sigma) with 10 000 U Penicillin,
10 mg Streptomycin, and 10 mg Gentamicin (Sigma) per
100 ml.
Immunization and challenge
Five thousand live L3 in IMDM/NCTC medium plus
antibodies were injected subcutaneously posterior to the
cervical area of each mouse. Booster immunizations of
5000 L3 were administered two weeks after the initial
immunization (Abraham et al. 1995).
Diffusion chambers were constructed as previously
described (Abraham et al. 1995). Briefly, 14-mm Lucite
rings were covered with 2.0 mm pore-sized polycarbonate
Isopore membranes (Millipore, Bedford, MA, USA) or
0.1 mm pore-sized Durapore membranes (Millipore).
Cement for binding the Lucite rings together and to the
Durapore membranes consisted of equal parts of 1,2-
dichloroethane (Sigma) and acryloid resin (Rohm and
Haas, Philadelphia, PA, USA). Cyanoacrylate adhesive
(Super Glue Corp., Hollis, NY, USA) was used for binding
the Isopore membranes to the Lucite rings. The diffusion
chambers were sterilized by exposure to ethylene oxide gas.
For challenge infections, 50 S. stercoralis L3, in IMDM/
NCTC medium with antibiotics, as above, were inoculated
into each diffusion chamber.
Immunized mice received challenge infections 21 days
after the initial immunization. Control mice received
diffusion chambers at the same time as immunized
mice, but had not been previously imunized with larvae.
Animals were anaesthetized with intraperitoneal injections
of a mixture of 10 mg/ml ketamine hydrochloride (Park-
Advise, Morris Plains, NJ, USA) and 0.02 mg/ml acepro-
mazine maleate (Aveco, Fort Dodge, IA, USA). The
anaesthetic solution was administered at 0.1 ml/10 g body
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Volume 19, Number 1, January 1997
weight. A single diffusion chamber was inserted into the
subcutaneous pocket formed in the rear flank of each
animal.
Recovery and analysis of diffusion chamber contents
All diffusion chambers were recovered one day after
implantation. Mice were anaesthetized with Methoxyflurane
(Pitman-Moore, Inc., Mundelein, IL, USA) and sacrificed
by exsanguination. Blood for cell differential counts was
collected at the time of death. Diffusion chambers were
removed from the mice and the total number of cells in the
diffusion chamber fluid was determined. This fluid was
centrifuged onto slides using a Cytospin 3 (Shandon Inc.,
Pittsburg, PA, USA). Slides were stained with DiffQuik
(Baxter Healthcare Corp., Dade Div., Miami, FL, USA).
Larvae recovered from diffusion chambers were considered
live if they were motile.
Cytokine depletion
The following rat MoAb were used in vivo cytokine deple-
tion: anti-mouse IL-5 (TRFK-5) (Coffman et al. 1989) and
anti-mouse IL-4 (11B11) (Ohara & Paul 1985) (cell lines
contributed by Dr R.L.Coffman, DNAX Corp, Palo Alto,
CA, USA). Rat IgG (Sigma Chemical Co., St. Louis, MO,
USA) was used as the IgG control antibody. Mice that were
challenged with L3 in diffusion chambers were injected
twice i.p. with each MoAb. The first dose was given seven
days before challenge, and the second dose at the time of
challenge. Doses of MoAb were as follows: TRFK-5, 1 mg/
mouse/dose. The initial dose of 11B11 and the rat control
Abs was 10 mg/mouse/dose, and the remaining treatments
were 5 mg/mouse/dose.
Depletion of T cell populations by MoAb treatment
The following rat MoAb were used for in vivo T cell
depletion: anti-mouse CD4 (GK1.5) (Dialynas et al. 1983,
Wofsy et al. 1985) and anti-mouse CD8 (H35-17.2) (Sar-
miento, Glasebrook & Fitch 1980). Mice that were chal-
lenged with L3 in diffusion chambers were injected i.p. with
six doses of anti-CD4 MoAb, or five doses of anti-CD8
MoAb. The first dose of GK1.5 MoAb was given seven
days, and second dose five days before initial immunization.
The first dose of H35-17.2 MoAb was given six days before
initial immunization. All other doses were given at the time
of initial immunization, and seven, 14, and 21 days after
initial immunization. Doses of the MoAb were as follows:
GK1.5, 1 mg/mouse/dose; and H35-17.2, 0.25 mg/mouse/
dose.
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IL-12 eliminates Th2-type immunity to S. stercoralis
Cytokine treatment
Groups of animals were injected i.p. daily for five days on,
and two days off, with 0.2 mg murine rIL-12 in PBS (specific
activity, 5:6 × 106 U/mg, Genetics Institute, Cambridge,
MA, USA). Treatment started on the day before initial
immunization with live S. stercoralis L3, and continued
for a total of three weeks.
ELISA procedures
Soluble antigen was prepared from S. stercoralis L3. Larvae
were boiled for ten min in distilled water containing 2%
SDS, and the preparation was then centrifuged at 14 000 rpm
at RT for 10 min. Tris buffer, pH 8.8, was added to the
supernatant, to a final concentration of 50 mM. The solution
was passed through a detergent-removing gel column
(Pierce, Rockford, IL, USA), and aliquots were collected
and used to coat ELISA plates. Protein concentration in the
aliquots was determined by staining with Coomassie Plus
protein assay reagent (Pierce, Rockford, IL, USA). The
ELISA was performed by coating each well of the ELISA
plate with 50 ml of antigen at a concentration of 10 mg
protein per ml of 50 mM Tris buffer, pH 8.8. The plates
were incubated at 378C for 2 h. Excess antigen was washed
away with distilled water. Unoccupied sites in the antigen-
coated wells were blocked using 200 ml of a blocking
solution containing 0.25% bovine serum albumin (BSA),
and 0.05% Tween-20 in PBS, pH 7.2 at 378C for 1 h. After
several washes as above, 50 ml of serum samples diluted in
blocking solution were added to each well. Serum-free
blocking solution served as a negative control. The plates
were reincubated at 378C for 2 h and then washed several
times with distilled water. Secondary and tertiary antibodies
were as follows: goat anti-mouse IgG1 (1 : 4000 dilution),
goat anti-mouse IgG2a (1 : 4000 dilution), biotinylated goat
anti-mouse IgM (1 : 4000 dilution), and biotinylated rabbit
anti-goat IgG (1 : 1000 dilution). All secondary and tertiary
antibodies were from Sigma ImmunoChemicals, and were
diluted in blocking solution. After each step, the plates were
incubated at 378C for 2 h, and washed with distilled water.
Peroxidase conjugated avidin (Cappel, Organon Teknika
Corp., Westchester, PA, USA) at a 1 : 4000 dilution in
blocking solution was added to the plates for 30 min at
378C. After thorough washing, the enzyme substrate
(ABTS substrate, Kirkegaard and Perry Laboratories, Inc.,
Gaithersburg, MA, USA) was added (75 ml per well). The
enzyme-substrate reaction was allowed to occur at 378C in
the dark for 20 min. The optical density of the contents of
each well was measured at 410 nm against the blank using a
Dynatech MR5000 ELISA reader (Dynatech Laboratories,
Inc., Chantilly, VA, USA).
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H.L.Rotman et al.
Surface staining
50 ml of undiluted pooled mouse serum was placed with
100–400 live S. stercoralis L3 at 378C for 2 h. Pooled
mouse serum was from 5–7 individual mice. One aliquot
of worms was placed in serum from mice that had been
immunized, and the other was placed in serum from
control mice. The worms were then washed five times
in PBS, and were placed in individual wells in a 96-well
round-bottomed ELISA plate. L3 were placed in rat anti-
mouse IgM diluted 1:20 in PBS for one hour at 378C,
followed by a similar incubation in goat anti-rat FITC
antibodies diluted 1:50 (Sigma). The worms were washed
four times in PBS to remove excess antibody, and once in
a 1% formalin-PBS solution. Worms were placed on
slides, and fluorescence was analysed using fluorescent
microscopy.
Worms recovered from 0.1 mm diffusion chambers were
tested for surface complement binding, 0.1 mm diffusion
chambers, which do not permit cell entry, allowed >90%
of implanted larvae to survive while in the host. Larvae
removed from diffusion chambers were fixed in 3%
formalin, aliquoted into 96-well round-bottom plates,
washed with PBS, and exposed to goat anti-mouse com-
plementary protein 3 (C3) (Cappel, Organon-Teknika
Corp., Durham, NC, USA) at a 1 : 20 dilution for 1 h.
After washing in PBS, biotin-conjugated rabbit anti-goat
IgG (Sigma) was added to each well for 1 h at dilutions of
1 : 50. After a final washing in PBS, avidin-FITC (1 : 100)
Figure 1 Effect of depleting CD4þ T cells on immune-mediated killing of challenge S. stercoralis larvae. Cells were depleted by MoAb treatment
with GK1.5, at 1 mg/mouse/dose. MoAb was given seven days before, five before, and at the time of initial immunization, and seven, 14 , and 21
days after initial immunization. Mean percentage 6 SD of larval recovery in diffusion chambers. n ¼ 5 mice per group. *P < 0:05: difference
between results in control and immunized mice in like-treated groups.
32
Parasite Immunology
(Sigma) was added to each well for 30 min. Worms were
placed on slides, and fluorescence was analysed as described
above.
Measurement of eosinophil peroxidase levels
The method of Strath, Warren & Sanderson 1985, as
modified by White et al. 1991 was used for this assay.
Briefly, 50 ml of diffusion chamber fluid was combined
with 75 ml of a 16 mM o-phenylenediamine solution in
100 nm Tris-HCl, pH 8.0 containing 0.1% Triton X-100
and 0.01% hydrogen peroxide. Polystyrene 96-well micro-
titre plates containing the reactions were placed at 378C
until a color change could be detected, and absorbance at
492 nm was measured using a Dynatech Microtiter Plate
Reader (Dynatech Laboratories, Inc., Chantilly, VA,
USA). Horseradish peroxidase (Sigma) was used as a
standard.
Statistics
All experiments were done a minimum of two times, and the
tables and graphs presented in this paper are representative
examples of experiments performed. Data were analysed
when there were two groups by Student’s t-test and when
there were multiple groups by ANOVA in Systat 5.2 (Systat,
Inc., Evanston, IL, USA). Probability values of less than
0.05 were considered significant.
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Volume 19, Number 1, January 1997
RESULTS
Depletion of CD4þ T cells ablates protective immunity
In order to determine which subset of T cells was
responsible for generating protective immunity to S.
stercoralis L3, CD4þ and CD8þ , T cells were depleted
in immunized animals, and larval killing in these animals
was assessed. CD4þ T cells were depleted by treating
BALB/cByJ mice with the MoAb GK1.5 (Wofsy et al.
1985). Significantly fewer challenge L3 were recovered
from diffusion chambers from immune mice than from
control mice. However, protective immunity in immunized
mice was almost completely ablated when mice were
depleted of their CD4þ T cells (Figure 1). Depletion of
CD8þ T cells by MoAb treatment with H35-17.2 did not
affect the protective immunity seen in immunized BALB/
cByJ mice (Figure 2). In addition, immunized b2 -micro-
globulin-deficient mice, which lack CD8þ T cells (Zijlstra
et al. 1990), still developed significant protective immu-
nity that was equivalent to that seen in intact immunized
mice of the same genetic background (C57/BL6) (Figure 2).
Eosinophil peroxidase levels are diminished in
anti-CD4 treated mice
Previously, it has been suggested that eosinophils are
Figure 2 Effect of depleting CD8þ T cells on immune-mediated
killing of challenge S. stercoralis larvae. Cells were depleted by
MoAb treatment with H35-17.2 or by use of b2-microglobulin
deficient mice. MoAb was given six days before and at the time of
initial immunization, and seven, 14, and 21 days after initial
immunization, at 1 mg/mouse/dose. Mean percentage 6 SD of larval
recovery in diffusion chambers. n ¼ 5 mice per group. *P < 0:05:
difference between results in control and immunized mice in like-
treated groups. A BALB/cByJ; B BALB/cByJ MoAb depletion; d
C57B1/6; c C57B1/6 b2 microglobulin deficient.
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IL-12 eliminates Th2-type immunity to S. stercoralis
necessary for protective immunity to S. stercoralis L3
(Rotman et al. 1996). Eosinophil numbers in the peripheral
blood and diffusion chambers of immunized mice treated
with anti-CD4 and anti-CD8 were equivalent to the levels
found in untreated immunized mice (data not shown).
Eosinophil peroxidase is an indicator of eosinophil numbers
and activation in tissue (Strath, Warren & Sanderson 1985).
Diffusion chamber fluid from chambers implanted in control
and immune mice was analysed for levels of eosinophil
peroxidase. Immune mice had eosinophil peroxidase levels
significantly higher than those seen in control mice, in all
animals tested. In immunized mice which received anti-
CD4 MoAb, however, eosinophil peroxidase levels dropped
to the levels seen in control mice (Figure 3). Immunized
mice which received anti-CD8 MoAb showed no diminish-
ment of eosinophil peroxidase levels when compared to
untreated immune mice peroxidase levels (Figure 3).
Depletion of IL-4 or IL-5 diminishes protective
immunity to S. stercoralis L3
Th-2-type CD4þ cells generate IL-4 and IL-5 (Mosmann et al.
1986), which are responsible for generating and localizing
eosinophils in tissue spaces. BALB/cByJ mice were treated
with MoAbs to eliminate either IL-4 or IL-5. Immunized
mice, untreated with MoAb, had a significant decrease in
parasite survival. Immunized mice treated with either anti-
IL-4 or anti-IL-5 had challenge worm recovery rates that
were not significantly different from those found in control
mice (Figures 4 and 5). The concentration of eosinophils
was elevated in immunized groups of mice that received no
MoAb treatment, but immunized mice which received anti-
IL-5 MoAb had reduced numbers of eosinophils (Figure 4).
No such eosinophil reduction was seen in mice given anti-
IL-4 MoAb, however (Figure 5). Immunized mice treated
with control Ab (rat IgG) had larval survival equivalent to
that seen in untreated immunized mice (data not shown).
IFN-g is not essential for protective immunity
BALB/cByJ and IFN-g knockout mice (BALB/c back-
ground) were immunized as described above. Larvae con-
tained within diffusion chambers were then implanted
subcutaneously in both naive and immunized mice, and
one day later the diffusion chambers were removed, and
larval survival was determined. Larval recoveries were as
follows: Control BALB/cByJ, 57 6 11%; Immune BALB/
cByJ, 3 6 4%; Control IFN-g knock-out, 36 6 20%;
Immune IFN-g-knockout, 3 6 2%. The concentration of
eosinophils was elevated in all immunized groups of mice
(data not shown).
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Figure 3 U/ml of eosinophil peroxidase found in diffusion chambers from anti-CD4 or anti-CD8 MoAb treated and untreated BALB/cByJ mice at
the time of larval challenge recovery of S. stercoralis L3 n ¼ 4 or 5 samples per group tested. *P < 0:05: difference between results in control and
immunized mice in like-treated groups. A; control; d immune.

Figure 4 Effect of depleting IL-5 on immune-mediated killing of challenge S. stercoralis larvae. IL-5 was neutralized by MoAb treatment with
TRFK-5, at 1 mg/mouse/dose. MoAb was given at the time of booster immunization, and at the time of parasite challenge. Mean percentage 6 SD
of larval recovery (left) and mean number 6 SD of eosinophils (right) found in diffusion chambers at the time of larval challenge recovery. n ¼ 7
mice per group. *P < 0:05: difference between results in control and immunized mice in like-treated groups. A control; d immune.

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Volume 19, Number 1, January 1997
Figure 5 Effect of depleting IL-4 on immune-mediated killing of challenge S. stercoralis larvae. IL-4 was neutralized by MoAb treatment with
11B11, at 10 mg/mouse/dose at the time of booster immunization, and at 5 mg/mouse/dose at the time of parasite challenge. Mean percentage
6 SD of larval recovery (left) and mean number 6 SD of eosinophils (right) found in diffusion chambers at the time of larval challenge recovery.
n ¼ 4 mice per group. *P < 0:05: difference between results in control and immunized mice in like-treated groups. A control; d immune.
Recombinant IL-12 therapy abolishes protective
immunity
The effect of shifting the Th-2 response to S. stercoralis L3
to a Th-1 response by rIL-12 treatment was analysed.
BALB/cByJ mice were treated with daily injections of
rIL-12 during the course of immunization with S. stercoralis
L3. Immunized mice, untreated with rIL-12, had a signifi-
cant decrease in parasite survival. In contrast, immunized
mice treated with rIL-12 had challenge worm recovery rates
that were not significantly different from those found in
control mice (Figure 6). The concentration of eosinophils
was elevated in immunized groups of mice, with the excep-
tion of those treated with rIL-12 (Figure 6).
The effect of recombinant IL-12 therapy on humoral
responses
In order to determine whether the Th-2 response had shifted
to a Th-1 response in IL-12-treated immunized animals,
individual mouse sera were tested for parasite-specific anti-
body reactivity using IgG1 and IgG2a-antigen-specific
ELISA. Immune sera from rIL-12-treated animals had an
eight-fold increase in IgG2a levels (Immune sera dropped to
background levels at a dilution of 1 : 16, while sera from
immunized mice treated with rIL-12 dropped to background
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IL-12 eliminates Th2-type immunity to S. stercoralis
levels at a dilution of 1 : 128), and a two-fold decrease in
IgG1 levels (Immune sera dropped to background levels at a
dilution of 1 : 512, while sera from immunized mice treated
with rIL-12 dropped to background levels at a dilution of
1 : 128), when compared to untreated immune sera, indicating
that a shift to a Th-1 type response had occurred. No
differences were seen between rIL-12 treated and untreated
control sera in regard to IgG1 and IgG2a levels (background
levels were seen in all control sera at a dilution of 1 : 16).
As parasite-specific IgM is essential for S. stercoralis L3
killing in this mouse model (Brigandi et al., 1996), IgM
antibody recognition of S. stercoralis L3 surface and soluble
antigens was analysed in sera, to determine whether these
responses had been altered by IL-12 treatment. IgM in sera
from rIL-12-treated and untreated immunized mice at dilu-
tions up to 1 : 50 recognized surface antigens of L3, while
IgM in both groups of control sera did not. Parasite-specific
ELISA, using soluble antigen preparation, also showed no
differences in IgM levels between IL-12-treated and
untreated immune serum groups, as both types of sera
dropped to background levels at serum dilutions of
1 : 20 000.
Complement has also been shown to be necessary for
L3 killing in the mouse model (Brigandi et al. 1996), and
so surface complement binding to L3 was analysed in
IL-12-treated animals. L3 recovered from 0.1 mm diffusion
35
H.L.Rotman et al. Parasite Immunology

Figure 6 Effect of administering rIL-12 on immune-mediated killing of challenge S. stercoralis larvae. Groups of animals were injected i.p. daily
for five days on, and two days off, with 0.2 mg murine rIL-12 in PBS. Therapy started on the day before initial immunization with live
S. stercoralis L3, and continued for a total of three weeks. Mean percentage 6SD of larval recovery found in diffusion chambers (left) and mean
number 6 SD of eosinophils (right) found at the time of larval challenge recovery. n ¼ 5 mice per group. *P < 0:05: difference between results in
control and immunized mice in like-treated groups. A control; d immune.

chambers implanted in rIL-12-treated and untreated immu-
nized mice showed equally strong surface fluorescence at
24 h post-implantation, whereas L3 recovered from both
treated and untreated control mice showed background
levels of surface fluorescence.
DISCUSSION
CD4þ T cells were found to be essential in immune-
mediated killing of S. stercoralis L3 in this model system,
while depletion of CD8þ T cells had no effect. CD4þ T cells
can be divided into Th-1 and Th-2 subsets, based on the
cytokines they produce. IL-5, a Th-2 cytokine, has been
shown to be essential for the generation and development of
eosinophils (Coffman et al. 1989). Eosinophils have been
demonstrated to be an essential component of the protective
immunity to S. stercoralis larvae in mice (Rotman et al.
1996). In this study, depletion of IL-5 was found to diminish
protective immunity to S. stercoralis L3 contained within
diffusion chambers. Immunized mice had eosinophils
reduced by treatment with MoAb to IL-5. Immunity to
several parasite species has been found to depend on IL-5,
including O. volvulus (Lange et al. 1994), A. cantonensis
(Sasaki et al. 1993), and S. venezuelensis (Korenaga et al.
1991, 1994). However, administration of anti-IL-5 to
animals with H. polygyrus (Urban et al. 1991), Trichinella
spiralis (Herndon & Kayes 1992), Schistosoma mansoni
(Sher et al. 1990), and Nippostrongylus brasiliensis (Coff-
man et al. 1989), has been shown to block the development
of eosinophilia in mice but does not prevent the develop-
ment of resistance to these infections.
In addition to IL-5, Th-2 cells produce several other
cytokines, including IL-4 (Mosmann et al. 1986). IL-4 has
many immune related functions, including inducing the
production of IgG1 and IgE isotypes (Snapper, Finkelman
& Paul 1988). Anti-IgG1 and anti-IgE treatments have
been found to have no effect on protective immunity to
S. stercoralis L3 (Brigandi et al. 1996), indicating that
alterations in IgG1 and IgE levels, which are potentially
affected by IL-4, are not relevant to protective immunity
in this model system. IL-4 also plays a role in the adhesion
and migration of eosinophils across vascular membranes
using human endothelial cells (Moser, Fehr & Bruijnzeel
1992).
Eosinophil numbers found in diffusion chambers and peri-
pheral blood are not altered in IL-4 depleted animals. This is
in agreement with other studies, in which neutralization of
IL-4 using MoAb had little effect on the influx of eosino-
phils during an antigenic challenge (Corry et al. 1996).
However, IL-4 has been shown to have an effect on

36 q 1997 Blackwell Science Ltd, Parasite Immunology, 19, 29–39

Volume 19, Number 1, January 1997
eosinophil localization and infiltration in other systems
(Tepper 1994). The demonstration that IL-4 is required
for protective immunity to S. stercoralis L3, yet has no
effect on eosinophil numbers at the site of parasite chal-
lenge, leaves unanswered the mechanism by which protec-
tion is diminished. Immunity to H. polygyrus is dependent
on IL-4, yet IL-4 operates in a mechanism independent of
IgE, mast cells, and eosinophils (Urban et al. 1992). Thus,
IL-4 is contributing some essential component to immunity
against S. stercoralis which has yet to be identified. IL-4
has an effect on many other components of the immune
system such as B cell proliferation (Howard et al. 1982),
IgG2a inhibition (Snapper & Paul 1987), cytotoxic and
helper T cell growth and differentiation (Trenn et al. 1988,
Fernandez-Botran et al. 1988), mast cell growth (Mosmann
et al. 1986), and macrophage cytotoxicity and inhibition
(McInnes & Rennick 1988, Crawford et al. 1987), and so it
is hypothesized that some other crucial component of the
protective immune response is being altered in anti-IL-4-
treated mice.
In order to measure differences in the number and func-
tion of eosinophils in this model system, the amount of
eosinophil peroxidase found within the diffusion chambers
of immunized and control animals was determined. Mea-
surement of eosinophil peroxidase activity has been utilized
as an assay of eosinophil function and number in biologic
fluids (White et al. 1991). Mice immunized against S.
stercoralis L3 showed increased EPO levels in diffusion
chamber fluid. When immune mice were depleted of CD4þ
T cells, EPO levels in diffusion chambers dropped to back-
ground levels. Depletion of CD8þ T cells had no negative
effect on EPO levels. These results further indicate that
depleting the T cells responsible for a Th-2-type response in
mice results in a lower number of eosinophils. As eosino-
phils are essential in immune-mediated killing of S. stercor-
alis L3, this reduction of eosinophils coincides with loss of
L3 killing ability.
Several studies have demonstrated that reduced eosino-
philia occurs when animals are given exogenous IL-12
during established Th-2 responses (Finkelman et al. 1994,
Wynn et al. 1994, Pearlman et al. 1995). However, when
rIL-12 is given before a Th-2 response has been established,
there is a more complete shift from Th-2 to Th-1 type
responses. When given at the beginning of some parasitic
infections, IL-12 can promote protection and prevent pathol-
ogy in host animals (Afonso et al. 1994, Finkelman et al.
1994, Heinzel et al. 1993, Stevenson et al. 1995, Gazzinelli
et al. 1993, Sypek et al. 1993). These findings all suggest
that IL-12, if given at the beginning of a developing immune
response, can change the response from one that is char-
acterized by the production of Th-2-associated cytokines,
along with associated eosinophilia, to one characterized by
q 1997 Blackwell Science Ltd, Parasite Immunology, 19, 29–39
IL-12 eliminates Th2-type immunity to S. stercoralis
the production of Th-1-associated cytokines. In addition, IL-
12 treatment has less of an effect once Th-2 responses have
already become established.
Because IL-12 induces IFN-g production and promotes
differentiation of Th-1 cells (Chan et al. 1991, Germann
et al. 1993), it was determined whether IL-12 treatment,
initiated one day prior to initial immunization, would affect
protective immunity to S. stercoralis L3 challenge. Indica-
tions that a Th-1-type response was generated in IL-12-
treated animals comes from the observation that parasite-
specific IgG2a levels increased eight-fold and IgG1 levels
decreased by two-fold, when compared to untreated immu-
nized animals. Larval killing was completely ablated in
immunized mice which received IL-12 therapy. Three
components essential for protective immunity to S. stercor-
alis in mice have been defined: eosinophils (Rotman et al.
1996), IgM (Brigandi et al. 1996), and complement (Brigandi
et al. 1996). C3 levels on worms recovered from immune
mice and IgM levels binding to soluble antigen and on the
surface of L3 were identical in both IL-12-treated and
untreated immunized mice. However, the decrease in protec-
tive immunity was associated with a decrease in the number
of eosinophils found both in diffusion chamber fluid and in
the peripheral blood. It appears that eosinophils decrease in
IL-12-treated animals due to the effects IL-12 has in dimin-
ishing Th-2 responses, whether directly or through induced
IFN-g production (Chan et al. 1991, Germann et al. 1993,
Kobayashi et al. 1989). IL-12 administration has also been
shown to induce neutropenia, lymphopenia, and anaemia in
mice (Brunda 1994, Eng et al. 1995), though eosinophils
were the only cell types to decrease in treated animals in this
system. The use of IFN-g knockout mice had no effect on
larval survival in immunized mice, indicating that this Th-1
cytokine was not essential in generating protective immunity
to S. stercoralis L3.
These results indicate that the generation of a Th-2 type
response is necessary to kill third-stage larvae in S. stercor-
alis-immunized mice. When this response is shifted to a
Th-1 response by administration of IL-12, protective immu-
nity is ablated. Eosinophil levels are altered in IL-12-treated
animals, while IgM levels and complement activation
appear unaltered. The reduction in eosinophils appears be
responsible for the loss of protective immunity seen in this
model system. These results indicate the importance of
generating the correct T-helper cell response to an infection,
and demonstrate how a shifting to the opposing T cell
response can completely ablate protective immunity.
Abbreviations
ANOVA, analysis of variance; ELISA, enzyme-linked
immunosorbant assay; EPO, eosinophil peroxidase; Ig,
37
H.L.Rotman et al.
immunoglobulin; IMDM, Iscove’s Modified Dulbecco’s
Medium; L3, infective third stage larvae; PBS, phosphate
buffered saline; NCTC-135, National Cancer Institute Tissue
Culture Medium-135; rIL-12, recombinant interleukin-12.
ACKNOWLEDGEMENTS
We thank Ofra Leon and De’Broski Herbert for expert
technical assistance, Dr Stanley Wolf and Dr Joseph
Sypek from the Genetics Institute, Inc. for the kind gift of
rIL-12, and Dr Bice Perussia for critical reading of the
manuscript. This work was funded in part by NIH grant
AI22662 and by grants from the Edna McDonnell Clark
Foundation.
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