EXPERIMENTAL PARASITOLOGY 73,500-511 (19%)
Heligmosomoides
polygyrus:
CD4+ but not CD8+ T Cells Regulate
the IgE Response and Protective Immunity in Mice’
JOSEPH F. URBAN,JR., *,* ILDY M. KATONA,~ AND FRED D. FINKELMANS
*Helminthic Diseases Laboratory,
Livestock and Poultry Sciences Institute, Agricultural Research Service,
U.S. Department of Agriculture, Beltsville, Maryland 20705-2350; and the TDepartment of Pediatrics and
#Medicine, F. Edward Herbert School of Medicine, Uniformed Services University of the Health Sciences,
Bethesda, Maryland 20888-4799
URBAN, J. F., JR., KATONA, I. M., AND FINKELMAN, F. D. 1991. Heligmosomoides
polygyrus: CD4+ but not CD8+ T cells regulate the IgE response and protective immunity
in mice. Experimental Parasitology 73, 500-511. Oral inoculation of BALB/c mice with
infective larvae of Heligmosomoides polygyrus resulted in chronic infection characterized
by the release of parasite eggs in the feces for several months. The actual number of eggs per
gram of feces was dependent on the dose of the inoculum. Serum IgE in infected mice
peaked at a level of >70 &ml during Weeks 3 through 6 following inoculation, and high
levels of IgE (>40 l&ml) persisted for over 14weeks. Protective immune responses resulted
in reduced egg production and the development of markedly fewer adult worms in the small
intestines following a challenge inoculation. The role of CD4+ and CD8+ T cells in these
responses was examined by depletion in vivo of either T cell subpopulation with rat mAb
specific for the appropriate determinants. Mice treated with anti-CD4 during a primary
infection had increased EPG which was due primarily to an increase in worm fecundity (eggs
produced per adult female). A challenge inoculation of mice that had been cleared of the
primary infection with an anthelmintic drug induced a protective response that reduced
development of new adult worms by 7080% and their fecundity by >90%. This protective
response was abrogated by injection of mice with anti-CD4. Serum IgE diminished when
adult worms were removed after anthelmintic treatment. A more precipitous drop in serum
IgE followed successive treatments of mice with an anthelmintic and anti-CM. In addition,
the anamnestic serum IgE response to a challenge inoculation was reduced by over 80% in
anti-CD4-treated mice. Anti-CD8 treatment had no appreciable effect on the immunological
or parasitological parameters measured following a challenge inoculation with H. polygyrus.
Thus, CD4+ T cells regulate host protective immunity, worm fecundity, and IgE levels in an
H. polygyrus infection. This experimental system may be particularly suitable for analysis
of chronic nematode infections of humans and livestock because of the responsiveness of
the parasite in vivo to changes in host immune function.
INDEX DESCRIPTORS AND ABBREVIATIONS: Nematode, Heligmosomoides polygyrus; L3,
Ensheathed, Infective third-stage larvae; Parasite eggs per gram of feces (EPG); Monoclonal
antibody (mAb); Rat monoclonal IgG2b (GKl.5) to CD4 determinants (anti-CD4); Rat
monoclonal IgG2b (2.43) to CD8 determinants (anti-CDI); Rabbit anti-mouse IgE (RaME);
Alkaline phosphatase conjugated goat anti-rabbit Ig (APGaRIg); Rat monoclonal IgGl (54-l)
to 4-hydroxy-3-nitrophenylacetyl (anti-NP).
i The U.S. Government’s right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, for governmental purposes, is acknowledged.
* To whom correspondence and reprint requests
should be addressed at: Helminthic Diseases Laboratory, LPSI, ARS, USDA, Building 1040 BARCEAST, Beltsville, MD 20705-2350.
INTRODUCTION
The requirement for T cells in protective
immunity to helminths is well documented
(Mitchell 1980). In addition, T cells have
been purported to regulate immune effector
elements associated with helminthiasis
500
0014-4894191$3.00
T CELL
REGULATION
OF IMMUNITY
TO
H. polygyrus
501
such as intestinal mastocytosis, eosino- sponsiveness that is not dependent on IgE
philia, and IgE production (Jarrett and Ba- or mucosal mast cells. In contrast, antizin 1974; Butterworth 1977; Befus et al. CD4 antibody had no effect on the protec1979; Urban et al. 1984). In spite of these tive response to a secondary, challenge infindings, there is still considerable debate fection with N. brusiliensis, even though it
over the precise role of T cells in immunity actively inhibited the anamnestic IgE reto parasites (Finkelman et al. 1990a). In sponse in vivo by >90%. These results invivo models have been developed where dicated that effector mechanisms that rethe injection of antibodies against specific T duce parasitic nematode survival may not
cell determinants will ablate T cell func- always be CD4+ T cell dependent. Retions in situ and allow examination of the moval of CD8+ T cells with appropriate in
role of T cell subpopulations in protective vivo antibody treatment did not atfect eximmunity. Kumar et al. (1989), for exam- pulsion of N. brusiliensis or IgE levels durple, showed that the injection of anti-CD4 ing the infection (Katona ef al. 1988). This
antibody could abrogate immunity in mice is in contrast to the clear role of CD8+ T
exposed to Plasmodium vinckei vinckei and cells in the killing of viral-infected cells folimmunity could be restored by the adoptive lowing recognition of viral antigen/class I
transfer of immune CD4+ T cells, but these determinant complex (Leist et al. 1989).
same cells could not transfer immunity to
The present study evaluated the in vivo
parasite-naive mice. It was postulated that role of CD4+ and CD8+ T cells in immunity
other cellular elements resident in the to another gastrointestinal parasitic nemapolygyrus.
The
spleen of the parasitized host were acting tode Heligmosomoides
synergistically with CD4+ T cells to ex- family Heligmosomidue are common parapress an effective immunity. In another ex- sites of wild rodents in North America,
perimental system, mice infected with Nipwestern Europe, and the USSR with the
postrongylus brasiliensis expressed T cell species H. polygyrus studied extensively,in
dependent immune responses differently
models of host parasite interaction; adult
worm burdens of ~200 have LD,,,/30 days
depending on the stage of the infection
when anti-CD4 antibody was given. Treat- of ~20% in laboratory mouse strains, but
ment of mice with anti-CD4 antibody prior these levels are not typically found in natto and during a primary infection with N. ural infections (Fort-ester 1971). Primary inbrasiliensis
larvae prevented the typical fections of mice with H. polygyrus are unspontaneous cure (expulsion) of adults like infections with N. brusiliensis because
from the intestine for as long as antibody there is no spontaneous cure of adults and
was administered and blocked the mucosal mucosal mastocytosis is absent with some
mastocytosis and IgE production which are systems (Dehlawi et al. 1987), but is incharacteristic of the infection (Katona et al. duced in others (Madden, Urban, Finkel1988). However, when anti-CD4 antibody man, Katona, unpublished data). However,
was given to N. brasiliensis-infected mice H. polygyrus does evoke persistent serum
IgE and eosinophil levels (Urban et al.
on Day 8 or 9 after a primary inoculation
(i.e., 1 to 2 days before worm expulsion), it 1991) that are typical of nematode infecpartially blocked spontaneous cure, even tions. Host protective responses are exthough IgE levels and mucosal mast cell pressed as decreased parasite egg producnumbers were elevated. This observation tion (fecundity), slow elimination of adults
demonstrated that functional CD4+ T cells during a primary infection, and prevention
may be required close to the point of expul- of development of adults from a secondary
sion, possibly to attain a threshold of re- challenge infection. The results of the
URBAN,KATONA,AND
502
present study indicate that CD4+ T cells
stimulate IgE production and inhibit worm
fecundity during a primary infection and
are required for the development of immune protection to a secondary challenge
infection. In contrast, CD8+ T cells do not
appear to play a direct role in any protective response.
MATERIALS
AND METHODS
Female BALB/c mice (Small Animal Division of the
National Cancer Institute, Frederick, MD) were used
for experimentation at 8 to 12 weeks of age. All experiments were conducted with five mice per group.
Blood samples were obtained from the orbital plexus;
serum was separated from clotted blood by centrifugation and stored at - 80°C until used.
Vermiculite-fecal cultures containing infective, ensheathed third-stage larvae of H. polygyrus (U.S. National Museum Helminthological
Collection No.
81930, specimens on tile at the U.S. National Parasite
Collection, Beltsville, MD) were originally obtained
through the cooperation of Dr. Lis Eriksen of the
Royal Veterinary and Agricultural University, Copenhagen, Denmark. Larvae were separated from culture
using a Bearmann apparatus, washed in saline,
counted, and stored at 4°C until used (generally within
4 weeks). Mice were inoculated orally using a balltipped feeding tube. Parasites were propagated by collecting the feces of infected mice and mixing them with
water, sterilized charcoal, and peat moss in 100-cm
petri dishes. Larvae were isolated at 7 to 10 days after
culture.
An anthelmintic was used to remove H. polygyrus
adults from a primary infection prior to secondary
challenge infection. Mice were given an oral dose of 1
to 2 mg of pyrantel pamoate (Strongid T, Pfizer Diagnostics, New York, NY) administered through a feeding tube.
Fecal egg counts were determined to monitor infection status. Feces were collected for approximately 1
to 2 hr from five mice per group housed in a cage with
a suspended wire floor. Feces were weighed and dispersed in a flask containing a measured amount of water and glass beads. Aliquots of the suspension were
removed in duplicate and the particulate material was
pelleted by centrifugation in collecting tubes. Eggs
were then floated in a solution of zinc sulfate (1.18
specific gravity) and counted after adhering to a glass
cover slip. The counts were expressed as eggs per
gram of feces (EPG). Adult worms and total eggs in the
intestines were determined at necropsy. The entire
small intestine was removed and opened into a tube
containing a measured amount of saline. The cecum
and large intestine were treated similarly in a separate
FINKELMAN
tube. Adult worms were counted and sexed by examining the small intestinal contents and mucosa under a
dissecting microscope. Aliquots of the small intestine
and the large intestine and cecum were processed as
above to determine the total number of eggs in the
alimentary track of each mouse. Worm fecundity was
expressed by dividing the total number of eggs recovered from each mouse by the total number of female
worms.
Several different antibodies were used for immunological assays or to remove T cell populations in vivo.
They were prepared and tested for specificity as previously described: Monoclonal rat IgG2b anti-mouse
CD4 (GK1.5) (Wilde et al. 1983) and anti-CD8 (2.43)
(Sarmiento et al. 1980), monoclonal rat IgG2a antimouse IgE (EM95) (Baniyash and Eshhar 1984)rabbit
anti-mouse IgE (RaME) (Katona ef al. 1983), alkaline
phosphatase conjugated goat anti-rabbit Ig (APGaRIg)
(Finkelman et al. 1983), monoclonal mouse IgE antiDNP (SPE-iv-7) (Eshhar et al. 1980). monoclonal rat
IgGl anti-4-hydroxy-3-nitrophenylacetyl
(54-l)
(Finkelman et al. 1988).
Serum levels of mouse IgGl and IgG2a were determined by radial immunodiffusion with standards and
Mancini plates purchased from Hazelton Laboratories
(Rockville, MD). Serum IgE levels were determined
by a micro-ELISA that uses monoclonal anti-IgE
(EM95) to capture IgE and a sandwich of RaME and
APGaRIg to label captured IgE. 4-Methylumbilliferyl
phosphate was used as a fluorogenic substrate and fluorescence was determined with a 3M FluoroFAST 96
well fluorometer (Trendcom, Sunnyvale, CA) (Finkelman et al. 1987). Purified mAb mouse IgE (SPE-iv-7)
was used as a standard.
Statistical significance between worm number and
egg counts was tested using an analysis of variance
procedure and separated by Duncan’s comparison.
RESULTS
Parasite egg excretion and IgE production were found to be dose dependent.
Doses of 100, 200, or 300 infective larvae
(L3) of H. polygyrus were inoculated orally
into mice to determine the level of increase
in EPG and serum IgE. Egg production, expressed as EPG, was directly related to the
inoculation dose (Fig. 1A). Peak egg production was at 23 days after inoculation
with doses of 100and 200 L3, and at 36 days
after inoculation with 300 L3. Egg production persisted at the highest level and for
the longest period after inoculation with 300
L3, but decreased to 10% or less of the peak
level between 50 to 57 days after inocula-
T CELL
REGULATION
OF IMMUNITY
Larvae Inoculated
0
0
I
14
21
28
15
12
Days Following Inoculation
FIG. 1. (A and B) Three groupsof five BALBlc female mice were inoculated orally with either 100,200,
or 300 Heligmosomoides polygyrus L3. The groups
were sampled periodically (A) for EPG from a pool of
feces from each group or (B) by bleeding weekly to
obtain serum for analysis of IgE. The IgE levels are
the geometric mean of five samples for each point.
tion at all doses tested. The serum IgE response to infection varied less with inoculation dose than did egg production; less
than a twofold difference in serum IgE concentration was seen among the three groups
at any given time point (Fig. 1B). The IgE
response increased through 21 days after
inoculation and persisted at a high level
through 42 days for all doses tested. All
subsequent infections were performed with
an inoculation dose of 200 L3 per mouse.
To determine whether serum IgE and
IgG levels and parasite egg excretion are
regulated by CD4+T cells during a primary
infection, mice were infected with H.
pofygyrus and injected with anti-CD4 on
Days 7, 14, and 21 after inoculation. This
period of antibody treatment corresponds
to the times just prior to, and after, the developing fourth-stage larvae emerge from
503
TO H. polygyrus
A
d-
Control
---M--
aCD4-Treated
14281256708498
Days Following Inoculation
FIG. 2. (A and B) Two groupsof five BALBlc female
mice were inoculated
with 200 Heligmosomoides
polygyrus L3. One group received 0.5 mg of monoclonal rat anti-mouse CD4 antibody iv on Days 7, 14, and
21 after inoculation (treated); the other group of mice
was not injected (control). (A) EPG levels from a pool
of feces collected periodically from each group and (B)
the geometric means of serum IgE from five mice in
each group were determined periodically from 7 to 120
days after inoculation.
the intestinal mucosa as adults (Bryant
1973). The activity of anti-CD4 antibody
treatment was indicated by suppression of
the polyclonal serum IgE response to infection (Fig. 2B). The suppression was reversible, however, because serum IgE levels
began to rise sometime between 14 and 56
days after the last injection of anti-CD4.
Anti-CD4-treated mice had only 12% of the
IgE response of untreated H. polygyrusinfected control mice even 56 days after the
last injection of anti-CD4 (77 days after inoculation) and only 28% of the level of control mice 24 days later (101 days after inoculation). Levels of EPG were also followed
in these mice as an indicator of parasite activity (Fig. 2A). Infected mice injected with
504
URBAN, KATONA,
anti-CD4 had consistently higher EPG from
Day 42 through 98 after inoculation with H.
polygyrus. At times the EPG level of antiCD4 treated mice was three to four times
that of the infected control mice.
To further explore the role of CD4+ T
cells in immunity to H. polygyrus, anti-CD4
was first administered to infected mice after
egg production and high serum IgE levels
were established. Mice injected with antiCD4 on Days 19, 26, and 33 after inoculation showed a precipitous drop in serum
IgE (Fig. 3B) and an increase in EPG (Fig.
3A) within the first week after anti-CD4
treatment. The anti-CD4 treated mice had
20% more adult worms at necropsy than
infected control mice, but this difference
30.
A
aCDCTreated
-
Control
aCD4 Antibodv lniection
1
4
1
20.
6
8o1
-t-
A
Uninfected
-
Ho-Infected
Days Following Inoculation
FIG. 3. (A and B) Two groups of five BALB/c female
mice were inoculated with 200 Heligmosomoides
polygyrus L3. One group received 0.5 mg of monoclonal rat anti-mouse CD4 antibody iv on Days 19, 26,
and 33 after inoculation (treated); the other group of
mice was not injected (control). (A) The EPG levels
from a pool of feces collected periodically from each
group and (B) geometric means of serum IgE levels of
five mice in each group were measured periodically
from Day 11 through 41 after inoculation.
AND FINKELMAN
was not significant (P s 0.05). However,
both IgE and IgGl levels in anti-CD4treated and -infected mice were markedly
lower than infected controls (Table I). Levels of IgG2a were similar among all infected
and uninfected groups.
To more definitively determine if increases in EPG levels in anti-CD4 treated
mice were due to a greater number of adults
in the intestines and/or to an increase in
their fecundity following treatment, a protocol was designed to remove CD4+ T cells
before and during the time that parasitic larvae invade the intestinal mucosa. Mice
were injected with anti-CD4 or a control
antibody (54-l) on Day 7 before, the same
day as, and 7 days after a primary inoculation with H. polygyrus L3, and necropsied
7 days later (Table II). Anti-CDCtreated
mice again had between 20-25% more adult
worms than control mice (not statistically
significant, P s 0.05), and worm fecundity
in anti-CD4+-treated mice was increased
approximately twofold. The proportion of
male:female worms did not differ appreciably among the groups examined (data not
shown).
The role of CD4+ T cells in protective
immunity to a secondary challenge infection was evaluated next. Mice inoculated
with H. polygyrus were treated with anthelmintic 14 days postinoculation to remove
adult worms and were either given a challenge inoculation 7 days later or not reinfected. Some groups of mice were also
given anti-CD4 antibodies on Days 19, 26,
and 33 after the primary inoculation. The
effect of anti-CD4 treatment on serum IgE
levels was evaluated weekly (Fig. 4) and
protective immunity was evaluated on the
basis of adult worms recovered from the
intestines 20 days after the challenge (Table
III). Mice treated sequentially with a primary inoculation, anthelmintic drug, and a
secondary-challenge inoculation (primary
and challenge group) had secondary serum
IgE and IgGl responses that were more
than threefold greater than previously par-
T CELL REGULATION
Adult Heligmosomoides
OF IMMUNITY
505
TO H. POfygyrUS
TABLE I
polygyrus Recovery and Serum Immunoglobulin Levels following Treatment of Mice
with anti-CD4 Antibody
Larval
inoculation
Anti-L3T4
treatment0
Adult
recoveryb
-
-
0
+
+
+
84.4 k 16
101.4 f 14
0.2 (1.8)
35.4 (1.2)
4.8 (1.2)
IgGl
mg/ml
IgG2a
mg/ml
0.3 (1.1)
12.3 (1.2)
3.1 (1.1)
0.3 (1.0
0.2 (1.0)
0.2 (1.1)
a Anti-CD4 (0.5 mg/mouse) was injected intravenously on Days 19, 26, and 33 after oral inoculation with 200
larvae; mice were necropsied 8 days after the last injection.
b Arithmetic mean recovery of adults from the small intestines of five mice per group + the standard error of
the mean.
c Geometric means of serum levels (the standard error of the mean) obtained 2 days before necropsy.
asite-naive mice given only a single inoculation (primary at time of challenge group)
(Table III). The primary and challenge
group had 70% fewer adult worms in their
intestines and EPG levels that were approximately 24 times lower than the challenge
only group. However, anti-CD4 antibody
treatment ablated this protective response
and inhibited secondary IgE and IgGl levels. Examination of the kinetics of serum
IgE responses (Fig. 4) demonstrated that
removal of adult worms with an anthelmintic drug resulted in a gradual decline in the
serum IgE level, and this decline was accelerated by concomitant injection of antiCD4 antibody. In addition, anti-CD4 antibody inhibited the peak secondary IgE response by over 85% when compared to
previously infected and anthelmintictreated mice that were given a challenge inoculation.
Because CD4+ T cells regulate the subpopulation of CD8+ CTL (Cantor and
Boyse 1975) that have been found in high
numbers in the intestinal mucosa (Fiocchi
er al. 1983), it was possible that anti-CD4
antibody could affect protective immunity
to a secondary challenge infection by interfering with H. polygyrus-specific CD8+
CTL function. This possibility was examined by injecting mice, whose primary infection was cleared by anthelmintic treatment, with anti-CD8 alone or in combina-
tion with anti-CD4 prior to and during a
secondary challenge infection. Protective
responses were determined by comparing
the total eggs produced, total adults, and
worm fecundity from the different groups.
Anti-CDCtreated mice that had received a
primary and challenge infection yielded total eggs, adult worms, and worm fecundity
levels similar to parasite-naive mice that received only a primary inoculation at the
time that the other mice were challengeexposed (Table IV). In contrast, similarly
TABLE II
Adult Heligmosomoides polygyrus Recovery and
Worm Fecundity following Treatment of Mice
with Anti-CD4 Antibody
MAb
treatment”
Total
adults
recovered
Total
ek33
produced
Eggs/female
(fecundity)b
None
Control
Anti-CD4
103 + 7
111 f 3
125 f 8
6494 f 623
4686 k 471
13494 * 119
110 + 12
19 + 6
213 k 11
0 Mice were untreated or injected iv with anti-CD4
(GKl.5) or a control antibody (54-l) against 4-hydroxy-3nitrophenylacetyl (NP) on Day 7, before, the
same day, and I days after inoculation with H. polygyrus; mice were necropsied 7 days after the last injection. Arithmetic means + the standard error of the
mean are expressed in all columns.
b Fecundity was determined by dividing the total
eggs found at necropsy in the small intestine, cecum,
and large intestine by the total number of female
worms.
506
URBAN,
KATONA,
AND
FINKELMAN
CD8 alone had no effect on the secondary
IgE response (Fig. 5).
DISCUSSION
1y
11
I
18
I
25
I
32
I
39
Days Following Inoculation
FIG. 4. Four groups of five BALBk
female mice
were inoculated with 200 Heligmosomoides polygyrus
L3 (HP); one uninfected group served as a control. All
groups were given an anthelmintic treatment (drug) at
14 days after the primary inoculation. Two groups
were given a secondary-challenge inoculation (Hp 2”)
with 200 H. polygyrus L3 on Day 21 and one of these
groups was also given anti-CD4 antibody 2 days before, 5 days after, and 12 days after the challenge
(Days 19, 26, and 33 after the primary inoculation).
The two remaining groups that had been given a primary inoculation and anthelmintic treatment were not
given a challenge inoculation (Hp 1”). One of these
groups was treated with anti-CD4 as described above.
The geometric means of serum IgE levels of five mice
in each group were determined weekly and expressed
as t&ml of serum.
challenge-exposed mice treated with antiCD8 antibody alone yielded numbers of
adult worms and eggs that were comparable
to or lower than those obtained from mice
that were not antibody-treated or were
treated with a control antibody. Mice
treated concurrently with anti-CD4 and
anti-CD8 responded like those given antiCD4 alone, i.e., protective immunity was
blocked. Mice treated with anti-CD4 antibody, either alone or in combination with
anti-CD8 had a peak secondary IgE response to infection that was 80% lower than
controls, while treatment of mice with anti-
These experiments demonstrate that parasite egg excretion and host IgE production
are good indicators of H. polygyrus activity
and T cell function in viva. Regulation of
the IgE response to H. polygyrus appears
to be comparable to that observed in an earlier study where the in vivo T cell response
to N. brasiliensis was modulated by specific mAb to T cell subpopulations (Katona
ef al. 1988). In that study the efficiency of
anti-CD4 and anti-CD8 antibody treatment
in depleting CD4+ and CD8+ cells, respectively, was measured indirectly by observing changes in the percentage of each T cell
subpopulation within the T cell compartment expressing Thy 1.2+ both before and
after mAb treatment. When anti-CD4 was
injected into mice on the same day and 7
days after inoculation, the percentage of
Thy 1.2+ cells expressing CDS+ (Lyt 2+)
changed from 28 to 91%. In turn, similar
treatment of mice with anti-CD8 (anti-Lyt
2) changed the percentage of Thy 1.2+ cells
expressing CD4+ from 74 to 98% (Katona
et al. 1988). The results of these specific
depletions in T cell subpopulations, although incomplete, indicate that CD4+ T
cells but not CD8 + T cells are required for
the induction and maintenance of an IgE
response following either a H. polygyrus or
a N. brasiliensis infection. Treatment of
mice with anti-CD4 antibody during a primary infection with N. brasiliensis totally
suppressed the IgE response. Adult worms
or their metabolic products are apparently
the major stimulus for IgE production because serum IgE levels fell markedly after
the normal immune expulsion of adult N.
brasiliensis (Katona et al. 1988) or after anthelmintic treatment to remove adult H.
polygyrus. Furthermore, the levels of serum IgE produced either during a patent
infection with H. polygyrus or following removal of adult worms of either species (Ka-
T CELL REGULATION
OF IMMUNITY
507
TO H. POlygyrUS
TABLE III
Effect of Anti-CD4 Antibody on Protective Immunity and the IgE and IgGl,
Response to Heligmosomoides polygyrus
Parasite exposure”
None
Primary alone
Primary alone plus anti-CD4
Primary and challenge
Primary and challenge
plus anti-CD4
Primary at time of challenge
Immunoglobulinsd
Anthehnintic
treatment
EPGb
Total
adults
recovered’
+
+
+
0
0
0
260
0
0
0
21 * 15
0.2 (1.8)
13.0 (1.2)
4.0 (1.2)
11.5(1.3)
0.3 (1.0)
ND
ND
27 (1.1)
+
-
5328
6218
84k 11
71 2 13
34 (1.2)
32 (1.2)
6 (1.2)
8 (1.2)
Ii@
I&~
Wl
mg/ml
a Mice were inoculated with H. polygyrus on Day 0 (primary) and some groups were challenge-exposed on
Day 21 after the primary inoculation (primary and challenge). One group received a primary inoculation at the
time that the others were challenge-exposed (primary at challenge). Some groups of mice were anthelmintictreated on Day 14 after the primary inoculation. Anti-CD4 (0.5 mg/mouse) was given iv to some groups of mice
on Days 19 and 26 and 33 days after the primary inoculation.
b The eggs per gram of feces was determined on the day of necropsy, 41 days after the primary inoculation.
c Arithmetic means of adults recovered from the small intestines of five mice per group + standard error.
d Geometric means of serum levels (standard error of the mean) obtained 39 days after the primary inoculation.
tona et al. 1988) were reduced more precipitously after comparable treatment of mice
with anti-CD4. It is notable that the anamnestic IgE response to H. polygyrus was
incompletely blocked by either anti-CD4,
or anti-CD4 plus anti-CD8 treatment, suggesting a reduced requirement for either T
cell subpopulation in the IgE memory response to this nematode. This observation
weakens the position that IgE has a critical
role in the host’s protective response to
worm infection because protection to a
challenge infection with H. polygyrus was
completely blocked by anti-CD4 treatment.
TABLE IV
Effect of Anti-CD4 and/or Anti-CD8 Antibodies on Protective Immunity to Heligmosomoides
Parasite exposure”
Primary
Primary
Primary
Primary
and
and
and
and
challenge
challenge
challenge
challenge
Primary and challenge
Primary at time of challenge
polygyrus
Antibody
treatmentb
Total eggs
produced
Total adults
recovered
Eggs/female
(fecundity)
None
Anti-CD4
Anti-CD8
Anti-CD8
Anti-CD4
Anti-NP
None
40 2 37
3249 rt: 547
525
2850 f 590
15 + 9
87 2 12
If1
83 2 16
2’2
702 11
121
65 f 12
180 f 167
2099 k 455
725
71 f 9
10 f 7
55 f 15
0 Mice were inoculated with H. polygyrus on Day 0 (primary) and challenge-exposed on Day 36 after the
primary inoculation; all mice were anthelmintic treated on Day 17. One group of mice received a primary
inoculation at the time that the others were challenge-exposed. Mice were necropsied on Day 55 after the
primary inoculation.
b Rat monoclonal antibodies against CD4, CDS, or NP were given iv on Days 22, 28, 36, 43, and 50 after the
primary inoculation.
c Worm fecundity was determined by dividing the total number of eggs recovered at necropsy from the small
and large intestines and cecum over the total female worms recovered. Arithmetic means 2 the standard error
of the mean are expressed in all columns.
508
URBAN, KATONA,
lnochion
I
Drug
Treatment
I
15
AND FINKELMAN
i
----___
,“.
_._._.*ALI
““i”,ected
Days ;6110win~slnoculat~n
FIG. 5. Five groups of five BALBlc female mice were inoculated with 200 Heligmosomoides
polygyrus L3 and were treated with an anthelmintic drug 17 days later; two groups were not infected.
Some groups were given anti-CD4 + anti-CD& anti-CD8 alone, control rat IgG2b anti-NP antibody,
or no antibody on Days 22, 28, 36,43, and 50 after a primary inoculation, and then given a secondary
challenge inoculation (Hp 2”) on Day 36 after the primary inoculation. One of the uninfected control
groups also was given a primary inoculation at the time that the other mice were challenge-exposed
(Hp 1”). The geometric means of the serum IgE levels of five mice in each group were determined
weekly and expressed as l&ml of serum.
In fact, treatment of H. polygyrus-infected
mice with an anti-mouse IgE mAb, which
neutralizes secreted IgE, does not inhibit
protective immunity (Katona, Madden,
Finkelman, Urban, unpublished data).
These studies are not definitive because
they have not examined IgE levels at the
site of infection in the intestine or the role
of parasite-specific IgE antibodies. They do
suggest, however, that other effects regulated by CD4+ T cells such as stimulation
of T cell and mucosal mast cell growth
(Lichtman et al. 1987), activation of cytotoxic T cells (Widmer and Grabstein 1987),
or the regulation of the more diverse components of enteric physiology such as goblet cells, epithelial function, smooth muscle
cells responses, etc. (Castro 1989; Kassai
1989), may contribute to protective immunity to H. polygyrus.
In neither infection model have CDS+ T
cells provided a protective function since
depletion of CD8+ cells by treatment with
anti-CD8 antibodies did not prevent protection. But our studies do raise the possibility
that CD8 T cells can mollify host resistance
because anti-CD8-treated mice yielded
fewer worms and eggs from a challenge infection with H. polygyrus than infectedcontrol mice injected with no antibody or a
control antibody. This issue will have to be
investigated further before definite conclusions can be drawn because of the large intragroup variation in the experiment.
The role of CD6bearing cells in protective immunity to N. brasiliensis and H.
polygyrus appeared to change depending on
the stage of the infection. Treatment of
mice with anti-CD4 1 to 2 days before expulsion of a primary N. brasiliensis infection blocked host-induced adult worm expulsion. The persistence of adults in the intestines of anti-CD4-treated
mice is
analogous to infected athymic nude mice
that inherently lack functional T cell activity (Jacobson and Reed 1974). Anti-CD4 antibody given to mice prior to and during a
primary infection with H. polygyrus also affected the host response to adult worms because their fecundity was increased as com-
T CELL
REGULATION
OF IMMUNITY
pared to worms from control-infected mice.
This indicates that changes in worm physiology in situ can have an immunological basis. Fecundity measurements have been
used to reflect changes in worm physiology
during an infection, as well as host related
changes in immunity (Ogilvie and Hockley
1968; Ogilvie and Jones 1973; Kerboeuf
1984). It now appears clear that CD4+ T
cells can play a critical role in the stability
and fecundity of gastrointestinal worm populations in situ.
While anti-CD4 treatment of mice prior
to a secondary infection with H. polygyrus
suppressed protective immunity, anti-CD4
treatment of mice given a secondary challenge infection with N. brasiliensis was
without affect (Katona et al. 1988). A possible explanation is that the parenteral migration of N. brasiliensis larvae through the
skin, blood vessels, and lungs activates
protective elements that are not exclusively
T-cell-regulated, but are capable of inhibiting the development of larvae from a challenge infection. In contrast, the strictly enteral life cycle of H. polygyrus interfaces
with host mucosal elements that have relatively short immunological
memory
(Newby 1984) or require T cell activated
renewal. It is possible, however, that the
enteral T cell dependent mechanism that is
protective against H. polygyrus is the same
one that when blocked by anti-CD4 treatment late in a primary infection with N.
brasiliensis
interferes with spontaneous
cure (Katona et al. 1988). This possibility
could be tested by direct transfer of adult
N. brasiliensis into the intestines of previously infected anti-CD4 treated mice, thus
precluding the parenteral phase of the infection, and determining if expulsion is now
CD4+ T cell dependent.
The precise mechanism of CD4+ T cell
regulation of protection against helminth infection is not known, but removal of CD4+
cells in vivo eliminates both Thl and Th2
subpopulations. Lymphokines produced by
both of these T cell subpopulations have
509
TO H. POlygyrUS
been implicated in protective immunity to
helminth parasites (Finkelman et al. 1990a).
Recent studies have begun to characterize
the relative contributions of CD4+ T cell
dependent phenomena in protective immunity to H. polygyrus. For example, the inability of anti-IL-5 mAb treatment to block
protective immunity to H. polygyrus, even
though parasite induced eosinophilia was
reduced to control levels, would argue
against a critical role for these cells in antinematode protective mechanisms (Urban et
al. 1991). Likewise, the role of parasite specific IgGl antibody in protection against H.
polygyrus
(Williams and Behnke 1983;
Pritchard et al. 1983) may not be absolute
because in vivo treatment of mice with antiIL-4 and anti-IL-4 receptor antibody,
which blocks IL-4 activity and suppresses
IgE but not IgGl production, interferes
with the protective response to a secondary
challenge infection (Urban et al. 1991;
Finkelman et al. 1990b).
The recent demonstration of an important role for IL-4 in protective immunity to
H. polygyrus (Urban et al. 1991) contrasts
markedly with its strong inhibitory effect
on protective immunity to Leishmaniu major (Sadick et al. 1990). These results suggest that production of a single cytokine
can be either protective or deleterious to
the host depending on the nature of the infectious agent. Because CD4+ T cells regulate the protective responses to both protozoan and nematode infections and these
cells are capable of differentiating into cells
that produce either large or limited quantities of IL-4 (Street and Mosmann 1991), the
mechanisms that regulates this process of
differentiation could have a critical role in
host resistance or susceptibility to a variety
of parasites.
ACKNOWLEDGMENTS
This work was supported
in part by the U.S. De-
partment of Agriculture CRIS 1265-34ooO-009,the National Institutes of Health Grant AI-26150, and the
Uniformed Services University of the Health Sciences
510
URBAN,
KATONA,
Research Protocols RO8308 and R086AB. The opinions and assertions contained herein are those of the
authors and are not to be construed as ofi%ial or reflecting the views of the Departments of Defense or
Agriculture or the Uniformed Services University of
the Health Sciences. The experiments reported herein
were conducted according to the principles set forth in
the Guide for the Care and Use of Laboratory Animals, Institute of Animal Resources National Research Council, Department of Health, Education and
Welfare Publication No. 78-23 (National Institutes of
Health).
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Received 11 December 1990; accepted with revision 3
August 1991