and interleukin-5 in antigen-induced eosinophil recruitment into the site of cutaneous late-phase reaction in mice. Itsuo Iwamoto,. Sanae. Tomoe, Hisao Tomioka ...
Role of CD4
T lymphocytes
and interleukin-5
antigen-induced eosinophil recruitment cutaneous late-phase reaction in mice Itsuo
Iwamoto,
Second Institute
Department for Medical
Abstract: recruitment (LPR) was this study,
Sanae
Tomoe,
Hisao
Tomioka,
of Internal Medicine, Chiba University Immunology, Kumamoto University
Previous studies suggested that the eosinophil into the site of cutaneous late-phase reaction dependent on IgE antibody and mast cells. In we determined the role of CD4 T cells and
sites
Key
J.
Words:
CD8
T cells
LPR 52: .
mast
is mediated 572-578; cells
.
by 1992.
mast
the
are
late-phase associated
reactions with
(LPRs) provoked intense eosinophil
572
was
Journal
an increase
of Leukocyte
in lymphocytes
Biology
infiltrating
Volume
52,
November
is
Moreover,
of LPR
not
yet
*Department
there
in the
known
is an
skin
of Biology,
increase
[6] and
whether
T
in CD4 [7].
airways
lymphocytes
and
AND
and
METHODS
immunization
Reagents anti-L3T4
mAb produced
factor
into
sites
Yoshida
and
BALB/c mice were purchased from Charles River Laboratonies (Shizuoka, Japan) and were used at 6-8 weeks of age. The mice were immunized intrapenitoneally twice with 1 g of ovalbumin (OVA) (Sigma Chemical Co., St. Louis, MO) in alum at a 2-week interval. At 10 to 14 days after the second immunization, OVA was administered subcutaneously as described below. The titer of anti-OVA IgE antibody in sera of immunized mice assessed by passive cutaneous anaphylaxis reaction was 80- to 160-fold dilution positive.
mAb
53.6-72
GK1.S
(IgG2a) the
from
(IgG2b)
[11] and
[12] were hybnidomas
rat
anti-Lyt-2
purified from by ammonium
ascites sulfate
precipitation. Rat anti--munine IL-S mAb was obtained from the hybnidoma NC17 and prepared as described previously [13]. Purified rat IgG was purchased fromJackson Immunoresearch Laboratories (West Grove, PA). Disodium cromoglycate was obtained from Fujisawa Pharmaceutical
aninto
Co. line
site
there
it
Mice
of antigen administration [1-3]. LPR occurs 3 to 8 h after antigen administration and persists for up to 48 h in a variety of tissues including the skin and airways. There is increasing evidence that the infiltrating eosinophils cause the tissue damage in LPR by releasing the cytotoxic granules and membrane products [4]. Previous studies suggested that the eosinophil infiltration in cutaneous LPR was dependent on immunoglobulin E (IgE) antibody and mast cells [1-3]. In addition to the infiltration of eosinophils, it was shown that
[2, 3, 5].
at the
MATERIALS
Rat
by specific infiltration
Sho
and neutralizing IL-S in vivo with their corresponding monoclonal antibodies (mAbs). We provide direct evidence that CD4 T cells and IL-S mediate the second peak of antigen-induced eosinophil recruitment of cutaneous LPR. In contrast, we have also demonstrated that the first peak of antigen-induced eosinophil recruitment of cutaneous LPR is mediated by mast cells and platelet-activating factor (PAF).
INTRODUCTION Allergic tigens
ofLPR
However,
cells
platelet-activating
and
their cytokines, especially interleukin-5 (IL-5) [8-10], mcdiate antigen-induced eosinophil recruitment into the site of LPR. To solve this issue, we studied the role ofCD4 T cells, CD8 T cells, and IL-S in antigen-induced eosinophil infiltration into mouse skin by depleting both T cell subsets
decreased the first peak of OVA-induced cutaneous eosinophilia in the mouse. These results indicate that CD4 T cells, but not CD8 T cells, cause the second peak of antigen-induced eosinophil recruitment of cutaneous LPR and that IL-S mediates this eosinophil recruitment. In contrast, the first peak of antigen-induced eosinophil of cutaneous Leukoc. Biol.
Takatsu,’
of Medicine, Chiba, Japan School, Kumamoto, Japan
T cells
CD8 T cells in causing antigen-induced eosinophil recruitment of LPR in mouse skin. Eosinophil infiltration into the subcutaneous tissue of ovalbumin (OVA)sensitized BALB/c mice was biphasic, reaching the first peak at 6 h after the subcutaneous challenge with OVA and the second peak at 24 to 48 h. The in vivo depletion of CD4 T cells by pretreatment with anti-L3T4 monoclonal antibody (mAb) significantly decreased the second peak (at 24 h and 48 h), but not the first peak (at 6 h), of OVA-induced eosinophil infiltration into the skin of OVA-sensitized mice. However, the depletion of CD8 T cells by pretreatment with anti-Lyt-2 mAb had no significant effect on either the first peak or second peak of OVA-induced cutaneous eosinophilia. Pretreatment with anti-murine interleukin-5 (IL-5) mAb also decreased the second peak, but not the first peak, of OVAinduced cutaneous eosinophilia. In contrast to the inhibitory effects of depletion of CD4 T cells and of anti-IL-S mAb on the second peak of antigen-induced cutaneous eosinophilia, disodium cromoglycate and a selective antagonist for platelet activating factor (PAF) CV-6209
recruitment and PAF.
into the site of
Kiyoshi School Medical
in
(Osaka, (PBS).
Japan) A selective
Abbreviations: activated phase
cell
DTH, sorter;
reaction;
delayed-type IgE,
mAb,
and dissolved in phosphate-buffered PAF antagonist, CV-6209 (2-[N-acetyl-
hypersensitivity;
immunoglobulin
monoclonal
E;
antibody;
OVA,
activating factor; PBS, phosphate-buffered Reprint requests: Itsuo Iwamoto, Second cine, Chiba University School of Medicine, Chiba
260,
Received
the
1992
4, 1992;
accepted
July
FACS, interleukin; ovalbumin;
fluorescenceLPR, PAF,
saline. Department of Internal 1-8-1 Inohana, Chiba
Japan.
May
IL,
1, 1992.
sa-
late-
plateletMcdi-
City,
N-(2-methoxy-3-octadecyl-carbamoyloxypropoxycarbonyl) aminomethyl]-1-ethylpynidinium chloride) from Takeda Pharmaceutical Co. (Osaka, solved in 0.9% saline.
[14], was Japan)
aminoobtained and dis-
Effect of inhibitor of mast cell degranulation antagonist of platelet-activating factor To
determine
philia
Antigen-induced
eosinophil
infiltration
in mouse
Eosinophil infiltration into the skin was induced cutaneous antigen challenge in sensitized mice, number of eosinophils infiltrating the subcutaneous was measured by the method described by Lawman [15]. Briefly, 0.9 ml of air was slowly injected into cutaneous tissue of the left flank of OVA-sensitized mice and
using then
syringe control,
a 1-ml 0.1 ml
tuberculin of OVA
syringe (50 g/ml)
with
a 26-gauge present in
by and
subthe tissue et al. the subBALB/c needle, same
successively introduced into the air bleb. As a alone was injected into the air bleb. At various intervals after the injection, the thin membrane of the air bleb was carefully excised after sacrifice by cervical dislocation and was stretched onto a glass slide. After drying, the specimen was fixed with methanol and stained with WrightGiemsa solution. The slides were coded and then five fields at a magnification of x400 were examined by one observer (S.T.), who was unaware of the identify of the individual specimens. The number of eosinophils was expressed as the sum of eosinophils in the five fields.
Effect
was PBS
of anti-L3T4
To determine
mAb
whether
and
anti-Lyt-2
T lymphocytes
are
T
cells
and
CD8
T
cells
involved
was
confirmed
by
of CD4
Pretreatment numbers of PBS-pnetreated
T cells and CD8 T cells, respectively. purified rat IgG induced no changes in either T cell population compared to those in mice. Eosinophil infiltration into the skin
with
was evaluated at 6, 24, and 48 h after the challenge in those three groups of mice.
Effect
of anti-murine
IL-5
we
examined
cutaneous
eosino-
the
of diso-
effect
cnornoglycate, an inhibitor of mast cell degranulation [16-18], on OVA-induced eosinophil infiltration in mouse skin. We also evaluated mast cell degnanulation according to the criteria described by Wershil et al. [19] by examining 200 mast cells in the dermis under a light microscope at a magnification of x 1000. In these experiments, disodium cromoglycate (10 tg) or vehicle (PBS) was administered subcutaneously
air
with
OVA
(S g;
total
volume
0.1
ml)
into
the
bleb.
To determine whether PAF mediates antigen-induced eosinophil infiltration into the skin, we examined the effect of a PAF antagonist, CV-6209 [14], on OVA-induced eosinophil infiltration in mouse skin. CV-6209 (1 jg/g) or vehicle (saline) was administered intrapenitoneally 30 mm before the subcutaneous injection of OVA (S pg). We chose this dose of CV-6209 because CV-6209 (1 g/g) completely protected mice against PAF-induced anaphylactic death [14]. Eosinophil infiltration was then evaluated at 6 and 24 h after the injection of OVA. The effect of CV-6209 was also examined at 48 h after the injection of OVA. In these expeniments, CV-6209 (1 g/g) was administered intraperitoneally 30 mm before and 24 h after the subcutaneous injection of
Data
in causing
fluorescence-activated cell sorter (FACS) analysis using spleen cells prepared from the mice 2 days after treatment with the anti-L3T4 mAb or anti-Lyt-2 mAb. The FACS analysis by use of another anti-L3T4 mAb YTS 191.1 and anti-Lyt-2 mAb YTS 169.4 revealed that the pretreatment with anti-L3T4 mAb and anti-Lyt-2 mAb caused complete depletion
antigen-induced
dependent,
OVA.
mAb
antigen-induced eosinophil infiltration into the tissue, we cxamined the effect of the in vivo depletion of CD4 T cells on CD8 T cells on antigen-induced cutaneous eosinophilia in sensitized mice. OVA-sensitized BALB/c mice were injected intrapenitoneally once with 1 mg ofanti-L3T4 mAb (GK1.5) [11] 2 days before the subcutaneous challenge with OVA (5 pg). Other OVA-sensitized mice were given 1 mg of anti-Lyt-2 mAb (53.6-72) [12] intrapenitoneally twice at a 2-day interval and then challenged subcutaneously with OVA 2 days later. As a control, OVA-sensitized mice were injected intnapenitoneally with purified nat IgG (1 mg) 2 days before the subcutaneous OVA challenge. The depletion of
CD4
cell
dium
skin
the
whether
is mast
and
subcutaneous
OVA
Analysis
Data are summarized as mean ± SD. Inhibition infiltration by a given treatment was expressed 1 [(OVA-induced response with the treatment induced nesponse)/(OVA-induced response responding vehicle PBS-induced response)]. analysis of the results was performed by analysis using Fisher’s least significant difference test comparisons [20]. P values < .05 were considered
RESULTS
Kinetics mouse
after after course
Since IL-S has been shown to be chemotactic for eosinophils in mice [10], the effect ofanti-munine IL-S mAb on antigeninduced cutaneous eosinophilia was examined in sensitized mice. An anti-murine IL-S mAb NC17 (1 mg) [13] on purified rat IgG (1 mg) was injected intrapenitoneally 24 h before the subcutaneous challenge with OVA (S sg). Eosinophil infiltration into the skin was then evaluated at 6, 24, and 48 h after the subcutaneous OVA challenge.
Iwamoto
et aL
of antigen-induced skin
eosinophil
infiltration
into
Subcutaneous administration of OVA caused biphasic infiltrations of eosinophils into the skin of OVA-sensitized mice. Eosinophil infiltration into the subcutaneous tissue of OVA-sensitized BALB/c mice occurred 4 h after the subcutaneous injection of OVA (S pg), reached the first peak 6 h after the injection, and then declined at 12 h (n = S mice at each time point) (Fig. 1). The antigen-induced eosinophil infiltration into mouse skin again increased at 24 and 48 h the
1). In
mAb
of eosinophil as percent of PBSwith a corStatistical of variance for multiple significant.
injection,
addition,
reaching
there
the subcutaneous of cutaneous
was
the
injection eosinophilia
Effect of depletion of CD4 antigen-induced cutaneous The
in vivo depletion of CD4 anti-L3T4 rnAb decreased the but not the first peak (at 6 h), eosinophilia in the mouse. The with anti-L3T4 mAb (GK1.S;
CD4
T cells
and
IL-S
peak (n = 5) (Fig. blood eosinophilia of OVA during the entire in the OVA-sensitized mice. second
no significant
T cells and eosinophilia
CD8
T cells
on
T cells by pretreatment with second peak (at 24 and 48 h), of antigen-induced cutaneous intrapenitoneal preinjection 1 mg) 2 days before the sub-
in antigen-induced
eosinophilia
573
Eosinophils (number/5 fields)
Eosinophils (number/5 fields) 100
120
80
100
80
60
60
40
40 20
20
0 6
12
18
24
Time
30
36
42
0
48
6h
(h) Fig.
Fig.
1.
skin.
OVA
Time
course
(5 g;
subcutaneous sinophils
of antigen-induced
solid
tissue
squares) into
the
Open
circles
sensitized
mice.
The
number
five
fields
SD
for
in
five
the
mice
PBS
of OVA-sensitized
infiltrating
injection. sinophils
or
eosinophil
skin
indicate
at each
was
(open
squares)
BALB/c
mice,
counted
at
OVA-induced
of eosinophils at
was
and
various
eosinophil
was
a magnification
time
infiltration
mouse
injected
the
into
number
intervals
x400.
Data
are
un-
of anti-murine
mice.
Data
on
antigen-induced
anti-L3T4
mAb
before
the
mice
(GKI.5)
(solid
subcutaneous
eosinophil
were
injected
columns)
or
challenge
with
in-
IgG OVA (5
rat
after
the
subcutaneous
are
means
±
mean
SD value
challenge
with
for six mice in each of the corresponding
PBS
in
OVA-sensitized
group. control
Significantly response
(rat
Eosinophils (number/5 fields)
mAb
induced eosinophil infiltrations into the mouse 48 h by 65 and 64%, respectively (n = 6, P < which were similar to the degrees of decrement pretreatment with anti-L3T4 mAb (Fig. 2). mAb
T cells
OVA-sensitized
the simultaneous subcutaneous administration of disodiurn cromoglycate (10 zg) with OVA decreased OVA-induced eosinophil infiltration into the mouse skin at 6 h by 55% (PBS 81.0 ± 12.2 vs. disodium cromoglycate 38.7 ± 6.6 eosinophils/five high-power fields, n = 6, P < .001) (Fig. 5). The administration of disodium cromoglycate also significantly decreased mast cell degranulation induced by subcutaneous OVA challenge at 6 h (Table 1). However, disodium cromoglycate only slightly affected OVA-induced cutaneous eosinophilia at 24 h (n = 6, P < .05) (Fig. 5). The intrapenitoneal pretreatment with the selective PAF antagonist CV-6209 (1 g/g) 30 mm before the subcutaneous injection of OVA also decreased OVA-induced eosinophil
Pretreatment with anti-murine IL-S mAb also decreased the second peak, but not the first peak, of antigen-induced cutaneous eosinophilia in the mouse. The intrapenitoneal pneinjection with anti-munine IL-S mAb (NC17; 1 mg) 24 h before the subcutaneous OVA challenge decreased OVA-
preinjection with anti-IL-S the first peak of OVA-induced skin (Fig. 4).
of CD4’ skin.
2 days
different from the IgG), P < .001.
point.
lL-5
with
columns)
infiltrating
±
cutaneous injection ofOVA had no significant effect on OVA (5 sg)-induced eosinophil infiltration into the skin of OVAsensitized mice at 6 h (n = 6) (Fig. 2). In contrast, the intrapenitoneal preinjection with anti-L3T4 mAb significantly decreased OVA-induced eosinophil infiltration into the skin at 24 h by 67% (control rat IgG 113.5 ± 9.8 vs. anti-L3T4 mAb 41.6 ± 6.1 eosinophils/five high-power fields, mean ± SD, n = 6, P < .001) (Fig.2). The intrapenitoneal preinjection with anti-L3T4 mAb also decreased the OVA-induced eosinophil infiltration at 48 h by 68% (control rat IgG 99.3 ± 12.5 vs. anti-L3T4 mAb 35.2 ± 4.5 eosinophils/five high-power fields, n = 6, P < .001) (Fig. 2). However, depletion of CD8 T cells by pretreatment with anti-Lyt-2 mAb (53.6-72; 1 mg, twice) had no significant effect on either the first or second peak of OVA-induced cutaneous eosinophilia in the mouse (Fig. 3).
Effect
mouse
48h
ag). The number of eosinophils infiltrating into the skin was then counted at 6, 24, and 48 h. Open columns indicate the number of eosinophils
of eo-
means
of depletion in
(hatched
the
in
Effect
traperitoneally
of eo-
as the sum
2.
infiltration
the
after
infiltration
expressed
of
into
24h
did not eosinophil
120 100
80
skin at 24 and .001) (Fig. 4), observed for However, the
significantly infiltration
60 40
affect in the
20 0
Effects
of disodium
In contrast
cromoglycate
and
6h
PAF antagonist Fig. 3. Effect of depletion infiltration in mouse
to the
inhibitory effects of anti-L3T4 mAb and anti-IL-S mAb on the second peak of antigen-induced cutaneous eosinophilia, disodium cromoglycate, an inhibitor of mast cell degranulation, decreased the first peak of antigen-induced cutaneous eosinophilia in the mouse. Thus,
574
Journal
of Leukocyte
Biology
Volume
52,
November
traperitoneally
twice
with
24h
of CD8’ T cells skin. OVA-sensitized anti-Lyt-2
mAb
on
antigen-induced mice were
(53.6-72)
2-day interval and then challenged subcutaneously Other experimental protocols and symbols are are means ± SD for six mice in each group.
1992
48h
(solid
with the
eosinophil injected in-
same
columns)
at
OVA
2 days
later.
as
Fig.
Data
in
2.
a
Eosinophils
Eosinophils (number/5
(number/5
fields)
I 20
fields) 120
-
1 00
100
80
80
60
60
40
40
20-
20
0-
0 6h
Fig.
4.
Effect
infiltration
of in
mouse
traperitoneally
IgG
with
(hatched
are
means
the
mean
anti-murine
±
24h
IL-S
skin.
mAb
on
antigen-induced
OVA-sensitized
anti-murine
IL-S
48h
mice
mAb
(NCI7)
were
value
of
the
corresponding
infiltration CV-6209
into the skin 40.1 ± 8.4 n = 5, P < .001) (Fig. not affect OVA-induced at 24 or at 48 h (n =
control
response
injected
(solid
columns) 24 h before the subcutaneous OVA SD for six mice in each group. Significantly (rat
6h
eosinophil columns)
Fig. in-
or
(Fig.
rat
challenge. Data different from IgG), P < .001.
sensitized
.
Injection
of
Di sodium
Cromoglycate
on
mice, at
T cell
and
6 and
24
the
number
h.
involvement
OVA
(S ;Lg)
‘Mast
cell
described
g)
4
PBS
+
Diso dium
degranulation
was
by
et
Wershil
(10 g)
Cromoglycate
classified al.
[191.
morphologically Data
are
means
as extensively ±
SD
for
recruitment our findings
Iwamoto
subcutaneously means the mean < .001.
eosinophil columns)
with infiltrating
±
SD
value
or
OVA into
for six mice of the
induction
of LPR
that
in LPR [6, 7], and they at 24 and 48 h. However,
there
is no significant antigen-induced
T cells
and
skin
6 h (Fig.
Antigen
at
-Induced
in
the
PBS OVA-
skin
in each
was
group.
corresponding
control
is in contrast
Mast
are
to
also consistent with our findings indicate
direct connection between eosinophil infiltration
into
CD4 the
2).
Cell
Degranulation cell
in
Mouse
degranulation
Skin
(%)
Slight/moderate
Extensive
91.9 47.9
±
4.8
5.7
±
2.1
±
1.3
±
6.5
20.4
±
4.7
31.7
±
5.6
78.2
±
9.2
10.5
±
3.5
11.3
±
4.7
degranulated, six
are
in the
None
(S
(solid
of eosinophils
Data
Mast
PBS
antigen-induced
cromoglycate
administered
different from P < .05,
.
OVA
Disodium
on
a previous notion that the entire LPR is due to IgE and mast cells [1-3]. Frew and Kay [6] found that numerous eosinophil infiltrates were present at 6 h at the site of allergen challenge in human skin of atopic patients and persisted for up to 48 h and that these infiltrates were associated with a significant increase in the infiltration of CD4 T cells at 6 to 48 h. Other indirect evidence of CD4 T cell involvement in antigen-induced eosinophil infiltration of LPR was an increase in both CD4 T cells and eosinophils in bronchoalveolan lavage fluid 48 h after segmental bronchial challenge [7]. These findings suggested that there might be some connection between the infiltrating CD4 T cells and the eosinophil
with
as
skin. was
24h
cromoglycate
of CD4 T cells and IL-S in the second peak of antigeninduced eosinophil recruitment of cutaneous LPR, the first peak is dependent on mast cells and PAF, as indicated by the inhibitory effects of disodium cromoglycate [16-18] and the selective PAF antagonist CV-6209 [14] at 6 h (Figs. S and 6). Our finding of the preventive effect of the depletion of CD4 T cells provides direct evidence that CD4 T cells are involved in the induction of antigen-induced eosinophil recruitment in cutaneous LPR. The present finding of CD4
6).
Effect
mouse
., * Significantly response (PBS),
of cutaneous LPR, as indicated by the of the in vivo depletion of CD4 T cells, but cells, on antigen-induced eosinophil infiltraskin at 24 and 48 h (Figs. 2 and 3). We also mediates this eosinophil recruitment in the LPR (Fig. 4). Finally, in contrast to the role
I.
disodium
columns)
counted
The development of mAb to T cell subsets has made it possible to deplete selectively specific T cell subsets in vivo [21, 22]. This approach has been used successfully in several cxpenimental models to assess the role of T cell subsets in the immunologic control of microbial infections [23], allognaft rejection [24], and autoimmunity [25, 26]. Therefore, since no direct evidence has been provided for the functional importance of CD4 and CD8 T cell subsets in causing antigen-induced eosinophil recruitment into the site of LPR, we have applied this approach to antigen-induced cutaneous eosinophilia in the mouse. In this study, we show that CD4 T cells play an important role in causing the second peak of antigen-induced eo-
TABLE
of
in
(hatched
DISCUSSION
sinophil recruitment preventive effect not of CD8 T tion into mouse show that IL-S second peak of
Effect
infiltration
at 6 h by 56% (saline 87.3 ± 14.4 vs. eosinophils/five high-power fields, 6). Pretreatment with CV-6209 did eosinophil infiltration in mouse skin 5)
5.
mice
et al.
in
slightly
each
CD4
to moderately
degranulated,
2.4
or
none
according
to the
criteria
group.
T cells
and
IL-S
in antigen-induced
eosinophilia
575
Eosinophils (number/5 fields)
cells dominantly Thl-type T cell
120
80 60 40
-
20
-
0-
6h 6. Effect mouse
with 30
a PAF
mm
of
skin.
means mean
and
SD
±
value
By
PAF
antagonist
OVA-sensitized
antagonist
before
CV-6209 24
h after
24h on
antigen-induced
mice
were
(solid
columns)
the
our
results
eosinophil
injected
for
the
“8
“
-?
I U
infiltration
intraperitoneally
or saline
subcutaneous
for five mice in each group. of the corresponding control
contrast,
OVA
twice
(hatched challenge.
Significantly different response (saline), P
