Pathology International 1995; 45: 914-924

Original Article

Experimental granulomatous vasculitis induced by sensitization

with Ascaris suum antigen in mice

Ntappiasse Lailo Alwie, Kunihiko Wakaki, Yoichi Korashige and Fumitomo Koizurni
Second Department of Pafhology, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama,
Japan

Experimental sensitization by repeated intramuscularinjection of Ascaris suum antigen (Ag-As) supplemented with
Freunds incomplete and complete adjuvants was carried
out in 50 BALBk CrSl c male mice (sensitized group) for
24 weeks, and the results were compared with those in a
control group of 25 mice. At the injection sites of the sensitized group, granulomatous angiitis with eosinophil infiltration was observed in all mice, and fibrinoid angiitis in
only four. By light and electron microscopic examinations
pulmonary granulomatous vasculitis with a few eosinophils
was observed at a high frequency (80%) after 12 experimental weeks. lmmunohistochemical examination revealed
pulmonary vascular and perivascular infiltration of L3/T4
(CR4) positive cells, B cells, IgG and C3 positive cells in
addition to activated macrophages, Thy-1 T cells, IgE positive cells, and lgM positive cells after 12 experimental
weeks. There were significant increases in the eosinophil
cell count of the peripheral blood, the hemagglutination
titers of the sheep erythrocytes, IgE and IgM antibodies to
Ag-As by ELISA and Western blotting after 8 experimental
weeks. After 12 experimental weeks the lgG antibody to the
Ag-As was low, but it increased significantly, and the sera
showed multiple precipitation lines to the Ag-As by the
Ouchterlony method. In conclusion, the pulmonary granulomatous vasculitis in this study is considered to consist
of allergic reactions of type IV and probably type 111 based
on type 1.

Key words: allergic angiitis, allergic granulomatosis,

animal model, Ascaris suum, eosinophilia, granulomatous
vasculitis,
hypersensitivity,
pulmonary
vasculitis,
sensitization

Correspondence: Kunihiko Wakaki, MD, Second Department of

Pathology, Faculty of Medicine, Toyarna Medical and
Pharmaceutical University, 2630 Sugitani, Toyama 930-01, Japan.
Received4 March 1994. Accepted for publication 8 August 1995.

Allergic granulomatosis and angiitis (AGA) defined by Churg

and Strauss in 1951 is a vasculitis syndrome characterized
by bronchial asthma, fever, hyperglobulinemia of IgE and
peripheral hypereosinophilia, and histologically multisystem
necrotizing vasculitis, granulomatous angiitis and extravascular granuloma with a marked eosinophil infiltration.
However, the etiology of AGA remains unclear, and there
has been only one case report of AGA (Churg-Strauss vasculitis) and ascaris infection? Furthermore, no adequate
animal model of AGA has yet been established?
To elucidate the pathogenesis of AGA, we have reported
an experimental granulomatous angiitis with eosinophil infiltration of pulmonary blood vessels in rabbits sensitized with
Ascaris suum antigen (Ag-As) emulsified with an equal
volume of Freunds incomplete or complete adjuvant (sensitization by the Ag-As) to induce allergic reactions type I
and type IV.8
The present study provides a new mouse model in which
the pathogenesis of an experimental granulomatous angiitis
with eosinophil infiltration can be analyzed more easily than
in the rabbit model.

MATERIALS AND METHODS

Homogenized Ascaris suum was centrifuged at 15 000 rpm
for 30 min at 4C. After separation from the sediment the
supernatant was filtered with a 0.45 km millipore filter,
adjusted to a protein content of 30 mg/mL by 0.01 molfL
pH 7.4 PBS and stocked in a deep freezer at -80C as the
Ag-As. The Ag-As was diluted to a protein content of 10 mgl
mL with 0.01 moVL PBS at use.
Seventy-five BALB/c CrSl c male mice aged 5 weeks were
divided into two groups, a sensitized group (50 mice) and a
control group (25 mice). In the sensitized group as shown in
Fig. 1, each mouse was injected intramuscularly into the
right posterior thigh with 0.2 mL of the Ag-As emulsified with
an equal volume of Freunds incomplete adjuvant (Nacalai

Experimental granulomatous vasculitis

sacrifice
10
10

10

10

10

(mice)

24

(weeks)

Semitized grwp

12

16

20

t t ~ t t t f t t T f f f f f t f t t f t t f f t
FCA

+ FIA
I

12

16

20

24

(weeks)

(mice)

Control group
sacrifice

7 : Ascarissourn antigen (2mg10.2mL i m . )

F I A : Freund's incomplete adjuvant (0.2mL im.)
FCA : Freund's complete adjuvant (0.2mL i.m.)
Figure 1 Experimentaldesign for sensitization.

Tesque Inc., Kyoto, Japan) at the first experimental week.

From the second experimental week, only 0.2 mL of Ag-As
was injected into each mouse once a week, and only at the
sixth experimental week each mouse was injected with 0.2
mL of the Ag-As emulsified with an equal volume of Freund's
complete adjuvant (Nacalai Tesque Inc.). From the 7th to
the twenty-fourth weeks, only 0.2 mL of the Ag-As was
injected into each mouse once a week. Ten mice were killed
at each of the 8th, 12th, 16th, 20th and 24th experimental
weeks by intracardiac puncture for blood collection under
anesthesia with diethyl ether.
In the control group each mouse was intramuscularly
injected with only 0.2 mL of Freund's incomplete adjuvant at
the first experimental week, and at the sixth week each one
was injectedwith 0.2 mL of Freund's complete adjuvant, and
followed up to 24 weeks. From the eighth experimental week
five mice were sacrificed at each of the 8th, 12th, 20th and
24th experimental weeks by the above-mentioned method
(Fig. 1).
Blood smear preparations were made just before death.
All organs were fixed in 7.4% formaldehyde for light microscopic examination. Sectioned specimens were stained with
hematoxylin-eosin (HE) and as needed by periodic acidSchiff reaction and elastica von Giesson stain. Also, several
deparaffinized sections of the injection site of the sensitized
group were immunohistochemically examined.
Small pieces of the lung were immediately frozen in
hexane with acetone-dry ice for immunohistochemicalexaminations by the avidin-biotin peroxidase complex (ABC)
method. Used antibodies were diluted as follows: anti-mouse
IgG (monoclonal antibody against mouse IgG, Sanbio BV)
1 : 100, anti-mouse IgM (monoclonal antibody against
mouse IgM, Sanbio EV) 1 : 500, anti-mouse IgE (monoclonal
antibody against mouse IgE, Sanbio BV) 1 : 500, anti-mouse
C3 (goat anti-mouse C3, Bethyl Lab Inc) 1 : 2000, antimouse Thy-1 (monoclonal antibody against Thy-1, Biosys
SA) 1 : 500, anti-B cells (monoclonal antibody against
mouse B cells, Biosys SA) 1 :500, anti-macrophage (mono-

915

clonal antibody against mouse macrophage, Biosys SA)

1 : 500, Anti-L3/T4 (CD4: monoclonal antibody against
mouse L3R4 on helpedinducer T cells, Biosys SA) 1 : 100,
anti-Lyt-1 T cell (CD5: monoclonal antibody against Lyt-I+T
cells, Biosys SA) 1:500, anti-Lyt-2 (CD8: monoclonal antibody against mouse Lyt-2 on suppressor/cytotoxic T-cells,
Biosys SA) 1 : 500, anti-mouse IL-1 alpha (monoclonal
hamster anti-mouse IL-la, Genzyme C o p ) 1 : 500, and
anti-mouse 11-2 (rat anti-murine 11-2 monoclonal, Endogen
Inc.) 1 : 100. In immunohistochemical examinations nontreated mice and two mice of the control group at each of
the 8th, 12th, 16th, 20th and 24th experimental weeks were
used as a control for specifity. The degree of appearance of
positive cells was judged as slight when 1-7 positive cells
were noted per 40 high power fields and as moderate when
eight or more positive cells were noted.
Other small pieces of the lung were fixed by conventional
methods for electron microscopic examinations. Collected
sera were used for detection of the anti Ag-As antibodies by
agar gel immunodiffusion (Ouchterlony method), and by
hemagglutination of the Ag-As coated sheep erythrocytes
using tannic acid. Measurement of each titer-of the anti AgAs antibody of IgE, IgM and IgG was determined by ELISA?
In addition, detection of each anti Ag-As antibody (IgE, IgM
and IgG) was carried out by western blotting."
Student's t-test was used for statisticalanalysis of the data
between the control group and sensitized group. Statistical
significance was defined as P < 0.05.

RESULTS

Eosinophil cell count

Regarding serial changes, in the eosinophil cell count in the
peripheral blood, each average cell count of the control
group was stationary at between 4% and 5% throughout the
experimental period. On the other hand, the eosinophil cell
count of the sensitized group significantly increased up to a
mean of 13% with a maximum of 22% at the twelfth experimental week, and then decreased gradually to the same
range as that of the control group at the twenty-fourth experimental week (Fig. 2).

Light microscopic and immunohlstochemicalff ndings

Histologically, in the muscular tissue of the injectionsites in
all mice of the sensitized group there appeared granulomatous angiitis and myositis associated diffusely with infiltration of eosinophils, lymphocytes, plasma cells,
macrophages and bizarre large monocytic cells (Fig. 3), and
scattered adjuvant granulomas.

916

M. L. Alwie et a/.

20

l16
8I

..

12

14.

12.
10.

8.

6.
4.

2.

OJ
16

20
-p<o 01, "P'O

24 weeks

02

Figure 2 Peripheral eosinophil cell counts. (0)Sensitized group,

W control group.

Deparaffinized sections were immunohistochemically

strongly positive for IgM (Fig. 4), IgE and moderately positive
for IgG, B cells, Thy-1 cells, and macrophages in the granulomatous angiitis. In a few cases (four mice) fibrinoid angiitis with marked perivascular infiltration of eosinophils,
macrophages and fibroblastic cells was also found (Fig. 5).
On the other hand, in the control group there appeared only
adjuvant granulomas with a few eosinophils, but no angiitis.
Besides the injection sites, granulomatous vasculitis
without fibrinoid necrosis appeared only in the lungs of the
sensitized group, and this vasculitis was observed in a high
proportionof the mice (28 of 35, 80%) after 12 experimental
weeks, when the eosinophil cell count of the peripheralblood
increased, whereas in the lungs of the control group, only
that of one mouse showed mild vasculitis without granulomatous cell infiltration at the 20th experimental week
(Table 1). This granulomatous vasculitis was characterized
by focal infiltration of lymphocytes, macrophages and a few
eosinophils in the vascular wall and perivascular area, and
also by swelling and slight proliferation of endothelial cells

Figure 3 (a) Angiitis and (b) myositis associated with marked infiltration of eosinophils, lymphocytes, plasma cells, macrophages. and
bizarre large monocytic cells in the local sensitized region (56th experimental day; HE),

Experimental granulomatous vasculitis

917

Figure 4 Many IgM positive cells in

the granulomatouslesion (ABC method,
Hematoxylin).

Figure 5 Fibrinoid angiitis and marked infiltration of eosinophils. macrophages, and fibroblastic cells in the local sensrtized region (112th
experimental day). (a) XIOO, (b)X Z O O (HE).

918

M. L. Alwie eta/.

Figure 6 Granulomatous vasculitis in a small pulmonary artery and arteriole of the sensitized group. (a) 84th experimental day, (b) 140th
experimental day (HE).

of the small pulmonary arteries, arterioles and small veins

(Fig. 6). The immunohistochemical findings of the frozen
sections of the lung were compared in the sensitized and
control groups. In the immunohistochemical findings we
could not strictly differentiate between positivities on the cell
membrane and in the cytoplasm. As shown in Table 2, in
the sensitized group, macrophages, Thy-1 T cells and IL-1
alpha, IgE and IgM positive cells were revealed in the perivascular regions at the eighth experimental week. On the
other hand, after 12 experimental weeks in the sensitized
group Lyt-l(CD5)T cells, Lm4(CD4)T cells, B cells, and
IL-2, IgG, and C3 positive cells were predominant in the vasTable 1 Frequency of pulmonary vasculitis: comparison between

control and sensitized groups

Groups

Vasculitis 8 w
-

Control
group

+
++
+++
-

Sensitized
group

++
+++

12 w 16 w 20 w 24 w Total(%)

5
0

5
0

4
0

4
1

5
0

0
0

0
0

0
0

0
0

0
0

3
2
3
0

0 li(37.8)
4 11(24.4)
5 15(33.3)
0 2(4.4)

1
0
0
0

2
3
3
1

2
2
4
1

23(95.8)
l(4.2)
0
0

+, slight; + +, moderate; + + +, marked; w, weeks; *, P <: 0.01.

cular and perivascular regions in addition to the abovementioned cells. Lyt-2(CDB)Tcells increased later than the
other cells (Table 2). Furthermore, the vascular walls with
vasculitis showed clear positivity for C3, IgM and IgE, and
faint positivity for IgG (Fig. 7). During the experimental
course, one mouse in the control group and five mice in the
sensitized group died, and so the data of these mice were
excluded from this result.

Electron microscopic findings

Electron microscopic findings of the vasculitis lesions in the
lung revealed an increase in pinocytic vesicles and degenerative changes of the endothelial cells. In addition, lymphocytes, plasma cells and macrophages were proved to be
inflammatory cells infiltrating the intra- and perivascular
areas (Figs 8 and 9).

Other findings by hemagglutination, Ouchterlony

method, western blotting and ELISA
In the hemagglutination findings by the Ag-As coated sheep
erythrocytes, the sera of the sensitized group showed high
titers up to 1 : 2650, compared with low titers in the control

919

Experimental granulomatous vasculitis

Table 2 lrnrnunohistochernical findings of the infiltrating cells in the vascular and perivascular regions of the lung: Comparison between
control and sensitized groups
Markers fantibodiesl
Groups

Weeks

Control
group

8-24

Sensitized
group

No.
cases

lo

vascular
perivascular

IgE

IgM

Lyt-2 LWT4 B cell

(CD8) (CD4)

IL-2

IgG

C3

--+

--+

--+

--+

--+

--+
-

--+

--+

--+

--+

vascular
perivascular

12

vascular
perivascular

16

vascular
perivascular

20

vascular
perivascular

+++
+++
+++

vascular
perivascular

+-++
++

24

+, slight;

Regions Macrophage Lyt-I Thy-I IL-la

of cell
(CD5)
infiltration

++

+ + +-+++-++
-

+
++ --+

+
+
+
+
--+
- - + +
+
+
+
--+ --+ --+
+
+
+ +-+++-++ ++ ++ + +-+++-+++-++
+
+
+
+
+
+ +-++ +
+
+
+ -- + + +
++ + + +-+++-+++-+++-++
+ + ++ +-++--++
+
+
+
+
+
+
+
+
+
+
+
+
++ + + ++ ++ ++ ++ + + ++ ++
+
+
+
+
+
+
+
+
+
+
+
+
+
+ + + + ++ ++ + + + + + + + + ++ ++ + +

+ +, moderate.

Figure 7 lrnmunohistcchemicalfindings of the infiltrating cells in the vascular and perivascular regions of the lung of the sensitized group.
*, 16 weeks; **, 24 weeks. (ABC method. Hematoxylin).

920

M. L. Alwie eta/.

Figure 8 Electron microscopic findings showing (a) increase of pinocytic vesicles and (b) degenerative change of the endothelial cells.
= 1 pm.

Bar

group. The titer of the sensitized group decreased to 1 : 640

at the 24th experimental week, compared with high titers at
the 8th and 12th experimental weeks (Table 3).
In the findings of the Ouchteriony method, the sera of the
sensitized group at the 8th experimental week showed a
single precipitation line to the Ag-As, whereas the respective
sera at the 12th, 16th, 20th, and 24th experimental weeks
showed multiple precipitation lines.
In the findings of western blotting, the sera of the sensitized group at the 8th, 12th, and 24th experimental weeks
disclosed the presence of distinctive IgE, IgM and IgG antibodies to the Ag-As (Fig. 10). The number of positive lines
of the IgG antibody to the Ag-As increased more at the
twelfth and twenty-fourth experimental weeks than at the
eighth experimental week.
In the findings of ELISA, the sera of the sensitized group
after eight experimental weeks showed a distinct increase in
the IgE and IgM antibodies to the Ag-As, as compared with
the control group (Fig. 11). The titers of IgE antibodies to
the Ag-As decreased significantly at the twenty-fourth experimental week, compared with those at the eighth experimen-

tal week. After 12 experimental weeks the titers of IgG

antibodies to the Ag-As in the sensitized group increased
significantly ( P < 0.02) in spite of the presence of low titers
throughout the experimental course (Fig. 11).

DISCUSSION
First, pulmonary vasculitis with slight eosinophil infiltration
and granulomatous perivascular infiltration of lymphocytes,
macrophages and plasma cells were observed at the 12th
experimental week in mice as well as in rabbits sensitized
by Ag-As. In addition, pulmonary vasculitis developed in a
higher proportion of the present mice (80%) than previously
reported in rabbits. This result may be due to inter-species
differences in sensitization to Ag-As.
Second, as to the relationship between pulmonary
vasculitis and peripheral hypereosinophilia or increase of
immunoglobulins in sera, hypereosinophilia (maximum 22%)
and high titers of IgE and IgM antibodies to the Ag-As by
ELSA and western blotting were observed already at the

Experimental granulomatous vasculitis

921

Figure 9 Electron microscopic findings showing various inflammatory

cells in the intra- and perivascular
areas of a pulmonary small vein. Bar =
10 pm.

Figure 10 Western blotting of Ascaris

suurn antigen (Ag-As). Lane 1 showing
marker proteins; lanes 2, 3 and 4
showing IgE antibody to AgAs (2 : Ow.,
3:12w., 4:24w.); lanes 5, 6 and 7
showing IgM antibody to Ag-As (5 : 8w..
6 : 12w., 7 : 24w.); lanes 8. 9 and 10
showing IgG antibody to Ag-As (8 : 8w..
9 : 12w.. 10 : 24w.); lane 11 showing Ag-

As.

Table 3 Hemagglutinationtiters of sheep erythrocytes coated with Ascaris suum antigen: Comparison betweencontrol and sensitized groups

Groups
Control group
Sensitized group

Weeks

<x40

x 80

x160

Titers
x 320

XMO

X1280

x2560

8 -24

11

8
12
16
20
24

0
0
0
0

0
0
0
0
0

0
0
0
0
1

0
0
1
0
1

0
0
3
2
3

6
3
0
1

1
3
2
2
0

M. t. Alwie et ai.

922

a%?,
10

9
8
7

5
.4

.3

2
.I

12

16

20

24

12

16

20

24

12
16
20
24 weeks
*:P<o.002 ' %i * : 0 . 0 0 2 < P < 0 . 0 2

Figure 11 Serial changes in anti-Ascaris suum mouse IgE, IgM and IgG by ELISA. (0)Sensitized group, W control group.

matous angiitis was not found in any organs. However, graneighth experimental week, when granulomatous vasculitis
ulomatous
vasculitis with slight eosinophil infiltration and
was not yet apparent in the lung. On the other hand, a sigwithout
fibrinoid
necrosis appeared only in the lung after 12
nificant increase in the IgG antibody to the Ag-As by ELSA
experimental weeks. This granulomatous vasculitis in the
and westem blotting was noted at the 12th experimental
lung may have been due to the small amount of circulating
week. Later, at the twenty-fourth experimental week, the
Ag-As. In the immunohistochemical examination of the lung
eosinophil cell count in the peripheral blood decreased and
at the 8th experimental week, the infiltrating cells in the perithe IgE antibody to the Ag-As in sera showed lower titers
vascular areas were predominantly macrophages, Thy-l T
than those at the eighth and twelfth experimental weeks.
cells, and IL-1 alpha, IgE and IgM positive cells. On the other
These findings indicate that type I allergic reaction was
hand, at the twelfth experimental week, when pulmonary
induced by the sensitization to Ag-As. The role of the eosinvasculitis
appeared, CD5 positive T cells, CD4 positive
ophils in the immune mechanism of angiitis remains unsetT cells, B cells, and IL-2, IgG, and C3 positive cells in additled, but circulating immune complex is associated with IgE
tion to the previously mentioned positive cells increased in
antibody.l'-l4 Increasingevidence suggests that eosinophilia
the vascular and perivascular areas. However, the degree
and IgE antibody production in certain parasitic infections
of appearance of IgM positive cells did not change between
are mediated by immune processes.'s25
the eighth and the twelfth experimental weeks. After the sixHistologically, except for adjuvant granulomas in the senteenth experimental week CD8 positive T cells increased.
sitized group after eight experimental weeks, there appeared
Although we did not examine IFNy, IL-4 or IL-5, from these
granulomatous angiitis and myositis with marked inflammaresults it may be suggested that after the Ag-As was phagotory cell infiltrationof eosinophils, lymphocytes, plasma cells
cytized by macrophages in the lung, the activated macroand macrophages in the local tissue injectedwith the Ag-As.
phages produced IL-1, which stimulatedtype 1 helper T cells
lmmunohistochemically, these inflammatory cells were pos(Th 1) and type 2 helper T cells (Th 2). Th 1 produce such
itive for antibodies against IgM, IgE, IgG, B cells, Thy-1
T cells, and macrophages. Fibrinoid angiitis was also
cytokines as IFNy, IL-2, IL-3, GM-CSF and TNF, and Th 2
revealed in four mice of the sensitized group. These histoproduce IL-3, IL-4, IL-5, IL-6, GM-CSF, and TNF.= IL-5 stimlogical findings are similar to those described in AGA?' IgE
ulated eosinophil proliferation, IL-2 and IL-4 stimulated 3
cells, IL-2 and IL-5 stimulated B cells to change into IgM
antibody might be involved in the production of the cell infiltrations mediated by the release of vasoactive amine, which
producing cells, IL-4 stimulated B cells to change into IgE
increases vascular permeability and thus facilitates the
producing cells and IL-4, IL-5 and IFNy stimulated B cells to
deposition of immune complex in the blood vessels.13-14~27change into IgG producing ce11s.30-34Furthermore, 11-2
The mechanism of granuloma formation is still unknown,
increased, followed by proliferation of activated macroalthough it may be induced by T cells relating to delayed
phages, CD4 positive T cells, B cells, IgG and C3 positive
hypersensitivity, as well as to activated macrophages?'-28-32 cells and late CD8 positive T cell^.^'^^^ Thus, the cell mediExcept for the injected local tissue, distinctive granuloated reaction of type IV allergic reaction might play a role in

Experimental granulomatous vasculitis

the development of pulmonary vasculitis, and many cytokines might participate in this r e a ~ t i o n . ~
The
. ~ ~electron
microscopic findings disclosed an increase in pinocytic vesicles and degenerative changes of the endothelial cells and
lymphocytes, plasma cells and macrophages proved to be
infiltrating cells in the intra- and perivascular areas.
After 12 experimental weeks, in the sensitized group a
significant increase in the IgG antibody to the Ag-As by
ELISA and an increase in the number of the positive lines
of precipitation by the Ouchterlony method and of the IgG
antibody to Ag-As by Western blotting were observed. It is
interesting that the increase in the IgG antibody to the AgAs may be correlated to the induction of pulmonary vasculitis. lmmunohistochemically, the vascular wall showed
distinctive positivity of C3,IgM, IgE and faint positivity of IgG.
Thus, pulmonary vasculitis in the present study may be
mediated by precipitation of immune complexes in capillary
walls.4 This might mean an allergic reaction of type 111,
although Ag-As on the vascular wall has not yet been
demonstrated. Studies are now in progress on the role of
allergic reaction of type 111.
In the hemagglutinationfindings of the sheep erythrocytes
coated with Ag-As, the titers at the 24th experimental week
were decreased as compared with those at the 8th and 12th
experimental weeks, similar to the serial changes in the level
of IgM antibody to the Ag-As. However, the relationship
between IgM and pulmonary vasculitis is not clear.
This mouse model in the present study is useful to analyze
the pathogenesis of an experimental granulomatous vasculitis with eosinophil infiltration.

ACKNOWLEDGMENTS
This work was supported by a Grant-in-Aid from the
Research Committee on Intractable Vasculitis, Ministry of
Health and Welfare of Japan.

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