Experimental Granulomatous Vasculitis Induced by Sensitization With Ascaris Suum Antigen

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 intramuscular injection of Ascaris suum antigen (Ag-As) supplemented with Freund‘s 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.

Received 4 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 extravas- cular 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 Freund‘s 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 4°C. 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 -80°C 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 Freund‘s incomplete adjuvant (Nacalai

Experimental granulomatous vasculitis 91 5

sacrifice 10 10 10 10 10 (mice)

Semitized grwp 4 8 12 16 20 24 (weeks)

I I I I I I I I

t t ~ t t t f t t T f f f f f t f t t f t t f f t + FIA + FCA

I I I I I I I 4 8 12 16 20 24 (weeks)

Control group 5 5 5 5 5 (mice)

sacrifice

7 F I A : Freund's incomplete adjuvant (0.2mL im.) FCA : Freund's complete adjuvant (0.2mL i.m.)

: Ascarissourn antigen (2mg10.2mL im.)

Figure 1 Experimental design 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 injected with 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 acid- Schiff 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 immunohistochemical examinations 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, anti- mouse 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-

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 non- treated 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 Ag- As 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 statistical analysis 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 immunohlstochemical ff ndings

Histologically, in the muscular tissue of the injection sites 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/.

14.

12.

10.

8.

6.

4.

2.

% 20

l 8 I 16 .. T

OJ 24 weeks 8 12 16 20

-p<o 01, "P'O 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 proportion of the mice (28 of 35, 80%) after 12 experimental weeks, when the eosinophil cell count of the peripheral blood 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),


Figure 4 Many IgM positive cells in the granulomatous lesion (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 vas-

Table 1 Frequency of pulmonary vasculitis: comparison between control and sensitized groups

Groups Vasculitis 8 w 12 w 16 w 20 w 24 w Total(%) -

Control + group ++

+++ -

Sensitized + group ++

+++

5 5 4 4 5 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0

1 0 2 2 3 0 0 3 2 2 4 0 3 4 3 5 0 1 1 0 0

3 23(95.8) l(4.2) 0 0

li’(37.8) 11 (24.4) 15(33.3) 2(4.4)

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

cular and perivascular regions in addition to the above- mentioned cells. Lyt-2(CDB)T cells 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


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 No. Regions Macrophage Lyt-I Thy-I IL-la IgE IgM Lyt-2 LWT4 B cell IL-2 IgG C3 cases of cell (CD5) (CD8) (CD4)

infiltration

vascular - - - - - - - ~ - - - -

lo perivascular - -+ --+ - -+ --+ --+ + --+ - -+ --+ --+ --+ - 8 -24 Control

group

Sensitized group

- - - - - - - vascular - - + - -

perivascular + + + + + +-+++-++ ++ --+ + + + - --+ vascular - - + + + + + - --+ --+ --+ + +

vascular + + + + +-++ + + + + -- + + +

vascular + + + + + + + + + + +

vascular +-++ + + + + + + + + + + +

8

12 3 perivascular +++ + +-+++-++ ++ ++ + +-+++-+++-++ + + 16 3 perivascular +++ ++ + + +-+++-+++-+++-++ + + + + +-++- -++ + 20 3 perivascular +++ ++ + + ++ ++ ++ ++ + + ++ ++ + +

24 perivascular ++ ++ + + ++ ++ + + + + + + ++ ++ ++ + + +, slight; + +, moderate.

Figure 7 lrnmunohistcchemical findings 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. Bar = 1 pm.

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 Hemagglutination titers of sheep erythrocytes coated with Ascaris suum antigen: Comparison between control and sensitized groups

Groups Titers

Weeks <x40 x 80 x160 x 320 X M O X1280 x2560

Control group 8 -24 11 3 0 0 0 0 0

Sensitized group 8 0 0 0 0 0 6 1 12 0 0 0 0 0 3 3 16 0 0 0 1 3 0 2 20 0 0 0 0 2 1 2 24 0 0 1 1 3 0 0

922 M. t. Alwie et ai.

a%?, 1 0

9

8

7

6

5

.4

.3

2

. I

0 8 12 16 20 24 8 12 16 20 24 8 12 16 20 24 weeks

*:P<o.002 ' %i *:0 .002<P<0.02

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

eighth experimental week, when granulomatous vasculitis was not yet apparent in the lung. On the other hand, a significant increase in the IgG antibody to the Ag-As by ELSA and westem blotting was noted at the 12th experimental week. Later, at the twenty-fourth experimental week, the eosinophil cell count in the peripheral blood decreased and the IgE antibody to the Ag-As in sera showed lower titers than those at the eighth and twelfth experimental weeks. These findings indicate that type I allergic reaction was induced by the sensitization to Ag-As. The role of the eosinophils in the immune mechanism of angiitis remains unset- tled, but circulating immune complex is associated with IgE antibody.l'-l4 Increasing evidence suggests that eosinophilia and IgE antibody production in certain parasitic infections are mediated by immune processes.'s25

Histologically, except for adjuvant granulomas in the sensitized group after eight experimental weeks, there appeared granulomatous angiitis and myositis with marked inflammatory cell infiltration of eosinophils, lymphocytes, plasma cells and macrophages in the local tissue injected with the Ag-As. lmmunohistochemically, these inflammatory cells were positive for antibodies against IgM, IgE, IgG, B cells, Thy-1 T cells, and macrophages. Fibrinoid angiitis was also revealed in four mice of the sensitized group. These histo- logical findings are similar to those described in AGA?' IgE antibody might be involved in the production of the cell infil- trations mediated by the release of vasoactive amine, which increases vascular permeability and thus facilitates the deposition of immune complex in the blood vessels.13-14~27 The mechanism of granuloma formation is still unknown, although it may be induced by T cells relating to delayed hypersensitivity, as well as to activated macrophages?'-28-32 Except for the injected local tissue, distinctive granulo-

matous angiitis was not found in any organs. However, granulomatous vasculitis with slight eosinophil infiltration and without fibrinoid necrosis appeared only in the lung after 12 experimental weeks. This granulomatous vasculitis in the lung may have been due to the small amount of circulating Ag-As. In the immunohistochemical examination of the lung at the 8th experimental week, the infiltrating cells in the perivascular areas were predominantly macrophages, Thy-l T cells, and IL-1 alpha, IgE and IgM positive cells. On the other hand, at the twelfth experimental week, when pulmonary vasculitis appeared, CD5 positive T cells, CD4 positive T cells, B cells, and IL-2, IgG, and C3 positive cells in addition to the previously mentioned positive cells increased in the vascular and perivascular areas. However, the degree of appearance of IgM positive cells did not change between the eighth and the twelfth experimental weeks. After the six- teenth experimental week CD8 positive T cells increased. Although we did not examine IFNy, IL-4 or IL-5, from these results it may be suggested that after the Ag-As was phago- cytized by macrophages in the lung, the activated macrophages produced IL-1, which stimulated type 1 helper T cells (Th 1) and type 2 helper T cells (Th 2). Th 1 produce such cytokines as IFNy, IL-2, IL-3, GM-CSF and TNF, and Th 2 produce IL-3, IL-4, IL-5, IL-6, GM-CSF, and TNF.= IL-5 stimulated eosinophil proliferation, IL-2 and IL-4 stimulated €3 cells, IL-2 and IL-5 stimulated B cells to change into IgM producing cells, IL-4 stimulated B cells to change into IgE producing cells and IL-4, IL-5 and IFNy stimulated B cells to change into IgG producing ce11s.30-34 Furthermore, 11-2 increased, followed by proliferation of activated macrophages, CD4 positive T cells, B cells, IgG and C3 positive cells and late CD8 positive T cell^.^'^^^ Thus, the cell mediated reaction of type IV allergic reaction might play a role in

Experimental granulomatous vasculitis 923

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 Ag- As 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 hemagglutination findings 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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Experimental Granulomatous Vasculitis Induced by Sensitization With Ascaris Suum Antigen

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