Mouse mammary tumor virus superantigens and murine autoimmune gastritis

Mouse Mammary Tumor Virus Superantigens Autoimmune Gastritis DIRK Cl_AEYS,*

OCHINE

HANS ACHA-ORBEA,§

KARAPETIAN,*

EMILIA

SARAGA,’ MAGALI

ANDRl? L. BLUM,” and JEAN-PIERRE

and Murine

SCHREYER,§

JACQUES

LOUIS,”

KRAEHENBUHL*

*Swiss Institute for Experimental Cancer Research and Institute of Biochemistry, University of Lausanne. Epalinges; tlnstitute of Pathology, University of Lausanne. Lausanne; “Ludwig Institute for Cancer Research, Lausanne Branch, Epalinges; “World Health Organization Immunology Research and Training Center, Institute of Biochemistry, Epalinges; and ‘Division of Gastroenterology, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland

Background/Aims: Neonatal thymectomy induces autoimmune gastritis in BALB/c (minor lymphocyte-stimulating antigen [ MIS]-lb) mice, whereas DBA/2 (MIS-~“) mice are resistant. Resistance has been linked to the Mls-1’ locus, which encodes a retroviral superantigen, and to superantigen reactive T cells that express V,6+ Tcell receptors. V,6+ T cells are known to be deleted in mice expressing MIS-1” superantigens. Methods: Neonatal thymectomized BALB/c and MIS-~” congenic BALB.D2.Mls-1” mice were analyzed to examine directly the role of MIS-1” self-superantigens and V,6’ T cells in autoimmune gastritis. Results: Autoimmune gastritis was detected in thymectomized BALB.D2.Mls1” mice with high incidence. Autoantibodies to the gastric H+,K+-adenosine triphosphatase were present independent of the MIS phenotype in sera of gastritic mice. Severe gastritis had already appeared 1 month after thymectomy in BALB.D2.Mls-1” mice. V,6+ T cells were deleted in the stomach lymph nodes of l-monthold gastritic BALB.D2.Mls-1” mice but could be de tected by immunocytochemistry in the stomach lesions. Conclusions: Endogenous MIS-1” self-superantigens and Mls-1” reactive V,6+ T cells are not involved in resistance to autoimmune gastritis in BALB.D2 mice.

T cells are not deleted the periphery

but migrate

before the suppressor

nTx at the critical

from the thymus

age of 3 days prevents

of the latter

T-cell

subset

autoimmune

response.”

the maturation

and consequently

The antigen

to

T cells. Presumably, induces

specificity

an

of the T

cells that transfer AIG is not known, but CD4+ T cells are required for induction of the disease.” Interestingly, it has been shown that the transgene expression of the p subunit of the H+,K+-ATPase in the thymus of BALB/c mice prevents the induction of AIG by nTx.” A genetic

study linked

the resistance

to murine

AIG

to the minor lymphocyte-stimulating (MIS-~“) locus, although the involvement of other genetic factors could not be ruled mammary

out.’ The Mls-1”

tumor

locus encodes

virus endogenous

superantigen

a mouse

(Mtv-

7). This self-antigen in conjunction with major histocompatibility complex class II molecules induces activation, deletion, or anergy of T cells with specific Vp segments in their T-cell receptor.” For example, peripheral CD4+ and CD8+ T cells expressing Vb6 are known to be deleted within 10 days after birth in the Mlsla-expressing

mice, whereas

these lymphocytes

are not

after neonatal

deleted in BALB/c mice (Mls-lb).‘” The resistance of DBA/2 mice (Mls-1”) to post-nTx AIG has been related to the lack of Vb6+ T cells, whereas clonally expanded

thymectomy (nTx) is the best defined animal model to study human type A chronic atrophic gastritis.’

Vg6+ T cells were proposed to mediate AIG in BALB/c mice.’ Later, these observations were contradicted by the

AIG in mice, as in humans, affects predominantly the mucosa of the body (fundus)2-4 and is accompanied by parietal cell-specific autoantibodies that recognize the cx and p subunits of the gastric proton pump (H+,K+adenosine triphosphatase lATPase)).5*6 The incidence of post-nTx AIG in BALB/c mice ranges between 30% and SO%, whereas DBA/2 mice are resistant.798 Several adoptive transfer experiments have shown that the autoreactive T cells able to induce gastritis are present but suppressed in normal adult mice.“,” These studies also have shown that in neonatal mice, the autoreactive

same investigators. To examine the role of Mls-1” and clonal deletion of Vp6’ T cells in the susceptibility to post-nTx AIG, we have performed thymectomy on both BALB/c and Mls1” congenic BALB.D2 mice, a strain that differs only at

M

urine

autoimmune

gastritis

(AIG)

14.15

Abbreviations usedin thispaper:AIG, autoimmune gastritis; FACS, fluorescence-activated cell sorter; MIS, minor lymphocytestimulating antigen; MW, mouse mammary tumor virus; nTx, neonatal thymectomy; PAS, periodic acid-Schiff. 0 1994 by the American Gastroenterological Association OOlS-5085/94/$3.00

SELF-SUPERANTIGENS

October 1994

the Mls-1”

locus

and

its adjacent

chromosome

region

from the BALB/c strain.” We analyzed the time course of deletion of V,6+ T cells and the appearance of gastric

lymph

nodes with the gastric

lesions. This experimental

design allowed the correlation

in the maintenance

in the

the onset of the disease.

Table1. Histopathology BALB/c

stomach

of the Stomach

(peripheral)

Body of Gastritic

GASTRITIS

925

lesions. Our data indicate

that resistance to murine AIG is not linked to the Mls1” locus and that V,6+ T cells seem not to be involved

of V,-,b’ T-cell

deletion

AND AUTOIMMUNE

Neonatally

Thymectomized

of AIG

but

may be implicated

and Sham-Operated

BALB.D2

in

and

Mice Inflammation

Mice

Age (mo)

Chronic

Acute

Diffuse

Nodular

Atrophy

Metaplasia

Mucins

Ratio

A

1

4 4 3 3 3 3 3 3 3 3 4 3 4 4 1 2 2 2 2 2 3 3 3 3 3 3 4 4 4 3 4 4 1 1 2 2 3 4 4 4 1 1 2

3 3 2 2 2 2 3 3 2 2 2 0 0 2 0 0 0 0 0 0 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 1 0 1 2 2 2 1 1

2 2 2 2 2 2 2 2 3 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 2 3 2 3 3 2 2 2 0 2 2 1 1 3 3 3 2 2 2

0 0 0 0 0 0 2 0 2 2 2 3 2 3 0 2 0 0 0 0 2 2 2 3 0 2 2 2 0 2 4 4 0 0 0 2 3 4 4 4 0 0 0

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

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

2 3 0 0 0 3 0 2 0 3 3 2 3 3 0 0 0 0 0 0 3 2 3 3 2 3 2 3 3 4 2 4 0 0 0 0 0 3 3 2 0 0 0

2:2

1.5

2

3.5

5

7

B

6 7

C

5

D

5

6~7

3:3

3:3

15:20

3:5

I:7 2:2 5:16

3:7

Anti-H+.K’-ATPase 0 1 2 0 1 1 2 2 2 2 2 2 2 2 1 2 2 2 0 2 2 2 2 2 2 2 2 1 2 2 2 2 0 0 0 2 2 2 2 2 0 0 0

NOTE. A, nTx-BALB.D2 mice; B, control BALB.D2 mice; C, nTx-BALB/c mice; and D, control BALB/c mice. The different histological observations on individual mice were graded as 1, minimal; 2, slight; 3, moderate; and 4, severe. The inflammation in the fundic mucosa was classified as chronic (mononuclear eels); acute (neutrophils); diffuse (homogeneously distributed throughout the lamina propria and submucosa); and nodular (lymphocytic aggregates). The other histological changes assessed were atrophy (destruction of acid-secreting glands); metaplasia (replacement of acid-secreting glands by mucus-secreting glands): and presence of mucins. The prevalence of mice with gastric lesions (ratio) is given. Mice that were scored negative (0) are not given. The presence of anti-H+,K+-ATPase autoantibodies was detected by Western blotting as 0, negative; 1, slightly positive; and 2, positive staining.

926

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GASTROENTEROLOGY Vol. 107, No. 4

ET AL.

Materials and Methods

dilutions bation

Animals

IgG (Protoblot

BALB/c/Cpb/Hsd The Netherlands).

mice were from Harlan CPB (Zeist,

The BALB.D2.Mls-I”

by Drs. Festenstein maintained

and Berumen.16

under conventional

the Institute

Mice have been bred and

conditions

in accordance

with

guidelines.

Thymectomy transcervical-retrosternal

incision.

and

underwent

BALB/c

mice)

sham

of nTx was confirmed

autopsy

(47 BALB.D2

operation. thymectomy.

macroscopically

Blood was recovered

At autopsy,

monoclonal

antibodies

curvatural

stomach

lymph

one half of the stomach embedded

or popliteal)

and the small

nodes were taken. The antrum

in paraffin wax, sectioned,

and stained

CD4 (H 129.19),20 CD8 (H35-17.2),2’

graded

blindly

by assigning

observations

Severity

numeric

as described

cance of difference

between

mice was calculated

by Mann-Whitney’s

mucins

were differentiated

changes

by periodic

and high-iron

on sections

diamine/Alcian

Detection

U test. The secreted showing

inflammatory

blue, pH 2.5.

against analysis.

the proton A vesicular

IgG (l/10)

the primary

antibodies

fraction

stomach

mice were prepared

were incubated from

for 3 hours at room temperature

nTx and control

and

mice at dilutions

(l/200)

with plasma similar

to

with

consisted

Im-

3-amino-9

of substitution

of

saline alone.

and small and nTx

on ice with the following

(1) either anti-VP6 (44-22-l) goat antirat

antirat

(rat IgG) and fluores-

(Caltag,

San Francisco,

(using

standard

methods)

Becton Dickinson

View, CA); and (4) phycoerythrin-labeled (Boehringer,

followed

sequentially

IgG (20 minutes),

utes) to block

Mannheim,

node cells were stained

and anti-VP1 1 hybridoma

free-binding

CA)

anti-VP6

(rat IgG); (3) phycoerythrin-

CD4 (rat IgG) (GK1.5;

mouse CD8 (rat IgG) (53-6.7)

(20 minutes)

cells (0.2monoclonal

IgG (l/50)

(2) anti-VP1 1 (RR3.15)

labeled antimouse

anti-VP6

saline, 0.2% bovine

and 0.1% sodium azide. Lymphoid

1 X 106) were incubated

(44-22-l);

from the peripheral

nodes of sham-operated

in phosphate-buffered

cyl sulfate-polyacrylamide

conditions

visualized

Controls

lymph

many). Briefly, lymph

membranes (Millipore Corp., with bovine serum albumin,

goat

at room temperature.‘”

with phosphate-buffered

Single cell suspensions curvatural

from hog gastric mucosa, which is highly enriched in H+,K+on 7.5% sodium dodeATPase,” was used for electrophoresis gels under reducing

azide. Briefly, air-dried

Flow Cytometric Analysis of Peripheral and Gastric Lymph Node Lymphoid Cells

Co., Mountain

transferred onto nitrocellulose Bedford, MA). Strips, blocked

was

or Auorescein-conjugated were identi-

bovine

was inhibited

(200 PgimL) (Sigma, St. Louis, MO) and H20z

for 5 minutes.

antibodies:

pump

with 0.1%

activity

stain-

by horse radish peroxidase-labeled

for 30 minutes reaction

ethylcarbazole

ceinated

membrane

(IgG)

CA). Nonspecific

the sections

VP8

by horse radish

immunoglobulin

sodium

rat

V,6 (44-22-1),22

peroxidase

H202 and 0.1%

serum albumin,

(PAS), Alcian blue, pH 2.5,

antirat

by blocking

munoperoxidase

values to the different of

IN) and with

CD3 (17 A2),”

and revealed

Burlingame,

Endogenous

hour (2O’C) followed antirat

in Table 1. The signifigroups

and of

sections were fixed in acetone for 10 minutes at room temperature and incubated with the rat monoclonal antibodies for 1

was

of Serum Autoantibodies

Autoantibodies fied by immunoblot

0.3%

isopentane

of gastritis

the values of different

acid-Schiff

TAGO,

ing was reduced

(0.06%)

with H&E.

The other body half was frozen in liquid Nz-cooled and used for immunohistochemistry. histological

and

body were fixed in 10% formaldehyde,

tissues

mice were em-

Inc., Elkhart,

against T-cell markers

Com-

exam-

WI).

were immunolabeled

goat

from the portal vein, and plasma

(inguinal

(Miles

sections

(mouse adsorbed;

in the presence of 0.1% sodium azide and frozen. peripheral

compound

Cryostat

with

ination.

in O.C.T.

snap-frozen.

serum albumin.

ages for histological

Madison,

body mucosal

peroxidase-conjugated

Histopathology

was prepared

of the stomach

lymph nodes from nTx BALB.D2

Sixty

at the time of

by incu-

goat antimouse

Blot, mouse; Promega,

(RR3.15)24

and 16 BALB/c mice).

Mice were killed at different

conjugated

(KJ16),23 and Vpll

Control mice (11 BALB.D2

BAL.B.D2 and 24 BALB/c mice underwent pleteness

by suction via a

Western

Samples the stomach bedded

on day 3 was performed

by others. ‘*6,1”,18This was followed

lmmunohistochemistry

strain was produced

nTx

7

reported

with alkaline-phosphatase

&

antiGer-

either with the

cell culture

supernatants

by fluorescein-labeled

goat

with an excess of rat IgG (10 minsites of the latter

antibody

and

Figure 1. Histology of gastric mucosa of mice undergoing sham thymectomy and mice with AIG after nTx. (A) A histological staining of the fundic mucosa of a sham-operated BALB/c mouse (PAS-H&E; original magnification x85). (B-E) Cross sections of the gastric mucosa of a BALB/c mouse with AIG. (s) The fundic glands are partially destroyed and replaced by an intense, diffuse, and nodular inflammatory infiltrate (atrophy) (H&E: original magnification x120). (C) The fundic glands are replaced by mucus-secreting cells (metaplasia) (H&E; original magnification x85). (0) High-power view of C showing mucus-secreting cells (H&E; original magnification x165). (E) High-power view of C showing the presence of neutral mucins in these cells after PAS staining. (Fand G) Cross sections of the gastric mucosa of a BALB.D2.Ml.s-1” mouse with AIG. (f) H&E staining showing inflammation, atrophy of glands, and metaplasia; (G) PAS-H&E staining showing the presence of neutral mucins in the metaplastic cells (original magnification x85).

October 1994

SELF-SUPERANTIGENS

AND AUTOIMMUNE

GASTRITIS

927

928

GASTROENTEROLOGY Vol. 107, No. 4

CIAEYS ET AL.

phycoerythrin-conjugated directly

with

erythrin-conjugated The samples

antibody

fluoresceinated antibody

were analyzed

sorter (FACS) (FACScan

to CD4 (20 minutes),

anti-VP6

antibody

to CD4 or CD8 (20 minutes). on a fluorescence-activated

flow cytometer;

Dead cells and erythrocytes

or

and phyco-

Becton

were excluded

of forward and 90” light scatter.

cell

Dickinson).

by a combination

A minimum

of 5000 events

were gated.

with

the blotted

had AIG

H+,K+-ATPase.

of varying

nTx Induces AIG in Adult BALB.D2.Mls-1” as in BALB/c (MIS-lb) Mice: Histopathological and Immunological Findings Post-nTx affected

mainly

was usually

AIG

in BALB.D2

the fundic

spared.

mucosa,

Sections

inflammation

flammation

(Figure

was predominant

BALB/c

whereas

throughout

showed the presence of a moderate chronic

and

mouse had slight chronic gastritis Next

we examined

bearing

mice

severe of the

mucosa and sometimes extended to the submucosa. The pattern of the inflammatory infiltrates was diffuse and nodular.

The cellular infiltrate

consisted

mainly

BALB.D2

number

with germinal of neutrophils

flammation.

centers

(not shown).

were detected

a small

in the gastric

At the site of dense inflammatory

and

mice. Cryostat

sections

with monoclonal

AIG-

of the fundus to the infilnTx

mice consisted

mainly

of T cells (CD3+) (not

with many CD4+ T cells and few CD8+ T cells

transfer

AIG to syngeneic

athymic

mice.3.10

nTx Induces AIG in BALB.D2 Mice at the Age of 4 Weeks As shown in Table and severe chronic gastritis cosa and submucosa

1, a combined

moderate

as early

as 4 weeks

of age. The ---

D

C AIG

acute

affected the gastric body mu-

__-

of mono-

Only

of CD4+

CD3, CD4, and CD8 antigens. The inflammatory trates in the fundic mucosa of 5-month-old

nuclear cells (T cells and plasma cells), which frequently formed lymphoid aggregates resembling follicles, sometimes

on immunoblot.

distribution

(Figure 3). This is consistenr with recent reports showing that CD4+ T cells, but not CD8+ T cells, adoptively

1). The in-

in the basal part

(Table 1). None of the

antibodies

shown),

the antrum

and sometimes

the

mice

One negative

CD8+ T cells in the gastric lesions of 5-month-old,

BALB.D2

the body mucosa

1 and Table

All the positive

of severity.

sera from control mice reacted positively

were immunolabeled

Results

degrees

-

+

in-

infiltrates,

the fundic glands were destroyed (indicated in Table 1 as atrophy). Mucus-secreting cells replaced parietal and chief cells in less inflamed

areas (metaplasia).

These cells

contained a mixture of neutral mucins (PAS-positive) and sialomucins (alcian blue-positive). Sulfomucins were never detected (data not shown). Histological signs of gastritis were present in 24 of the 31 nTx BALB.D2 mice older than 2 months (Table l), but six of the positive mice had only slight chronic gastritis without signs of atrophy and metaplasia. Unspecific gastric lesions, i.e., a minimal chronic and slight acute inflammation, were detected in 3 of the 9 sham-thymectomized BALB.D2 mice. The chronic gastritis observed in nTx BALB.D2 mice was significantly different (P < 0.001, MannWhitney V test) from the lesions in control mice. The histological modifications of the AIG induced in the BALB.D2 mice were essentially the same as those observed in 5 of 16 nTx BALB/c mice (Table 1). AIG in adult BALB.D2 and BALB/c panied by serum autoantibodies to the of the gastric H+,K+-ATPase as detected ting (Figure 2 and Table 1). At autopsy, samples from the nTx BALB.D2 mice

mice was accoma and p subunits by immunoblot23 of 31 plasma reacted positively

Figure 2. lmmunoblots of hog gastric membrane vesicles, enriched in the a subunit (c&U) and S subunit (!%w) of the H’,K+-ATPase, using sera from thymectomized and sham-thymectomized mice. A a Coo massie blue B stained with sodium dodecylsulfate-PAGE gel of the proton pump-enriched vesicular fraction; nitrocellulose strips containing about 0.5 pg of total protein were immunostained with sera from nTx BALB/c. 6 sham-nTx BALB/c; C nTx BALB.D2.Ml.s-1”; and D mice with (+) and without (-) AIG.

SELF-SUPERANTIGENS

October 1994

AND AUTOIMMUNE

GASTRITIS

929

Figure 3. lmmunoperoxidase staining of T ceils in the gastric inflammatory infiltrates of 5-month-old BALB.D2.Mls-1” mice with AIG using two adjacent sections. (A) Many CD4’ and (6) few CD8’ T cells infiltrate the fundic mucosa.

histopathology

dominant

was

chronic

significantly different from the prelesions observed in adult mice. The

inflammatory infiltrates consisted mainly cells, but neutrophils were also present.

week-old gastritic mice were negative or only slightly positive on immunoblots for the H+,K+-ATPase (Table

of mononuclear A nodular pat-

1). The lower correlation between H+,K+-ATPase autoantibodies and histopathology in young nTx BALB.D2

tern of the cellular infiltrates was rarely observed. In contrast, partial destruction of the acid-secreting glands

mice compared with adults may have resulted, however, from the low sensitivity of the immunoblotting tech-

as well as their metaplastic replacement by mucus-secreting glands was already observed 4-6 weeks after nTx. The different histological signs of the chronic gastritis in 4-6-week-old mice were scored (Table l), and the

nique.

numeric

values were used to compare 4-6-week-old,

gas-

tritic BALB.D2 mice with adult gastritic BALB.D2 mice by the Mann-Whitney’s U test. No significant differences were found for the following histological changes: chronic inflammation (P = 0.63), atrophy (P = 0.36), and metaplasia (mucins) (P = 0.25). However, significantly less nodular lymphocytic aggregates (P = 0.006) and more neutrophils (P = 0.001) were observed in the gastric lesions of young mice. Some of the sera of 4-6-

nTx BALB.D2 Mice Develop AIG Despite the Early Deletion of Vp6’ CD4’ Peripheral T Cells Our results clearly establish that Mls-1” mice on a BALB/c background develop post-nTx AIG in contrast to the nonsusceptible Mls-1” DBA/2 mice.’ It has been shown that peripheral Vp6+ T cells are deleted rapidly in BALB.D2 mice and in nTx-BALB.D2 mice.25 Therefore, we analyzed the frequency of Vp6+ CD4+ and Vb6’ CD8+ T cells in the stomach lymph nodes of nTxBALB.D2 and control mice at different ages by FACS.

930

CLAEYS ET AL.

GASTROENTEROLOGY Vol. 107. No. 4

Table 2. Deletion of Vb6+ T cells in the Stomach Lymph Nodes From Neonatal Thymectomized BALB.D2, BALB/c and Sham-Operated Mice Vp6+

CD4’ plus CD8’

Age Mice

(W

n

% CD4

% CD8

(%)

Sham BALB.D2 nTx BALB.D2

3 2

2 3

0.2 !Y 0.2 2.0 k 0.1 (0.001) 0.7 k 0.2 (0.023) 0.5 t 0.3 (0.264) 1.0 2 0.2 (0.013) 0.4 2 0.3 (0.540) 11.6 2 0.5 10.2 t 1.7

0.8 2 0.7 4.2 -c 0.5 (0.009) 1.0 ? 0.3 (0.458) 0.9 2 0.7 (0.865) 1.5 2 0.3 (0.211) ND

73 -c 1 17 + 2

Sham BALB/c nTx BALB/c

4

6

6

7

8

3

22

7

22 22

7 5

20 k 3 28 2 9 30 ? 3

Vp6+ CD4+ T cells in the periphery after nTx did not prevent BALB.D2

V,G+, Vg8+, Vgl Sections pared

of Vb6’ CD4+

sham-operated

adult

these lymph

nodes

because

the inflamed

mucosa

the stomach

sections

through

body mucosa the stomach

4). We used the staining

control

because this subset is not deleted

15% Vg8+ T cells among

CD4+

were comlymph

node

of Vg8+ T cells as a (approximately

T cells) in normal

or

mice.25 It has been shown that CD4+ and Vpl 1 are deleted in adult BALBi

antigens encoded by the endogenous retroviruses MN-8,9 expressed in these mice.26 Because Vpl 1+ T cells can be

(Table

they were

significantly enlarged in gastritic mice when compared with nondiseased nTx mice or controls, indicating that they were draining

mice by immunocytochemistry.

67 2 3 38 k 9

nTx and seven

BALB/c mice was also determined

2). We selected

through

nTx-BALB.D2

CD8’,

c and BALB.D2 mice because of their reactivity with super-

T cells in the stomach

nodes of the five diseased

nTx BALB.D2

with

of CD4+,

(Figure

readily detected in the periphery of adult nTx-BALB/c that develop AIG with high incidence,“-‘” examined

of the fundus.

However, gastric lymph nodes were not enlarged, and there were no signs of a macroscopic gastritis in 2-weekold nTx-BALB.D2 mice in contrast to older diseased mice. Single cell suspensions of lymph nodes were doublestained with anti-CD4+ or anti-CD8+ and anti-Vp6. The percentages of Vp6+ T cells are given in Table 2. The

this subset.

The frequency

mice

we have also

of Vpl l+ T cells

(2 + 0.7% of CD4’ P/US CD8+ T cells, n = 7) in the gastric lymph nodes of 5-month-old

nTx-BALB.D2

mice

was determined by FACS analysis and was higher than in sham-nTx mice (0.5 t- 0.1% of CD4+ P/US CD8+ T cells,

lymph

the distribution

29 2 6

NOTE. Dissociated lymphocytes from the stomach lymph nodes were labeled with the monoclonal antibodies, and their cell distribution was analyzed by FACS. The T cells expressing V,6 were calculated as a percentage of the indicated T-cell subsets (mean ? SD). The percentages of CD4+ plus CD8’ 1 cells are expressed as percentage of total gated cells. Parentheses indicate the significance of difference, calculated by Student’s t test, between the percentages of V,S’ T cells of nTx-BALB.D2 mice and those of sham BALB.D2.

The frequency

in

1+ T cells in the gastric lesions of 4-6-

CD8+ T cells expressing

ND ND

of AIG

mice.

We then analyzed week-old

as early as 4 weeks

the development

n = 7), confirming

the data

reported.27-29

The

increase of Vpll+ T cells occurred mainly in the CD8+ subset (5.4 + 2.8%) in agreement with recent findings.‘* As in adult mice, the lymphocytic infiltrates in the stomach

fundus

consisted ingly,

of 4-6-week-old

mainly

of CD4+

nTx-BALB.D2

T cells (Figure

Vp6+ T cells could still be detected

mice

4). Surprisin the gastric

lesions of young mice (4-6 weeks of age) despite their deletion in the periphery. We also found that Vpl It T cells, which have been proposed to be involved in postnTx autoimmune diseases, ‘7-29 are abundant in the fundus of gastritic 4-week-old mice.

Discussion Autoimmune

diseases are thought

to be mediated

percentages of CD4+ plus CD8+ T cells were significantly decreased in nTx animals when compared with

by nontolerized autoreactive lymphocytes. Clonal tion of self-reactive lymphocytes in the thymus

controls, reflecting the effect of nTx. As shown in Table 2, a similar percentage of Vp6+ cells within the CD4+ T cell population was found in the stomach lymph nodes of adult AIG-bearing BALB/c and normal mice. In contrast, Vp6+ CD4+ T cells were deleted in the stomach lymph nodes of4-week-old nTx-BALB.D2 mice. In addition, we found that only 2% of the CD4+ T cells from the gastric lymph nodes expressed Vp6+-TCR in 2-weekold nTx-BALB.D2 mice. The deletion of Vp6+ CD8+ T cells was slightly delayed when compared with the CD4+ T-cell subset. These results indicate that the deletion of

important mechanism to establish self-tolerance. 3” However, because CD4+ lymphocytes from the thymus of neonatal and adult BALB/c mice can transfer AIG to athymic nude BALB/c mice,‘O it is likely that the selfreactive cells responsible for AIG originate in the thymus, are not deleted, and migrate to the periphery. The autoreactivity of these cells appears to be suppressed by a yet unidentified T-cell subset (CD4+) in normal, but not in nTx-BALB/c, mice.’ It has been proposed that resistance to post-nTx AIG in mice is linked to a locus encoding a retroviral (Mtv-7) superantigen (Mls- la),’

deleis an

October 1994

ms~

4. lmmunoperoxidase staining of (A and 8) CD4+, (C and D) CD8+, (E and F) V,6’, (G and H) V&t’, and (I and J) V,8+ T cells on cryosections of the (A, C, E, G, and I) stomach body and (B, D, F, H, and J) stomach lymph node of 4weekold. AIG-bearing BALB.D2 mice (B, D-H, and J, original magnification x150; A, C, and I, original magnification x60).

SELF-SUPERANTIGENS

AND AUTOIMMUNE

GASTRITIS

931

932

CLAMS

which

ET AL.

GASTROENTEROLOGY Vol. 107, No. 4

causes deletion

of most

T cells expressing

V,6

elements. This link was further supported by the observation that peripheral Vp6+ T cells increased in number in gastritic

BALB/c mice (MIS-lb).’ However,

the role of

Vp6+ T cells in AIG remains controversial. Recently, it was reported that anti-CD4 antibodies, when injected into nTx-BALB/c

mice, could prevent

the development

of AIG. This was not the case for injected anti-VP6

antibodies,

the respective

although

subsets

the peripheral

cell subsets

(VpG+), thereby

examined

at the very early stages (2-6

the H+,K+-ATPase or

gen can activate

deletion

of

be excluded

the

the number

of

weeks post-nTx)

of the disease. One important question regarding the fundic Vp6+ T cells is whether gastric self-antigens (e.g.,

anti-CD8

did occur.14 To test directly

enhancing

pathogenic effector cells. The link between Vp6+ T cells in the fundus and the onset of AIG now has to be better

stomach

p subunit)

or the MIS-1” superanti-

these T cells. Thus, at present,

that the persistence

mucosa

of young

it cannot

of Vg6+ T cells in the

nTx mice is unrelated

to the

involvement of Mtv-7 and Vp6+ T cells in the murine AIG, we thymectomized 3-day-old, MIS-1” congenic

pathogenesis of AIG. This being so, the Vs6+ T cells would infiltrate the fundus along with the pathogenic

BALB.D2 mice, known to delete rapidly peripheral T cells. 25

(CD4+) contrast

We

found

developed

that

in nTx-BALB/c

BALB.D2

nTx-BALB.D2

of

(MIS-~“) mice

an AIG identical

link between

DBA/2

mice

the MIS-1” locus and the to

post-nTx

mice, the gastric lesions appeared

thymectomy.

to that

(MIS- 1 b, mice. These results clearly ques-

tion the proposed resistance

adult

with high incidence

Vp6+

A substantial

inflammatory

AIG.’ rapidly infiltrate

In after was

observed in the fundic mucosa of 4-week-old, nTxBALB.D2 mice which consisted mainly of CD4+ T cells and neutrophils and only a few CD8+ T cells. This is in agreement with adoptive transfer studies that presume that the CD4+ T-cell population It is interesting of the gastritic expression

initiates

the disease.”

that the time course for the appearance lesions (day 14-30) parallels that of the

of the H+,K+-ATPase

during

ontogeny

(day

l-30):

i.e., low protein levels until day 15, adult levels indicate that the by day 30.31 Recent experiments H+,K+-ATPase plays a crucial role as an autoantigen in the initiation of autoimmune gastritis. Mice transgenic for the H+,K+-ATPase p subunit develop thymic tolerance to the autoantigen and become resistant to the development

of autoimmune

gastritis

after nTx.”

We analyzed by immunocytochemistry the Vp6+ T cell population in the gastric body mucosa and the stomach lymph nodes of 4-6-week-old, At this age, Vp6+ T cells still

nTx-BALB.D2 mice. infiltrated the gastric

mucosa, whereas in the draining lymph nodes, they were deleted. The persistence of autoreactive Vp6+ T cells in the gastric mucosa of 4-6-week-old, nTx-BALB.D2 mice suggests that they might play a role in the onset of the disease. These findings also indicate the importance of analyzing the T cells within the autoimmune lesions itself rather than in the draining lymph nodes. It has been shown that the incidence of AIG is significantly increased after injection of irradiated Mls-1” spleen cells from DBA/2 mice into nTx-BALB/c mice,’ suggesting that the Mls-1” antigen activated particular T-

T cells at the onset of the disease. Then, in to the more rapid deletion of Vb6+ T cells in

the periphery,

their

deletion

would

be delayed

in the

stomach because of a possible lack of tolerizing selfsuperantigens in the fundic mucosa of young mice. Finally, the potential MIS-1” reactive Vb6+ T cells in the gastric lesions may have an extrathymic (intestinal) origin. Indeed,

T cells with Mls-1” specific Vg segments

in

their T-cell receptor can be detected in the intestinal intraepithelial-lymphocyte compartment of MIS-1” selfsuperantigen expressing mice.32.33 We are currently investigating if thymic-independent T cells may be involved in the initiation of AIG in mice. Clearly, further analysis and quantification of the T-cell subsets in the fundic mucosa at the onset of AIG will be necessary to characterize the direct role of Mtv encoded self-superantigens and their reactive T-cell subsets in experimental autoimmune gastritis in BALB/c and BALB.D2 mice.

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Received May 21, 1993. Accepted June 13, 1994. Address requests for reprints to: Jean-Pierre Kraehenbuhl, M.D., Swiss Institute for Experimental Cancer Research, Chemin des Boveresses 155, W-1066 Epalinges, Switzerland. Fax: (41) 21-652-6933. Supported by grant 32-30011.90 from the Swiss National Science Foundation. Dr. Acha-Orbea was a recipient of a START fellowship from the Swiss National Science Foundation. The authors thank Dr. Irene Corthisy-Theulaz for help with statistical analysis, Dr. George Sachs for kindly supplying the hog H’,K’adenosine triphosphatase, Dr. H. Robson MacDonald for providing the BALB.DZ mouse strain, and Nicole de Montmollin for typing the manuscript.