close

Вход

Забыли?

вход по аккаунту

?

Presence of microchimerism in labial salivary glands in systemic sclerosis but not in Sjgren's syndrome.

код для вставкиСкачать
ARTHRITIS & RHEUMATISM
Vol. 46, No. 4, April 2002, pp 1039–1043
DOI 10.1002/art.10137
© 2002, American College of Rheumatology
Presence of Microchimerism in Labial Salivary Glands in
Systemic Sclerosis but Not in Sjögren’s Syndrome
Sélim Aractingi,1 Jean Sibilia,2 Véronique Meignin,3 David Launay,4 Eric Hachulla,4
Caroline Le Danff,1 Anne Janin,3 and Xavier Mariette5
Objective. To determine whether microchimerism
can be implicated in Sjögren’s syndrome (SS) by studying minor salivary glands, one of the targets of the
disease.
Methods. Labial salivary gland (LSG) biopsy
specimens from 16 female patients with primary SS and
11 with systemic sclerosis (SSc) (a disease in which
microchimerism is frequently detected) were analyzed.
All 27 women had a history of pregnancy with a male
baby. Specimens were microdissected, and polymerase
chain reaction (PCR) was performed using the unique
sex-determining region Y gene probe.
Results. The sensitivity of PCR for detecting male
cells in LSG was high; the presence of 3 male cells was
consistently detected in DNA extracted from a normal
female LSG specimen to which male DNA had been
added, and 1 male cell was detected in 50% of specimens
analyzed. Male DNA was not found in any of the
specimens from the 16 SS patients but was detected in 5
(45%) of 11 SSc specimens (P ⴝ 0.006). No differences
in the rate of detection were found between patients with
diffuse and limited SSc (male DNA detected in 2 of 3
and 3 of 8, respectively; P ⴝ 0.55) or between patients
with and those without secondary SS (1 of 6 and 4 of 5,
respectively; P ⴝ 0.08).
Conclusion. The results of our study strengthen
the possibility that microchimerism is implicated in
SSc. This is the first study to demonstrate the presence
of chimeric cells in LSG from 45% of SSc patients,
independent of the presence of secondary SS. However,
microchimerism was not detected in LSG from patients
with primary SS, suggesting that the pathogenesis of the
2 diseases is different.
Sjögren’s syndrome (SS) is an autoimmune disorder characterized by lymphocytic infiltration of the
exocrine glands, including salivary and lacrimal glands,
leading to xerostomia and xerophthalmia and to systemic production of autoantibodies (1). In SS, early
mononuclear cell (mainly CD4⫹ T cells) infiltration of
these glands is followed by the destruction of epithelial
cells through activation of several apoptotic pathways
(2). This disease is either isolated (primary SS) or
secondary to another autoimmune systemic disease such
as rheumatoid arthritis, lupus, polymyositis, dermatomyositis, or scleroderma (systemic sclerosis; SSc).
The pathogenesis of SS remains unclear. Like
numerous other autoimmune diseases, SS affects women
more frequently, with a female-to-male ratio of 9:1. The
reasons for this female predominance are unknown. Sex
hormones have been suspected to play a role, but such
hormonal influence alone cannot explain the female
predominance in SS (3).
Recently, the presence of high levels of peripheral microchimerism of male origin was demonstrated
in women with SSc, suggesting involvement of fetal cells
from past pregnancies (4–6). Like SSc, Sjögren’s syndrome occurs mainly in women, with the highest incidence in those older than age 40 years. This population
usually has experienced pregnancy, a condition in which
persistent microchimerism is implicated (7). In addition,
clinical and pathologic features resembling those of
chronic graft-versus-host disease (GVHD) develop in
both SSc and SS (8). Because chimeric cells seem to be
associated with the pathogenesis of spontaneously oc-
1
Sélim Aractingi, MD, Caroline Le Danff: Hôpital Tenon,
Hôpitaux de Paris, and Hôpital Saint Louis, Paris, France; 2Jean
Sibilia, MD: Hôpital de Hautepierre, Strasbourg, France; 3Véronique
Meignin, MD, Anne Janin, MD, PhD: Hôpital Saint Louis, Paris,
France; 4David Launay, MD, Eric Hachulla, MD: Hôpital Claude
Huriez, Lille, France; 5Xavier Mariette, MD, PhD: Hôpital de Bicêtre,
Hôpitaux de Paris, Université Paris XI, Le Kremlin Bicêtre, France.
Address correspondence and reprint requests to Xavier Mariette, MD, Service de Rhumatologie, Hôpital de Bicêtre, 78 rue du
Général Leclerc, 94275 Le Kremlin Bicêtre, France. E-mail:
xavier.mariette@bct.ap-hop-paris.fr.
Submitted for publication June 19, 2001; accepted in revised
form October 24, 2001.
1039
1040
ARACTINGI ET AL
curring diseases that are similar to chronic GVHD, we
sought to determine whether this type of phenomenon is
involved in primary SS by studying minor salivary glands,
which are one of the targets of the disease.
MATERIALS AND METHODS
The databases from 2 pathology departments were
searched for archival labial salivary gland (LSG) biopsy specimens from female patients with primary SS. The diagnosis of
primary SS was made according to revised European Community (EC) criteria (9). Only specimens that had been obtained
from adult women with a history of pregnancy with a male
baby were selected. From these specimens, only those with a
focus score ⱖ1 (according to the Chisholm scale) (10) were
included. Control LSG specimens were obtained from adult
women with SSc (a disease in which microchimerism is frequently detected in different organs) and a history of pregnancy with a male baby. The diagnosis of SSc was based on the
American College of Rheumatology (formerly, the American
Rheumatism Association) criteria (11). SSc was considered to
be limited as opposed to diffuse if 3 of the 5 components of the
CREST syndrome (calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, telangiectasias) were present.
Some patients with SSc also had secondary SS, defined according to the 1993 EC criteria (12).
All experiments were performed using 6 ␮m–thick
paraffin-embedded LSG tissue sections. All sections were
prepared by the same female technician, who used a new slide
box and a sterile knife each time. Sections were deparaffinized
in toluene for 10 minutes, rinsed in 4 successive ethanol
solutions (pure, 75%, 50%, and 30%), and then rinsed in
water. After drying, the sections were incubated in 2.5%
glycerol for 3 minutes, allowed to dry again, and were then
ready for microdissection.
The microdissection procedure was performed according to a previously described technique (13). Sections were
stained with hematoxylin and eosin and examined under the
light microscope at a magnitude of 10⫻. Because of the small
diameter of the LSG specimens, microdissection was directed
at the whole section. Therefore, the section was gently scraped
with the tip of a sterile, 25-gauge needle until the material from
the entire section was detached. The cells of interest were then
procured from material attached to the needle. This procedure
avoided selection bias due to dissected cells, because whole
cells present in the section were obtained and analyzed. A
trained pathologist (VM) manually counted several samples
and found that in each section dissected, the total number of
cells (⬃5,000) was the same in both groups.
After microdissection, procured cells were immediately resuspended in a mixture of 40 ␮l Tris (pH 8.5), 50
mmoles/liter, EDTA, 1 mmole/liter, 0.5% Tween 20, and 0.2
mg/ml proteinase K and then incubated overnight at 37°C. The
mixture was boiled for 10 minutes at 99°C to inactivate the
proteinase K and then centrifuged for 10 minutes at 14,000
revolutions per minute. Five to 10 ␮l of the supernatant was
used for the polymerase chain reaction (PCR). PCR for the
unique sex-determined region Y (SRY) gene was performed
under class II containment conditions. The SRY primers were
upstream 5⬘-TCC-ACT-TTA-TTC-CAG-GCC-TGT-CC-3⬘
and downstream 3⬘-TTG-AAT-GGA-ATG-GGA-ACG-AATGG-5⬘. PCR amplification (40 cycles) was performed for 30
seconds at 94°C, for 30 seconds at 60°C, and for 30 seconds at
72°C. Amplified DNA was run on an agarose gel, transferred
on a nitrocellulose filter, and hybridized with the internal
32
P-labeled probe 5⬘-ATC-CCG-CTT-CGG-TAC-TCT-GC3⬘. All experiments were performed twice. Amplification of
HLA–DR␣ was performed on every specimen as a DNA
quality control.
In order to assess the sensitivity of detection of male
cells in the tissues, similar microdissections were performed on
Table 1. Characteristics of patients with primary Sjögren’s syndrome*
Age,
years
No. of
male
children
Duration of
disease, years
Systemic involvement
Autoantibodies
46
30
36
40
36
42
43
50
35
42
43
66
63
37
77
20
2
2
2
1
3
1
2
1
1
2
1
1
1
1
2
1
5
12
3
8
7
18
18
11
6
8
1
3
4
2
23
1
Arthritis
Arthritis
–
Vasculitis, arthralgia
Arthralgia, thrombosis
Arthritis, myalgia
Arthritis, neuropathy
Arthralgia
Arthralgia
Myalgia, vasculitis, neuropathy
Autoimmune thrombocytopenia
Marginal-zone lymphoma
–
Arthralgia
Arthritis
Arthritis
Ro, RF
Ro/La, RF
Ro/La, RF
Ro/La
Ro/La, RF
Ro
Ro/La, RF
Ro, RF
Ro/La
Ro/La
Ro
RF
Ro/La
Ro
* PCR ⫽ polymerase chain reaction; SRY ⫽ sex-determining region Y; RF ⫽ rheumatoid factor.
† According to the Chisholm-Mason classification (10).
Glandular
histology†
Glandular
DNA by
PCR SRY
III
IV
IV
IV
IV
III
IV
IV
IV
IV
IV
IV
IV
III
IV
III
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
POSSIBLE ROLE OF MICROCHIMERISM IN SYSTEMIC SCLEROSIS
1041
Table 2. Characteristics of patients with systemic sclerosis*
Age, years
Diffuse disease
47
41
73
Limited disease
73
57
61
36
60
35
58
71
No. of
male
children
Disease
duration,
years
1
2
1
9
5
8
2
2
1
1
9
8
9
2
2
1¶
2
1
2
6
10
25
Glandular
histology§
Glandular
DNA by
PCR SRY
Systemic involvement
Autoantibodies
Secondary
SS‡
Arthritis, mild pulmonary fibrosis
Severe pulmonary fibrosis
Renal, mild pulmonary fibrosis
Scl-70, Ro
Scl-70
Scl-70
⫹
⫺
⫺
III
II
I
⫺
⫹
⫹
CREST
REST
CRST
RST
Mild pulmonary fibrosis
0
Mild pulmonary fibrosis
0
⫹
⫺
⫺
⫹
IV
III
I
III
⫹
⫹
⫹
⫺
REST
RST
ERS
CREST
0
0
Arthritis
PBC
ACA
ACA
ACA
ANA
1/320
ACA
ACA
ANA
ACA
Scl-70
⫹
⫹
⫺
⫹
IV
IV
III
IV
⫺
⫺
⫺
⫺
CREST†
* SS ⫽ Sjögren’s syndrome; ACA ⫽ anticentromere antibodies; ANA ⫽ antinuclear antibodies; PBC ⫽ primary biliary cirrhosis (see Table 1 for
other definitions).
† CREST: C ⫽ calcinosis, R ⫽ Raynaud’s phenomenon, E ⫽ esophageal dysmotility, S ⫽ sclerodactyly, T ⫽ telangiectasias.
‡ According to the 1993 European criteria (12).
§ According to the Chisholm-Mason classification (10).
¶ Patient had 10 spontaneous fetal losses since onset of CREST.
DNA extracted from a female LSG specimen without inflammatory infiltration. After overnight incubation with proteinase
K, the male DNA was added to the female salivary lysate in
decreasing amounts (the highest amount being 125 pg [equivalent to 25 male cells] and the lowest being 5 pg [equivalent to
1 male cell]). Each of these dilution samples was studied in
duplicate.
Statistical analysis was performed using Fisher’s exact
test.
RESULTS
Biopsy specimens from 16 women with primary
SS and 11 women with SSc were obtained and analyzed
as described in Materials and Methods. Among the SSc
patients from whom specimens were obtained, 3 had
diffuse disease and positive anti–Scl-70 antibodies. The
other 8 patients had limited SSc, and 6 of them had
anticentromere antibodies. In addition, 6 of the 11 SSc
patients (5 with CREST syndrome and 1 with diffuse
SSc) fulfilled criteria for a diagnosis of secondary SS.
The clinical characteristics of patients with SS and those
with SSc are shown in Tables 1 and 2, respectively.
The PCR performed on DNA extracted from the
normal female LSG specimen to which decreasing
amounts of male DNA were added consistently demonstrated the presence of 3 male cells, while 1 male cell was
detected in 1 of the duplicates (Figure 1). In 2 analyses of
specimens from patients with SS, male signal was never
detected. In contrast, male DNA was detected in 5 (45%)
of 11 women with SSc (P ⫽ 0.006) (Figure 1). No difference in the rate of detection was found between patients
with diffuse SSc and those with the limited form (2 of 3 and
3 of 8, respectively, positive for male DNA) (P ⫽ 0.55)
(Table 2). In patients with SSc, male DNA was detected
more frequently in the absence of secondary SS (4 of 5
versus 1 of 6), but this difference did not reach statistical
significance (P ⫽ 0.08). Water and 100-ng samples of
female DNA, which were used as controls, were consistently negative for male DNA, demonstrating the absence
of contamination during the procedures. Amplification of
HLA–DR␣ yielded positive results in every case, thereby
demonstrating the presence of amplifiable DNA in every
specimen (Figure 1).
DISCUSSION
Microchimerism is defined by the presence within
an individual of a very low level of cells derived from a
different individual (7). In 1996, Bianchi et al demonstrated that male fetal CD34⫹ cells could be detected in
maternal blood as long as 27 years postpartum (14). The
prolonged presence of fetal cells could represent a
source of allogeneic cells in some individuals. Other
research has focused on the role of microchimerism in
conditions that closely resemble GVHD and are characterized by a female predominance, because pregnancy is
the major source of microchimerism. The first such
1042
ARACTINGI ET AL
Figure 1. Analysis of DNA extracted from microdissected sections of salivary glands. Sections were amplified with sex-hormone Y region (SRY)
primers and, as a control, with DR␣ primers, then transferred on nitrocellulose filters and hybridized with a radiolabeled probe (see text for
sequences). Male DNA was absent in labial salivary gland specimens from patients with Sjögren’s syndrome and was present in 5 specimens from
patients with systemic sclerosis.
disease to be assessed for this phenomenon was SSc, in
which a high level of fetal cells was found in peripheral
blood as well as in skin lesional tissue (4–6). Interestingly, analysis of HLA in families in which a female
member had SSc showed that a “compatibility from the
mother’s view” occurred in the females with SSc significantly more frequently than in control females from the
same families (4,15). This type of compatibility had
previously been shown to be a reliable predictor of
GVHD development after red blood cell transfusion
(16). Taken together, these results provided evidence to
support the hypothesis that microchimerism could represent a key triggering event in the development of SSc.
Subsequently, several studies addressed the possible role of microchimerism in other autoimmune diseases with features similar to those of GVHD, i.e.,
primary biliary cirrhosis and juvenile dermatomyositis.
In primary biliary cirrhosis, the absence of microchimerism was repeatedly demonstrated whenever this phenomenon was studied in liver cells or in peripheral blood
lymphocytes (7). In contrast, microchimerism of maternal origin was frequently displayed in juvenile dermatomyositis (17,18). Researchers have therefore targeted
the possible reverse microchimerism induced by pregnancy, known as maternal–fetal trafficking.
Our study demonstrates the absence of male cells
in salivary glands from 16 women with primary SS and a
history of pregnancy with a male baby. The sensitivity of
PCR for detecting male cells in this tissue was high,
because DNA from 3 male cells that was added to DNA
extracted from a normal female LSG specimen was
consistently detected, and addition of DNA from 1 male
cell was detected in 1 of the duplicates. In addition, the
fact that male DNA was found in 5 of 11 LSG specimens
from patients with SSc, a disease known to be associated
with microchimerism, demonstrated that the technique
we used to detect microchimerism in tissues was adequate. Because all 16 females with primary SS had been
previously pregnant with a male baby, the above results
suggest that microchimerism is not implicated in the
pathogenesis of SS. These results are in accordance
with those reported by another group, which recently
showed that microchimerism was not detected in the
peripheral blood or in CD34-enriched fraction from
women with primary SS (19). Interestingly, microchimerism was present in LSG from 45% of women with
SSc, regardless of the presence of secondary SS.
This is the first study to demonstrate that microchimerism is detected in salivary glands of women with
SSc. Our findings are in accordance with those of a
recent study showing that, in women with scleroderma,
the presence of male cells could be detected in various
other tissues (20). LSG microchimerism was present
both in patients with the diffuse form of SSc and in those
with CREST syndrome. Subtypes of SSc have not been
distinguished in previous studies investigating microchimerism. Recently, it has been suggested that pregnancy
microchimerism could be preferentially associated with
limited rather than diffuse SSc (21).
In conclusion, our study strengthens the notion
POSSIBLE ROLE OF MICROCHIMERISM IN SYSTEMIC SCLEROSIS
that microchimerism may be implicated in SSc, by
demonstrating the presence of chimeric cells in LSG
independent of the presence of secondary SS. However,
microchimerism was not detected in the same target
organ in primary SS, suggesting a different pathogenesis
for the 2 diseases.
REFERENCES
1. Moutsopoulos HM, Chused TM, Mann DL, Klippel JH, Fauci AS,
Frank MM, et al. Sjögren’s syndrome (sicca syndrome): current
issues. Ann Intern Med 1980;92:212–26.
2. Kong L, Ogawa N, Nakabayashi T, Liu GT, D’Souza E, McGuff
HS, et al. Fas and Fas ligand expression in the salivary glands of
patients with primary Sjögren’s syndrome. Arthritis Rheum 1997;
40:87–97.
3. Sullivan DA. Sex hormones and Sjögren’s syndrome. J Rheumatol
1997;24 Suppl 50:17–32.
4. Nelson JL, Furst DE, Maloney S, Gooley T, Evans P, Smith A, et
al. Microchimerism and HLA compatible relationships of pregnancy in scleroderma. Lancet 1998;351:559–62.
5. Artlett CM, Smith JB, Jimenez SA. Identification of fetal DNA
and cells in skin lesions. N Engl J Med 1998;338:1186–91.
6. Artlett CM, Cox LA, Jimenez SA. Detection of cellular microchimerism of male or female origin in systemic sclerosis patients by
polymerase chain reaction analysis of HLA–Cw antigens. Arthritis
Rheum 2000;43:1062–7.
7. Aractingi S, Uzan S, Dausset J, Carosella ED. Microchimerism in
human diseases. Immunol Today 2000;21:116–8.
8. Aractingi S, Chosidow O. Cutaneous graft-versus-host disease.
Arch Dermatol 1998;134:602–12.
9. Vitali C, Bombardieri S, Moutsopoulos HM, Coll J, Gerli R,
Hatron PY, et al. The European Study Group on Diagnostic
Criteria for Sjögren’s Syndrome. Assessment of the European
classification criteria for Sjögren’s syndrome in a series of clinically
defined cases: results of a prospective multicentre study. Ann
Rheum Dis 1996;55:116–21.
1043
10. Chisholm DM, Mason DK. Labial salivary gland biopsy in Sjögren’s syndrome. J Clin Pathol 1968;21:656–60.
11. Subcommittee for Scleroderma Criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee. Preliminary criteria for the classification of systemic sclerosis
(scleroderma). Arthritis Rheum 1980;23:581–90.
12. Vitali C, Bombardieri S, Moutsopoulos HM, Balestrieri G, Bencivelli
W, Bernstein RM, et al. Preliminary criteria for the classification of
Sjögren’s syndrome. Arthritis Rheum 1993;36:340–7.
13. Zhuang Z, Bertheau P, Emmert-Bück MR, Liotta LA, Gnarra J,
Linehan WM, et al. A microdissection technique for archival DNA
analysis of specific cell populations in lesions ⬍1 mm in size. Am J
Pathol 1995;146:620–5.
14. Bianchi DW, Zickwolf G, Weil G, Sylvester S, DeMaria MA. Male
fetal progenitor cells persist in maternal blood for as long as 27
years postpartum. Proc Natl Acad Sci U S A 1996;93:705–8.
15. Artlett CM, Welsh KI, Black CM, Jimenez SA. Fetal-maternal
HLA compatibility confers susceptibility to systemic sclerosis.
Immunogenetics 1997;47:17–22.
16. McMilin KD, Johnson RL. HLA homozygosity and the risk of
related-donor transfusion-associated graft-versus-host disease.
Transfus Med Rev 1993;7:37–41.
17. Artlett CM, Ramos R, Jiminez SA, Patterson K, Miller FW, Rider
LG, Childhood Myositis Heterogeneity Collaborative Group. Chimeric cells of maternal origin in juvenile idiopathic inflammatory
myopathies. Lancet 2000;356:2155–6.
18. Reed AM, Picornell YJ, Harwood A, Kredich DW. Chimerism in
children with juvenile dermatomyositis. Lancet 2000;356:2156–7.
19. Toda I, Kuwana M, Tsubota K, Kawakami Y. Lack of evidence for
an increased microchimerism in the circulation of patients with
Sjögren’s syndrome. Ann Rheum Dis 2001;60:248–53.
20. Johnson KL, Nelson JL, Furst DE, McSweeney PA, Roberts DJ,
Zhen DK, et al. Fetal cell microchimerism in tissue from multiple
sites in women with systemic sclerosis. Arthritis Rheum 2001;44:
1848–54.
21. Launay D, Hebbar M, Hatron PY, Michon-Pasturel U, Queyrel V,
Hachulla E, et al. Relationship between parity and clinical and
biological features in patients with systemic sclerosis. J Rheumatol
2001;28:509–13.
Документ
Категория
Без категории
Просмотров
3
Размер файла
116 Кб
Теги
presence, microchimerism, syndrome, labial, sjgren, systemic, gland, salivary, sclerosis
1/--страниц
Пожаловаться на содержимое документа