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: firstname.lastname@example.org. 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. 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