increase of rp105-lacking activated b cells in the

Increase of RP105-lacking activated B cells in the peripheral blood and salivary glands in patients with Sjögren’s syndrome

Y. Kikuchi1,6, S. Koarada1, S. Nakamura2, N. Yonemitsu3, Y. Tada1, Y. Haruta1, F. Morito1, A. Ohta4, K. Miyake5, T. Horiuchi6, K. Nagasawa1

1Department of Internal Medicine, Faculty of Medicine, Saga University; 2Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic and Surgical Sciences, Faculty of Dental Science, Kyushu University; 3Department of Pathology, Faculty of

Medicine, Saga University; 4Department of Nursing, Faculty of Medicine, Saga University; 5Division of Infectious Genetics, Department of Microbiology and Immunology, Institute of Medical 5Division of Infectious Genetics, Department of Microbiology and Immunology, Institute of Medical 5

Science, University of Tokyo; 6Department of Medicine and Biosystemic Science, Kyushu University, 6Department of Medicine and Biosystemic Science, Kyushu University, 6

Graduate School of Medical Sciences, Fukuoka, Japan.

AbstractObjective

To quantify the activated B cells in the peripheral blood and salivary glands of patients with Sjögren’s syndrome (SS) by ’s syndrome (SS) by ’analyzing the expression of RP105 molecule on the B cells.

MethodsThe expression of RP105 on the peripheral blood B cells of patients with SS (19 cases) was analyzed by fl ow cytometry.

RP105-positive and negative B cells were sorted and cultured in vitro and the amount of immunoglobulins (IgG and IgM) produced in the supernatant was measured by enzyme-linked immunosorbent assay (ELISA). Salivary gland biopsy samples

from 9 SS patients were histologically evaluated and the sequential frozen sections were separately immunostained by anti-RP105 and anti-CD20 monoclonal antibodies.

ResultsA signifi cantly higher proportion of peripheral blood RP105-negative B cells was found in SS patients than in healthy

individuals. RP105-negative, but not positive, B cells from SS patients were capable of producing IgG and IgM spontaneously in vitro, which was enhanced by the addition of Staphylococcus aureus Cowan I strain (SAC) or I strain (SAC) or I IL-6.

Salivary glands from 2 of 9 SS patients were found to have lymphoid follicles whose germinal centers consisted of RP105-negative B cells. Moreover, a larger proportion of B cells extensively infi ltrating the area other than lymphoid

follicles was also RP105-negative.

ConclusionRP105-negative B cells, a subset of highly activated and well differentiated B cells, which are increased in number in

the peripheral blood and extensively infi ltrate salivary glands, may be responsible for the production of class-switched immunoglobulin in SS. In addition, those cells might be associated with the infl ammation and tissue damage of the

salivary glands.

Key wordsSjögren’s syndrome, RP105-negative B cells, salivary glands, germinal center.

Clinical and Experimental Rheumatology 2008; 26: 5-12.

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RP105-negative B cells in patients with Sjögren’s syndrome / Y. Kikuchi et al.

Yuji Kikuchi, MD; Syuichi Koarada, MD; Seiji Nakamura, Dentist; Nobuhisa Yonemitsu, MD; Yoshifumi Tada, MD; Yoshio Haruta, MD; Fumitaka Morito, PhD; Akihide Ohta, Professor; Kensuke Miyake, MD; Takahiko Horiuchi, MD; Kohei Nagasawa, Professor.This work was supported in part by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 16590985).Please address correspondence to: Yuji Kikuchi, MD, Department of Internal Medicine, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga 849-8501, Japan.E-mail; [email protected] on October 16, 2006; accepted in revised form on May 4, 2007.© Copyright © Copyright © CLINICAL AND EXPERIMENTAL RHEUMATOLOGY 2008.EXPERIMENTAL RHEUMATOLOGY 2008.EXPERIMENTAL RHEUMATOLOGY

Competing interests: none declared.

IntroductionSjögren’s syndrome (SS) is a chronic infl ammatory disorder characterized by lymphocytic infi ltration in lacrimal and salivary glands, which results in dry eyes and dry mouth, and occasional presence of serum autoantibodies such as anti-SS-A/Ro and anti-SS-B/La (1-5). Clusters of T and B cells resembling germinal centers (GCs) and numerous plasma cells are found in the salivary glands of SS patients, which produce immunoglobulins (Igs) including au-toantibodies (3, 6-11). SS appears as a primary or a secondary condition in association with rheumatoid arthritis, systemic lupus erythematosus (SLE), or systemic sclerosis (1, 2). Intense B cell activity such as germinal center formation occurs in exocrine glands of SS patients, placing them in a high risk category for the development of lym-phomas (12, 13). However, the role of B cells and autoantibodies in the patho-genesis of SS remains unclear.RP105, a novel molecule on B cells detected in 1994 (14), transmits an ac-tivation signal that leads to B cell pro-liferation and resistance against apop-tosis in mice. The extracellular domain of RP105 is structurally similar to Toll-like receptors, suggesting that RP105 senses pathogen invasion and activates B cells (15-17). Mice devoid of RP105 have recently been generated, and it has been shown that RP105 regulates li-popolysaccharide (LPS) signaling in B cells (18). In contrast to these fi ndings in mice, little is known about the func-tion of RP105 in humans although the human homologue of RP105 has been identifi ed and its monoclonal antibod-ies have been established (19-21).It is known that virtually all mature B cells have RP105 molecules on their surface in humans as well as in mice (22). We have previously demonstrated that the peripheral blood B cells lack-ing RP105 are signifi cantly increased in patients with SLE (22) and der-matomyositis (23, 24), whereas most of the B cells in normal subjects carry that molecule. RP105-negative B cells have been identifi ed as IgD-negative, CD95-positive, CD86-positive and CD38-bright, which is consistent with activated B cells or GC B cells (22).

Particularly in SLE, we have suggested that RP105-negative B cells, but not positive B cells, are capable of produc-ing Igs without stimulation and even anti-double-stranded DNA antibodies with activated T cell help (25). This prompted us to investigate the ex-pression of RP105 on B cells both in the peripheral blood and in the salivary glands in SS patients. In this study, we demonstrated RP105-negative B cells from SS patients were increased in the salivary glands as well as in the periph-eral blood.

Patients and methodsPatientsNineteen patients (17 female and 2 male) who fulfi lled the diagnostic cri-teria for SS proposed by the European Community Study Group were included in this study (26). Their age ranged from 18 to 85 years (mean = 48 years). Until the time of examination, no patient had received immunosuppressive therapy. As controls, 32 patients (31 female, 1 male, mean age = 33 years; range 16 – 69 years) with SLE and 37 normal – 69 years) with SLE and 37 normal –subjects (21 female, 16 male, mean age = 30 years; range 23 – 52 years) were – 52 years) were –tested for the expression of RP105 on peripheral blood B cells. Immunohisto-chemical studies on the salivary gland biopsy specimens from the lower labial mucosa were performed in 9 SS patients (8 female, 1 male, mean age = 44 years; range 17 – 60 years).– 60 years).–

Cell preparationSamples of peripheral venous blood from SS patients and SLE patients and normal subjects were collected in tubes containing heparin. Peripheral blood mononuclear cells (PBMC) were sepa-rated by centrifugation over Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) gradient.

Immunofl uorescence analysis of RP105 expression on B cells.PBMC were stained with fl uorescein isothiocyanate (FITC)-conjugated anti-human RP105 monoclonal antibody (mAb) (IgG1κ) (21) and phycoeryth-rin (PE)-conjugated anti-human CD19 mAb (BD PharMingen, San Diego, CA), and analyzed using FACScan.

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Statistical analyses were performed us-ing Cell Quest software. (Becton Dick-inson, Mountain View, CA)Cells from 4 SS patients were then sort-ed into the 2 fractions, RP105-negative CD19-positive B cells and RP105-posi-tive CD19-positive B cells, using the Epics Elite cell sorter (Coulter, Hialeah, FL).

Preparation of immunoglobulin-coated platesGoat anti-human immunoglobulin (γ, α, μ, light chain) (Biosource, Fleurus, Bel-gium) at a dilution of 1:2,000 (1.25 μg/ml) in phosphate buffered saline (PBS) was coated on 96-well Nunc maxisorb immunoplates (Nalgene Nunc), and incubated overnight at 4°C. The plates were washed with 0.05% Tween 20 in PBS 3 times. The plates were blocked with 1% bovine serum albumin in PBS for one hour at room temperature. The plates were washed with 0.05% Tween 20 in PBS 3 times.

Production of total IgG and IgMRP105-positive and RP105-negative B cells were separately suspended in RPMI 1640 containing 10% fetal calf serum and incubated in a 5% CO2 in-cubator at 37°C in 96-well plates (Nal-gene Nunc, Milwaukee, WI). Cells (2 x 104) were cultured for 5 days without stimulation or stimulated with: 0.001% Staphylococcus aureus Cowan I strain (SAC), or 1 ng/ml recombinant human IL-6 (rIL-6) (Genzyme, Cambridge, MA) with or without 50 μg/ml mono-clonal mouse anti-human IL-6 antibody (Genzyme, Cambridge, MA). The cul-tured supernatants were harvested on day 3 and day 5 and added to prepared immunoplates with control human IgG and IgM (Chemicon, Hofheim, Ger-many) and incubated overnight at 4°C. After discarding the supernatants and washing with 0.05% Tween in PBS 3 times, the bound human immunoglobu-lins were detected with peroxidase-la-beled goat anti-human IgG γ chain and IgM μ chain (BioSource) at a dilution of 1:2,000 in PBS. The plates were washed again and developed with o-phenylendiamine. The reaction was stopped with 3N HCl solution, and the optical density (OD) at 490 nm was

read with an ImmunoMini NJ-2300 microplate reader (System Instruments, Tokyo, Japan).

Grading standard for labial salivary gland biopsyIn order to standardize the degree of histopathological change, the grad-ing standard described in Table I was employed. Whole minor salivary gland tissue in the section was examined and scanned for the presence of mononu-clear cells. Grades (+) ~ (+++) fulfi ll one item (histopathological diagnosis) of the diagnostic criteria of SS.

ImmunohistochemistryA normal tonsil tissue obtained by ton-sillectomy was embedded in Tissue-Tek OCT compound, frozen in liquid nitrogen, and stored at –70°C. Acetone-fi xed cryostat sections of 5 μm were incubated with avidin solution for 10 minutes. The avidin solution was then rinsed off and the slide was incubated with Biotin Solution for 10 minutes. The biotin solution was washed off, (DAKO Biotin Blocking System) and proceed the fi rst step of staining pro-cedure. The slide was stained with the anti-RP105 mAb overnight at 4°C. Af-ter washes, LSAB2/HRP kit (DAKO Japan, Inc, Tokyo, Japan) was added. Briefl y, biotinylated anti-mouse im-munoglobulins were applied for 10 minutes and washed with PBS. Strep-toavidin conjugated to horseradish peroxidase (HRP) was applied for 10 minutes and washed with PBS. 3,3’-diaminobenzidine (DAB) substrate was applied for 5 minutes. After rinse with distilled water, the slide was stained with hematoxylin.The biopsy tissues of labial salivary glands were embedded in Tissue-Tek OCT compound, and stored at –70°C

until used. Tissue sections (5 μm thick) were cut and mounted on glass slides precoated with aminopropyltriethox-ysilane. The sequential sections were separately stained with mouse anti-hu-man CD3 mAb (UCHL1), mouse anti-human CD20 mAb (DAKO Japan, Inc, Tokyo, Japan) or mouse anti-human RP105 mAb (21) overnight at 4°C. Af-ter washes, immunostaining was visual-ized as described above. Biotin block-ing was performed similarly.RP105-negative B cells were detected as negative staining of RP105 among CD20 positive cells. The percentage of infi ltrating RP105-negative B cells were obtained by counting those cells out of 1000 CD20-positive cells under microscope.

StatisticsThe Mann-Whitney U-test was used to compare the RP105-negative B cells (%) between groups of diseases. A value of pvalue of pvalue of < 0.05 was taken to indicate signifi cance.

ResultsRP105-negative B cells in the peripheral blood in SS patientsRP105-negative B cells in the peripheral blood were quantitatively assessed fi rst. Figures 1A, B and C show representa-tive results from a patient with SS and SLE and a normal subject. As shown in these fi gures, numbers of RP105-nega-tive B cells, which were very few in a normal subject (3.3%), were increased in a SS patient (14.1%) and in a SLE patient (16.9%). Percentages of RP105-negative B cells from 19 SS patients, 32 SLE patients and 37 normal subjects were plotted in Figure 2. In comparison with a small percentage of RP105-neg-ative B cells in normal subjects, they appeared in signifi cant numbers in SS

Table I. Grading standard for labial salivary gland biopsy.

Grade Mononuclear cells around an intralobular duct

(-) Absent

(±) Slight infi ltration (An aggregate of less than 50 cells)

(+) Moderate infi ltration (One or two lesions with infi ltration of 50 or more cells)

(++) Many lesions (more than 2) with infi ltration of 50 or more cells, although destruction

of exocrine glands is within half of lobe

(+++) Severe lymphocytic infi ltration destroys more than half of lobe

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patients as well as in SLE patients as had been previously described (22). The mean ± SD percentage of RP105-negative B cells was 13.3 ± 8.5 in 19 SS patients, being signifi cantly higher than 4.0 ± 2.1 (p 2.1 (p 2.1 ( < 0.005) in 37 normal subjects and comparable to 17.3 ± 9.5 in 32 SLE patients.

IgG and IgM production in vitro by B cells from SS patientsRP105-positive and negative B cells in the peripheral blood from 4 SS patients were separately cultured without stimula-tion and IgG and IgM produced in the su-pernatants were measured on day 5. The RP105-positive and the RP105-negative B cell purity were determined by FACS-can (Becton Dickinson, Mountain View, CA). As had been expected, RP105-negative B cells spontaneously produced certain amount of IgG and IgM. On the other hand, RP105-positive B cells did not produce any amount of IgG or IgM (detection threshold for IgG 150 ng/ml and for IgM 15 ng/ml). As shown in Table II, the amount of Igs produced by RP105-negative B cells widely varied de-pending on individual patients. Whereas RP105-negative B cells from patients 3 and 4 produced considerable amount of Igs (IgG: 5.16 and 58.6 μg/ml, IgM: 196 and 432 ng/ml, respectively) comparable to SLE patients (25), those from patients 1 and 2 did only a small amount of Igs (IgG: 0.98 and 2.35 μg/ml, IgM: 27 and 59 ng/ml, respectively).

RP105-positive and negative B cells from patients 1 and 4 were stimulated with SAC or rIL-6 in vitro and the amount of IgG and IgM in the super-natant was assayed. Figure 3A and B show the results of RP105-negative B cells from patient 1 and 4, respectively. The addition of 0.001% SAC or even 1 ng/ml rIL-6 enhanced the total IgG and IgM production by RP105-negative B cells from both patients although the absolute amount of Igs in patient 1

was much smaller than in patient 4. To confi rm the effect of IL-6, anti-IL-6 antibody was added in this system. As shown, anti-IL-6 antibody completely eliminated the effect of IL-6 and even reduced Igs production to a lower level than without stimulation. In contrast, RP105-positive B cells from patients 1 and 4 did not respond to the stimula-tion with SAC or IL-6 and no Igs were detected in the supernatant (data not shown).

Fig. 1. Expression of RP105 on peripheral blood B cells from a representative SS patient (A), a SLE patient (B) and a normal subject (C). Peripheral blood mononulear cells were stained with anti-RP105 monoclonal antibody (mAb) and anti-CD19 antibody.

Fig. 2. Proportion of RP105-negative B cells in the peripheral blood of SS patients, SLE patients and normal subjects. RP105 expression on B cells from 19 SS and 32 SLE and 37 normal subjects was analyzed by fl ow cytometry. The percentage of RP105-negative B cells in SS and SLE patients was larger than in normal subjects (plarger than in normal subjects (plarger than in normal subjects ( < 0.005). Bars show the mean ± SD.

A B C

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RP105 expression in a normal human tonsilAs a comparison with salivary glands, an immunohistochemical study was done for a normal human tonsil. It was found that RP105 was expressed mainly on B cells in mantle zones as had been described (21). Almost all the cells in GCs were either dull or nega-tive for RP105 (Fig. 4).

RP105 expression on B cells in the salivary glands of SS patientsThe immunological and histological data of 9 SS patients whose salivary glands were examined are shown in Table III. Antinuclear antibody was detected in 7 out of 8 patients tested. Seven out of 9 patients had anti-SS-A/Ro antibod-ies and 4 had anti-SS-B/La antibodies. Serum levels of IgG were all elevated ranging from 1880 to 4337 mg/dl (mean = 3065 ± 897). All 9 patients fulfi lled the histopathological diagnostic criteria of SS. Seven patients showed grade (++) and the remaining 2 patients, grade (+) and (+++), respectively. In patients 2 and 6, infi ltrating mononuclear cells (IMC) formed GC-like lesions (Fig. 5). To de-termine the phenotype of lymphocytes infi ltrating the salivary glands, immuno-histochemical analysis with monoclonal antibodies was conducted. As shown in Figure 6, T cells (CD3+ cells) were rarely seen and almost all the IMC were comprised by B cells (CD20+ cells) in this area. RP105 expression was almost negative in the same section, suggest-ing that IMC in this lesion consisted of RP105-negative B cells.The percentage of RP105-negative B cells infi ltrating salivary glands was as-sessed by the count of RP105-negative cells out of 1000 CD20-positive cells. It was found that a large percentage of infi ltrating B cells of SS were RP105-negative, ranging from 20.5% to 100% (mean = 57 ± 30%) (Table III). GC-like lesions seen in 2 patients showed that RP105 was mainly expressed on B cells in the mantle zone and that the cells in GC were either dull or negative for RP105 similar to the normal tonsil (Fig. 5).All the patients tested had elevated se-rum levels of IgG (Table III), which tended to correlate with the percentage

Table II. Spontaneous IgG and IgM production by RP105-negative B cells from patients with Sjögren’s syndrome.

Patient no. 1 2 3 4

Peripheral RP-105-negative B cells (%) 14 5.5 16 27

RP105-positive B IgG (μg/ml) ND ND ND ND IgM (ng/ml) ND ND ND ND

RP105-negative B IgG (μg/ml) 0.98 2.35 5.16 58.6 IgM (ng/ml) 27 59 196 432

ND: not detected.

Fig. 3. Immunoglobulin production by RP105-negative B cells from patient 1 (A) and patient 4 (B) in vitro. Highly purifi ed RP105-positive and RP105-negative B cells were cultured separately for 5 days without stimulation or stimulated with: 0.001% Staphylococcus aureus Cowan I strain (SAC) or 1 ng/ml recombinant human IL-6 (rIL-6) with or without 50 μg/ml anti-IL-6 antibody. Supernatants were harvested after 5 days of culture and IgG and IgM secretion was measured by enzyme-linked immu-nosorbent assay. RP105-positive B cells produced neither IgG nor IgM with SAC or rIL-6 stimulation (not shown), and fi gures only show IgG and IgM production by RP105-negative B cells. Dotted lines (A) show detective threshold (IgG: 150 ng/ml, IgM: 15 ng/ml).

Fig. 4. RP105 expression in a human tonsil. Fro-zen sections from a human tonsil were stained with anti-RP105 mAb. RP105 was mainly expressed on B cells in the man-tle zone. (Origi-nal magnifi cation x100).

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of RP105-negative B cells infi ltrating the salivary glands although not signif-icant (Pearson’s correlation coeffi cient r = 0.17, p > 0.05). Of note is that in 4 patients (patients 6-9) who had both anti-SS-A/Ro and anti-SS-B/La anti-bodies, a markedly higher percentage of RP105-negative B cells infi ltrated the salivary glands (61.2–100%).

DiscussionSS is one of the systemic autoimmune diseases characterized by hyperacti-vation of B cells which produce large amount of polyclonal Igs and autoan-tibodies such as anti-SS-A/Ro and anti-SS-B/La. In this study we found a signifi cantly increased proportion of RP105-negative B cells in the periph-eral blood of SS patients comparable to SLE patients. We have recently sug-gested that RP105-negative B cells, but not RP105-positive B cells, in the pe-ripheral blood of SLE patients have the ability to produce Igs including autoan-tibodies such as anti-double stranded and single stranded DNA antibodies (25). RP105-negative B cells from SS patients were found to be able to pro-duce class-switched immunoglobulin spontaneously in vitro, although the amount varied widely depending on individual patients, compared to SLE patients. In contrast to RP105-positive B cells which did not respond to SAC or IL-6, Igs production by RP105-nega-tive B cells was signifi cantly enhanced by SAC or IL-6, indicating that the lat-ter are also ready to respond to exoge-neous stimulations. It is of great interest

that anti-IL-6 antibody not only abol-ished the effect of IL-6, but reduced the Igs production to even lower lev-els than control (without stimulation). This suggests that RP105-negative B cells may secrete and respond to IL-6 in an autocrine manner. It is possible

that RP105-negative B cells from SS patients may also produce disease-specifi c autoantibodies such as anti-SS-A/Ro or anti-SS-B/La antibody as those cells from SLE patients produced anti-DNA antibodies. We could not, however, detect such autoantibodies in

Table III. Serum immunological parameters and immunohistochemistry of the salivary glands in patients with Sjögren’s syndrome.

Patient Sex Age at IgG ANA Anti-SS-A/Ro Anti-SS-B/La Histological GC-like Salivary gland no. diagnosis (mg/dl) antibody antibody grade structures RP105-negative B cells (%)

1 M 53 2210 (+) (+) (-) (++) (-) 20.52 F 48 3519 (+) (-) (-) (++) (+) 24.73 F 28 3467 (+) (+) (-) (++) (-) 37.54 F 17 4337 (+) (+) (-) (++) (-) 43.85 F 75 2211 (-) (-) (-) (++) (-) 47.36 F 48 2401 (+) (+) (+) (+++) (+) 61.27 F 60 1880 (+) (+) (+) (++) (-) 75.28 F 45 4030 (+) (+) (+) (++) (-) 1009 F 22 3528 ND (+) (+) (+) (-) 100

ANA: antinuclear antibodies; GC: germinal center; M: male; F: female; ND: not done.

Fig. 5. Germinal center (GC)-like cell clusters in la-bial salivary gland biopsies from pa-tient 2. The same samples were im-munostained with anti-CD20 (Top) or anti-RP105 mAb (Bottom). RP105 was main-ly expressed on B cells in the mantle zone and GC B cells were nega-tive for RP105 as similar to a hu-man tonsil. (Orig-inal magnifi cation x200 in both).

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this in vitro system so far. Halse et al. indicated the presence of anti-SS-A/Ro and anti-SS-B/La antibody-producing cells in the peripheral blood of patients with SS (4). These results indicate that RP105-negative B cells in SS patients also have a similar character to those found in SLE, being concerned in the pathogenesis of SS.It is considered that SS patients do not have as many circulating B cells spon-taneously secreting polyclonal Igs as SLE patients despite a similarly hyper-active B cell function in both diseases (2, 6). In SS patients, however, those hyperactive B cells may be sequestered in lymphoid organs such as spleen, lymph nodes and bone marrow (2) and probably in salivary and/or lacrimal glands (6). On the other hand, based on the Banchereau’s classifi cation (Bm1-Bm5), which determines the differen-tiation of B cells (27), Bohnhorst et al. have recently demonstrated that SS pa-tients have higher percentage of activat-ed B cells in the peripheral blood than normal subjects (28). Our results seem to be consistent with this description although the activated and Ig-secreting B cells in SS patients tended to be a bit smaller in number compared to SLE pa-tients. In consideration of marked infi l-tration of activated B cells in the target organs, salivary and lacrimal glands, in SS, there may be a similar or even more

number of activated B cells in SS pa-tients compared to SLE patients.The histopathological examinations of the salivary glands of SS patients re-vealed a marked infi ltration of mononu-clear cells of which RP105-negative B cells were predominant. It is known that infi ltrating cells are comprised predomi-nantly of CD4+ T cells and that B cell in-fi ltration becomes prominent as the dis-ease progresses (7-10). Our results sug-gest that these B cells are activated and RP105-negative. In 2 patients there were even GC-like structures that were com-prised mainly of RP105-negative B cells as seen in tonsils. GCs are considered to be a major site for the production of autoantibodies (3, 10). Of the 2 patients, however, patient 2 was negative for anti-SS-A/Ro and anti-SS-B/La antibodies, although patient 6 had both antibodies. It should be noted that the patients who were positive for both anti-SS-A/Ro and anti-SS-B/La antibodies had extremely high percentage of RP105-negative B cells in the salivary glands (Table III). Moreover, all the patients examined had signifi cant numbers of RP105-negative B cells in the salivary glands and el-evated serum levels of IgG. It is possible that infi ltrating RP105-negative B cells may secrete Igs and autoantibodies in the salivary glands. Tengnér et al. have demonstrated the presence of SS-A/Ro and SS-B/La

autoantibody-producing cells in the salivary glands of SS patients and in-dicated their potential involvement in the autoimmune exocrinopathy of SS (9). Although the relationship between these cells and RP105-negative B cells is not known, both cell types could be similar in the character. It is also known that infi ltrating B cells contain plasma cells producing antibodies of the IgG and IgM isotypes (6-8).Very recently, Groom et al. have dem-onstrated a BAFF-mediated B cell hy-peractivity in the SS model mice (29). According to their description, those in-fi ltrated and accumulated B cells in the salivary glands are phenotypically simi-lar to marginal zone (MZ) B cells. They have also suggested the role of MZ B cells in the pathogenesis of human SS as well, based on the results of the markedly elevated serum levels of BAFF and the accumulation of cells expressing BAFF in the salivary glands of SS patients. On the other hand, RP105-negative B cells focused on in this study seem to be a lit-tle more differentiated than those MZ B cells since the former are phenotypi-cally close to GC B cells (22). However, the relationship between both cell types remains to be investigated.In addition, as suggested in the in vitroexaminations, RP105-negative B cells may secrete IL-6 and these hyperactive B cells may possibly affect salivary or

Fig. 6. Immunohistochemical staining of a labial salivay gland biopsy specimens from patient 9. This lesion revealed infi ltrating mononuclear cells (IMC) around the acinar and ductal epithelium (A). The same sample was immunostained with (B) anti-CD3, (C) anti-CD20 or (A, D) anti-RP105 mAb. (Original magnifi cation x100 in A, x400 in B, C and D) AE: acinar epithelium, DE: ductal epithelium, IMC: infi ltrating mononuclear cells.

A B C D

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lacrimal glands directly or indirectly through cytokines and/or chemokines. It remains to be elucidated, however, how RP105-negative B cells emerge in SS. Many attempts to convert RP105-positive B cells to negative ones in vitro have not succeeded yet (data not shown).In conclusion, it is now evident that the emergence of RP105-negative B cells, which are hyperactive and well differ-entiated, is responsible for the devel-opment of hypergammaglobulinemia, and may play a role in the formation of pathophysiology of SS.

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increase of rp105-lacking activated b cells in the

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