Rheumatology 2009;48:12791282

Advance Access publication 11 August 2009

doi:10.1093/rheumatology/kep228

Usefulness of initial histological features for stratifying Sjogrens

syndrome responders to mizoribine therapy
Shingo Nakayamada1, Takashi Fujimoto2, Akitaka Nonomura3, Kazuyoshi Saito1, Shinobu Nakamura2
and Yoshiya Tanaka1

KEY

WORDS:

Sjogrens syndrome, Mizoribine, Xerostomia, Salivary gland biopsy.

findings of minor salivary glands before treatment could be

useful in predicting the response to immunosuppressor therapy.
Although histological evaluation of minor salivary glands has
been used as a tool for the diagnosis of SS, no attempt has been
made to determine the method of treatment for SS based on
histological features.
The present study was undertaken to assess the pre-treatment
histological features of minor salivary glands in responders to
mizoribine therapy and the validity of histological classification
as a means to predict the response to treatment.

Introduction
SS is a systemic disorder with features of both systemic and organspecific autoimmunity affecting the salivary and lacrimal glands.
The disease is mainly characterized by dry eyes and dry mouth,
and patients may also have diverse systemic lesions (extraglandular symptoms) such as interstitial pneumonia, neuropathies and
interstitial nephritis [1, 2]. At the sites of the lesions, a marked
periductal autoreactive lymphocytic infiltration progresses into
destruction of the acini and fibrosis in the lacrimal and salivary
glands. Thus, control of such excessive lymphocyte activation is
attractive as a target of treatment. However, despite marked
reduction of the patients quality of life (QOL) due to glandular
lesions associated with SS, no radical therapy for correcting
immunological abnormalities is available, and currently patients
are primarily given symptomatic treatment using cevimeline
hydrochloride, pilocarpine, etc. [3, 4].
SS can be characterized by hypergammaglobulinaemia and
excessive autoantibody production due to B-cell activation [5].
Mizoribine, an immunosuppressor whose basic activity is suppression of the proliferation of activated B cells, is expected to be
effective in SS patients. We previously reported that mizoribine
increased salivary secretion, reduced serum IgG and improved
the patients assessments of dry mouth and dry eyes; besides, we
confirmed the effectiveness and safety of this drug in multicentre
studies [6, 7].
However, the results obtained to date suggest that there are
both responders and non-responders to mizoribine. It has also
been shown that in patients subjected to pathological examination
of oral and labial biopsy specimens at relatively early stages before
treatment, mizoribine produced marked alleviation of histological
signs of inflammation. Therefore, it seems that histological

Patients and methods

Patients
The study involved patients definitely diagnosed as having SS who
presented subjective sicca symptoms. The diagnosis of SS was
based on the revised diagnostic criteria established by the committee on SS of the Ministry of Health and Welfare of Japan and
the criteria proposed by the AmericanEuropean consensus group
[8, 9]. Untreated patients matched for age, baseline salivary secretion and other background variables served as controls. The study
excluded patients who had begun treatment with steroids,
immunosuppressors or DMARDs within the previous 3 months.
A washout period, lasting at least 4 weeks, was set for patients
receiving treatment with anti-SS drugs such as cevimeline hydrochloride and anethole trithione. During the study period, the use
of any drug which could affect the aforementioned drugs or the
salivary secretion was prohibited. The continuous use of drugs
employed before the start of this study to treat an underlying
disease or complications was permitted provided their dose
levels were as far as possible kept unchanged. After the study
was approved by the ethics committee of our institutes, 40 patients
who gave their informed consent to participate in the study were
prescribed oral mizoribine three times daily (after each meal)
at a daily dose of 150 mg.

The First Department of Internal Medicine, University of Occupational and

Environmental Health, School of Medicine, Kitakyushu, 2Department of General
Medicine and 3Department of Diagnostic Pathology, Nara Medical University,
School of Medicine, Kashihara, Japan.

Histological evaluation of minor salivary glands

Submitted 14 April 2009; revised version accepted 30 June 2009.

After written informed consent was obtained, oral/labial biopsy

was performed in each patient before the start of the treatment.
The histological features of minor salivary glands were classified
as follows. (i) Activity of the disease was rated on a three-grade

Correspondence to: Yoshiya Tanaka, First Department of Internal Medicine,

University of Occupational and Environmental Health, School of Medicine, 1-1
Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan.
E-mail: tanaka@med.uoeh-u.ac.jp

1279
The Author 2009. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

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Objectives. To evaluate the response of patients with SS to mizoribine therapy in relation to histological features of minor salivary glands.
Methods. Forty patients definitely diagnosed as having SS were treated with mizoribine (150 mg/day). Thirty-four untreated patients matched
for age, baseline salivary secretion, etc., served as controls. Salivary secretion volume (measured by the Saxon test) and serum IgG level
were measured before and after 24 weeks of treatment. Each histological finding (lymphocytic infiltration, acinar atrophy and intralobular
fibrosis) was graded at baseline and the therapeutic responses were compared with the grade at 24 weeks in both the groups.
Results. There was a significant increase of the salivary secretion volume after treatment with mizoribine as compared with the untreated
control group. The effect of mizoribine on salivary secretion was more marked in patients having moderate lymphocytic infiltration and
moderate or less severe acinar atrophy and intralobular fibrosis at baseline.
Conclusions. Mizoribine was clinically effective in patients with SS whose minor salivary glands had moderate cell infiltration and were free
of intralobular fibrosis. The drug was less effective in patients who presented intralobular fibrosis. Histological evaluation of the minor salivary
glands may serve to predict the response of SS patients to mizoribine therapy.

Shingo Nakayamada et al.

1280

scale according to the degree of inflammatory cell infiltration

per lobule: Grade A (mild infiltration, with less than one focus;
corresponding to Greenspans Grades 0, 1 and 2 [10, 11]); Grade B
(moderate infiltration with one or more focuses; corresponding to
Greenspans Grades 3 and 4); and Grade C (infiltration severe
enough to form lymph follicles). (ii) Severity of acinar atrophy
was rated on a three-grade scale according to the extent of
atrophy: Grade A (less than one-third within a lobule); Grade B
(over one-third and less than two-thirds); and Grade C (over twothirds). (iii) Degree of chronic nature of the disease was rated on
a three-grade scale according to the extent of intralobular fibrosis:
Grade A (less than one-third within a lobule); Grade B (over
one-third and less than two-thirds); and Grade C (over twothirds). Histological features were evaluated in a blind manner
by two rheumatologists and one pathologist. We decided the
histological categories by a majority of three doctors.

Evaluation of efficacy

Statistical analysis
For statistical analysis of efficacy, the intra- and inter-group
differences between values at baseline and at 24 weeks were subjected to Wilcoxon signed-rank test and MannWhitney U-test.
P < 0.05 (two-sided) was regarded as statistically significant.

Responses analysed in relation to histological classification

Salivary secretion volume increased significantly following treatment with mizoribine in patients in whom lymphocytic infiltration
had been Grade B and acinar atrophy and intralobular fibrosis
Grade A or B before treatment. In the control group, patients
with similar grades at the start of the study, showed no change
in salivary secretion volume after the 24-week period. Thus, there
was a significant difference in salivary secretion volume between
the two groups (Table 3). On the other hand, in patients in whom
lymphocytic infiltration had been Grade A and acinar atrophy
and intralobular fibrosis Grade C before treatment, i.e. patients
with chronic lesions (acinar atrophy and fibrosis were more
marked than inflammatory cell infiltration), no particular
response to treatment was noted. The percent changes in salivary
secretion volume from baseline (100) are shown in Fig. 1. This
parameter also differed significantly between the two groups when
comparison was confined to cases where lymphocytic infiltration
had been Grade B and acinar atrophy and intralobular fibrosis
Grade A or B before treatment (Fig. 1ac). Serum IgG level
TABLE 1. Characteristics of the patients

Results

Untreated
patients,
n 34

Background variables
Table 1 summarizes the background variables of the patients.
There was no significant difference between the mizoribine-treated
group and the untreated control group in terms of sex, age, disease
type (primary/secondary), duration of sickness, pre-treatment
baseline salivary secretion volume or serum IgG level. The pretreatment baseline grades of histological features of minor salivary
glands (lymphocytic infiltration, intralobular fibrosis and
acinar atrophy) showed an approximately similar distribution in
both the groups, with no statistically significant inter-group
differences.

Efficacy
No serious adverse reaction was noted in any group. Table 2
shows the time course of salivary secretion volume and serum
IgG level in each group. In the untreated group, salivary secretion
volume at 24 weeks (1.6  1.6 mg/2 min) did not differ from that at
baseline (1.7  1.6 g/2 min; P 0.87). In the mizoribine-treated
group, on the other hand, salivary secretion volume increased

Characteristic
Men/women, n/n
Age, mean  S.D., years
Primary/secondary, n/n
Disease duration, mean  S.D.,
years
Salivary secretion volume,
mean  S.D., g/2 min
Serum IgG, mean  S.D., mg/dl
Histological variables at biopsy
Lymphoid infiltration, n (%)
A (mild)
B (moderate)
C (severe)
Intralobular fibrosis, n (%)
A (mild)
B (moderate)
C (severe)
Acinar atrophy, n (%)
A (mild)
B (moderate)
C (severe)

Mizoribine-treated
patients,
n 40

P-value

0/34
54.1  16.5
28/6
2.9  3.7

1/39
53.5  14.7
31/9
1.9  2.6

0.35
0.48
0.60
0.17

1.7  1.6

1.6  1.5

0.84

1955.7  713.7

1939.7  755.9

0.58

5 (15)
17 (50)
12 (35)

5 (13)
20 (50)
15 (37)

16 (47)
14 (41)
4 (12)

20 (50)
12 (30)
8 (20)

15 (44)
15 (44)
4 (12)

20 (50)
12 (30)
8 (20)

0.79

0.84

0.97

TABLE 2. Changes relative to baseline in salivary function and serum IgG level in patients treated with or without mizoribine
Untreated patients, n 34
Baseline

Week 24

Salivary secretion volume, mean  S.D., g/2 min

Total
1.7  1.6
1.6  1.6
Primary
1.7  1.7
1.7  1.6
Secondary
1.5  1.2
1.4  1.1
Serum IgG, mean  S.D., mg/dl
Total
1955.7  713.7
1924.3  742.4
Primary
2055.3  729.8
2016.0  748.6
Secondary
1507.7  442.1
1429.0  515.2

Mizoribine-treated patients (n 40)

P-value

Baseline

Week 24

P-value

P-value for
each group

0.87
0.46
0.05

1.6  1.5
1.7  1.6
0.9  1.0

2.1  2.2
2.3  2.4
1.5  1.3

<0.01
<0.01
0.02

<0.01
0.02
<0.01

0.53
0.48
0.69

1939.7  755.9
1906.6  767.0
2053.6  748.9

1886.2  733.3
1818.9  749.5
2117.7  661.0

0.04
<0.01
0.37

0.52
0.29
0.55

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Salivary secretion volume was determined by Saxon test according

to the method reported by Kohler and Winter [12]. The mean of
two measurements was adopted. Serum IgG level was also
measured. Each parameter was measured at the start and after
24 weeks of treatment and compared within each group and
between the two groups.

significantly from baseline (1.6  1.5 g/2 min) to 24 weeks

(2.1  2.2 g/2 min; P < 0.01). As for the serum IgG level, the
untreated group showed no significant change (from
1955.7  713.7 to 1924.3  742.4 mg/dl, P 0.53); whereas, in the
mizoribine treated group, this was significantly decreased after 24
weeks of treatment (from 1939.7  755.9 to 1886.2  733.3 mg/dl,
P 0.04). We compared the efficacy among primary and secondary SS. Although the level of serum IgG had a tendency to
decrease in primary SS, there were no significant differences
between primary and secondary groups.

Histological evaluation in SS

1281

TABLE 3. Salivary secretion volume (g/2 min) in patients distributed according to histological variables
Untreated patients

Lymphoid infiltration
A (mild)
B (moderate)
C (severe)
Intralobular fibrosis
A (mild)
B (moderate)
C (severe)
Acinar atrophy
A (mild)
B (moderate)
C (severe)

Mizoribine-treated patients

Baseline

Week 24

P-value

Baseline

Week 24

P-value

P-value for
each group

3.0  2.4
1.9  1.5
0.7  0.5

3.0  2.0
1.9  1.5
0.7  0.5

0.50
0.98
0.18

3.4  2.1
1.4  1.4
1.1  1.2

5.0  4.2
1.9  1.5
1.5  1.4

0.22
<0.01
0.06

0.35
0.03
0.03

2.2  1.8
1.4  1.3
0.6  0.5

2.2  1.7
1.4  1.4
0.5  0.6

0.62
0.97
0.47

2.2  1.8
1.1  1.1
0.6  0.7

3.0  2.7
1.7  1.2
0.7  0.7

0.01
0.02
0.78

0.01
0.03
0.44

2.2  1.8
1.4  1.4
0.6  0.5

2.2  1.7
1.4  1.4
0.5  0.6

0.32
0.56
0.47

2.1  1.8
1.2  1.1
0.6  0.7

2.9  2.7
1.8  1.2
0.7  0.7

0.01
0.01
0.78

0.05
0.01
0.44

(c)

Mean percent change from baseline

Mean percent change from baseline

(b)

**

**

FIG. 1. Mean  S.D. changes of salivary secretion in patients distributed according

to histological variables. Each histological variable was assessed using a threedegree scale (A, B and C). The percent change in salivary secretion volume from
the pre-treatment baseline in the untreated group is represented by the white bars
and that in the mizoribine-treated group by black bars. (a) Lymphocyte infiltration.
(b) Intralobular fibrosis. (c) Acinar atrophy. *P < 0.05, **P < 0.01.

decreased significantly following treatment with mizoribine in

patients with Grade A acinar atrophy and intralobular fibrosis,
but there was no significant difference in this parameter between
the treated and untreated groups (Table 4).

Discussion
Although SS can markedly reduce the QOL of patients because
of symptoms related to dryness, no radical therapy aimed at
correcting the immunological abnormalities associated with this
disease is available as yet. Mizoribine was discovered in the culture
broth of Eupenicillium brefeldianum isolated from soil from the
Hachijo Island, Japan, and has been developed as an anti-fungal
agent. It is currently used as an immunosuppressant for the
control of graft rejection associated with renal transplants and
for the treatment of lupus nephritis, RA and primary nephrotic
syndrome. Mizoribine is known to act upon the pathway of purine
synthesis in cells via the specific competitive inhibition of inosine
monophosphate (IMP) dehydrogenase, thereby exerting suppressive effects on T- and B lymphocytes [13, 14]. In patients with SS,
a marked periductal autoreactive lymphocyte infiltration in the
lacrimal and salivary glands arises, and acinar destruction and
fibrosis ensue, thus leading to a diminished lacrimal and salivary
gland function. These infiltrating lymphocytes largely comprise
CD4 T lymphocytes at an early stage and, as the lesions progress,
B lymphocytes predominate. The resultant excessive B-cell activation is accompanied by hypergammaglobulinaemia, and the
production of autoantibodies, such as the anti-SS-A/Ro antibody
and the anti-SS-B/La antibody, has been noted. Since mizoribine
has been shown to strongly suppress the proliferation of B lymphocytes [15, 16], this drug is expected to be clinically useful in the
treatment of SS. We previously conducted a multicentre study in
which 59 patients with primary SS were treated with mizoribine
(150 mg/day) for 16 weeks. In that study, significant improvement
or alleviation was noted in the patients self-assessment of oral and
eye dryness, the physicians assessment of oral sicca symptoms,
labioangular sicca symptoms and the physicians overall assessment [7]. However, that study was designed as an open study.
To demonstrate the usefulness of mizoribine in the treatment of
SS, it is necessary to conduct a placebo-controlled randomized
study. Furthermore, detailed evaluation is needed on the effects
of mizoribine on extraglandular lesions and its long-term
effects [17].
In the present study, the response to treatment with mizoribine
was analysed in relation to pre-treatment histological features of
minor salivary glands. To our knowledge, there is no published
analysis of responses of SS to immunosuppressor therapy in
relation to histological features of minor salivary gland. In this
study, mizoribine-induced increase of salivary secretion volume
was particularly marked in patients who had moderate lymphocyte infiltration without acinar atrophy or intralobular fibrosis
before treatment. In those in whom the disease had become
chronic (showing structural destruction of glandular tissue such
as acinar atrophy and intralobular fibrosis), the response to treatment with this drug was poor, suggesting that the response to this
drug depends on the functional state of the exocrine glands.

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(a)

Mean percent change from baseline

Values are represented as mean  S.D.

Shingo Nakayamada et al.

1282

TABLE 4. Serum IgG level (mg/dl) in patients distributed according to histological variables
Untreated patients

Lymphoid infiltration
A (mild)
B (moderate)
C (severe)
Intralobular fibrosis
A (mild)
B (moderate)
C (severe)
Acinar atrophy
A (mild)
B (moderate)
C (severe)

Mizoribine-treated patients

Baseline

Week 24

P-value

Baseline

Week 24

P-value

P-value for
each group

1601.6  281.6
1945.7  556.5
2128.5  983.3

1621.8  374.3
1889.4  623.7
2093.8  965.8

0.89
0.36
0.93

1544.6  376.5
1738.4  629.2
2339.7  853.8

1480.2  328.1
1695.5  630.4
2275.7  809.0

0.69
0.18
0.17

0.60
0.66
0.28

1792.3  488.4
1985.4  496.0
2510.5  1667.3

1711.9  472.8
1970.5  583.0
2570.3  1581.8

0.16
0.94
0.47

1637.3  560.2
2315.1  859.1
2132.4  791.5

1559.8  539.2
2264.8  823.2
2134.1  722.1

0.04
0.16
0.89

0.84
0.38
0.87

1809.3  483.2
1965.7  509.3
2510.5  1667.3

1729.3  468.7
1950.5  595.6
2570.3  1581.8

0.16
0.94
0.47

1681.7  605.9
2241.1  863.7
2132.4  791.5

1601.8  579.2
2194.8  836.3
2134.1  722.1

0.04
0.16
0.89

0.89
0.41
0.87

Values are represented as mean  S.D.

Rheumatology key messages

 Mizoribine was effective in SS patients whose salivary glands had
moderate cell infiltration and were free of intralobular fibrosis.
 Histological evaluation may serve to predict response of SS
patients to mizoribine.

Acknowledgements
Funding: This work was supported in part by Research Grants-InAid for Scientific Research from the Ministry of Health, Labor
and Welfare of Japan, the Ministry of Education, Culture, Sports,
Science and Technology of Japan and University of Occupational
and Environmental Health, Japan.

Disclosure statement: The authors have declared no conflicts of

interest.

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Another finding was that in patients showing severe lymphocytic

infiltration (Grade C), treatment with mizoribine tended to
improve salivary secretion volume. The severity of histological
abnormalities of minor salivary glands is usually assessed based
on the degree of lymphocytic infiltration. Some investigators
reported that the response to treatment with cevimeline hydrochloride was poor when SS patients showed severe lymphocytic
infiltration around the conduits [18]. However, considering the
mechanism of action of mizoribine, a response is expected even
in patients with active lesions presenting marked lymphocytic
infiltration. In other words, we may say that mizoribine is
expected to be effective when SS is at relatively early stages
(absence of fibrosis despite the presence of lymphocytic
infiltration). Our findings suggest the validity of the view that
lymphocytic infiltration not only plays an important role in
the pathogenesis of SS but that it also is a valid target of SS
treatment.
In conclusion, mizoribine is unlikely to cause serious adverse
reactions and it improves salivary secretion in patients with SS.
The results suggest that a pathological evaluation is useful in
determining the indication of this drug in individual cases and
that early therapeutic intervention with this drug is important.
It is indispensable to conduct a placebo-controlled randomized
study to confirm the efficacy of mizoribine against SS and to
evaluate its long-term effects.