Download Elevated serum BAFF levels in patients with sarcoidosis: association

Rheumatology 2013;52:1658–1666
doi:10.1093/rheumatology/ket186
Advance Access publication 17 May 2013
RHEUMATOLOGY
Original article
Elevated serum BAFF levels in patients with
sarcoidosis: association with disease activity
Ikuko Ueda-Hayakawa1, Hirotsugu Tanimura2, Manabu Osawa2,
Hiroshi Iwasaka2, Shuichi Ohe2, Fumikazu Yamazaki2, Kana Mizuno2 and
Hiroyuki Okamoto1,2
Abstract
Objective. The purpose of this study was to determine serum levels of B-cell-activating factor (BAFF) and
its clinical association in patients with sarcoidosis.
Methods. Serum levels of BAFF from 37 patients and 21 healthy subjects were examined by ELISA.
Serum angiotensin-converting enzyme (ACE), lysozyme and IFN-g levels in sarcoidosis patients were
also measured. Isolated monocytes cultured with IFN-g, IL-4 or IL-10 and their expression of membrane
and soluble BAFF were analysed by flow cytometry or ELISA. Peripheral B cell subsets were analysed by
flow cytometry. BAFF expression in the granuloma of the skin was examined by immunohistochemistry.
ANAs were determined by indirect IF using HEp-2 cells as a substrate.
CLINICAL
SCIENCE
Results. Serum BAFF levels were significantly elevated in sarcoidosis patients when compared with
healthy controls. The frequency of skin and eye involvement was significantly higher in patients with
elevated serum BAFF than in patients with normal levels. Serum BAFF levels were correlated
with serum levels of ACE, lysozyme and IFN-g. Immunostaining of anti-BAFF in the skin revealed BAFF
expression by epithelioid cells of granuloma. In vitro, IFN-g induced membrane-bound BAFF expression
on monocytes and secretion of soluble BAFF by isolated monocytes. In the peripheral blood, sarcoidosis
patients showed increased naı̈ve B cells with a reciprocal decrease in memory B cells and plasmablasts.
Seventeen of 26 (65%) sarcoidosis patients exhibited ANA positivity.
Conclusion. Serum BAFF levels can be used as a surrogate marker of disease activity in sarcoidosis
patients. Increased BAFF may be related to the pathogenesis of sarcoidosis.
Key words: sarcoidosis, B-cell-activating factor (BAFF), B cell, monocyte.
Introduction
Sarcoidosis is a systemic granulomatous disorder that is
characterized by pathological lesions with a discrete,
compact, non-caseating epithelioid cell granuloma that
are commonly observed in the lungs, lymph nodes, eyes
and skin [1]. Once mononuclear inflammatory cells accumulate in the target organ, macrophages aggregate and
differentiate into epithelioid and multinucleated giant cells
[2]. In addition to predominant involvement of cellular
1
Department of Dermatology, Kansai Medical University, Hirakata,
Japan and 2Department of Dermatology, Kansai Medical University,
Moriguchi, Japan.
Submitted 18 July 2012; revised version accepted 4 April 2013.
Correspondence to: Ikuko Ueda-Hayakawa, Department of
Dermatology, Kansai Medical University, 2-5-1 Shinmachi, Hirakata,
Osaka 573-1010, Japan. E-mail: [email protected]
immunity in the pathogenesis of sarcoidosis, an involvement of the humoral immune system in the inflammatory
process is indicated by frequently observed hypergammaglobulinaemia [3, 4], autoantibody production [5] and
circulating immune complexes [6]. Moreover, the clinical
and pathological features of sarcoidosis mimic those of
many systemic autoimmune diseases [7].
The TNF family ligand B-cell-activating factor (BAFF)
has critical roles in B cell development and function [8].
A role for BAFF and BAFF receptor (BAFFR) in the survival
of B cells during B cell maturation has been shown in
BAFF- and BAFFR-deficient animals [9, 10]. As BAFF promotes B cell survival, its overexpression could potentially
break B cell tolerance by rescuing self-reactive B cells
from deletion. Indeed, BAFF-transgenic mice develop
severe autoimmune symptoms that are similar to SLE
and SS characterized by hypergammaglobulinaemia,
! The Author 2013. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: [email protected]
Elevated serum BAFF levels in sarcoidosis patients
high titres of anti-dsDNA antibodies and GN [11, 12]. In
humans, serum levels of BAFF are high in patients with
systemic autoimmune diseases such as SLE, RA, SS and
SSc [12–14].
In this study, we showed that serum BAFF levels were
elevated in patients with sarcoidosis. High serum BAFF
levels were associated with skin and eye involvement.
Serum BAFF levels were also significantly correlated
with the serum levels of both angiotensin-converting
enzyme (ACE) and lysozyme, as well as with serum interferon (IFN)-g levels. In vitro, IFN-g enhanced membranebound BAFF expression and secretion of the soluble form
of BAFF by monocytes from healthy controls, suggesting
that IFN-g-induced BAFF expression by monocytes may
play a role for elevated serum BAFF production.
Immunohistostaining of skin biopsies revealed a local
production of BAFF by epithelioid cells in the granuloma.
We also showed altered B lymphocyte homeostasis
characterized by an expanded frequency of naı̈ve B
cells (CD19+CD27 ) and reduced frequency of both
memory B cells (CD19+CD27+) and plasmablasts
(CD19+/ CD27high) in sarcoidosis patients. A high frequency of ANA positivity was observed in sarcoidosis patients, though there was no correlation between BAFF
levels and ANA positivity. From these observations, we
suggest that elevated BAFF production may cause B
cell alterations and contribute to the pathogenesis in
sarcoidosis.
Materials and methods
Patients
Serum samples were obtained from 37 Japanese sarcoidosis patients (5 males and 32 females). The age
(mean ± SD) of these patients was 61 ± 12 years. Fresh
venous blood samples were centrifuged shortly after clot
formation and all samples were stored at 80 C before
use. For comparison, serum from 21 age- and sexmatched healthy Japanese volunteers was collected and
stored in the same manner. The diagnosis of sarcoidosis
was established on the basis of clinical and radiological
findings, supported by histological evidence in one or
more organs of noncaseating epithelioid cell granulomas
in the absence of organisms or particles. Patients with
both sarcoidosis and SSc were excluded because BAFF
is known to be elevated in CTDs such as SSc.
Patients underwent a clinical assessment. Involvement
of their organ systems, including skin, lung, heart, eye and
muscle, was evaluated as described previously [15].
Cutaneous involvement was assessed by skin biopsy; pulmonary involvement included bilateral hilar lymphadenopathy (BHL), interstitial infiltrate or pulmonary fibrosis
based on a chest radiogram and high-resolution CT; cardiac involvement included 18FDG PET and/or 67Ga scintigraphy uptake for inducible arrhythmias; eye
involvement included anterior and/or posterior uveitis
determined by slit-lamp and funduscopic examination.
Involvement of others organs including muscle, liver,
spleen, kidney and bone was examined by imaging with
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18
FDG PET and/or 67Ga scintigraphy. ACE activity was
measured using a method based on colourimetry of the
quinoneimine dye produced from the substrate hippuryl-Lhistidyl-L-leucine [16]. Lysozyme activity was measured
using a turbidimetric method [17]. ACE and lysozyme
were considered elevated when the values were
>20.0 U/l and 10 mg/ml, respectively. ANAs were determined by indirect IF using HEp-2 cells as a substrate,
and a titre of 1:80 or higher was considered positive
(MBL, Nagoya, Japan). This study was approved by the
ethics committee on human research of Kansai Medical
University and informed consent was obtained from all
patients.
Enzyme-linked immunosorbent assay
The levels of BAFF in sera and supernatant of monocytes
culture were assessed by an ELISA using commercial kits
for the assay of human BAFF (R&D Systems, Minneapolis,
MN) according to the manufacturer’s protocol. The mean
minimum detectable dose of BAFF was 3.38 pg/ml. Serum
IFN-g levels were measured by a commercial ELISA kit
(Human IFN-gamma Quantikine ELISA kit system, R&D
Systems, sensitivity >8.0 pg/ml) according to the manufacturer’s protocol. All samples were analysed in duplicates. Absorbance was measured at 450 nm.
Flow cytometric analysis
Heparinized whole blood samples from both healthy
donors (n = 11) and sarcoidosis patients (n = 15) were obtained for FACS analysis. Peripheral blood mononuclear
cells (PBMCs) were isolated by centrifugation over
Lymphoprep (Axis-Shield PoC AS, Oslo, Norway) and
washed in PBS. Erythrocytes were lysed using red blood
cell lysis buffer (BD Pharmingen, Franklin Lakes, NJ)
before use. Cells were stained in ice-cold PBS supplemented with 5% bovine calf serum using a combination
of a FITC-conjugated antibody, phycoerythrin (PE)-conjugated antibody, allophycocyanin (APC)-conjugated
antibody and 7-amino-actinomycin D (7AAD) (Invitrogen
Life Technologies, Carlsbad, CA). Antibodies used in
this study included FITC-conjugated anti-CD19 (HIB19;
eBioscience, San Diego, CA), FITC-conjugated BAFF
(1D6; eBioscience), APC-conjugated anti-CD27 (LG7.F9;
eBioscience),
PE-conjugated
CD14
(M5E2;
BD
Pharmingen) and APC-conjugated anti-CD3 (UCHT1;
eBioscience). Dead and damaged cells were labelled
with 7AAD and were eliminated from the analysis. Cells
were analysed on a FACSCalibur (BD Biosciences, Franklin Lakes, NJ).
Monocyte isolation and cell cultures
Monocytes from healthy individuals were isolated from
PBMC by negative selection employing antibodies
labelled with magnetic beads according to the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach,
Germany). For the analysis of BAFF secretion, monocytes
were FACS sorted after using a negative monocyte isolation kit. Cell sorting was performed on a FACSAlia III
(BD Biosciences). Monocytes were resuspended in
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Ikuko Ueda-Hayakawa et al.
RPMI 1640 culture medium (Sigma Aldrich, St Louis, MO,
USA) containing 10% fetal calf serum, 100 units/ml penicillin and 100 mg/ml streptomycin and maintained at 37 C
and 5% CO2. The following recombinant human reagents
were used: 5–100 ng/ml IFN-g (PeproTech Inc., Rocky Hill,
NJ, USA), 100 ng/ml IL-4 (PeproTech Inc.) and 100 ng/ml
IL-10 (R&D Systems).
Immunohistochemical staining for BAFF
BAFF expression was examined by immunohistochemical
staining in skin biopsies from both sarcoidosis patients
with elevated serum BAFF levels (n = 3; three females)
and five normal controls. Sections were stained with rat
monoclonal IgG2a antibody specific for human BAFF
(Buffy-1, LifeSpan Biosciences, Inc., Seattle, WA, USA),
then with horseradish peroxidase-conjugated avidinbiotin complexes (Vectastatin avidin-biotin Complex
Methods, Vector Laboratories, Burlingame, CA, USA).
Sections were finally developed with DAB [Histofine
SAB-PO (M), Nichirei Bioscience, Tokyo, Japan] and
counterstained with haematoxylin.
Statistical analysis
Statistical analysis was performed using the Mann–
Whitney U test for determining the level of significance
of differences between sample means. Fisher’s exact
probability test for comparison of frequency and Bonferroni’s test for multiple comparisons were also used.
Spearman’s rank correlation coefficient was used to
examine the relationship between two continuous variables. P values < 0.05 were considered statistically
significant.
Results
Elevated serum BAFF levels in patients with
sarcoidosis
Serum BAFF levels were measured in 37 sarcoidosis patients and in 21 healthy controls. Serum BAFF levels were
significantly higher in patients with sarcoidosis than in
healthy controls (mean ± S.D. of 1.59 ± 0.92 ng/ml vs
0.97 ± 0.21 ng/ml, P < 0.0005, Fig. 1A).
Correlation of serum BAFF levels with both ACE
and lysozyme levels
Because serum ACE and lysozyme have been commonly
used as markers of sarcoidosis activity [18, 19], we examined serum ACE and lysozyme levels in patients with sarcoidosis. We found that serum BAFF levels were
significantly correlated with both serum ACE and lysozyme levels (P < 0.01 and P < 0.0001, respectively,
Fig. 1B). No significant correlation between serum levels
of BAFF and serum gammaglobulin levels was observed.
These findings indicate that serum BAFF levels are associated with disease activity in sarcoidosis.
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Clinical and laboratory characteristics in patients
with sarcoidosis showing normal and elevated
serum BAFF levels
Clinical and laboratory findings were compared between
sarcoidosis patients with elevated BAFF levels and those
with normal levels. BAFF values higher than the
mean + 2 S.D. (1.40 ng/ml) of the control serum samples
were considered to be elevated in this study. Elevated
serum BAFF levels were found in 38% (14/37) of the patients (Table 1). The frequency of skin involvement was
significantly higher in patients with elevated BAFF compared with those with normal levels (P < 0.0005, Table 1).
In addition, patients with elevated serum BAFF levels had
more frequently eye involvement than those patients with
normal levels (P < 0.05). No significant difference was
observed in BHL and in muscle involvement between patients with elevated BAFF levels and those without. The
frequency of diffuse lung involvement and heart involvement tended to be higher in patients with elevated BAFF
than those without. Serum ACE, lysozyme and gammaglobulins except IgM levels also tended to be higher in
patients with elevated BAFF levels compared with patients with normal BAFF levels, although no significant difference was observed between the two groups. Serum
calcium levels were similar between these two groups.
Correlation between serum BAFF and serum IFN-g
levels in patients with sarcoidosis
Several cytokines have been reported to increase BAFF
production [20, 21]. IFN-g has been known to play important roles in the formation of sarcoid lesions in active patients [22]. Thus, we examined the association between
levels of serum BAFF and IFN-g. Serum IFN-g levels were
significantly elevated in patients with increased BAFF
levels compared with those with normal levels (Table 1),
and serum levels of BAFF significantly correlated with
those of IFN-g (r2 = 0.51, P < 0.0001, Fig. 1C). Thus,
these findings suggest that increased IFN-g contributed
to the enhanced BAFF production in sarcoidosis patients.
IFN-g induced membrane-bound BAFF expression
on monocytes and soluble BAFF production
by monocytes
As it has been reported that IFN-g induced BAFF expression on monocytes [21], we next investigated whether the
presence of IFN-g, IL-4 or IL-10 affected BAFF expression
on monocytes from healthy controls. These cytokines are
known to have differential effects on monocytes: IFN-g is
the prototype of activating cytokines, whereas IL-10 and
IL-4 exhibit inhibitory effects. Monocytes were enriched
by negative selection using the MACS system and were
cultured at 1–5 105 per well in a 96-well plate for 3 days
in the presence of IFN-g, IL-4 or IL-10. After 3 days of
cytokine treatment, cells were stained with BAFF- and
CD14-specific antibodies to determine whether these
cytokines could regulate BAFF expression on monocytes.
BAFF-specific immunostaining on monocytes was enhanced by treatment with IFN-g but not with either IL-4 or IL10 (supplementary Table S1, available at Rheumatology
www.rheumatology.oxfordjournals.org
Elevated serum BAFF levels in sarcoidosis patients
FIG. 1 Serum BAFF level and its correlation with disease activity in patients with sarcoidosis.
(A) Serum levels of BAFF in patients with sarcoidosis and in healthy controls. Serum levels of BAFF were determined by
specific ELISA. The dashed lines indicate the cut-off value (mean + 2 S.D. of the control samples). The lines indicate the
median. Serum levels of BAFF were significantly elevated in sarcoidosis patients when compared with healthy controls.
(B) Correlation of serum BAFF levels with both ACE and lysozyme. Serum ACE and lysozyme levels were significantly
correlated with serum BAFF levels in sarcoidosis patients: r2 = 0.20; P < 0.01 and r2 = 0.62; P < 0.0001, respectively.
(C) Correlation between serum IFN-g and serum BAFF levels in sarcoidosis patients. Serum IFN-g levels were significantly
correlated with serum BAFF levels in sarcoidosis patients: r2 = 0.51; P < 0.0001.
Online). Next, we examined production of the soluble form
of BAFF by monocytes. Monocytes from healthy controls
were purified and cultured with the cytokines for 4 days,
and the soluble form of BAFF in the supernatant was
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quantified by ELISA. Levels of the soluble forms of BAFF
were significantly elevated in supernatant of monocytes
treated with IFN-g (Fig. 2A). Moreover, the numbers of
monocytes treated with IFN-g were significantly increased
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Ikuko Ueda-Hayakawa et al.
TABLE 1 Clinical and laboratory observations in patients
with sarcoidosis showing elevated and normal serum
BAFF levels
Elevated
BAFF
(n = 14)
FIG. 2 IFN-g mediates enhanced BAFF production and
proliferation of monocytes.
Normal
BAFF
(n = 23)
Age, years
65.8 (9.6)
58.3 (12.7)
Sex, n, male:female
1:13
3:20
Disease duration, years
6.1 (5.4)
4.4 (3.6)
Skin, %
100***
86
Lung, %
91
67
Radiographic stage 0/I/II/III
1/5/2/3/(3)
6/11/0/1/(5)
or IV/(not determined), n
BHL, %
67
70
Diffuse lung, %
17
6
Eyes, %
67*
41
Liver, %
0
0
Nervous system, %
0
0
Heart, %
33
9
Kidney, %
0
0
Bones, %
0
0
Muscles, %
17
14
Lymphadenopathy, %
67
67
ACE, IU/l
20.7 (11.6)
16.3 (7.9)
Lysozyme, mg/ml
12.2 (13.2)
6.3 (2.9)
Ca, mg/dl
9.3 (0.6)
9.4 (0.3)
IgG, mg/dl
1465 (559)
1416 (381)
IgA, mg/dl
279 (109)
228 (93)
IgM, mg/dl
66 (35)
93 (44)
IFN-g, pg/ml
4.9 (6.4)**
0.0 (0.1)
Current treatment
Oral steroids, n
7
5
MTX, n
1
0
Except where indicated otherwise, values are mean (S.D.).
*P < 0.05, **P < 0.0005, ***P < 0.0005 vs sarcoidosis patients
with normal BAFF levels.
compared with controls, although the numbers of monocytes cultured with IL-4 or IL-10 were comparable to the
control (Fig. 2B). These results suggest that IFN-g plays
an important role in enhancing BAFF production by
monocytes.
(A) Production of the soluble form of BAFF by monocytes.
Monocytes from healthy controls were purified and cultured with cytokines for 4 days, and the soluble form of
BAFF in the supernatant was quantified by ELISA. The
level of the soluble form of BAFF was significantly elevated in supernatant of monocytes treated with IFN-g.
(B) Proliferation of monocytes by IFN-g. The number of
monocytes from healthy individuals treated with IFN-g
showed a significant increase compared with the control,
whereas the number of monocytes cultured with IL-4 or
IL-10 was comparable to the control.
Immunohistochemistry of BAFF in sarcoid skin lesions
To determine the role of BAFF in the pathogenesis of
granuloma, we examined BAFF expression in skin biopsies from three sarcoidosis patients by immunohistochemistry. BAFF staining was observed in granuloma
(Fig. 3A). The majority of BAFF-expressing cells were
CD68+ epithelioid cells (Fig. 3A and B), which are thought
to be derived from monocyte-macrophage lineage cells
[2, 23]. On the other hand, BAFF expression was rarely
seen in infiltrated lymphocytes (Fig. 3A, C and D).
Alteration of B cells subsets in sarcoidosis patients
Since BAFF has been known to be an important player in
the survival and differentiation of B cells, we examined the
frequency and numbers of B cells to determine whether
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there were alterations in the peripheral B cell populations
in patients with sarcoidosis. Blood samples from healthy
donors and patients were analysed by flow cytometry.
PBMCs were stained with CD19- and CD27-specific antibodies and classified into three subsets: CD19+CD27
naı̈ve B cell, CD19+CD27+ memory B cell and
CD19+/ CD27high plasmablast, as previously described
[24, 25]. The frequency of total lymphocytes and absolute
number of CD19+ B cells were similar between healthy
donors and patients (Table 2). Compared with healthy
donors, a significantly higher frequency of naı̈ve B cell
(CD19+CD27 ) was observed in sarcoidosis patients
(P < 0.05). In contrast, the frequency of memory B cells
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Elevated serum BAFF levels in sarcoidosis patients
FIG. 3 Immunohistochemistry of BAFF in sarcoid skin lesions.
Representative picture of BAFF staining in skin biopsies from sarcoidosis patients with elevated serum BAFF levels.
(A) BAFF, (B) negative control, (C) CD68, (D) CD3, (E) CD20. BAFF staining was observed in granuloma (A). The majority
of BAFF-expressing cells were CD68+ epithelioid cells (A, C). BAFF expression was rarely seen in infiltrated lymphocytes
(D, E).
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Ikuko Ueda-Hayakawa et al.
TABLE 2 Frequency and number of B cells in healthy donors and sarcoidosis patients
Healthy donors (n = 11)
Age, years
Male:female
CD19+ B cell
Frequency, % of lymphocytes
Absolute number, 104/ml
B cell subset, % of total B cells
CD19+CD27 (naı̈ve)
CD19+CD27+ (memory)
CD19+/ CD27high (plasmablast)
Sarcoidosis patients (n = 15)
50.3 (13.1)
3:8
58.2 (11.3)
1:14
4.72 (2.05)
86 (41)
5.30 (3.43)
84 (65)
69.41 (7.97)
27.30 (6.98)
3.29 (1.41)
78.55 (15.69)*
19.43 (14.66)*
2.01 (1.33)*
Values are mean (S.D.). B cells were gated on CD19+ cells and further distinguished by surface
markers as indicated. *P < 0.05.
TABLE 3 Characteristics of ANA positivity in 26 sarcoidosis patients
Pt
Age, years
Sex
ANA titre
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
60
63
62
50
47
59
42
77
59
62
68
72
63
74
75
37
73
62
60
79
46
74
75
61
70
47
F
F
F
F
F
F
F
F
F
F
F
F
F
F
M
F
F
M
F
F
F
F
F
F
F
F
<40
<40
<40
<40
40
40
40
40
40
80
80
80
80
80
80
80
80
80
80
80
80
160
160
640
1280
1280
ANA pattern
Specific antibody
Sp, H
Sp, H
dsDNA
Sp, H
Sp, H
Sp, H
Sp
Sp, H
Sp
Sp
Sp, H
Sp
Sp (80), DS (160)
Sp
Sp
DS
DS
U1RNP, SSA, SSB
ACA
ACA
Serum BAFF
levels, ng/ml
IFN-c, pg/ml
2.38
0.98
1.23
1.03
1.03
1.19
1.27
2.34
3.39
0
0
0
9.31
3.13
1.13
2.1
1.38
1.18
1.02
1.93
1.53
2.54
5.55
0.78
1.44
1.9
1.86
0.92
2.15
1.47
0
7.32
0.59
0
0
0
14.68
1.27
19.34
0
0
8.48
1.75
0
0
0
Sp: speckled; H: homogeneous; DS: discrete speckled.
(CD19+CD27+) and plasmablasts (CD19+/ CD27high) was
significantly reduced (P < 0.05 for each).
ANAs in sarcoidosis patients
Since overexpression of BAFF could potentially break B
cell tolerance by rescuing self-reactive B cells from deletion, we performed an ANA test by indirect IF. We examined ANA in 26 sarcoidosis patients, and a titre of 1:80 or
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higher was considered positive (Table 3). Seventeen of the
26 patients (65%) had ANA positivity. Fifteen of these
were female. Several IF patterns were observed, including
fine speckled (in 14 sera), homogeneous (in 7 sera) and
discrete speckled (in 3 sera). Additionally, four patients
exhibited some autoantibody specificities including
anti-dsDNA, anti-U1RNP, anti-SSA, anti-SSB and ACA
antibodies. No correlation was found between serum
BAFF levels and the presence of these autoantibodies.
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Elevated serum BAFF levels in sarcoidosis patients
Positive ANA titres ranged from 1:80 to 1:1280. None of
the patients showed any clinical findings of other CTDs.
None of the healthy controls was positive for ANA.
Discussion
In the current study, serum levels of BAFF were significantly elevated in patients with sarcoidosis (Fig. 1).
Elevated ACE and lysozyme, which have been used as
activation markers [18, 19], were significantly associated
with serum BAFF levels (Fig. 2). In addition, the frequency
of skin and eye involvement was significantly higher in
patients with elevated BAFF levels compared with those
patients with normal levels (Table 1). Close association of
elevated serum BAFF with disease activity could provide a
novel mechanism for the pathogenesis of sarcoidosis.
There was a significant correlation between BAFF and
IFN-g in the serum levels of sarcoidosis patients (Fig. 1C),
suggesting a linkage between BAFF and IFN-g production. Previously we reported an increase in CD14+CD16+
monocytes coincident with high ACE levels in sarcoidosis
patients [26]. Stimulation with IFN-g induced the production of the soluble form of BAFF by monocytes (Fig. 2A)
with increased numbers (Fig. 2B), suggesting that IFN-g
mediated BAFF production by monocytes underlie the aberrant BAFF levels in sarcoidosis patients.
Examination of BAFF expression in the skin from sarcoidosis patients revealed considerable levels of BAFF in
granulomas (Fig. 3). The majority of BAFF-expressing cells
was CD68+ epithelioid cells (Fig. 3A and B). BAFF expression was rarely seen in infiltrated lymphocytes (Fig. 3A, C
and D). BAFF production from the granulomas in the skin
could contribute to the increase in serum BAFF levels. We
did not find any differences, including the size of granuloma and the infiltration of B and T cells among these
patients.
Since BAFF is known to be important in the survival and
differentiation of B cells [27], we examined the frequency
and numbers of B cell subsets in the blood of patients with
sarcoidosis. It revealed that there was an increase in the
frequency of naı̈ve B cells (CD19+CD27 ) and reciprocal
decreases in the frequency of both memory B cells
(CD19+CD27+) and plasmablasts (CD19+/ CD27high) in
patients with sarcoidosis (Table 2). While our paper was
under review, Saussine et al. [28] published similar reports
that BAFF serum levels were significantly higher in patients with chronic active sarcoidosis. Consistent with
our study, naı̈ve B cells tended to be increased, whereas
memory B cells were decreased. Interestingly, Saussine
et al. also showed an increase in transitional B cells. Thus,
increased naı̈ve B cell numbers in active sarcoidosis patients could be the result of expansion of transitional B
cells, which is supported by high levels of serum BAFF.
Moreover, Saussine et al. [29] showed an increase in IL10-producing regulatory B cells (Bregs) in active sarcoidosis patients and proposed anti-inflammatory roles for
granuloma progression.
It has been shown that aberrant expression of BAFF
causes the defective selection of autoreactive B cells
[30]. Increased serum and/or plasma levels of
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BAFF have been documented in SLE, RA, SSc and SS
[12–14, 31]. In this study, we detected low levels of but
significantly higher ANA positivity in our patients with sarcoidosis. Consistent with our observation, Weinberg et al.
[5] reported that ANA positivity was evident in 10 out of 34
(29%) of their sarcoidosis patients. These observations
could be the result of dysregulation of self-tolerance.
Currently the role of autoantibody production in the
pathogenesis of sarcoidosis is unclear. Further study
should be required to identify the relationship between
the autoantibody production and the pathogenesis of
sarcoidosis.
Sarcoidosis outcome cannot be predicted from the initial presentation, making it difficult to determine how to
treat patients. The monoclonal antibody against BAFF
(Belimumab) has completed a placebo-controlled phase
III trial in SLE and has been approved by the FDA for the
treatment of SLE [32, 33]. Although a precise understanding of the immune processes that initiate the disease is
still unclear, we propose that BAFF might be a pathogenic
factor in sarcoidosis and that targeting BAFF may represent a new therapeutic strategy for sarcoidosis patients.
Rheumatology key messages
Serum BAFF levels were significantly elevated in
sarcoidosis patients.
. Serum BAFF levels can be used as a surrogate
marker of disease activity in sarcoidosis.
. Increased BAFF may be related to the pathogenesis
of sarcoidosis.
.
Funding: This work was supported by a Grant-in-Aid for
Young Scientists (B) from the Ministry of Education,
Culture, Sports, Science and Technology.
Disclosure statement: The authors have declared no conflicts of interest.
Supplementary data
Supplementary data are available at Rheumatology
Online.
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