Lupus (2015) 0, 1–11

http://lup.sagepub.com

PAPER

Association of BAFF, APRIL serum levels, BAFF-R, TACI and

BCMA expression on peripheral B-cell subsets with clinical
manifestations in systemic lupus erythematosus
DC Salazar-Camarena1, PC Ortiz-Lazareno2, A Cruz1, E Oregon-Romero1, JR Machado-Contreras1,
JF Muñoz-Valle1, M Orozco-López1, M Marı́n-Rosales1 and CA Palafox-Sánchez1
1
Instituto de Investigación en Ciencias Biomédicas (IICB), Departamento de Clı́nicas Médicas, Centro Universitario de Ciencias de la Salud,
Universidad de Guadalajara, Guadalajara, Jalisco, México; and 2División de Inmunologı́a, Centro de Investigación Biomédica de Occidente
(CIBO), Instituto Mexicano del Seguro Social IMSS, Guadalajara, Jalisco, México

Objective: B-cell-activating factor (BAFF) and a proliferation-inducing ligand (APRIL) sig-

naling pathways regulate B-cell survival through interactions with their receptors BAFF-R,
TACI and BCMA. We evaluated the association of these ligands/receptors on B-cell subsets
according to clinical manifestations of systemic lupus erythematosus (SLE). Methods: BAFF
and APRIL serum concentrations were measured in 30 SLE patients by enzyme-linked
immunosorbent assay. The BAFF-R, TACI and BCMA expression was analyzed on
each B cell subset (CD19 þ CD27-CD38–/ þ naı̈ve; CD19 þ CD27 þ CD38–/ þ memory;
CD19 þ CD27-CD38 þ þ immature and CD19 þ CD27 þ CD38 þ þ plasma cells) by flow
cytometry, and compared among patients with different clinical manifestations as well as
healthy controls (HCs). Results: Serum BAFF and APRIL levels were high in SLE patients
and correlated with the Mex-SLEDAI disease activity index (r ¼ 0.584; p ¼ 0.001 and
r ¼ 0.456; p ¼ 0.011, respectively). The SLE patients showed an increased proportion of
memory and plasma B cells (p < 0.05). BAFF-R, TACI and BCMA expression in SLE patients
was decreased in almost all B cell subsets compared to HCs (p < 0.05). A lower BCMA
expression was associated with severe disease activity, glomerulonephritis, serositis and hemo-
lytic anemia (p < 0.01). BCMA expression showed a negative correlation with Mex-SLEDAI
score (r ¼ –0.494, p ¼ 0.006). Conclusions: Decreased BCMA expression on peripheral B cells
according to severe disease activity suggests that BCMA plays an important regulating
role in B-cell hyperactivity and immune tolerance homeostasis in SLE patients. Lupus
(2015) 0, 1–11.

Key words: Systemic lupus erythematosus; BAFF; BAFF-R; BCMA; BAFF receptors; B cell
subsets; disease activity

Introduction increased autoantibody production, defective elim-

ination of apoptotic remnants, tissue deposition of
Systemic lupus erythematosus (SLE) is the proto- immune complexes, complement activation and
type of systemic autoimmune disease, characterized cytokine overproduction are involved in the patho-
by overactive B and T-cell responses and loss genesis of SLE.
of immune tolerance against self-antigens. The B-cell-activating factor (BAFF) and a
underlying causes remain unclear; however, proliferation-inducing ligand (APRIL) are cyto-
kines belonging to the tumor necrosis factor
(TNF)-ligand superfamily that have an important
Correspondence to: Claudia Azucena Palafox-Sánchez, Instituto de role in the selection, maturation and survival of B
Investigación en Ciencias Biomédicas (IICB), Departamento de
Clı́nicas Médicas, Centro Universitario de Ciencias de la Salud,
cells.1,2 Both cytokines bind with different affinities
Universidad de Guadalajara, Sierra Mojada 950, Edificio Q, 1er. to three receptors expressed on B cells: BAFF
Piso, Colonia Independencia, C.P. 44340, Guadalajara, Jalisco, receptor (BAFF-R), transmembrane activator
México. and cyclophilin ligand interactor (TACI) and B-
Email: kklaumx@yahoo.com cell maturation antigen (BCMA). BAFF and
Received 22 January 2015; accepted 2 September 2015
! The Author(s), 2015. Reprints and permissions: http://www.sagepub.co.uk/journalsPermissions.nav 10.1177/0961203315608254

Downloaded from lup.sagepub.com at UNIV NEBRASKA LIBRARIES on October 5, 2015

 BAFF, APRIL, BAFF-R, TACI and BCMA expression in SLE
DC Salazar-Camarena et al.
2
APRIL bind both with TACI and BCMA,
whereas BAFF-R is exclusive for BAFF.3 The
signal transduced by BAFF and APRIL depends
on binding receptors as well as the cellular popu-
lation where those are expressed. In general,
BAFF-R is involved in germinal center (GC) for-
mation, as well as in the selection and survival of B
cells,4 and like TACI can induce immunoglobulin-
class switching,5,6 whereas BCMA promotes sur-
vival of plasma B cells.4,7
Circulating levels of BAFF, APRIL and mem-
brane-bound BAFF, increase in response to Toll-
like receptor (TLR)9 activation on human B cells,8
type I interferons (IFNs), IFNg, interleukin (IL)-10
and granulocyte colony-stimulating factor (G-CSF).
BAFF and APRIL have been found to be elevated
both in humans with SLE and lupus-prone
mice,9–13 as well as other autoimmune diseases,
such as Sjögren’s syndrome14–16 and rheumatoid
arthritis.17,18 Furthermore, increased BAFF serum
levels have been associated with higher disease
activity and anti-double-stranded DNA antibodies
(dsDNA) in SLE patients,19,20 and recently inhib-
ition of BAFF with monoclonal antibody (beli-
mumab) has been approved for treatment of SLE
patients with mild or moderate disease.21–23
In relation to receptors, there have been efforts
to analyze their expression in SLE patients and
associate them with clinical characteristics, show-
ing controversial results.24–27 Therefore, it is
important to study the expression of BAFF and
APRIL together with their cognate receptors on
B-cell subsets according to clinical spectrums in
SLE patients.
Materials and methods
Patients and healthy controls (HCs)
Thirty SLE patients were recruited from the
Rheumatology Department of the Hospital
General de Occidente in Zapopan, Jalisco,
Mexico. The clinical diagnosis in all patients was
made in accordance with the American College of
Rheumatology (ACR) 1997 revised criteria for
SLE.28 The Mexican version of the Systemic
Lupus Erythematosus Disease Activity Index
(Mex-SLEDAI)29 and the Systemic Lupus
International Collaborating Clinics index
(SLICC)30 were applied to SLE patients at the
enrollment of the study. Patients were subdivided
into four groups: inactive (Mex-SLEDAI <2)
(n ¼ 11), mild and moderate (Mex-SLEDAI 3–7)
(n ¼ 9) and severe disease activity (Mex-SLEDAI
Lupus
Downloaded from lup.sagepub.com at UNIV NEBRASKA LIBRARIES on October 5, 2015
8) (n ¼ 7) and the treatment-naı̈ve group, which
included three newly diagnosed patients with severe
disease activity. The first three groups received
standard-of-care therapy for their disease, and the
last group (treatment-naı̈ve), had not received prior
steroid or immunosuppressive therapy. Fifteen
HCs matched by age and gender were recruited
from personnel at the Hospital General de
Occidente and the Centro Universitario de
Ciencias de la Salud, Universidad de Guadalajara.
The study was performed according the ethics prin-
ciples for experiments involving humans stated in
the Declaration of Helsinki, and it was also evalu-
ated and approved by the ethics committee (C.I.
083-2014).
Laboratory assessments
A clinical assessment was performed in all eligible
patients at the time of enrollment, including deter-
minations of complete blood count (CELL-DYN
3500R; Abbott Diagnostics, Abbott Park, IL,
USA) and erythrocyte sedimentation rate (ESR)
(Wintrobe method).
Antibodies and reagents
Anti-human allophycocyanin/Cy7 (APC/Cy7)-
conjugated anti-CD3, peridinin chlorophyll pro-
tein-conjugated (PerCP)-anti-CD19, fluorescein
isothiocyanate-conjugated (FITC) anti-CD27,
phycoerythrin/Cy7 (PE/Cy7)-conjugated anti-
CD38, phycoerythrin (PE)-conjugated anti-268 -
(BAFF-R), PE-conjugated anti-267 (TACI) and
their corresponding isotypes were all purchased
from BioLegend Inc (San Diego, CA, USA).
Allophycocyanin (APC)-conjugated anti-269
(BCMA) and isotype were purchased from R&D
Systems (Minneapolis, MN, USA).
Flow cytometry
For identification of B-cell subsets (CD19 þ CD27-
CD38–/ þ naı̈ve; CD19 þ CD27 þ CD38–/ þ
memory; CD19 þ CD27-CD38 þ þ immature and
CD19 þ CD27 þ CD38 þ þ plasma cells) and
BAFF-R, TACI and BCMA expression in these,
total blood (300 ml) was stained with the appropri-
ate combinations of fluorochrome-conjugated anti-
bodies to CD3, CD19, CD27, CD38, BAFF-R,
TACI and BCMA. At the end of the labeling, red
cells were lysed by adding 1X BD FACS Lysing
solution (BD Biosciences, Franklin Lakes, NJ,
USA) to each sample. After 15 minutes of incuba-
tion at room temperature, cells were pelleted and
washed twice before acquisition on a FACS Aria I

cytometer (BD Biosciences, San Jose, CA, USA).
For each sample, at least 50,000 lymphocytes were
acquired. Appropriate isotype and Fluorescence
Minus One (FMO) controls were used to adjust
for background fluorescence and gates, and results
are reported as the percentage (%) of expression or
geometric mean fluorescence intensity (MFI). Data
were processed with FACSDiva software (BD
Biosciences).
Enzyme-linked immunosorbent assay (ELISA)
All assays were performed on stored (–80 C) serum
samples. Serum BAFF concentration was deter-
mined with quantitative sandwich enzyme immuno-
assay technique (DBLYS0B; R&D Systems, MN,
USA) and according to recommendations made by
the manufacturer. Standard curves were derived
from serial dilutions of 40 ng of recombinant
human BAFF, and the sensitivity of the assay
ranged from 1.01 to 6.44 pg/ml. Serum concentra-
tions were measured in a microplate reader at
450 nm (BioTek Instruments Inc, Winooski, VT,
USA).
The concentration of human APRIL serum was
measured using the human APRIL DuoSet ELISA
Kit (DY884; R&D Systems, MN, USA). Briefly,
polystyrene microplate wells were coated with
100 ml per well of the diluted capture antibody
and incubated overnight at room temperature.
The next day, serum samples and standards were
added and incubated for two hours. A seven-point
standard curve using two-fold serial dilutions of
60,000 pg/ml of recombinant human APRIL was
created. As secondary antibody, biotinylated
mouse anti-human APRIL was used and incubated
for two hours. The plate was washed three times
and then 100 ml of the working dilution of strepta-
vidin-horseradish peroxidase (HRP) was added to
each well. After 20 minutes of incubation the reac-
tion was developed with substrate solution. After
adding the stop solution, the intensity was mea-
sured in an automated microplate reader at
450 nm (BioTek Instruments Inc, Winooski, VT,
USA).
Autoantibodies determination
Serum levels of biologic markers including
dsDNA, anti-Sm (Binazyme, The Binding
Site Ltd., Birmingham, UK), anti-Ro, anti-La
(ORGENTEC Diagnostika GmbH, Mainz,
Germany) autoantibodies were tested in SLE
patients using an ELISA kit. For dsDNA antibo-
dies, the range of detection was 12.3–1000 IU/ ml,
and sensitivity of the assay was 4.6 IU/ ml, for
Downloaded from lup.sagepub.com at UNIV NEBRASKA LIBRARIES on October 5, 2015
BAFF, APRIL, BAFF-R, TACI and BCMA expression in SLE
DC Salazar-Camarena et al.
3
anti-Sm sensitivity was 4.0 UI/ ml, while for anti-
Ro and anti-La, sensitivity of the assay was 1.0 IU/
ml. Determinations were performed according to the
instructions indicated by the manufacturer.
Statistical analyses
Data analysis was performed using PASW
Statistics 18 software (IBM Corporation,
Armonk, NY, USA) and GraphPad Prism v.6 soft-
ware (GraphPad Software Inc, San Diego, CA,
USA). Mann-Whitney U test and Kruskal-Wallis
analysis of variance were used for nonparametric
distribution data. Correlations were examined by
Spearman’s rank correlation test. P values  0.05
were considered significant.
Results
Demographic and clinical characteristics
All SLE patients included in this study were
females. The average age was 34 years old (range
18–67) having 7.3 years of disease duration (0–21
years). SLE patients presented an activity disease
score of 4.5 (range 0–18) for Mex-SLEDAI and
0.8 (range 0–3) score for SLICC damage index.
Hematological, renal and mucocutaneous involve-
ments were the major clinical manifestations. The
treatment of SLE patients included immunosup-
pressive drugs (azathioprine, methotrexate, cyclo-
phosphamide and mycophenolate), chloroquine
and prednisone (Table 1).
Peripheral B-lymphocyte subsets
and BAFF receptors
Figure 1(a) shows a representative strategy example
for the analysis of B-cell subsets (CD19 þ
CD27-CD38–/ þ naı̈ve; CD19 þ CD27 þ CD38–/
þ memory; CD19 þ CD27-CD38 þ þ immature
and CD19 þ CD27 þ CD38 þ þ plasma cells) in
an HC and a treatment-naı̈ve SLE patient.
Distribution of peripheral B-cell subsets was differ-
ent in SLE compared to HCs. The percentage of B
cells (CD3-CD19 þ ) was lower in the SLE group
(6.72  5.55) compared to HCs (9.43  2.96;
p ¼ 0.007) except in new-onset treatment-naı̈ve
patients, who had an elevated percentage of B
cells (18.21  9.08). We analyzed the expression of
BAFF-R, TACI and BCMA in B (CD3-CD19 þ )
cells in SLE patients and HCs. Both the BAFF-
R expression rate in B cells (94.96  9.56
vs 99.45  1.12%, p ¼ 0.003) and mean fluores-
cence intensity (MFI) (15,525.5  13,783.8
Lupus
 BAFF, APRIL, BAFF-R, TACI and BCMA expression in SLE
DC Salazar-Camarena et al.
4

Table 1 Demographic and clinical characteristics in SLE subpopulation in both groups corresponded to
patients immature B lymphocytes and showed no differ-
SLE, n ¼ 30
ences. The naı̈ve B subpopulation was the largest;
however, it was decreased in the SLE group
Male/Female 0/100 (52.30  17.22%) compared to HCs (68.54 
Age, yearsa 34  12 (18–67) 9.28%), p ¼ 0.003. Subpopulation distribution of per-
Disease duration, yearsa 7.3  6 (0–21)
Clinical assessment
ipheral memory B cells was expanded in SLE patients
Mex-SLEDAI, scorea 4.5  4 (0–18) (39.54  18.18%) in comparison with HCs
SLICC, scorea 0.8  0.9 (0–3) (25.77  8.61%), p ¼ 0.013 (Figure 1(c)). Patients
Clinical manifestations with active disease (Mex-SLEDAI3) did not differ
Hematologic
in their frequency of memory B cells compared to
Cytopeniasb, n (%) 27 (90)
Hemolytic anemia, n (%) 4 (13.3) those with inactive disease (Mex-SLEDAI 0-2),
Renal p ¼ 0. 478. Finally, as has been reported previously,
Renal activity, n (%) 10 (33.3) SLE patients had an increased proportion of plasma
Proteinuria, n (%) 7 (23.3) cells (3.86  4.15%) compared to HCs (1.30  1.19%,
Mucocutaneousc 11 (36.6)
Serositis, n (%) 4 (13.3)
p ¼ 0.001) (Figure (1c)), and those with severe disease
Arthritis, n (%) 4 (13.3) activity showed the highest percentage (6.00  7.88 vs
Neurologicd, n (%) 2 (6.7) 1.3  1.1% in HCs), p ¼ 0.023.
Autoantibodies Afterward, expression of BAFF-R, TACI and
ANA, n (%) 30 (100) BCMA in these B-cell subsets of SLE patients
Anti-dsDNA, n (%) 16 (52)
Anti-Sm, n (%) 11 (35.3)
and HCs was analyzed. In SLE patients, we
Anti-Ro, n (%) 7 (23.1) observed a lower BAFF-R expression in all B-cell
Anti-La, n (%) 7 (23.1) subsets, except memory cells (p < 0.05). TACI
Treatment showed decreased expression in memory and
Prednisone, n (%) 20 (66.7)
plasma cells of SLE patients, but no differences
Azathioprine, n (%) 18 (60)
Antimalarial, n (%) 20 (66.6)
were observed in immature or naı̈ve cells
Cyclophosphamide, n (%) 8 (26.7) (p < 0.05) in comparison with controls. Finally,
Methotrexate, n (%) 2 (6.7) we found a lower BCMA expression in naı̈ve,
Mycophenolate, n (%) 2 (6.7) memory and plasma B-cell subsets in SLE patients
SLE: systemic lupus erythematosus; Mex-SLEDAI: Mexican version compared to HCs (p < 0.05) (Figure 1(d)).
of the Systemic Lupus Erythematosus Disease Activity Index; SLICC:
Systemic Lupus International Collaborating Clinics; ANA: antinuclear BAFF-R, TACI and BCMA B-cell expression and
antibodies; dsDNA: double-stranded DNA. SLE features
a
Data provided in mean  standard deviation, minimum and
maximum.
b
In order to evaluate the possible effects of BAFF/
Cytopenias included leukopenia, lymphopenia and thrombocytopenia. APRIL levels and receptor expression in disease
c
Mucocutaneous manifestations included: malar erythema, discoid
lupus, oral ulcers and photosensitivity.
activity, we divided patients into four groups: inac-
d
Neurologic clinical manifestations included neurologic damage, tive, mild-moderate, and severe disease activity,
psychosis and convulsions. and new-onset treatment-naı̈ve patients. As shown
b
c
Cytopenias included leucopenia, lymphopenia and thrombocytopenia. in Table 2, severe disease activity and treatment-
Mucocutaneous manifestations included: malar erythema, discoid
naı̈ve SLE patients had lower BAFF-R and
lupus, oral ulcers and photosensitivity.
d
Neurologic clinical manifestations included neurologic damage, BCMA expression rates than the other
psychosis, and convulsions. groups (p < 0.05). TACI expression was similar
between SLE patients and HCs, but in stratified
groups, we found that the expression of TACI in
HCs (45.41  25.28%) and inactive patients
(45.58  15.82%) was similar, while the expression
vs 45,477.5  2,9321.8, p ¼ 0.013) were down-regu- rate in treatment-naı̈ve SLE patients was signifi-
lated significantly in SLE patients in comparison cantly decreased (23.97  13.82%) (p ¼ 0.049)
with HCs. BCMA expression was also decreased (Table 2). Notably, BCMA showed very low
in the SLE group (6.16  9.69%) in comparison expression in severe disease activity (Table 2) and
with HCs (17.33  8.48%), p < 0.001. No difference treatment-naı̈ve SLE patients compared to HCs
was observed in TACI expression (Figure 1(b)). and inactive patients (1.94  3.87% and
Subsequently, the different B-cell subsets in SLE 0.33  0.32% vs 17.3  8.4% and 9.37  11.89%,
patients and HCs were evaluated. The smallest respectively) (p < 0.05).
Lupus

Downloaded from lup.sagepub.com at UNIV NEBRASKA LIBRARIES on October 5, 2015

 BAFF, APRIL, BAFF-R, TACI and BCMA expression in SLE
DC Salazar-Camarena et al.
5

Figure 1 Expression of BAFF-R, TACI and BCMA on peripheral subpopulations of B cells in SLE patients and healthy controls.
(a) Representative analyses of subpopulations of B cells (CD19 þ CD27-CD38–/ þ naı̈ve; CD19 þ CD27 þ CD38–/ þ memory;
CD19 þ CD27-CD38 þ þ immature and CD19 þ CD27 þ CD38 þ þ plasma cells) in an HC and treatment-naı̈ve SLE patient.
(b) Percentage of expression of BAFF-R, TACI and BCMA on B cells were compared between SLE and HCs.
(c) Subpopulations of B cells (naı̈ve, memory, immature and plasma cells) were compared between SLE and HCs.
(d) Expression of BAFF-R, TACI and BCMA in naı̈ve, memory, immature and plasma cells in SLE and HCs were analyzed.
BAFF-R: B-cell activating factor receptor; TACI: transmembrane activator and calcium modulator and cyclophilin ligand inter-
actor; BCMA: B-cell maturation antigen; SLE: systemic erythematosus lupus; HC: healthy controls.
Statistical analysis was performed using Mann-Whitney U test. Values are represented as mean  SD. * p < 0.05 vs HCs. **p < 0.01
vs HCs.

Lupus

Downloaded from lup.sagepub.com at UNIV NEBRASKA LIBRARIES on October 5, 2015

 BAFF, APRIL, BAFF-R, TACI and BCMA expression in SLE
DC Salazar-Camarena et al.
6

Moreover, we found that BCMA expression Figure 3(c), APRIL correlated both with Mex-
negatively correlated with Mex-SLEDAI score SLEDAI (r ¼ 0.456, p ¼ 0.011) and SLICC scores
(r ¼ –0.494, p ¼ 0.006) (Figure 2). Decreased (r ¼ 0.407, p ¼ 0.026). Regarding the SLE groups,
BCMA was associated with different clinical set- those with severe disease activity and treatment-
tings. When we compared the expression of
BCMA on B cells, those patients with clinical mani-
festations showed a lower rate. As shown in Table 3,
patients with renal activity (0.28  0.32% vs
9.07  10.79%, p ¼ 0.001), serositis (0.16  0.05%
vs 7.06  10.12%, p < 0.01) and hemolytic anemia
(0.30  0.21% vs 7.37  10.46%, p < 0.01) displayed
a down-regulated expression of BCMA compared to
SLE patients without those clinical manifestations.

BAFF and APRIL and SLE features

As shown in Figure 3(a), elevated serum BAFF
levels were found in SLE compared to HCs
(3.19  4.26 vs 0.97  0.21 ng/ml, p < 0.05). BAFF
was correlated with the percentage of CD19 þ B
cells (r ¼ 0.407, p ¼ 0.025) in SLE patients and dis-
ease activity score evaluated by Mex-SLEDAI
(r ¼ 0.584, p ¼ 0.001) (Figure 3(b)). Treatment-
naı̈ve patients had the highest BAFF levels and
the lowest BAFF-R expression detected on B cells Figure 2 Correlation of the expression rate of BCMA in
(Table 2), which might indicate over-activation or CD19 þ B cells with Mex-SLEDAI score in SLE patients.
deregulation of the BAFF axis in this group. Serum Statistical analysis was performed using Spearman’s correl-
ation test.
APRIL levels were also increased in SLE compared BCMA: B-cell maturation antigen; Mex-SLEDAI: Mexican
to HCs (31.42  27.42 vs 13.41  9.84 ng/ml, version of the Systemic Lupus Erythematosus Disease
p < 0.05) (Figure 3(a), right panel). As shown in Activity Index.

Table 2 Expression of BAFF/APRIL levels and BAFF-R, TACI and BCMA on B cells in systemic lupus erythematosus patients
according to disease activity index and HCs
SLE

HCs Mild-moderate Severe disease Treatment-naı¨ve

n ¼ 15 Inactive n ¼ 11 p disease activity n ¼ 9 p activity n ¼ 7 p patientsa n ¼ 3 p

CD19þ B cells (%) 9.43  2.96 6.63  4.46 0.03 d 5.27  1.95 <0.01 b 3.80  2.01 <0.01 b 18.21  9.08 <0.05f,g
b b
þ þ
CD19 /BAFF-R (%) 99.4  1.1 92.3  14.2 <0.01 99.1  0.7 0.17 95.4  6.2 0.03 91.0  7.4 <0.05 b,f
MFI of BAFF-Rþ 36,426.6  28,653.6 22,213.5  16,297.0 0.29 10,796.3  11,582.5 0.05 b 10,686.2  7243.4 0.05 9,702.0  2,904.7 0.16
CD19þ/BCMAþ (%) 17.3  8.4 9.37  11.89 <0.05 b,d 7.45  10.39 0.02 b 1.94  3.87 <0.05 b,d 0.33  0.32 <0.01 b
MFI of BCMAþ 8232.7  5,398.2 9836.8  10,673.7 0.66 7231.0  5285.1 0.13 7542.0  5584.9 0.45 8625.5  2,865.9 0.61
CD19þ/TACIþ (%) 45.4  25.2 45.5  15.8 0.95 35.3  11.7 0.16 31.6  11.9 0.70 23.9  13.8 <0.05 b
MFI of TACIþ 5592.5  9,448.6 2,929.1  517.7 0.90 3554.2  2004.4 0.45 3152.7  2361.4 0.27 2,685.5  747.4 0.90
BAFF (ng/ml) 0.97  0.21 1.52  0.55 <0.01 b,d 1.60  0.89 <0.05 b,e 3.67  1.95 <0.05 b,d 13.01  8.71 <0.05 b,f
APRIL (ng/ml) 13.41  19.84 21.55  22.18 0.32 24.69  26.69 0.31 43.76  29.35 <0.01 b 58.92  24.58 0.01 b

BAFF: B-cell activating factor; APRIL: a proliferation-inducing ligand-R: B-cell activating factor receptor; TACI: transmembrane activator and
calcium modulator and cyclophilin ligand interactor; BCMA: B-cell maturation antigen; HCs: healthy controls; SLE: systemic lupus erythemato-
sus; MFI: geometric mean fluorescence intensity; ng/ml: nanograms per milliliter. Bold values represent p < 0.05.
a
Treatment-naı̈ve patients included newly diagnosed patients who had not received prior steroid or immunosuppressive therapy.
b
Comparison between each SLE group and HCs.
c
Comparison between inactive vs severe disease activity patients.
d
Comparison between inactive vs treatment-naı̈ve patients.
e
Comparison between mild-moderate vs severe disease activity patients.
f
Comparison between mild-moderate disease activity vs treatment-naı̈ve patients.
g
Comparison between severe disease activity vs treatment-naı̈ve patients.

Lupus

Downloaded from lup.sagepub.com at UNIV NEBRASKA LIBRARIES on October 5, 2015

 BAFF, APRIL, BAFF-R, TACI and BCMA expression in SLE
DC Salazar-Camarena et al.
7

Table 3 Comparison of serum BAFF/APRIL levels and BAFF-R, TACI, and BCMA expression on B cells of SLE patients
according to clinical manifestations
Clinical manifestations

Renal activity Serositis Hemolytic anemia

Present n ¼ 10 Absent n ¼ 20 p Present n ¼ 4 Absent n ¼ 26 p Present n ¼ 4 Absent n ¼ 26 p

BAFF-R (%) 99.02  0.87 92.96  11.25 0.027 90.80  7.05 95.30  9.97 0.09 92.42  8.34 95.30  9.97 0.59
BCMA (%) 0.28  0.32 9.07  10.79 <0.01 0.16  0.05 7.06  10.12 <0.01 0.30  0.21 7.37  10.46 <0.01
TACI (%) 28.74  35.79 45.70  26.72 0.07 20.20  25.38 42.77  30.29 0.07 30.75  17.47 42.81  32.19 0.65
BAFF (ng/ml) 4.13  4.53 2.73  4.16 0.21 11.22  7.96 1.96  1.27 <0.01 2.87  1.06 3.33  4.65 0.19
APRIL (ng/ml) 47.23  32.30 23.50  21.36 0.06 47.43  30.51 28.95  26.71 0.24 45.05  17.8 29.89  28.71 0.20

BAFF: B-cell activating factor; APRIL: a proliferation-inducing ligand; BAFF-R: B-cell activating factor receptor; TACI: transmembrane acti-
vator and calcium modulator and cyclophilin ligand interactor; BCMA: B-cell maturation antigen; SLE: systemic lupus erythematosus; ng/ml:
nanograms per milliliter.
Bold values represent p < 0.05.

naı̈ve SLE patients showed higher BAFF- and
APRIL-soluble levels than HCs and other SLE
groups (p < 0.05) (Table 2).
Discussion
SLE is an autoimmune disease characterized by B-cell
hyperactivity, and the production of autoantibodies
against nuclear components is the hallmark of the
disease. Because these cells are centrally involved in
the pathogenesis of SLE, we analyzed the expression
of B-cell receptors (BCRs) BAFF-R, TACI and
BCMA in four different peripheral B cell subsets to
determine their possible correlations with clinical par-
ameters and disease activity in SLE patients.
Our study found low B-cell levels in SLE
patients, while for HCs percentages were similar
to those reported in other studies.26,31,32 Inside
inactive B-cell compartments, patients with SLE
had a significantly lower percentage of naı̈ve B
cells compared to HCs.
Notably, we detected an increased frequency of
circulating antigen-experienced B cells, like plasma
cells and our work, demonstrates that this subset is
notably expanded in SLE patients, even more in
those with active disease. These findings, in add-
ition to those of other studies, emphasize the cen-
tral role of these cells in the pathogenesis of SLE,
mainly due to their ability to produce pathogeni-
cally relevant autoantibodies, such as anti-dsDNA
antibodies.33,34 Previously, plasma B cells from
SLE patients have been shown to be less susceptible
to immunosuppressive therapy.35 Also, the memory
B-cell subpopulation was significantly elevated in
SLE patients, which is consistent with previous
analyses. 36–38 Contributing to these abnormalities,
ectopic GC structures could be involved in T cell-
independent activation of memory B cells. In this
pathway, TLR engagement and activation of TACI
promote the recruitment of myeloid differentiation
primary-response protein 88 (MYD88), which
induces the expression of activation-induced cyti-
dine deaminase (AID).6
Altogether, an expansion in the subpopulation of
memory B cells involves a high risk for autoimmun-
ity because these cells already exceed negative selec-
tion checkpoints of the immune system and have
low levels of activation, allowing them to differen-
tiate rapidly in antibody-producing B cells. The dif-
ferences found in the B-cell subsets suggest
alterations in selection mechanisms and maturation
stage control; however, we could not establish if the
defects were intrinsic or related to the inflammatory
milieu.
On the other hand, previous studies have
reported BAFF-R as the BAFF receptor with the
highest expression rate on peripheral B cells, while
BCMA had the lowest expression rate.24–26 We
found both receptors were decreased in SLE
patients, while TACI showed no differences in
expression rates or MFI between SLE and HCs.
Previous studies from SLE patients show discord-
ant findings, lower BAFF-R expression and a
higher or no differences in TACI and BCMA
expression.26,27,39 Our work demonstrated that
BAFF-R, TACI and BCMA could be co-expressed
on the B-cell membrane and vary according to the
stage of differentiation of the cell. However,
because these three receptors can be occupied by
both cytokines, further work is necessary to eluci-
date which one of the ligands binds preferentially
in vivo according to the differentiation stage. Our
results are consistent with other studies: BAFF-R is

Lupus

Downloaded from lup.sagepub.com at UNIV NEBRASKA LIBRARIES on October 5, 2015

 BAFF, APRIL, BAFF-R, TACI and BCMA expression in SLE
DC Salazar-Camarena et al.
8

Figure 3 Serum levels of BAFF and APRIL and their association with SLE.
(a) Left and right panel show increased BAFF and APRIL serum levels in SLE patients compared to HCs.
(b) Left panel displays a positive correlation between disease activity and BAFF serum levels in SLE patients. In the right panel a
positive correlation of peripheral percentage of B cells and BAFF serum levels in SLE patients is shown.
(c) A positive correlation between disease activity and organ damage with APRIL serum levels in SLE patients is shown in the right
and left panel, respectively.
BAFF: B-cell activating factor; APRIL: a proliferation-inducing ligand; SLE: systemic lupus erythematosus; Mex-SLEDAI:
Mexican version of the Systemic Lupus Erythematosus Disease Activity Index; SLICC: Systemic Lupus International
Collaborating Clinics index; HC: healthy controls.
Statistical analysis was performed using Mann-Whitney U test for (a) and Spearman’s correlation test for (b) and (c). Horizontal
lines and error bars represent median with interquartile range, respectively.

Lupus

Downloaded from lup.sagepub.com at UNIV NEBRASKA LIBRARIES on October 5, 2015


the receptor that showed the highest expression rate
during almost all subsets, having elevated levels on
immature, naı̈ve, and memory B subsets.24–26,40
TACI and BCMA raised their expression rates in
later differentiation B-cell stages,24–26 probably
because their functions are more dependent at
these stages and are also related to immunoglobulin
class switching and plasma B-cell maintenance.7,41
It would be interesting to further analyze the
BAFF-R and BCMA expression relationship or
even their possible counter-regulation, as the
study showed the decreased expression of
BAFF-R coincided with the acquisition of BCMA
at later B cell differentiation stages (memory and
plasma cells). The analysis of the molecular mech-
anisms involved in this phenomenon will help elu-
cidate if it is only an event required at the stage of
differentiation of B cells, or even more, help deter-
mine the biological involvement of BCMA in B-cell
homeostasis.
Because of the systemic characteristic of the dis-
ease and the multiple clinical and serological mani-
festations of SLE patients, it is important to evaluate
the expression of BAFF-R, TACI and BCMA with
the disease activity score. In general, the level of
expression of the receptors decreases according to
the increase in disease activity. MFI of BAFF-R
was decreased in SLE patients with low-mild and
severe disease activity, which had been previously
reported.26,27,39 Regarding TACI, the expression of
the receptor is similar between HCs and SLE inac-
tive patients, and the expression rate decreased grad-
ually as disease activity increased.
BCMA is a receptor for BAFF that under
physiological conditions is important for sustaining
enduring antibody protection by mediating survival
of long-lived plasma cells, but is not reported to be
required for B-cell maturation and homeostasis.4,7
Two previous studies of B cells from SLE patients
have shown increased levels of BCMA expression
compared with those in B cells from healthy
donors.27,41 Kim et al. showed increased BCMA
expression and antinuclear antibodies (ANA) secre-
tion after TLR9 stimulation on B cells isolated
from SLE patients.27 These studies suggest that
increased BCMA expression levels on peripheral
human B cells correlates with B-cell activation
and responsiveness to BAFF and APRIL, which
can contribute to autoantibody production from
autoreactive B cells activated through TLR9.
No difference in MFI of BCMA among study
groups was found; nonetheless, expression rates on
BCMA receptor in SLE patients with severe disease
activity, including treatment-naı̈ve patients, were
low or null. This finding is relevant because so far
Downloaded from lup.sagepub.com at UNIV NEBRASKA LIBRARIES on October 5, 2015
BAFF, APRIL, BAFF-R, TACI and BCMA expression in SLE
DC Salazar-Camarena et al.
9
27
it differs from other data published in humans.
Recently, a study reported the importance of
BCMA in the negative regulation of T follicular
helper (TFH) cell expansion, because BCMA defi-
ciency in T cells promotes TFH expansion, GC for-
mation, autoantibody production and IFNg
production by TFH cells through BAFF-R in a
murine model of SLE.42 These findings suggest the
participation of BCMA in addition to BAFF-R for
the maintenance of B- and T-cell homeostasis.
Furthermore, when we compared BCMA expres-
sion in SLE patients according to clinical manifest-
ations, those SLE patients with renal activity,
serositis and hemolytic anemia had the lowest levels.
This could indicate the participation of BCMA in SLE
pathogenesis is more important than previously attrib-
uted. Similar to our results, other work in a murine
SLE model demonstrated that lack of BCMA pro-
motes lymphoproliferation in mature B cells, an
increased number of short- and long-lived plasma
cells, elevated titers of ANA and immune complex
deposition in kidneys.43 Overall, these results suggest
that lack of BCMA expression exacerbates the devel-
opment of lymphoproliferation and autoimmunity
manifestations in SLE; additionally it could indicate
that BCMA is a checkpoint of B cell homeostasis
and self-tolerance in systemic autoimmunity.
Serum BAFF levels were elevated in SLE patients
and correlated with disease activity and the B-cell
count, which led to the explanation of the biological
importance of this cytokine in the B-cell function.
The overexpression of BAFF could counterbalance
the pro-apoptotic signals induced by BCR in auto-
reactive B cells, allowing them to survive and be
activated by autoantigens contributing to auto-
immunity. Other authors have reported the associ-
ation of higher levels of BAFF with anti-dsDNA
autoantibody titers and musculoskeletal manifest-
ations in SLE patients,11–13,24 suggesting that dysre-
gulation of BAFF could be involved in SLE
pathogenesis. On the other hand, serum APRIL
levels were also increased in the SLE group and
correlated with disease activity and SLICC damage
score. Previous reports have shown the association
of disease activity, musculoskeletal manifest-
ations9,10,44,45 and neuropsychiatric SLE with
APRIL levels.20 Recently intrarenal APRIL messen-
ger RNA (mRNA) levels were associated with pro-
teinuria and with resistance to treatment in SLE
patients.46 These results suggest that APRIL may
be an important marker of disease activity and prog-
nosis in patients with SLE.
In summary, even though there are few studies of
BAFF and APRIL receptors in humans, TACI and
particularly BAFF-R have been implicated in the
Lupus
 BAFF, APRIL, BAFF-R, TACI and BCMA expression in SLE
DC Salazar-Camarena et al.
10
differentiation, maturation and homeostasis of B
cells. The third receptor of these ligands, BCMA,
has been poorly studied and associated with the
maintenance of long-lived plasma cells. Our find-
ings demonstrate that BCMA is not only expressed
in plasma cells, even if this particularly subset had
the highest expression rate. Moreover, BCMA
decreased expression rate or absence may have a
relevant role in the development or exacerbation
of SLE, and may be a useful marker of disease.
These findings emphasize the relevance of BCMA
in addition to BAFF-R in B-cell homeostasis.
To our knowledge, this is the first study in
humans that demonstrates the importance of
BCMA expression and its relation with disease
activity in SLE patients.
Funding
The authors disclosed receipt of the following
financial support for the research, authorship,
and/or publication of this article: This work was
supported by the National Council of Science and
Technology (grant number 115567).
Declaration of Conflicting Interests
The authors declared no potential conflicts of inter-
est with respect to the research, authorship, and/or
publication of this article.
References
1 Mackay F, Schneider P. Cracking the BAFF code. Nat Rev Immunol
2009; 9: 491–502.
2 Vincent FB, Saulep-Easton D, Figgett WA, et al. The BAFF/
APRIL system: Emerging functions beyond B cell biology and auto-
immunity. Cytokine Growth Factor Rev 2013; 24: 203–215.
3 Thompson JS, Bixler SA, Qian F, et al. BAFF-R, a newly identified
TNF receptor that specifically interacts with BAFF. Science 2001;
293: 2108–2111.
4 Schneider P. The role of APRIL and BAFF in lymphocyte activa-
tion. Curr Opin Immunol 2005; 17: 282–289.
5 Castigli E, Wilson SA, Scott S, et al. TACI and BAFF-R mediate
isotype switching in B cells. J Exp Med 2005; 201: 35–39.
6 He B, Santamaria R, Xu W, et al. The transmembrane
activator TACI triggers immunoglobulin class switching by activat-
ing B cells through the adaptor MyD88. Nat Immunol 2010; 11:
836–845.
7 O’Connor BP, Raman VS, Erickson LD, et al. BCMA is essential
for the survival of long-lived bone marrow plasma cells. J Exp Med
2004; 199: 91–98.
8 Abu-Rish EY, Amrani Y, Browning MJ. Toll-like receptor 9 acti-
vation induces expression of membrane-bound B-cell activating
factor (BAFF) on human B cells and leads to increased proliferation
in response to both soluble and membrane-bound BAFF.
Rheumatology (Oxford) 2013; 52: 190–201.
Lupus
Downloaded from lup.sagepub.com at UNIV NEBRASKA LIBRARIES on October 5, 2015
9 Koyama T, Tsukamoto H, Miyagi Y, et al. Raised serum APRIL
levels in patients with systemic lupus erythematosus. Ann Rheum
Dis 2005; 64: 1065–1067.
10 Becker-Merok A, Nikolaisen C, Nossent HC. B-lymphocyte acti-
vating factor in systemic lupus erythematosus and rheumatoid
arthritis in relation to autoantibody levels, disease measures and
time. Lupus 2006; 15: 570–576.
11 Zhang J, Roschke V, Baker KP, et al. Cutting edge: A role for B
lymphocyte stimulator in systemic lupus erythematosus. J Immunol
2001; 166: 6–10.
12 Pers JO, Daridon C, Devauchelle V, et al. BAFF overexpression is
associated with autoantibody production in autoimmune diseases.
Ann N Y Acad Sci 2005; 1050: 34–39.
13 Stohl W, Metyas S, Tan SM, et al. B lymphocyte stimulator over-
expression in patients with systemic lupus erythematosus:
Longitudinal observations. Arthritis Rheum 2003; 48: 3475–3486.
14 Groom J, Kalled SL, Cutler AH, et al. Association of BAFF/BLyS
overexpression and altered B cell differentiation with Sjögren’s syn-
drome. J Clin Invest 2002; 109: 59–68.
15 Daridon C, Devauchelle V, Hutin P, et al. Aberrant expression of
BAFF by B lymphocytes infiltrating the salivary glands of patients
with primary Sjögren’s syndrome. Arthritis Rheum 2007; 56:
1134–1144.
16 Candon S, Gottenberg JE, Bengoufa D, Chatenoud L, Mariette X.
Quantitative assessment of antibodies to ribonucleoproteins in pri-
mary Sjögren syndrome: Correlation with B-cell biomarkers and
disease activity. Ann Rheum Dis 2009; 68: 1208–1212.
17 Alsaleh G, Messer L, Semaan N, et al. BAFF synthesis by rheuma-
toid synoviocytes is positively controlled by alpha5beta1 integrin
stimulation and is negatively regulated by tumor necrosis factor
alpha and Toll-like receptor ligands. Arthritis Rheum 2007; 56:
3202–3214.
18 Moura RA, Canhão H, Polido-Pereira J, et al. BAFF and TACI
gene expression are increased in patients with untreated very early
rheumatoid arthritis. J Rheumatol 2013; 40: 1293–1302.
19 Petri M, Stohl W, Chatham W, et al. Association of plasma B
lymphocyte stimulator levels and disease activity in systemic
lupus erythematosus. Arthritis Rheum 2008; 58: 2453–2459.
20 George-Chandy A, Trysberg E, Eriksson K. Raised intrathecal
levels of APRIL and BAFF in patients with systemic lupus erythe-
matosus: Relationship to neuropsychiatric symptoms. Arthritis Res
Ther 2008; 10: R97.
21 Sanz I, Yasothan U, Kirkpatrick P. Belimumab. Nat Rev Drug
Discov 2011; 10: 335–336.
22 Navarra SV, Guzmán RM, Gallacher AE, et al. Efficacy and safety
of belimumab in patients with active systemic lupus erythematosus:
A randomised, placebo-controlled, phase 3 trial. Lancet 2011; 377:
721–731.
23 Furie R, Petri M, Zamani O, et al. A phase III, randomized, pla-
cebo-controlled study of belimumab, a monoclonal antibody that
inhibits B lymphocyte stimulator, in patients with systemic lupus
erythematosus. Arthritis Rheum 2011; 63: 3918–3930.
24 Carter RH, Zhao H, Liu X, et al. Expression and occupancy of
BAFF-R on B cells in systemic lupus erythematosus. Arthritis
Rheum 2005; 52: 3943–3954.
25 Darce JR, Arendt BK, Wu X, et al. Regulated expression of
BAFF-binding receptors during human B cell differentiation. J
Immunol 2007; 179: 7276–7286.
26 Zhao LD, Li Y, Smith MF, et al. Expressions of BAFF/BAFF
receptors and their correlation with disease activity in Chinese
SLE patients. Lupus 2010; 19: 1534–1549.
27 Kim J, Gross JA, Dillon SR, et al. Increased BCMA expression in
lupus marks activated B cells, and BCMA receptor engagement
enhances the response to TLR9 stimulation. Autoimmunity 2011;
44: 69–81.
28 Hochberg MC. Updating the American College of Rheumatology
revised criteria for the classification of systemic lupus erythemato-
sus. Arthritis Rheum 1997; 40: 1725.
29 Guzmán J, Cardiel MH, Arce-Salinas A, et al. Measurement of
disease activity in systemic lupus erythematosus. Prospective val-
idation of 3 clinical indices. J Rheumatol 1992; 19: 1551–1558.
30 Gladman D, Ginzler E, Goldsmith C, et al. The development and
initial validation of the Systemic Lupus International

Collaborating Clinics/American College of Rheumatology damage
index for systemic lupus erythematosus. Arthritis Rheum 1996; 39:
363–369.
31 Odendahl M, Jacobi A, Hansen A, et al. Disturbed peripheral B
lymphocyte homeostasis in systemic lupus erythematosus. J
Immunol 2000; 165: 5970–5979.
32 Hostmann A, Jacobi AM, Mei H, Hiepe F, et al. Peripheral B cell
abnormalities and disease activity in systemic lupus erythematosus.
Lupus 2008; 17: 1064–1069.
33 Cheng Q, Mumtaz IM, Khodadadi L, et al. Autoantibodies from
long-lived ‘‘memory’’ plasma cells of NZB/W mice drive immune
complex nephritis. Ann Rheum Dis 2013; 72: 2011–2017.
34 Jacobi AM, Odendahl M, Reiter K, et al. Correlation between
circulating CD27high plasma cells and disease activity in patients
with systemic lupus erythematosus. Arthritis Rheum 2003; 48:
1332–1342.
35 Dörner T, Lipsky PE. Correlation of circulating CD27high plasma
cells and disease activity in systemic lupus erythematosus. Lupus
2004; 13: 283–289.
36 Wei C, Anolik J, Cappione A, et al. A new population of cells
lacking expression of CD27 represents a notable component of
the B cell memory compartment in systemic lupus erythematosus.
J Immunol 2007; 178: 6624–6633.
37 Jacobi AM, Reiter K, Mackay M, et al. Activated memory B cell
subsets correlate with disease activity in systemic lupus erythema-
tosus: Delineation by expression of CD27, IgD, and CD95.
Arthritis Rheum 2008; 58: 1762–1773.
38 Sellam J, Miceli-Richard C, Gottenberg JE, et al. Decreased B cell
activating factor receptor expression on peripheral lymphocytes
Downloaded from lup.sagepub.com at UNIV NEBRASKA LIBRARIES on October 5, 2015
BAFF, APRIL, BAFF-R, TACI and BCMA expression in SLE
DC Salazar-Camarena et al.
11
associated with increased disease activity in primary Sjögren’s syn-
drome and systemic lupus erythematosus. Ann Rheum Dis 2007; 66:
790–797.
39 Zhang M, Ko KH, Lam QL, et al. Expression and function of
TNF family member B cell-activating factor in the development
of autoimmune arthritis. Int Immunol 2005; 17: 1081–1092.
40 Cancro MP. Signalling crosstalk in B cells: Managing worth and
need. Nat Rev Immunol 2009; 9: 657–661.
41 Koarada S, Tada Y, Suematsu R. Phenotyping of P105-negative B
cells subsets in patients with systemic lupus erythematosus. Clin
Dev Immunol 2012; 2012: 198206.
42 Coquery CM, Loo WM, Wade NS, et al. BAFF regulates T fol-
licular helper cells and affects accumulation and IFNg production
in autoimmunity. Arthritis Rheumatol 2015; 67: 773–784.
43 Jiang C, Loo WM, Greenley EJ, et al. B cell maturation antigen
deficiency exacerbates lymphoproliferation and autoimmunity in
murine lupus. J Immunol 2011; 186: 136–147.
44 Hegazy M, Darwish H, Darweesh H, et al. Raised serum level of
APRIL in patients with systemic lupus erythematosus:
Correlations with disease activity indices. Clin Immunol 2010;
135: 118–124.
45 Boghdadi G, Elewa EA. Increased serum APRIL differentially cor-
relates with distinct cytokine profiles and disease activity in sys-
temic lupus erythematosus patients. Rheumatol Int 2014; 34:
1217–1223.
46 Treamtrakanpon W, Tantivitayakul P, Benjachat T, et al. APRIL,
a proliferation-inducing ligand, as a potential marker of lupus
nephritis. Arthritis Res Ther 2012; 14: R252.
Lupus