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J. Exp. Med. Vol. 206 No. 9 1971-1982
The emergence of autoimmunity is often cou-
pled with aging, and is suggested to be linked to
activation of the innate immune system in indi-
viduals suffering from bacterial and viral infec-
tions (Baccala et al., 2007; Groom et al., 2007;
Krieg and Vollmer 2007; Rothlin et al., 2007).
Toll-like receptors (TLRs) expressed in leuko-
cytes of the innate immune system play indis-
pensable roles in the sensing of viral and bacterial
invasion through binding pathogen-associated
molecular patterns, which leads to efficient T
cell–mediated inflammatory responses (Akira
et al., 2001; Iwasaki and Medzhitov, 2004). The
TLR-mediated priming of inflammation and
production of neutralizing antibodies against
pathogens should be strictly regulated, otherwise
there is the possibility of the development of au-
toimmune diseases (Marsland and Kopf, 2007).
The mechanisms underlying the efficient TLR-
mediated activation of the innate and adaptive
immune systems with prevention of reactivity to
autologous tissues remain elusive.
Examples of critical cells that express TLRs
and could potentially link the innate and adap-
tive immune systems are relatively primitive
B cells, B-1 cells, found mainly in the peritoneal
and pleural cavities. In contrast to recirculating
follicular B cells (or conventional B or B-2
cells), B-1 cells are characterized by B220lowIg
MhighCD23CD43+IgDlow cells (Berland and
Wortis, 2002; Tung and Herzenberg, 2007).
Although it has been pointed out by many re-
searchers that innate B-1 cells but not conven-
tional B cells are producers of natural antibodies
against pathogens (Ochsenbein et al., 1999),
accumulating lines of evidence suggest that
a major source of autoantibodies is also those
B-1 cells (Baumgarth et al., 2005; Carroll and
Holers, 2005), but it has been a matter of debate.
Abbreviations used: 2-GPI,
2–glycoprotein I; Btk, Bru-
ton’s tyrosine kinase; ds, double
stranded; HRP, horseradish
peroxidase; ITIM, immunore-
ceptor tyrosine-based inhibitory
motif; MAPK, mitogen-acti-
vated protein kinase; PAS, peri-
odic acid–Schiff; PIR-B, paired
Ig-like receptor B; PVDF, poly-
vinylidene difluoride; RF, rheu-
matoid factor; SHP-1, SH2
phosphatase 1; ss, single
stranded; TLR, Toll-like
Augmented TLR9-induced Btk activation in
PIR-B–deficient B-1 cells provokes excessive
autoantibody production and autoimmunity
Tomohiro Kubo,1,2 Yuki Uchida,1 Yuko Watanabe,1 Masahiro Abe,1
Akira Nakamura,1 Masao Ono,3 Shizuo Akira,4 and Toshiyuki Takai1
1Department of Experimental Immunology, Institute of Development, Aging, and Cancer, Tohoku University,
Sendai 980-8575, Japan
2Department of Pediatrics, Self Defense Force Sendai Hospital, Sendai 983-0041, Japan
3Department of Pathology, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan
4Laboratory of Host Defense, World Premiere International Immunology Frontier Research Center, Osaka University,
Suita, Osaka 565-0871, Japan
Pathogens are sensed by Toll-like receptors (TLRs) expressed in leukocytes in the innate
immune system. However, excess stimulation of TLR pathways is supposed to be connected
with provocation of autoimmunity. We show that paired immunoglobulin (Ig)-like receptor
B (PIR-B), an immunoreceptor tyrosine-based inhibitory motif–harboring receptor for major
histocompatibility class I molecules, on relatively primitive B cells, B-1 cells, suppresses
TLR9 signaling via Bruton’s tyrosine kinase (Btk) dephosphorylation, which leads to attenu-
ated activation of nuclear factor B p65RelA but not p38 or Erk, and blocks the produc-
tion of natural IgM antibodies, including anti-IgG Fc autoantibodies, particularly
rheumatoid factor. The autoantibody production in PIR-B–deficient (Pirb/) mice was
further augmented in combination with the Faslpr mutation, which might be linked to the
development of autoimmune glomerulonephritis. These results show the critical link be-
tween TLR9-mediated sensing and a simultaneously evoked, PIR-B–mediated inhibitory
circuit with a Btk intersection in B-1 cells, and suggest a novel way toward preventing
pathogenic natural autoantibody production.
© 2009 Kubo et al. This article is distributed under the terms of an Attribu-
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The Journal of Experimental Medicine
PIR-B REGULATES TLR9-INDUCED BTK–NF-B CASCADE | Kubo et al.
B-1 cells, such as through down-regulation of Btk activation
or enhancement of PIR-B–mediated B-1 cell regulation.
PIR-B deficiency with Faslpr mutation characteristically
augments autoantibody production associated
with autoimmune glomerulonephritis
Pirb/ mice were grossly normal and survived well to at least
50 wk of age (Fig. 1 A) without any abnormalities in the histology
of their joint components (synovium and cartilage; Fig. S1 A),
skin, lungs, and glomeruli (not depicted). However, Pirb/Faslpr
mice were found to be markedly short-lived, with only about
half of them surviving at 40 wk of age (Fig. 1 A). Microscopic
examination of their organ samples revealed that the combined
mutant mice did not develop histopathological traits in the lung,
salivary glands (not depicted), and joints (Fig. S1 A), but that in
the kidney they readily developed diffuse glomerulopathy with
a mild increase in the size and cellularity (mesangial area) of the
glomerulus (Fig. 1 B, periodic acid–Schiff [PAS] stain). Immuno-
fluorescence analysis revealed substantial deposition of IgG,
IgM, and C3 in Pirb/Faslpr mice (Fig. 1 B). These pathological
changes were not observed in wild-type B6 or Faslpr mice, indi-
cating that autoimmune glomerulonephritis occurs as a com-
bined effect of PIR-B and Fas mutations (Fig. S1 B). Consistent
with no arthritis and Sjögren-like disease in the double-mutant
mice, anti–SS-B/La antibodies were below the detection limit
(Fig. S1 C, left). We also found that, although the levels of IgM-
and IgG-RF were high in Faslpr mice, these levels were further
elevated in Pirb/Faslpr mice (Fig. 1 C), indicating that PIR-B
deficiency has a marked impact on the augmentation of RF
production in vivo. In addition, anti–double-stranded (ds) DNA
autoantibody production was more augmented in Pirb/Faslpr
mice than in Faslpr animals, although the difference did not reach
a statistically significant level (Fig. 1 D). We also measured by
ELISA the levels of autoantibodies in sera against single-stranded
(ss) DNA and 2–glycoprotein I (2-GPI), but we did not
observe a significant elevation of anti-ssDNA production in
Pirb/Faslpr mice tested compared with Faslpr animals (Fig. 1 D,
right), nor we did not detect anti–2-GPI (Fig. S1 C, right).
Examinations of hematocrit values failed to demonstrate an au-
toimmune hemolytic anemia in Pirb/Faslpr and other mice
(Table S1). The PIR-B deficiency did not result in any obvious
changes in the staining pattern by antinuclear autoantibodies
(Fig. S1 D). The peritoneal B-1 cell (Fig. 1 E, top) and splenic
plasma cell (Fig. 1 E, bottom) populations were also markedly
increased in Pirb/Faslpr mice. With these data collectively, we
concluded that PIR-B deficiency renders mice susceptible to
the development of autoimmune glomerulonephritis in combi-
nation with Faslpr, which accompanies with a markedly elevated
RF production, with a robust production of anti-dsDNA and
Elevated RF production in Pirb/ mice administered
with CpG-B DNA
RF is one of the autoreactive natural antibodies whose primary
role is believed to be the first-line defense against infection.
By stimulation via different TLRs, the B-1 cell population in
the peritoneal cavity has been enlarged and B-1 cell–mediated
autoantibody production augmented (Murakami et al., 1995).
This could be partly because B-1 cells express a set of TLRs,
including TLR4, TLR7, and TLR9 (Gururajan et al., 2007),
and are more prone to differentiate into plasma cells than
B-2 cells upon TLR-mediated stimulation, although B-2 cells
similarly possess a range of TLRs (Genestier et al., 2007). For
example, Murakami et al. (1995) have shown, in anti–red
blood cell autoantibody transgenic mice, that the susceptibil-
ity to autoimmune hemolytic anemia was significantly in-
creased when the mice were transferred from germ-free or
specific pathogen-free conditions to conventional conditions
or injected with a TLR4 ligand, LPS, with a concomitant in-
crease in the peritoneal B-1 cell population, whereas almost
all B-2 cells are constitutively deleted in the transgenic mice.
These findings again suggest the importance of the regulation
of TLR signaling in B-1 cells, which prevents overstimulation
of TLRs so as not to evoke overproduction of natural anti-
bodies, including potentially harmful autoantibodies. There-
fore, what mechanisms may regulate the overstimulation of
the TLR signal, particularly in B-1 cells?
We speculated that paired Ig-like receptor B (PIR-B;
Hayami et al., 1997; Kubagawa et al., 1997) could participate
in the regulation of B-1 cells. Recruitment of SH2 domain–
containing tyrosine phosphatase 1 (SHP-1) to phosphotyro-
sylated immunoreceptor tyrosine-based inhibitory motifs
(ITIMs) in the cytoplasmic portion of PIR-B was shown to
be critical for PIR-B–mediated inhibitory signaling in gen-
eral (Ho et al., 1999; Maeda et al., 1999), and this inhibition
is achieved, at least in part, via constitutive binding of PIR-B
to its ligand, i.e., MHC class I molecules, expressed on the
same cell surface (Masuda et al., 2007). Interestingly, in PIR-
B–deficient (Pirb/) mice, the peritoneal B-1 cell popula-
tion significantly increased, particularly with aging, compared
with wild-type mice (Ujike et al., 2002). However, it has not
been determined how the B-1 cell compartment is physio-
logically regulated by PIR-B or what the physiological or
pathological consequence of the expanded B-1 cells is in the
contexts of infection and autoimmunity.
In this paper, we show that PIR-B can inhibit TLR9-
mediated signaling via regulation of Bruton’s tyrosine kinase
(Btk) phosphorylation in peritoneal B-1 cells. We found that
TLR9 activation immediately activates an Src family kinase,
Lyn, which then phosphorylates PIR-B cytoplasmic ITIMs.
In the absence of PIR-B, B-1 cells become hyperproducers
of natural IgM antibodies, including anti-IgG Fc autoanti-
bodies, i.e., rheumatoid factor (RF), via unmethylated CpG-
B oligodeoxynucleotide (CpG-B) stimulation in vitro and in
vivo or upon aging. These phenotypes caused by PIR-B de-
ficiency were further exaggerated in combination with the
Faslpr mutation, which caused the Pirb/Faslpr mutant mice
to be short-lived mainly because of autoimmune glomerulo-
nephritis with immune complex depositions. Our findings
may provide a novel strategy for preventing autoimmunity
by reducing the production of pathogenic autoantibodies by
JEM VOL. 206, August 31, 2009
mune diseases, which are otherwise evoked by too strong or
sustained innate responses against microbial or viral infections
(Murakami et al., 1995; Akira et al., 2001; De Re et al.,
2006; Krieg and Vollmer, 2007; Marsland and Kopf, 2007). To
The main source of such autoantibodies is considered to be B-
1 cells (Montecino-Rodriguez and Dorshkind, 2006). Ade-
quate regulation of the production of natural autoantibodies,
including RF, is proposed to be crucial for avoiding autoim-
Figure 1. Pirb/ with the Faslpr mutation is sufficient in mice for the development of glomerulonephritis with marked elevation of RF pro-
duction. (A) Pirb/Faslpr mice were remarkably short-lived. The survival curves for male and female B6.Pirb/Faslpr (n = 20 mice per group), B6.Faslpr
(n = 20), B6.Pirb/ (n = 20), and B6 (n = 20) mice until 50 wk of age are shown. (B) Pirb/Faslpr mice develop diffuse glomerulonephritis with a mild
increase in the size and cellularity of the glomerulus, and depositions of IgG, IgM, and complement C3. Kidney sections from each strain of mice at 24 wk
of age were stained with PAS or Alexa Fluor 488 anti–mouse IgG, IgM, and C3. Each panel is representative of samples from five mice per group with similar
staining profiles in a single staining experiment. Bars, 10 µm. (C) Both the IgM- and IgG-RF levels were significantly more elevated in Pirb/Faslpr mice
than those in Faslpr mice. RF in sera from each strain of mice at 24 wk of age was measured by ELISA. Data are shown as means ± SEM (n = 8 mice per
group) and are representative of three separate experiments with similar results. Statistical analyses were performed using a Student’s t test. *, P < 0.05;
**, P < 0.01. (D) The anti-dsDNA and anti-ssDNA autoantibody levels were augmented but not significantly elevated in the Pirb/Faslpr mice tested com-
pared with Faslpr mice. Anti-DNA in sera from each strain of mice at 24 wk of age for anti-dsDNA or at 4–10 mo of age for anti-ssDNA was measured by
ELISA. Data are shown as means ± SEM (n = 8 mice per group) and are representative of three (anti-dsDNA) or two (anti-ssDNA) separate experiments
with similar results. Statistical analyses were performed using a Student’s t test. *, P < 0.05; **, P < 0.01. (E) Markedly enlarged peritoneal B-1 cell and
splenic plasma cell populations in Pirb/Faslpr mice. Peritoneal cells isolated from 24-wk-old mice were stained with anti-B220, CD5, and CD3.
B220+CD5+CD3 B-1 cells (circled) with the percentages are shown (top). Staining of peritoneal cells from Pirb/ mice with anti-CD19, instead of B220,
gave a B-1 cell population with a similar size (not depicted in the figure). Splenocytes isolated from 24-wk-old mice were stained with anti-CD138, B220,
IgM, and IgD. CD138+B220+IgMIgD plasma cells (circled) with the percentages are shown (bottom). The figure is representative of five mice per group.
PIR-B REGULATES TLR9-INDUCED BTK–NF-B CASCADE | Kubo et al.
characterize the precise sources of RF, we next examined the
RF levels in sera from B6 and Pirb/ mice in the absence
of a potentially large influence by Faslpr mutation. Although
8- and 40-wk-old B6 wild-type mice did not show notable
production of either IgM-RF or IgG-RF, Pirb/ mice at
40 wk of age exhibited significantly higher levels of IgM-
RF and slightly elevated IgG-RF levels (Fig. 2 A). When
we intraperitoneally injected 3 nmol CpG-B DNA, a ligand
for TLR9, into 8-wk-old B6 and Pirb/ mice, and deter-
mined IgM-RF and IgG-RF levels in their sera after 48 and
120 h, respectively, we found that the RF of both classes
was significantly elevated in Pirb/ but not in B6 mice
(Fig. 2 B), suggesting that CpG-B–responsive cells in the
peritoneal cavity produce RF. Diminishing B-1 cells, as
well as B-2 cells, in the peritoneum by repeated injection of
water (Fig. S2; Murakami et al., 1995) resulted in a signifi-
cant reduction of the total IgM level and a nearly complete
abrogation of IgM-RF production in Pirb/ mice compa-
rable to that in wild-type mice (Fig. 2 C). These results sug-
gest that peritoneal B cells are a major source of IgM-RF
autoantibodies in Pirb/ mice.
Augmented CpG-B responses in PIR-B–deficient B-1
cells in vitro
Next, we aimed at examining more precisely the PIR-B–
mediated regulation of RF production by B-1 cells in vitro. We
sorted B220+CD11b+ B-1 cells from peritoneal cells of B6 and
Pirb/ mice, and examined them for their proliferation upon
stimulation with various TLR ligands. B-1 cells from both B6
and Pirb/ mice were found to respond well to LPS (TLR4
ligand) and CpG-B (TLR9 ligand), but not to poly(I:C) (TLR3
ligand) or ssRNA (TLR7/8 ligand; Fig. 3 A), indicating the
significant roles of TLR4 and TLR9 in the B-1 cell function,
as pointed out previously (Gururajan et al., 2007). Interest-
ingly, however, stimulation of Pirb/ B-1 cells with CpG-B
provoked higher proliferation than that of wild-type cells,
whereas the LPS-mediated responses were comparable. The
CpG-B–mediated augmented proliferation seen for Pirb/
B-1 cells was not observed for splenic B-2 cells (Fig. 3 B, left),
as in the case of LPS (Fig. 3 B, right). These results indicate that
Pirb/ peritoneal B-1 cells, but not the splenic B-2 cells, were
more sensitive to CpG-B stimulation and divided more vigor-
ously than those from wild-type mice. We observed that B-1
cells express about sixfold more PIR-B molecules on their sur-
face than splenic B-2 cells, whereas the TLR9 expression levels
are comparable between B-1 and B-2 cells when estimated by
flow cytometry (Fig. S2, A and B). We also noted that the ex-
pression level of Btk, one of the signal mediators downstream
of PIR-B (Maeda et al., 1999), is higher in B-1 cells than that
in B-2 cells (Fig. S3, C and D). These observations may ac-
count for, at least partly, the difference in the sensitivity to
CpG-B between B-1 and B-2 cells.
This observation of CpG-B–sensitive Pirb/ B-1 cells
prompted us to examine whether other possible B-1 cell
products (Baumgarth et al., 2005; Carroll and Holers, 2005;
Gururajan et al., 2007) could also be elevated. A culture
Figure 2. Enhanced RF production in Pirb/ mice. (A) The IgM-RF
levels are significantly elevated in Pirb/, aged mice in particular. Sera
from wild-type B6 and Pirb/ mice at 8 and 40 wk of age were subjected
to measurement of the IgM- and IgG-RF levels by ELISA. Data are shown
as means ± SEM (n = 5 mice per group) and are representative of three
separate experiments with similar results. Statistical analyses were per-
formed using a Student’s t test. *, P < 0.05. (B) Both the IgM- and IgG-RF
levels in sera were elevated in Pirb/ mice after CpG-B administration
into the peritoneum of the animals. B6 and Pirb/ mice at 8 wk of age
were injected with 3 nmol CpG-B into the peritoneal cavity. Serum sam-
ples collected after 48 and 120 h were analyzed by ELISA for IgM- and
IgG-RF, respectively. Data are shown as means ± SEM (n = 4 mice per
group) and are representative of three separate experiments with similar
results. Statistical analyses were performed using a Student’s t test. *, P <
0.05. (C) IgM-RF is produced preferentially by peritoneal B cells. B6 and
Pirb/ mice at 5 wk of age were injected intraperitoneally with water or
PBS as a negative control to achieve relatively specific elimination of the
B-1 cells (Murakami et al., 1995). After four rounds of water injection, the
total IgM level was significantly decreased and the IgM-RF was substan-
tially lost in sera from Pirb/ mice. Data are shown as means ± SEM (n =
3 mice per group) and are representative of three separate experiments
with similar results. Statistical analyses were performed using a Student’s
t test. *, P < 0.05.
JEM VOL. 206, August 31, 2009
Figure 3. Enhanced responses in peritoneal B-1 cells from Pirb/ mice upon CpG-B stimulation in vitro. (A and B) Proliferation of peritoneal
B-1 cells (A) or splenic B-2 cells (B) from wild-type B6 and Pirb/ mice (n = 5 per group) with various stimuli. Peritoneal B-1 cells and splenic B-2 cells
were pulse labeled with [3H]thymidine during the last 8 h of the 48-h culture period with or without a stimulant. Data are shown as means ± SEM of
triplicate cultures and are representative of three independent experiments. Statistical analyses were performed using two-way analysis of variance.
***, P < 0.001. (C) Production of Igs, autoantibodies, and a cytokine by peritoneal B-1 cells from B6 and Pirb/ mice (n = 5 per group). B-1 cells were
stimulated with 1 µM CpG-B or GpC control, and assessed for total IgM, IgM-RF, IgG-RF, anti-dsDNA, and IL-10 by ELISA. Data are shown as means ±
SEM of triplicate cultures, and are representative of three independent experiments. Statistical analyses were performed using a Student’s t test. ***, P <
0.001. Provided standard and assay diluent supplemented in the ELISA kit were served as positive control (Pc) and negative control (Nc), respectively.
PIR-B REGULATES TLR9-INDUCED BTK–NF-B CASCADE | Kubo et al.
cells or cultured macrophages, which give a grossly similar
PIR-B signal upon CpG-B stimulation as B-1 cells (unpub-
lished data), in some analyses of signaling events. PIR-B
phosphorylation in peritoneal cells was augmented, reached
a maximum as early as 5 min after CpG-B addition, and
lasted for at least 20 min (Fig. 4 A). Notably, PIR-B in peri-
toneal cells in the nonstimulated state was already tyrosine
phosphorylated, as in the cases of other cells such as splenic
B cells and macrophages (Fig. 4, B and C, B-1 cells and mac-
rophages, respectively; Ho et al., 1999; Kubagawa et al.,
1999; Maeda et al., 1999), possibly because of constitutive
PIR-B ligation to MHC class I molecules on the same cell
surface (Masuda et al., 2007). CpG-B–induced augmenta-
tion of PIR-B phosphorylation indeed also occurred in puri-
fied B-1 cells 5 min after the stimulation, and was accompanied
by a significant increase in SHP-1 recruitment (Fig. 4 B).
Importantly, although we also found augmented phosphoryla-
tion of PIR-B and enhanced recruitment of SHP-1 upon
stimulation of cultured macrophages with CpG-B, but not
GpC control DNA (Fig. 4 C, left), these events were not
observed in TLR9-deficient cells (Fig. 4 C, right), indicating
that CpG-B–mediated PIR-B phosphorylation is dependent
supernatant was collected after a 20–72-h stimulation of B-1
cells with 1 µM CpG-B, which was subjected to measure-
ment of the contents of autoantibodies and a cytokine by
ELISA (Fig. 3 C). We found that total IgM, IgM-RF, and
IL-10 release, but not IgG-RF or anti-dsDNA, were signifi-
cantly more elevated in Pirb/ B-1 cells than in wild-type
cells. Collectively, these data indicate that CpG-B stimula-
tion of Pirb/ peritoneal B-1 cells resulted in augmented
proliferation, enhanced IgM antibody production (including
autoreactive IgM-RF), and IL-10 release in vitro. Although
B-1 cells express PIR-B molecules more abundantly on their
surface than splenic B-2 cells (Fig. S3 A), the expression of
PIR-A, an activating counterpart of PIR-B, is not detected
on B-1 cells (Fig. S4, A and B), and the TLR9 level is not
augmented in Pirb/ B-1 cells (Fig. S4 C), suggestive of the
presence of any PIR-B–mediated inhibitory signaling that
specifically regulates TLR9 in B-1 cells.
Augmented phosphorylation of PIR-B and SHP-1
recruitment upon CpG-B stimulation of B-1 cells
Because of the very limited number of purified B-1 cells that
we can sort from peritoneal cells, we used whole peritoneal
Figure 4. Augmented phosphorylation of PIR-B and enhanced recruitment of SHP-1 in peritoneal B-1 cells stimulated with CpG-B. (A) Time
course of tyrosine phosphorylation of PIR-B in peritoneal cells from B6 and Pirb/ mice (n = 8 per group) stimulated with CpG-B. After stimulation with
1 µM CpG-B, samples were taken at various time points and first immunoprecipitated (IP) with anti–PIR-B and then immunoblotted (IB) to visualize PIR-B
phosphotyrosine (top), followed by reprobing with anti–PIR-B (bottom). Data are representative of three separate experiments with similar results. (B) Aug-
mented tyrosine phosphorylation of PIR-B and enhanced recruitment of SHP-1 in peritoneal B-1 cells stimulated with CpG-B. B-1 cells from wild-type B6
mice (n = 12) were stimulated with 1 µM CpG-B and subjected to immunoprecipitation and protein blot analysis (IP-Western) to visualize the phosphotyro-
sylation of PIR-B (top), SHP-1 (middle), and PIR-B (bottom). Data are representative of three separate experiments with similar results. (C) Phosphotyrosyl-
ation of PIR-B upon CpG stimulation is dependent on TLR9. Bone marrow–derived cultured macrophages (BMMs) from wild-type B6 mice (left) and
TLR9-deficient (Tlr9/) mice (right; n = 4 per group) were stimulated with 1 µM CpG-B or GpC control DNA, and subjected to IP-Western to visualize phos-
photyrosylated PIR-B (top), SHP-1 (second from top), Lyn (second from bottom; arrow), and PIR-B (bottom). Data are representative of three separate experi-
ments with similar results. The intensity for each band was estimated by densitometric scanning with normalization as to loading control.
JEM VOL. 206, August 31, 2009
Enhanced activation of NF-B but not the mitogen-
activated protein kinase (MAPK) pathway in PIR-B–
deficient B-1 cells after CpG-B stimulation
Next we aimed at elucidating the molecular events occurring
in two major downstream cascades of TLR9 activation upon
CpG-B stimulation of Pirb/ B-1 cells. Generally, TLR9-
mediated signaling leads to activation of NF-B as well as the
MAPK pathway (Akira et al., 2001; Baccala et al., 2007;
Krieg and Vollmer, 2007). We observed that the phosphoryla-
tion levels of p38 MAPK and Erk were significantly aug-
mented, particularly at 30 min after CpG-B addition, in both
wild-type and Pirb/ B-1 cells (Fig. 6, A–C). However, the
phosphorylation kinetics and levels were comparable between
these cells, suggesting that these phosphorylation events are
independent of PIR-B–mediated regulation. Interestingly, how-
ever, p65 NF-B phosphorylation was found to be markedly
augmented in Pirb/ B-1 cells as early as 5 min after CpG-B
addition (Fig. 6, A and D). These results indicate that the
NF-B p65RelA subunit could be the target molecule lo-
cated the most downstream of regulation through Lyn-medi-
ated PIR-B phosphorylation, whereas the MAPK pathway is
not linked to the PIR-B pathway.
Btk can link the PIR-B pathway and NF-B cascade
Which molecule acts as an intersection between the PIR-B–
SHP-1–initiated inhibitory signaling pathway and the TLR9–
NF-B cascade? Maeda et al. (1999) reported that, in a B cell
on TLR9. Interestingly, stimulation of macrophages with
CpG-B but not GpC induced coprecipitation of PIR-B and
Lyn, whereas the stimulation of TLR9-deficient macro-
phages did not (Fig. 4 C, second from bottom), suggestive of
an interaction of Lyn and PIR-B upon TLR9 activation (see
Up-regulated phosphorylation of PIR-B is mediated,
at least partly, by Lyn
Preceding studies by others indicated that, in splenic B cells,
Lyn is the major Src family kinase that constitutively phos-
phorylates PIR-B (Ho et al., 1999). To elucidate the mo-
lecular mechanisms underlying TLR9-mediated PIR-B
phosphorylation, we first examined activation of Lyn in TLR9-
sufficient or -deficient B-1 cells stimulated with CpG-B.
The phosphorylation level of Lyn was found to be aug-
mented as early as 5 min after CpG-B stimulation of B-1
cells regardless of the presence or absence of PIR-B (Fig. 5,
A and B). However, augmented phosphorylation of Lyn
was not observed in TLR9-deficient B-1 cells (Fig. 5,
C and D). In addition, as noted in the previous paragraph,
Lyn–PIR-B coprecipitation was detected in a macrophage
lysate prepared after stimulation with CpG-B (Fig. 4 C, sec-
ond from bottom). These results indicate that Lyn is rapidly
activated downstream of TLR9 and strongly suggest that
Lyn is a candidate, if not the sole, Src family kinase that
Figure 5. Up-regulated phosphorylation of Lyn upon CpG-B stimulation of wild-type and Pirb/ B-1 cells but not Tlr9/ cells.
(A and B) Peritoneal B-1 cells from wild-type B6 and Pirb/ mice (n = 12 per group) were stimulated with 1 µM CpG-B and subjected to IP-Western
analysis to visualize the phosphprylated Lyn (A). The phosphorylation levels of Lyn were estimated by densitometric scanning with normalization as to
loading control and are depicted as a bar graph (B). Lyn phosphorylation was augmented upon CpG stimulation regardless of the presence or absence of
PIR-B. Data are shown as means ± SEM of three separate experiments. (C and D) Peritoneal B-1 cells from wild-type B6 and Tlr9/ mice (n = 12 per
group) were stimulated with 1 µM CpG-B and subjected to IP-Western analysis to visualize the phosphorylated Lyn (C). The phosphorylation levels of Lyn
were estimated by densitometric scanning with normalization as to loading control and are depicted as a bar graph (D). Augmented Lyn phosphorylation
was not observed in Tlr9/ B-1 cells. Data are shown as means ± SEM of three separate experiments. Control bands of Lyn (56 kD) were obscured by
40–60-kD diffuse bands originated from protein A released from immunoprecipitation beads. *, P < 0.05.
PIR-B REGULATES TLR9-INDUCED BTK–NF-B CASCADE | Kubo et al.
observed that a slight increase in the phosphorylation level of
Btk 5 min after CpG-B stimulation was partially blocked by
pretreatment with the inhibitors (Fig. 8, A and B). The re-
duction of the phosphorylation was more evident in the case
of NF-B p65RelA (Fig. 8, A and C). These results indicate
that Btk phosphorylation is dependent, at least partly, on an
Src family kinase, most likely Lyn, and that NF-B p65RelA
phosphorylation is also a critical event downstream of Src
family kinase activation.
Recent studies have suggested several feedback regulators for
TLR9 activation, including SOCS1 (Rothlin et al., 2007),
ATF3 (Whitmore et al., 2007), IRF-4 (Martin et al., 2007),
and SHP-1 (An et al., 2008). For example, receptor tyrosine-
based activation motif–induced, SOCS1-mediated feedback
regulation seems to occur at later stages, such as hours, after
TLR9 activation, followed by inflammatory cytokine induc-
tion (Rothlin et al., 2007). In contrast, the PIR-B–mediated
suppression revealed in this study seems to take place as early
as 5 min after CpG-B addition to B-1 cells. Therefore, PIR-B
does not seem to fit well into the category of feedback regu-
latory elements for TLR9. Rather, it plays an important reg-
ulatory role in immediate-early cellular responses to TLR9
line, IIA1.6, Syk, and Btk were the substrates for SHP-1 re-
cruited to PIR-B ITIMs upon stimulation of B cell receptors.
This observation prompted us to examine the possible in-
volvement of these two molecules in the TLR9 pathway. Ex-
amination of the phosphorylation status of Syk upon CpG-B
addition to B-1 cells did not reveal significant enhancement
of the phosphorylation in either wild-type or Pirb/ B-1 cells
(Fig. 7, A and B), indicating that Syk does not seem to be a
critical substrate for SHP-1 recruited to PIR-B ITIMs in B-1
cells. Instead, we found that Btk phosphorylation was mark-
edly augmented 5 min after CpG-B addition in both wild-
type and Pirb/ B-1 cells, and the augmentation did not
show a marked decrease at least up to 30 min after CpG-B
stimulation in Pirb/ B-1 cells (Fig. 7, A and C), indicating
that Btk is a major substrate for SHP-1.
Pretreatment of B-1 cells with Src family kinase inhibitors
can suppress phosphorylation of Btk and NF-B p65RelA
after CpG-B stimulation
To verify that Lyn could be the most upstream effector for
Btk and NF-B p65RelA phosphorylation, wild-type B-1 cells
were pretreated with Src family kinase inhibitors (SU6656 or
PP2), stimulated with CpG-B, and examined to determine
the phosphorylation levels of Btk and NF-B p65RelA. We
Figure 6. p38 and Erk phosphorylation is not grossly influenced by PIR-B, whereas NF-B p65RelA phosphorylation is markedly augmented
in Pirb/ B-1 cells. (A–D) Immunoblot analysis of phospho-p38, phospho-Erk, and phospho-p65RelA after CpG stimulation of peritoneal B-1 cells from
wild-type B6 and Pirb/ mice (n = 10–14 per group). Although the time courses and augmentation magnitudes of the phosphorylation of p38 and Erk
were not grossly different between wild-type and Pirb/ B-1 cells (A–C), phosphorylation of NF-B p65RelA was markedly augmented in Pirb/ B-1
cells (A and D). The intensity for each band was estimated by densitometric scanning with normalization as to loading control. Data are shown as
means ± SEM of three separate experiments. *, P < 0.05.
JEM VOL. 206, August 31, 2009
As is well known, Btk is a critical kinase for the develop-
ment and function of B cells, including B-1 cells, by assur-
ing antigen receptor signaling, as has been demonstrated in
immunocompromised Xid mice and human X-linked agam-
maglobulinemia, in which Btk is dysfunctional (Bradley et al.,
1994; Hashimoto et al., 1996). Our current finding that Btk
is a critical link between the TLR9 cascade and the PIR-B–
mediated regulatory loop, particularly in B-1 cells expressing
abundant PIR-B (Fig. S3 A), provides an insight into the
mechanism underlying crosstalk of the innate immune system
with ITIM-harboring receptors.
Given that PIR-B might be a negative regulator for
TLR9 in B-1 cells, PIR-B–mediated inhibition of TLR9 ac-
tivation could become potentially harmful for host mammals
if their immune system should engage with invading patho-
gens that otherwise would cause serious infectious diseases.
Therefore, it is expected that this regulatory system might be
tightly controlled during the course of the capture, sensing,
activation. PIR-B is constitutively tyrosine phosphorylated
at the ITIMs and associates with SHP-1 (Ho et al., 1999;
Kubagawa et al., 1999; Maeda et al., 1999), which is critical
for maintaining steady-state levels of intracellular phosphoty-
rosylated proteins. In this study, we found a novel and im-
mediately initiated regulatory circuit for TLR9, namely that
PIR-B phosphorylation is immediately augmented by TLR9-
initiated Lyn activation, and the concomitant enhancement
of SHP-1 recruitment to augmented phospho-ITIMs of PIR-B
leads to Btk dephosphorylation, which then attenuates the
phosphorylation level of NF-B p65RelA. The molecular
mechanism for the TLR9-initiated Lyn activation, which aug-
ments Btk phosphorylation, is currently not known. Interest-
ingly, on the other hand, SHP-1 has recently been demonstrated
to be a critical regulator for type I interferon production by
macrophages and dendritic cells through inhibition of IRAK1
activation downstream of TLR4 (An et al., 2008), whereas
augmented IRAK1 activation is not observed in CpG-B–
stimulated Pirb/ B-1 cells (Fig. S5), suggesting that B-1 cells
preferentially use Btk as a key intersecting molecule for the in-
Recent studies have shown that Btk is an important
mediator for the TLR9 cascade in addition to MyD88 and
TRAF (Doyle et al., 2007; Gilliet et al., 2008). Btk is re-
quired for NF-B activation, participating in the pathway
leading to increased phosphorylation of p65RelA activated
by TLR8 and TLR9 (Doyle et al., 2007; Lee et al., 2008).
Figure 7. Phosphorylation of Btk but not Syk is markedly up-reg-
ulated upon CpG-B stimulation of Pirb/ B-1 cells. (A–C) Peritoneal
B-1 cells from B6 and Pirb/ mice (n = 10–14 per group) were stimu-
lated with 1 µM CpG-B and subjected to immunoblot analysis for visual-
ization of phospho-Syk and phospho-Btk (A), which was estimated by
densitometric scanning with normalization as to actin (B and C). Data are
shown as means ± SEM of three separate experiments. *, P < 0.05.
Figure 8. CpG-B–induced phosphorylation of Btk and NF-B
p65RelA in peritoneal B-1 cells is down-regulated by Src family
kinase inhibitors. (A–C) Peritoneal B-1 cells from wild-type B6 mice (n =
12) were treated with 10 µM SU6656 or PP2 for 30 min before stimula-
tion with 1 µM CpG-B. 5 min later, cell lysates were subjected to immuno-
blot analysis to visualize the phosphorylation of Btk (top), NF-B
p65RelA (middle), and loading control -actin (bottom; A). The phos-
phorylation levels of Btk and p65RelA were estimated by densitometric
scanning with normalization as to actin and are depicted as bar graphs
(B and C). Data are representative of three separate experiments with
PIR-B REGULATES TLR9-INDUCED BTK–NF-B CASCADE | Kubo et al.
basement membranes of glomeruli. Considering these obser-
vations, it may increasingly become important to manipulate
the PIR-B–mediated inhibitory system in the regulation of
autoimmune diseases while maintaining the integrity of TLR9-
mediated microbial sensing, in such cases as rheumatoid ar-
thritis (Newkirk, 2002). Understanding of the PIR-B system
in B-1 cells may lead to the development of novel and effec-
tive ways toward controlling autoimmune diseases.
MATERIALS AND METHODS
Mice. C57BL/6 (B6) mice were purchased from Charles River Laborato-
ries. Pirb/ mice were generated (Ujike et al., 2002) and backcrossed with
B6 mice for 12 generations. Pirb/Faslpr mice were generated by crossing
B6.Faslpr mice (C57BL/6J Jms Slc-lpr/lpr; The Jackson Laboratory) with
B6.Pirb/ mice and by intercrossing between the littermates. TLR9-deficient
(Tlr9/) mice were purchased from Oriental BioService, Inc. Mice were
maintained and bred in the Animal Facility of the Institute of Development,
Aging, and Cancer, Tohoku University, an environmentally controlled and
specific pathogen-free facility, according to guidelines for experimental ani-
mals defined by the university, and animal protocols were reviewed and ap-
proved by the Animal Studies Committee of the university. All experiments
were performed on 6–50-wk-old age-matched male and female mice.
Reagents and antibodies. LPS (B8) was purchased from Sigma-Aldrich.
Poly(I:C) and ssRNA/LyoVec were obtained from InvivoGen. Anti-IgM
F(ab)2 was purchased from Invitrogen. Phosphothioate–CpG-B oligode-
oxynucleotide (ODN1826; 5-TCCATGACGTTCCTGACGTT-3) and
phosphothioate-GpC (5-TCCATGAGCTTCCTGAGCTT-3) were syn-
thesized by Hokkaido System Science Co., Ltd. Src kinase inhibitors SU6656
and PP2 were obtained from EMD. Antibodies to phospho-Syk (Tyr352),
Syk, Lyn, phospho–p44/42 MAPK (Thr202/Tyr204 and D13.14.4E), phos-
pho-p38 (Thr180/Tyr182), p38 (5F11), phospho–NF-kB p65 (Ser536), and
NF-B p65 were obtained from Cell Signaling Technology. Phospho-Btk
(Tyr550) antibody was obtained from Abcam. Antibodies to Btk, Erk, PIR-
B (p91), anti-IRAK1, anti–mouse monoclonal Btk (E-9), and SHP-1 were
purchased from Santa Cruz Biotechnology, Inc. The horseradish peroxidase
(HRP)–conjugated antiphosphotyrosine mAb (RC20) was purchased from
BD. The antibody to -actin was obtained from Sigma-Aldrich.
Cell separation. For separation of splenic B cells, B220+ cells were purified
with a magnetic-activated cell sorter (MACS; Miltenyi Biotec). For prepara-
tion of peritoneal B-1 cells, peritoneal cells were isolated by washing of the
peritoneal cavity with ice-cold RPMI 1640 containing 10% FCS. Peritoneal
cells were first blocked with anti–mouse FcR mAb (2.4G2; BD) and then
stained with allophycocyanin (APC)-labeled anti-B220 (RA3-6B2; BD) and
FITC-labeled anti-CD11b (M1/70; BD) mAbs, and B220+CD11b+ cells
were sorted using a FACSAria (BD). The purity of the sorted populations
was consistently >95%, as determined by the B220+CD11b+ phenotype.
Bone marrow cells from femurs were cultured in -MEM containing 10%
FCS and 20 ng/ml M-CSF (PeproTech) at 37°C. After 7 d, the adherent
cells were used as bone marrow–derived macrophages (BMMs).
Proliferation assay. 105 splenic B cells per well and 2.5 × 104 peritoneal
B-1 cells per well were cultured in 96-well flat plates (Corning) with RPMI
1640 containing 10% FCS, 1 mM sodium pyruvate, and supplemented anti-
biotics. The cells were activated with the reagents described in Reagents and
antibodies. After 48 h of stimulation, proliferation was determined as
Measurement of antibodies and a cytokine. 5 × 104 peritoneal B-1 cells
per well were cultured in 96-well flat plates with or without stimulants. The
supernatants of 20–72-h-cultured peritoneal B-1 cells were collected, and the
amounts of anti-dsDNA antibodies, RF, IgM, and IL-10 were determined
processing, and elimination of virulent microbes. How is
PIR-B–initiated TLR9 regulation controlled? Although we
do not currently have an answer to this question, it is con-
ceivable that cell-surface expression of PIR-B might be down-
regulated or the expression of PIR-A with possibly the
opposite function, namely the activation of TLR9, might be
up-regulated, or both. Another possibility is that spatial com-
munication between PIR-B and TLR9 on, or near, the cell
membrane might be uncoupled during serious infections by
means of any unknown intracellular mechanism that controls
the dynamics of endosomal/lysosomal TLR9 as well as the
dynamics of PIR-B. It may be possible that PIR-B on plasma
membrane could translocate partially to the endosomal/lyso-
somal compartment of B-1 cells upon CpG stimulation. Our
preliminary confocal laser-scanning microscopic analysis of
B-1 cells stimulated with CpG suggests that it could be the
case (Fig. S6). Such translocation may account for, at least
partly, the reason why endosomal/lysosomal TLR9 can com-
municate intimately with PIR-B in terms of CpG-initiated,
TLR9-mediated signaling and may imply a presence of any
uncoupling mechanism for the TLR9–PIR-B communica-
tion. On the other hand, although preceding studies have
demonstrated the critical roles of inhibition mediated by
PIR-B in the maintenance of immune homeostasis (Wheadon
et al., 2002; Ujike et al., 2002; Nakamura et al., 2004; Pereira
et al., 2004; Zhang et al., 2005; Masuda et al., 2007), the
function of the activating counterpart, PIR-A, remains to be
determined, but it also can bind to MHC class I molecules
(Nakamura et al., 2004). Control mechanisms for balanced,
or in some cases unbalanced, PIR-B and PIR-A gene expres-
sion are also not known. One notable observation is that PIR-B
expression on activated B cells is reduced by IL-4 (Rudge
et al., 2002). Another interesting notion is that PIR-A expres-
sion on host dendritic cells is significantly augmented during
the induction phase of experimental acute graft-versus-
disease induced in Pirb/ mice (Nakamura et al., 2004). Ob-
viously, we need more precise knowledge on the regulation
of PIR-A and PIR-B, and the spatial communication be-
tween membrane PIR-B and endosomal/lysosomal TLR9.
CD5+ B-1 cells are considered to be involved in some
autoimmune diseases in which RF is frequently detected or
RF production is directly coupled to the disease (Hardy et al.,
1987; Sowden et al., 1987; Youinou et al., 1990). Also, RF+
B cells are effectively activated via the IgG2a–chromatin im-
mune complex and synergistic stimulation of TLR9-mediated
signaling (Leadbetter et al., 2002). It is known that RF can bind
to anti-dsDNA autoantibodies and can form immune com-
plexes (Izui and Eisenberg, 1980). Also, it has been reported
that RF itself has cryoglobulin activity and causes glomerulo-
nephritis without any other additional factors (Gyotoku et al.,
1987). Our observations on glomerulonephritis in Pirb/Faslpr
mice with markedly and specifically elevated IgM- and IgG-RF
productions, but not other autoantibodies examined, suggest
that IgM- and IgG-RF autoantibodies, namely anti-IgG Fc
antibodies, are factors that worsen the disease, presumably by
accelerating the deposition of IgG autoantibodies against
JEM VOL. 206, August 31, 2009
through a graded ethanol series. After washing with distilled water, sections
were incubated with trypsin (Nichirei) for 50 min at 37°C and washed three
times with PBS. Sections were incubated with Alexa Fluor 488–conjugated
anti–mouse IgG, IgM (Invitrogen), goat IgG fraction to mouse complement
C3 (Cappel; MP Biomedicals), or Alexa Fluor 488–conjugated anti–goat
IgG (Invitrogen). To detect antinuclear antibodies in sera, HEp2 cell plates
(FLUORO HEPANA test; MBL International) were stained with sera from
B6 Pirb/, Faslpr, Pirb/Faslpr, and Fcgr2b/Faslpr (Yajima et al., 2003) mice
at different dilutions, and detected with FITC-conjugated anti–mouse IgG
(Cappel; MP Biomedicals). Samples were examined under microscopes (BX50
[Olympus] and VB-7000 [Keyence]).
Renal function test. Serum from mice of each strain at 40 wk of age was
collected, and the amounts of blood urea nitrogen and creatinine were de-
termined with a dry chemistry analyzer (Fujifilm).
Confocal laser-scanning microscopic analysis. MACS-sorted perito-
neal B-1 cells (CD3, F4/80, CD23, and IgM+) from B6 mice were cul-
tured with Cy5-conjugated CpG-B (ODN1826; Nihon Gene Research
Laboratories Inc.) and LysoTracker Green DND-26 (Invitrogen) for 30 min.
After fixation with Cytofix/Cytoperm, cells were permeabilized with Perm/
Wash and stained with biotin-conjugated anti–mouse PIR-A/B (6C1) or
rat IgG1 isotype control (BD) and Alexa Fluor 546–conjugated streptavi-
din (Invitrogen). Samples were examined under a microscope (Fluoview
Statistical analysis. Statistical analyses were performed using two-way
analysis of variance or Student’s t test. P < 0.05 is considered as statisti-
Online supplemental material. Fig. S1 presents additional data showing
that PIR-B deletion leads to renal dysfunction when combined with the
Faslpr mutation but does not have an impact on arthritis development, or anti-
nuclear, anti–SS-B/La, and anti–2-GPI autoantibody production. Fig. S2
shows diminishment of peritoneal B cells in Pirb/ mice by repeated water
injection. Fig. S3 shows that peritoneal B-1 cells express much more PIR-B
than splenic B-2 cells, that TLR9 expression does not differ between B-1
and B-2 cells, and that Btk expression is higher in B-1 than B-2 cells. Fig. S4
shows that PIR-A expression is not detected on B-1 cells and that TLR9
expression does not change. Fig. S5 shows that the IRAK1 degradation level
was not significantly altered in PIR-B–deficient B-1 cells. Fig. S6 shows that
PIR-B can translocate into the endolysosomal compartment after CpG-B
stimulation of B-1 cells. Table S1 presents hematocrit values for Pirb/ and
Pirb/Faslpr mice. Online supplemental material is available at http://www
We thank N. Halewood for editorial assistance, and Y. Ito and A. Sugahara-Tobinai
for the outstanding technical support.
This work was supported in part by the Core Research for Evolutional Science
and Technology Program of the Japan Science and Technology Agency, a Grant-in-
Aid from the Ministry of Education, Culture, Sports, Science, and Technology of
Japan, the Kanae Foundation for the Promotion of Medical Science, and a grant
from the 21st Century Center of Excellence (COE) Program for Innovative
Therapeutic Development Toward the Conquest of Signal Transduction Diseases and
the Global COE Program for Network Medicine.
The authors have no conflicting financial interest.
Submitted: 23 October 2008
Accepted: 28 July 2009
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Immunoblot analysis. Peritoneal B-1 cells were solubilized in a lysis buf-
fer (1% NP-40, 20 mM Tris [pH 7.3], 150 mM NaCl, 10 mM EDTA, and
10% glycerol) containing 2 mM sodium vanadate and 50 mM sodium fluo-
ride, and supplemented with protease inhibitors. Cell lysates were separated
by SDS-PAGE and transferred to a PVDF membrane, followed by detection
with the appropriate antibodies. Signal intensities of each protein were esti-
mated by densitometric scanning (Dolphin View band tool; KURABO).
Flow cytometry. For flow cytometric analysis, the following mAbs ob-
tained from BD were used: purified FITC-, PE-, APC-, or biotin-conjugated
antibodies against mouse B220 (RA3-6B2), IgM (R6-60.2), IgD (11-26),
CD3 (145-2C11), CD5 (Ly-1), CD11b (M1/70), and CD138 (281-2); rat
IgG1 (A101-1); rat IgG2a (R35-95); PECy5-conjugated streptavidin; APC-
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the appropriate antibodies for the surface antigens of B cells. For intracellular
TLR9 or Btk staining, peritoneal cells or splenocytes were fixed with Cyto-
fix/Cytoperm (BD). After cells had been washed in a permeabilization buffer
(Perm/Wash; BD), they were stained with biotin-conjugated anti–mouse
TLR9 (M9.D6; eBioscience) and APC-conjugated streptavidin or rabbit anti–
mouse Btk antibody (M-138; Santa Cruz Biotechnology Inc.) and Alexa
Fluor 647–conjugated goat anti–rabbit IgG (Invitrogen). Cell surfaces were
stained by standard techniques, and flow cytometry was performed with a
FACSCalibur (BD) and analyzed with FlowJo software (Tree Star, Inc.).
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we used the method described by Murakami et al. (1995). In brief, distilled
water or PBS, as a negative control, was injected every week into the perito-
neal cavity of mice. The doses of injected water and PBS depended on the
size of the mice as follows: 1 ml for 4–8 wk of age or 2 ml for 8 wk of age
and older. Blood was collected after the fourth injection of water or PBS.
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foot joints were fixed with 4% neutral-buffered formalin-PBS, embedded in
paraffin, sectioned at 2 µm, and stained with PAS or hematoxylin and eosin.
To assay for immune complex deposition, formalin-fixed and paraffin-
embedded kidney sections were deparaffinized in xylene and dehydrated
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