degradation (3, 10–12). PGRN also functions as
a neurotrophic factor (13), and mutations in the
Grn gene cause frontotemporal dementia (14–16 ).
Despite such a variety of roles, efforts to exploit the actions of PGRN and understand the
mechanisms involved have been substantially
hampered by our inability to identify its binding receptor(s) (2).
TNFa-TNFR signaling has received great attention owing to its position at the apex of the
proinflammatory cytokine cascade and its dominance in the pathogenesis of various disease processes, and in particular, autoimmune disorders
(17). TNFa blockers, including etanercept (Enbrel),
infliximab (Remicade), and adalimumab (Humira),
are effective anti-inflammatory therapies (18, 19).
TNFR1 is expressed ubiquitously, whereas TNFR2
expression is tightly regulated and found predominantly in hematopoietic cells (20).
In a search for PGRN-associated proteins,
we screened a yeast two-hybrid (Y2H) cDNA library using the construct pDBleu-PGRN (
amino acids 21 to 588) encoding PGRN lacking
signal peptide as bait, and isolated 12 positive
clones among 2.5 million clones. Sequencing
data showed that two of them were cell surface
TNFR2 (TNFRSF1B/CD120b). We then verified
the interaction between PGRN and TNFR2 in
yeast by repeating the Y2H assay. The interaction
between PGRN and TNFR in human chondrocytes was demonstrated by coimmunoprecipita-tion (Co-IP) (Fig. 1A and fig. S1A). Recombinant
human PGRN (rhPGRN) demonstrated dose-dependent binding and saturation to the extracellular domains of TNFR1 and TNFR2 from the
liquid phase (fig. S1B). Kinetic binding studies revealed that rhPGRN exhibited comparable binding affinity for TNFR1 and TNFR2 and had higher
affinity for TNF receptors, especially TNFR2,
when compared to TNFa (Fig. 1B).
The finding that PGRN directly binds to
TNFR prompted us to determine whether PGRN
affected the TNFa-TNFR interaction. rhPGRN
demonstrated dose-dependent inhibition of TNFa
1Department of Orthopaedic Surgery, New York University
School of Medicine and NYU Hospital for Joint Diseases, New
York, NY 10003, USA. 2Institute of Pathogenic Biology, Shandong
University School of Medicine, Jinan, Shandong 250012, China.
3Cytovance Biologics, Oklahoma City, OK 73104, USA. 4Depart-
ment of Microbiology and Immunology, Weill Medical College of
Cornell University, New York, NY 10065, USA. 5Department of
Pathology, New York University School of Medicine, New York, NY
10016, USA. 6Section of Rheumatology, Department of Medicine, Yale University School of Medicine, New Haven, CT
06520, USA. 7College of Life Sciences, Nankai University,
Tianjin 300071, China. 8Baylor College of Dentistry, Texas A&M
Health Science Center, Dallas, TX 75246, USA. 9Division of
Rheumatology, New York University School of Medicine and
NYU Hospital for Joint Diseases, New York, NY 10003, USA.
10Department of Cell Biology, New York University School of
Medicine, New York, NY 10016, USA.
*These authors contributed equally to this work.
†Present address: Chongqing Medical University, Chongqing,
‡Present address: SRI International, 333 Ravenswood Avenue, Menlo Park, CA 94025, USA.
§To whom correspondence should be addressed. E-mail:
binding to TNFR1 and TNFR2 (Fig. 1, C and D),
which suggested that PGRN may act as a physiological antagonist of TNFa signaling. Indeed,
PGRN potently inhibits TNF-mediated neutrophil activation (8) and cartilage degradation (10).
We observed a significant increase in TNFa-
stimulated hydrogen peroxide in neutrophils and
nitric oxide in bone marrow–derived macrophages
(BMDMs) from PGRN-deficient mice (Fig. 1, E
and F). We previously reported that TNFa
induces the degradation of COMP (21), a prominent noncollagenous component of cartilage that
helps stabilize the cartilage matrix (22, 23) and is
heavily degraded in both osteoarthritis and rheumatoid arthritis (21, 24, 25). Using the same
model system, we observed that deletion of PGRN
results in a marked increase in TNFa-induced
COMP degradation (fig. S2).
To determine whether PGRN affects TNFa
signaling in human cells, we next examined whether treatment of human regulatory T cells [Tregs;
phenotypically TNFR2+ TNFR1− (26 )] with PGRN
may protect Treg cells from negative regulation
by TNFa (26, 27). PGRN protected Tregs from
negative regulation by TNFa (fig. S3) and promoted the differentiation of Tregs from naïve T
cells (fig. S4). Furthermore, TNFa up-regulated,
whereas PGRN down-regulated, interferon-g
(IFN-g) secretion in effector T cells (Teff) (fig.
S5). TNFR1 blocking antibodies largely inhibited
TNFa-induced up-regulation of IFN-g secretion,
but did not affect PGRN-mediated suppression;
in contrast, TNFR2 blocking antibodies abolished
PGRN-mediated down-regulation of IFNg
production (fig. S5). These data indicate that the regulation of TNFa and PGRN on Teff cells primarily
depends on TNFR1 and TNFR2, respectively.
To examine the role of endogenous PGRN
during inflammation in vivo, we investigated the
clinical and histopathological features of PGRN-deficient C57BL/6 mice (Grn−/−) in a mouse
model of collagen-induced arthritis (CIA) (28, 29),
which shares both immunological and pathological features with human rheumatoid arthritis.
Grn−/− mice developed more severe inflammatory arthritis and increased bone and joint destruction as compared with their control littermates
(Fig. 2, A and B). We also observed a significant
increase in the arthritis severity score (Fig. 2C), a
reduced time to disease onset, and a greater incidence of arthritis in Grn−/− mice compared to
control mice (Fig. 2D). Histological and quantitative analysis of whole ankle joints demonstrated
a significant increase in synovitis, pannus formation,
and destruction of bone and cartilage in Grn−/−
mice, compared with controls (Fig. 2E). Other
hallmarks of arthritis, such as loss of matrix staining in the articular cartilage and an increase in
bone-resorbing osteoclasts, were exacerbated in
Grn−/− mice (fig. S6, A and B).
To determine whether the inflammatory arthritis of collagen II–challenged PGRN-deficient
mice can be neutralized by recombinant PGRN,
we administered rhPGRN to these PGRN-deficient
mice for 11 weeks [supporting online materials
(SOM) Materials and Methods]. rhPGRN completely blocked disease progression (Fig. 2F). No
visible symptoms of CIA were observed in any
individual mouse, which was manifested by both
a 0% incidence and an arthritis score of “0” in
PGRN-deficient mice treated with rhPGRN (Fig.
2, G and H). rhPGRN also significantly inhibited
synovitis, pannus formation, tissue destruction
(fig. S6C), and the loss of cartilage matrix (fig.
S6D). Notably, the number of osteoclasts was
reduced in PGRN-deficient mice treated with
rhPGRN when compared to untreated PGRN-deficient mice (fig. S6E). Collectively, these data
suggest that the loss of PGRN expression in vivo
results in enhanced susceptibility to collagen-induced arthritis, which can be entirely reversed
by the administration of recombinant PGRN.
To determine whether the anti-inflammatory
actions of PGRN occur through the suppression
of TNFa signaling in vivo, we deleted the gene
that encodes PGRN in mice that express a human
TNFa transgene (TNF-Tg) (30). TNF-Tg mice
develop an inflammatory arthritis phenotype spontaneously (30, 31). We generated TNF-Tg /Grn+/−
and TNF-Tg/Grn−/− mice and found that the deletion of PGRN hastened the onset of arthritis
and resulted in a worse clinical score in a gene
dosage-dependent manner (Fig. 3, A and B).
Twelve-week-old TNF-Tg/Grn−/− and TNF-Tg/Grn+/− mice developed severe swelling and
joint deformation (fig. S7A), significantly increased synovitis, pannus formation, destruction
of the wrist joints (fig. S7B), and loss of cartilage matrix (fig. S7C). Overexpression of TNFa
resulted in prominent calvarial osteoclast activity of TNF-Tg mice, and deletion of PGRN
further enhanced this activity (fig. S7D). These
results suggest that PGRN may also be a negative regulator of TNFa-induced osteoclastogene-sis and a mediator of bone integrity during the
Next, we sought to examine the effects of applying rhPGRN to TNF-Tg mice. We administered rhPGRN (SOM Materials and Methods)
to TNF-Tg mice with established mild arthritis.
Treatment with rhPGRN resulted in the elimination of any visual signs of arthritis (Fig. 3C) and a
reduced arthritis severity score (Fig. 3D). To confirm that these effects were due to the inhibitory
effects of PGRN, we discontinued rhPGRN administration and continued to evaluate the TNF-Tg mice for signs of arthritis. At 7 days after the
cessation of rhPGRN treatment, signs of arthritis
began to develop (Fig. 3D). In contrast, application of rhPGRN to TNF-Tg mice in the phosphate-buffered saline (PBS)–treated group resulted in a
marked reduction of severe arthritis signs. Taken
together, these data suggest that PGRN may exert
its anti-inflammatory effects through inhibition
of TNF–TNFR signaling in vivo.
To identify the domains of PGRN required
for its interaction with TNF receptors, we constructed cDNA segments encoding a series of
PGRN mutants and analyzed their interactions
with TNFR2 using Y2H assays. No single granulin