Fig. 1. PGRN directly binds to TNFR and antagonizes TNFa actions. (A) PGRN interacts with TNFR2 in
chondrocytes (Co-IP assay). The cell lysates of human
chondrocytes were incubated with anti-PGRN, anti-
TNFR2, or control immunoglobulin G (IgG) antibodies, and bound protein was examined by Western
blotting (WB) with the corresponding antibodies, as
indicated. (B) FastStep Kinetic Assay for binding of
PGRN and TNFa to TNFR1 and TNFR2. Samples were
injected by FastStep injection, and dissociation of
analyte-ligand complexes was monitored. Dissociation constant (KD) for each interaction is indicated.
(C) PGRN inhibits the binding of TNFa to TNFR1 and
TNFR2 (solid-phase binding). Microtiter plate coated
with TNFa was incubated with TNFR1 or TNFR2 in the
presence of various amounts of rhPGRN, and the
bound TNFR to TNFa was detected by corresponding
antibodies. Values are means T SD (D) Flow cytom-etry analysis of Raw264.7 cells after staining with
50 ng of biotinylated human TNFa (Bt-TNFa) and
different doses of rhPGRN pretreatment. (E) PGRN
deletion potentiates TNFa-induced H2O2
production (neutrophil activation). Wild-type (WT) or
PGRN-deficient (KO) neutrophils were treated with
TNFa, and H2O2 production was measured. Values
are means T SD. P < 0.01; n = 4 assays. (F) PGRN
deletion potentiates TNFa-induced nitrite production in BMDMs. M-CSF pretreated WT or PGRN-deficient (KO) BMDMs were incubated with TNFa,
and the supernatants were tested for NO production.
Values are means T SD. P <0.001; n = 4 assays.
Fig. 2. PGRN-deficient
mice are highly susceptible to collagen-induced
arthritis, and administration of PGRN reverses the
severe inflammatory arthritis seen in collagen-challenged PGRN-deficient
mice. (A) Paws of wild-type (WT) and Grn−/− (KO)
mice derived from C57BL/6
strain (n = 10 mice per
group) were immunized
with collagen II for 15
weeks. (B) Radiography
of ankle joints of WT and
KO collagen II–immunized
mice. Arrow indicates areas
of severe joint destruction in PGRN-deficient
CIA mice. (C) Clinical arthritis scores in WT and
KO mice with CIA. The data are presented as the
mean clinical score T SEM.
*P < 0.05, P < 0.01 versus the control WT group.
(D) Incidence of arthritis
in the indicated groups.
(E) Hematoxylin and eosin (H&E)–stained sections and evaluation of
synovitis, pannus, and erosion of ankle joints in WT and KO mice with CIA 15
weeks after primary immunization. Scale bar, 200 mm. Values are means T SD.
*P < 0.05, P < 0.01 versus the control WT group. (F) Paws of KO mice treated
with PBS or rhPGRN from 4 to 15 weeks after collagen II immunization. (G)
Clinical arthritis scores in KO mice with CIA treated with PBS or rhPGRN (n =
10 mice per group). Data are presented as the mean clinical score T SEM. P <
0.05, P < 0.001 versus the control PBS group. (H) Incidence of arthritis in
each experimental group.