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. 2012 Nov;64(11):3638-48.
doi: 10.1002/art.34610.

Glycoprotein 96 Perpetuates the Persistent Inflammation of Rheumatoid Arthritis

Free PMC article

Glycoprotein 96 Perpetuates the Persistent Inflammation of Rheumatoid Arthritis

Qi-Quan Huang et al. Arthritis Rheum. .
Free PMC article


Objective: The mechanisms that contribute to the persistent activation of macrophages in rheumatoid arthritis (RA) are incompletely understood. The aim of this study was to determine the contribution of endogenous gp96 in Toll-like receptor (TLR)-mediated macrophage activation in RA.

Methods: RA synovial fluid was used to activate macrophages and HEK-TLR-2 and HEK-TLR-4 cells. Neutralizing antibodies to TLR-2, TLR-4, and gp96 were used to inhibit activation. RA synovial fluid macrophages were isolated by CD14 negative selection. Cell activation was measured by the expression of tumor necrosis factor α (TNFα) or interleukin-8 messenger RNA. Arthritis was induced in mice by K/BxN serum transfer. The expression of gp96 was determined by immunoblot analysis, enzyme-linked immunosorbent assay, and immunohistochemistry. Arthritis was treated with neutralizing anti-gp96 antiserum or control serum.

Results: RA synovial fluid induced the activation of macrophages and HEK-TLR-2 and HEK-TLR-4 cells. RA synovial fluid-induced macrophage and HEK-TLR-2 activation was suppressed by neutralizing anti-gp96 antibodies only in the presence of high (>800 ng/ml) rather than low (<400 ng/ml) concentrations of gp96. Neutralization of RA synovial fluid macrophage cell surface gp96 inhibited the constitutive expression of TNFα. Supporting the role of gp96 in RA, joint tissue gp96 expression was induced in mice with the K/BxN serum-induced arthritis, and neutralizing antibodies to gp96 ameliorated joint inflammation, as determined by clinical and histologic examination.

Conclusion: These observations support the notion that gp96 plays a role as an endogenous TLR-2 ligand in RA and identify the TLR-2 pathway as a therapeutic target.

Conflict of interest statement

Conflict of Interest: No support from commercial sources, no conflict of interest


Figure 1
Figure 1. Macrophage activation by RA SFs is suppressed by neutralizing antibodies to TLR2, TLR4 and gp96
(A). Macrophages were incubated with RA SF (SF), selected for their ability to activate macrophages, diluted 1:4 in RPMI-1640 medium plus IgG control antibody for 4 hours. The mRNA expression of TNFα and IL-8 was determined employing qRT-PCR, presented as fold expression compared to medium alone (None). (B). Suppression of macrophage activation by neutralizing antibodies to TLR2 or TLR4 (10 µg/ml) is presented as the percentage of TNFα and IL-8 in the presence of anti-TLR antibodies compared to IgG controls (100%). n=7 RA SFs. (C). RA SFs were pre-incubated with control rabbit serum or rabbit anti-gp96 antiserum for 30 minutes prior to incubation with macrophages. The suppression of TNFα and IL-8 was presented as the percentage in the presence of anti-gp96 compared to control rabbit serum (100%). The fluids were grouped according to the gp96 concentration determined by ELISA as low gp96 (< 400 ng/ml) (mean 136±49, n=7) or high gp96 (> 800 ng/ml) (mean 1350±256, n=4). All experiments were repeated >2 times, and the mean of the results was employed for analysis. All values present are mean ± SEM. * represents p < 0.05 and *** p< 0.001 between the indicated groups. (D). Gp96 from the 4 RA SFs with >800ng/ml gp96 were immunoprecipitated by anti-gp96 antiserum or control rabbit serum. The immunoprecipitates were analyzed employing Immunoblot analysis probing with a second anti-gp96 antibody.
Figure 2
Figure 2. HEK-TLR2 activation by RA SF is suppressed by anti-gp96
HEK-TLR2 or HEK -TLR4 cells were incubated with RA SF diluted 1:2 for 20 hours and activation was determined by the expression of IL-8 employing qRT-PCR (left, panels A, B). To determine the ability of gp96 to activated the HEK cells, SFs with gp96 <400 and > 800 ng/ml were pre-incubated with control rabbit serum or rabbit anti-gp96 antiserum for 30 min prior to incubation with HEK-TLR2 or HEK-TLR4 cells (right, panels A, B). The percentage of inhibition (right panel) by anti-gp96 was determined by comparing with the normal rabbit serum control (100%). All values represent the mean ± SEM from 2–3 repeated experiments for each individual fluid. * p represents < 0.05.
Figure 3
Figure 3. Blocking cell surface gp96 on RA SF macrophages suppresses cell activation
(A). Representative flow cytometry histograms examining gp96 on the cell surface of mononuclear cells isolated from peripheral blood (PB) of healthy controls and patients with RA, and RA SF s (SF). Cells were incubated with a rat anti-gp96 monoclonal antibody or a rat monoclonal IgG control followed by PE-labeled anti-rat IgG. Monocytes/macrophages were defined employing FITC-labeled anti-CD14 (B). The intensity of gp96 expression (MFI) on the surface of CD14+ or CD14− mononuclear cells from peripheral blood of healthy controls (n = 7), patients with RA (n=10), and RA SF macrophages (MФs) (n = 9). The values represent the mean ± SEM. (C). CD14+ macrophages isolated from RA SF are spontaneously activated compared to control in vitro differentiated macrophage (n=4). Cell activation was determined by fold induction of TNFα mRNA by qRT-PCR, compared to control macrophages. (D). The activation of CD14+ macrophages from RA SF was suppressed by pre-incubation of cells with neutralizing anti-gp96 antiserum (n=4), as determined by qRT-PCR compared to cells incubated with control normal rabbit serum (100%). * represents p < 0.05, ** p < 0.01 and *** p < 0.001.
Figure 4
Figure 4. Synergistic activation of RA SF macrophages by TLR2 and TLR4 activation
In vitro differentiated control macrophages or CD14+ macrophages isolated from RA SFs were incubated with a suboptimal concentration of LPS (0.1ng/ml) or PGN (0.2 µg/ml) individually or in combination for 4 hours. The TNFα (panel A) and IL-6 (panel B) in culture medium was determined by ELISA. n= 5 control and 4 RA SF macrophages (mean ± SEM). The synergistic effect is presented as fold increase of the combination compared to the sum of the response to the individual ligands. The values represent the mean ± SEM. * represents p < 0.05 and ** p< 0.01 between the indicated groups.
Figure 5
Figure 5. Gp96 expression in the inflamed ankle of anti-GPI induced arthritis correlates with disease activity
(A) The serum transfer model of arthritis was induced by intraperitoneal injection of anti-GPI serum (150 µl) on days 0 and 2 and joint swelling measured over time as ankle thickness. Nineteen mice were injected with anti-GPI serum, and 3 mice were sacrificed at each time point, except day 19 when 4 mice were sacrificed. One ankle from each mouse was collected at the indicated time points and homogenized to determine the concentration of gp96 by ELISA and by immunoblot analysis (B). The other ankle was employed for immunohistochemistry employing anti-gp96 antibody or control IgG (B). Serial sections were also stained with hematoxylin and eosin (H&E). (C) The concentration of gp96 correlated with ankle thickness. (D) The concentration of IL-1β determined by ELISA was increased and correlated with the expression of gp96. The values in panels A, C and D represent the mean ± SEM. N.D, not determined.
Figure 6
Figure 6. Neutralizing anti-gp96 antiserum ameliorates the progression of anti-GPI induced arthritis
(A). The serum transfer model of arthritis was induced with a single intraperitoneal injection of 100 µl anti-GPI serum and the course of the arthritis was determined by the clinical activity score (maximum 12) and the swelling of the 2 hind ankles (ankle thickness). The arrows indicate the time of intraperitoneal treatment with either the control or the anti-gp96 antiserum. There were 12 mice in each group and the results represent the mean of two independent experiments. (B) Pathologic analysis of inflammation, pannus, and bone erosion on ankles collected at day12 post arthritis in 8 mice. The values represent the mean ± SEM. (C) Representative H&E staining sections from mice that received control rabbit serum or anti-gp96 antiserum, harvested at day 12 post-arthritis induction. The brackets identify articular (A) inflammation and extra-articular (EA) inflammation. * represents p<0.05 and ** p<0.01 between the two groups.

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