Background/Purpose:

The mechanisms that contribute to the persistent activation of macrophages in rheumatoid arthritis (RA) are incompletely understood.  Toll-like receptors (TLRs) have been implicated in the regulation of innate immunity and inflammation, and to play an important role in the pathogenesis of RA.  We have recently identified that gp96 as an endogenous TLR2 ligand that contributes to the persistent inflammation of RA.  Employing RA synovial fluid (SF) macrophages, we demonstrated synergistic activation between the microbial TLR2 ligand peptidoglycan (PGN) and the TLR4 ligand lipopolysaccharide (LPS).  This study was performed to determine the synergistic effect of gp96 in macrophages activation and in the K/BxN serum transfer model of arthritis. 

Methods:

Recombinant N’ terminal fragment (aa 22-337) of gp96 was expressed from E.coli and purified with extensive endotoxin removing procedures (final product < 1EU/mg protein).  Suboptimal concentrations of LPS, Pam3CSK4, PGN, and RA SFs containing low concentrations of gp96 ( <300ng/ml) were employed individually or in combination to activate control or RA SF macrophages.  Except when LPS was added, cell activation was performed in media supplemented with endotoxin inhibitor Polymyxin-B.  Macrophage activation was determined by the expression of TNFa mRNA employing quantitative RT-PCR or by examining culture supernatants for TNFa and IL6 by ELISA.  Arthritis was induced employing the K/BxN serum transfer model in wild type C57Bl/6 mice.  Gp96 (20 mg/in 5ml of PBS) was injected intraarticularly into mice on days 3 and 10, with/without injection of K/BxN serum on day 0.  Heat inactivated gp96 (65^oc for 2 hour) severed as the negative control. Arthritis was evaluated by ankle swelling and clinical score from day 0 to 17 post-induction. 

Results:

Macrophage activation by low concentrations of gp96 (2.5-5 mg/ml) were synergistically enhanced by very low or trace amount of LPS, Pam3CSK4 and PGN.  Pre-incubation of RA SFs (10% in RPMI medium) with gp96 (1mg/ml) synergistically increased TNFa  expression in control and RA SF macrophages, compared with SF only or SF preincubated with heat inactivated gp96.  C57Bl/6 mice that received K/BxN serum plus gp96 intraarticularly developed more severe arthritis, compared with the group received K/BxN serum plus heat inactivated gp96.  Although gp96 or heat inactivated gp96 was administered at day 3 and boosted at day 10 post arthritis induction, the difference between these two groups was not observed until day 10 and afterward.  Gp96 or heat inactive gp96 ankle injection alone only caused mild ankle swelling at 24 hours that resolved in 2-3 days. 

Conclusion:

These observations suggest that gp96 synergizes with low levels of microbial TLR2 and TLR4 ligands in promoting of macrophage activation.  The intraarticular injection of gp96 exacerbated serum transfer arthritis. Gp96 may also interact with endogenous TLR ligands or other inflammatory molecules present in RA SF resulting in enhanced macrophage activation.  These observations suggest that gp96 does not act alone, but cooperates with other factors within the joint to promote inflammation and joint destruction.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/gp96-exacerbate-the-inflammation-of-rheumatoid-arthritis/