Pain in these mice was similar to WT mice receiving i.pl. disease, but not once they are established. TNF-driven inflammatory pain and TNF-driven arthritic pain and disease are dependent on GM-CSF and mechanistically require the same downstream pathway involving GM-CSFCCL17 formation via JMJD3-regulated IRF4 production, indicating that GM-CSF and CCL17 can mediate some of the proinflammatory and algesic actions of TNF. Given we found that TNF appears important only early in arthritic pain and disease progression, targeting a downstream mediator, such as CCL17, which appears to act throughout the course of disease, could be effective at ameliorating chronic inflammatory conditions where TNF is implicated. mice are reported to show reduced pain compared with WT mice following i.a. zymosan injection (24). Using mice we firstly determined that i.pl. zymosan-induced pain, as measured by a change in weight distribution (using the well-validated incapacitance meter method (23, 25C27), was TNF dependent (Figure 1A), with some reduced footpad swelling seen at 5C6 hours after injection (Figure 1B). Zymosan injection (i.pl.) upregulated (also known as mRNA expression (by quantitative PCR [qPCR]) in WT mice (Figure 1C) (23). As some indication of the mechanism governing the TNF dependence, in the absence of TNF, the increased expression of and mRNA seen following zymosan injection in WT mice was reduced (Figure 1C). expression levels were lower in the saline-injected footpad of versus WT mice (Figure 1C); following i.pl. zymosan injection, there was an approximately 2-fold increase in expression in the footpads of both and WT mice (Figure 1C), thereby maintaining the difference in expression levels seen between the strains in the steady state. Open in a separate window Figure 1 TNF is required for zymosan-induced inflammatory pain.(ACC) WT and mice received an intraplantar (i.pl.) injection of zymosan, and (A) pain (incapacitance meter [ratio of weight bearing on injected relative to noninjected hindlimb]; a value 100 indicates pain), (B) swelling, and (C) footpad mRNA expression (6 hours zymosan or saline) were measured (= 6C10 mice/group). Results are shown as mean SEM. values were obtained using a 2-way ANOVA. * 0.05, **** 0.0001, WT versus mice. mice also developed significantly less zymosan-induced arthritis (ZIA) pain and disease (Figure 2A) and the increased mRNA expression in the arthritic joints was also significantly reduced in the absence of TNF (Figure 2B), noting that, unlike for the footpad, mRNA expression could not be detected in saline-injected joints from either WT or mice and that there was no difference in mRNA expression in the saline-injected joints from versus WT mice. Interestingly, prophylactic (Figure 3A), but not therapeutic (Figure 3B), treatment with anti-TNF mAb was able to ameliorate pain and disease (histology), whereas both prophylactic (Figure 3A) and therapeutic (Figure 3B) treatment with antiCGM-CSF mAb was able to do so. Therapeutic treatment with anti-CCL17 mAb was also able to ameliorate pain and disease (Figure 3B), paralleling the prior findings seen using mice (23). Open in a separate window Figure 2 TNF is required for zymosan-induced arthritic pain and optimal disease.(A and B) WT and mice received an intra-articular (i.a.) injection of zymosan; (A) pain and arthritis (histology, day 7) and (B) joint mRNA expression (day 7 zymosan or saline) were measured (= 5C10 mice/group). For histology images, original magnification 60. (+)-Apogossypol Results are shown as mean SEM. values were obtained using a 2-way ANOVA test for pain (weight distribution) readings (A) and gene expression (B), and Mann-Whitney test (+)-Apogossypol for histology (+)-Apogossypol (A). ** 0.01, **** 0.0001, WT versus mice. Open in a separate window Figure 3 TNF is not required for the maintenance of zymosan-induced arthritic pain and disease.(A and B) WT mice received an intra-articular (i.a.) injection of zymosan and anti-TNF mAb, antiCgranulocyte macrophage-colony stimulating factor (antiCGM-CSF) mAb, IgG1 or IgG2a isotype control mAbs (150 g i.p.), either Rabbit polyclonal to ZNF268 (A) prophylactically (on days C2 and 0) (= 4C5 mice/group) or (B) therapeutically (day 1) (= 5C10 mice/group), and pain and arthritis (histology, day 7) were measured. Mice were also treated (B) therapeutically with anti-CCL17 mAb (150 g i.p.) (day 1) (= 5 mice/group). For histology images, original magnification 60. Results are shown as mean SEM. values were obtained using a 2-way ANOVA test for pain (weight distribution) readings, and a 1-way ANOVA test for histology. * 0.05, *** 0.001, **** 0.0001, anti-TNF versus IgG1 isotype; # 0.05, ## 0.01, ### 0.001, #### 0.0001, antiCGM-CSF versus IgG2a isotype; 0.05, 0.01, anti-CCL17 versus IgG2a isotype. Thus, in the zymosan-induced models studied,.