Key Points Plg and Pla induce macrophage reprogramming and promote resolution of acute inflammation. Plg and Pla enhance the efferocytic capacity of macrophages and override the prosurvival effect of LPS on neutrophils via annexin A1.

The plasminogen (Plg)/plasmin (Pla) system is associated with a variety of biological activities beyond the classical dissolution of fibrin clots, including cell migration, tissue repair, and inflammation. Although the capacity of Plg/Pla to induce cell migration is well defined, the mechanism underlying this process in vivo is elusive. In this study, we show that Pla induces in vitro migration of murine fibroblasts and macrophages (RAW 264.7) dependent on the MEK/ERK pathway and by requiring its proteolytic activity and lysine binding sites. Plasmin injection into the pleural cavity of BALB/c mice induced a time-dependent influx of mononuclear cells that was associated with augmented ERK1/2 and IκB-α phosphorylation and increased levels of CCL2 and IL-6 in pleural exudates. The inhibition of protease activity by using a serine protease inhibitor leupeptin or two structurally different protease-activated receptor-1 antagonists (SCH79797 and RWJ56110) abolished Pla-induced mononuclear recruitment and ERK1/2 and IκB-α phosphorylation. Interestingly, inhibition of the MEK/ERK pathway abolished Pla-induced CCL2 upregulation and mononuclear cell influx. In agreement with a requirement for the CCL2/CCR2 axis to Pla-induced cell migration, the use of a CCR2 antagonist (RS504393) prevented the Plg/Pla-induced recruitment of mononuclear cells to the pleural cavity and migration of macrophages at transwell plates. Therefore, Pla-induced mononuclear cell recruitment in vivo was dependent on protease-activated receptor-1 activation of the MEK/ERK/NF-κB pathway, which led to the release of CCL2 and activation of CCR2.

Annexin A1 (AnxA1) is a glucocorticoid-regulated protein known for its anti-inflammatory and pro-resolving effects. We have shown previously that the cAMP-enhancing compounds rolipram (ROL; a PDE4 inhibitor) and BtcAMP (a cAMP mimetic) drive caspase-dependent resolution of neutrophilic inflammation. In this follow-up study, we investigated whether AnxA1 could be involved in the pro-resolving properties of these compounds using a model of LPS-induced inflammation in BALB/c mice. The treatment with ROL or BtcAMP at the peak of inflammation shortened resolution intervals, improved resolution indices, and increased AnxA1 expression. studies showed that ROL and BtcAMP induced AnxA1 expression and phosphorylation, and this effect was prevented by PKA inhibitors, suggesting the involvement of PKA in ROL-induced AnxA1 expression. Akin to these findings, H89 prevented ROL- and BtcAMP-induced resolution of inflammation, and it was associated with decreased levels of intact AnxA1. Moreover, two different strategies to block the AnxA1 pathway (by using -Boc-Met-Leu-Phe, a nonselective AnxA1 receptor antagonist, or by using an anti-AnxA1 neutralizing antiserum) prevented ROL- and BtcAMP-induced resolution and neutrophil apoptosis. Likewise, the ability of ROL or BtcAMP to induce neutrophil apoptosis was impaired in AnxA-knock-out mice. Finally, in settings, ROL and BtcAMP overrode the survival-inducing effect of LPS in human neutrophils in an AnxA1-dependent manner. Our results show that AnxA1 is at least one of the endogenous determinants mediating the pro-resolving properties of cAMP-elevating agents and cAMP-mimetic drugs.

No abstract