CIRP Induces Neutrophil Reverse Transendothelial Migration in Sepsis

Abstract

Extracellular cold-inducible RNA-binding protein (CIRP) exaggerates inflammation in sepsis. Neutrophil reverse transendothelial migration (rTEM) allows neutrophils to migrate from tissues into the circulation. The phenotype of neutrophils following reverse migration is CD54CXCR1. We hypothesize that CIRP induces neutrophil rTEM in sepsis. Sepsis was induced in male C57BL/6 mice by cecal ligation and puncture (CLP), and at 5, 10, or 20 h after CLP, the frequencies of reversely migrated (RM) neutrophils were assessed in the blood by flow cytometry. Since 20 h of CLP showed highest increase in the frequency of RM neutrophils, we further assessed RM neutrophils in the blood of WT and CIRP mice at this time point. The effect of CIRP on neutrophil rTEM was determined by injecting mice with recombinant mouse CIRP (rmCIRP) intratracheally (i.t.) and assessed the frequencies of RM neutrophils. The expression of neutrophil elastase (NE) and junctional adhesion molecule-C (JAM-C) in the lungs were measured by Western blot. The mean frequency of RM neutrophils in sham mice was 0.4%, while the frequencies were significantly increased to 1%, 3%, and 7% at 5, 10, and 20 h of CLP, respectively. The mean frequency of RM neutrophils in the blood of CIRP mice was significantly lower than that of WT mice at 20 h of CLP. The RM neutrophils in the blood was significantly increased after administration of rmCIRP i.t. into mice in a time- and dose-dependent manners. NE expression was upregulated while JAM-C expression was downregulated in the lungs after CLP or rmCIRP administration. For the first time, we have showed that CIRP induces neutrophil rTEM in sepsis by increasing NE and decreasing JAM-C.

Extracellular CIRP (eCIRP) and inflammation

“…In the murine cremaster muscle ischemia and reperfusion injury model, lipid chemoattractant leukotriene B4 (LTB4) up‐regulated in the inflamed tissues induced neutrophil production of the proteolytic enzyme neutrophil elastase (NE), which disrupted JAM‐C at EC junction, thus facilitating neutrophil rTEM . This observation is in line with a recent study that showed increased NE and decreased JAM‐C in the lungs of mice with sepsis or injected with recombinant CIRP . In vitro neutrophil stimulation with recombinant CIRP significantly induced NE in cell culture supernatants, and the supernatant of eCIRP‐stimulated neutrophils dramatically decreased EC surface expression of JAM‐C, but not when NE blocking antibodies were added to the conditioned media .…”

“…A recent study revealed that eCIRP also induces neutrophil rTEM. Septic WT mice had significantly more RM neutrophils in the circulation than septic CIRP −/− mice . Additionally, circulating RM neutrophils increased after intratracheal administration of recombinant CIRP to healthy mice, demonstrating that eCIRP is sufficient to induce neutrophil rTEM …”

“…eCIRP‐induced lung injury is characterized by increased infiltration of neutrophils into the lungs, increased vascular endothelial permeability, and activation of vascular endothelial cells . The effects of eCIRP and its mechanism of action have already been studied in a number of immune cells, including macrophages, lymphocytes, neutrophils, and dendritic cells . eCIRP's effects have also been studied in endothelial and epithelial cells (Fig.…”

“…Although neutrophils have traditionally been considered to be a relatively homogeneous population, emerging evidence indicates the existence of phenotypic and functional heterogeneity among neutrophils . eCIRP induces deleterious subsets of neutrophils during sepsis . The effect of eCIRP on the phenotype and function of neutrophils is described below.…”

“…eCIRP then acts as a DAMP increasing macrophage production of proinflammatory cytokines . Since its original discovery as a DAMP, eCIRP's emerging proinflammatory role has been progressively extended to include additional target cells, such as neutrophils, lymphocytes, epithelial cells, and endothelial cells . eCIRP promotes inflammation by binding to the TLR4‐MD2 receptor complex, leading to the activation and nuclear translocation of the transcription factor NF‐κB .…”