CEP: a new β₂ integrin ligand in inflamed tissue

In this issue of Blood, Yakubenko et al report on 2-(ω-carboxyethyl)pyrrolle (CEP), a lipid oxidation product generated by extravasated neutrophils within inflamed tissue, where CEP seems to serve as a guidance cue for infiltrating macrophages during the second wave of innate immune cell invasion by interacting with β₂ integrins α_Mβ₂ (Mac-1) and α_Dβ₂.¹ 

Leukocyte recruitment during sterile inflammation follows a well-defined cascade of adhesion and activation events that enable circulating leukocytes to leave the intravascular compartment and extravasate into tissue to reach the location of sterile injury.²  During recruitment, neutrophils are the first leukocyte subset to arrive at the site of inflammation. This first wave of infiltrating leukocytes follows a second wave of monocytes/macrophages that further execute immune defense functions and eventually induce resolution of inflammation. Although the basic mechanisms of innate immune cell recruitment have been intensively investigated, the crosstalk between extravasated neutrophils and macrophages within inflamed tissue is still not entirely clear. Yakubenko and colleagues introduce CEP as a new player that regulates neutrophil-monocyte crosstalk and trafficking within inflamed tissue.

CEP is generated as a lipid oxidation product of docosahexaenoic acid (an n-3 polyunsaturated fatty acid) and is found under in vivo conditions as an adduct of proteins and phospholipids.³  Using CEP-modified proteins, anti-CEP antibodies were raised and were critical for the subsequent analysis of CEP expression and the discovery of CEP-dependent biological functions.⁴  Several studies closely linked expression of CEP to inflamed tissue and identified CEP as a ligand for the scavenger receptor CD36 as well as for TLR2 and TLR9. In fact, binding of CEP to endothelial-derived TLR2 exerted a pronounced induction of VEGF-independent angiogenesis in a mouse wound healing model.⁵  Furthermore, through interacting with TLR9 on platelets and TLR2 on macrophages, CEP was also identified as a prothrombotic and a proinflammatory factor, respectively.^6,7 

In their article, Yakubenko et al report on the role of CEP in leukocyte trafficking during sterile inflammation. They showed in the inflamed peritoneal cavity of mice (after stimulation of the peritoneal cavity with thioglycollate over 72 hours) a strong upregulation of CEP expression compared with that in untreated tissue. Using CEP-blocking antibodies, they demonstrated that early neutrophil extravasation was not affected by blocking CEP, but subsequent macrophage infiltration was significantly reduced. From these results, the authors concluded that infiltrated neutrophils might be involved in the generation of CEP within the peritoneal cavity, which in turn might help support the second wave of cell infiltration by circulating monocytes as well as macrophages from the local environment. To demonstrate that neutrophils are indeed responsible for the generation of CEP in inflamed tissue, the authors set up a 3-dimensional (3D) fibrin gel assay in the Boyden chamber. Using this setup, they found that fMLF-dependent neutrophil migration in the gel led to CEP generation within the gel. Interestingly, CEP production could be prevented in part by blocking neutrophil-derived myeloperoxidase (MPO), which suggests that MPO is contributing to the generation of CEP. The role of neutrophil-derived MPO in CEP generation was further confirmed in a mouse peritonitis model in which neutrophil but not monocyte/macrophage depletion led to a strong reduction in peritoneal CEP expression. Furthermore, CEP expression was strongly reduced in a wound-healing assay in MPO-deficient mice compared with wild-type mice. Finally, coincubation of fibrinogen, DHA, and MPO in vitro generated CEP adducts on fibrinogen. Having elaborated the molecular mechanisms of generating CEP in the inflamed peritoneal cavity, the authors then investigated how CEP regulates monocyte/macrophage recruitment. Although CEP did not exert any chemoattractant or integrin-activating function, immobilized CEP was shown to bind to macrophage-expressed β₂ integrins in a macrophage static adhesion assay. Using HEK293 cells transfected with different β₂ integrins, the authors found that α_Mβ₂ (Mac-1) and α_Dβ₂ integrins bound CEP, but α_Lβ₂ (LFA-1) did not interact with CEP. These results were further confirmed in biochemical assays using α_L-, α_M-, and α_D-I-domain-containing integrin fragments. Finally, the authors again used the Boyden chamber and a 3D fibrin gel matrix to investigate migration of isolated peritoneal macrophages in the presence or absence of CEP. Intriguingly, they found that the presence of CEP strongly supported β₂ integrin-dependent migration of macrophages under 3D conditions, but neutrophils did not change their migration behavior in the presence of CEP.

In summary, Yakubenko and colleagues have revealed a new and interesting mechanism of indirect crosstalk between neutrophils and macrophages during sterile inflammation. After neutrophils have arrived in inflamed tissue, they release MPO into the local environment, which leads to the formation of CEP-decorated extracellular matrix components that serve as guidance cues for macrophage migration through binding to macrophage-expressed α_Mβ₂ and α_Dβ₂ integrins. Furthermore, interactions between CEP and β₂ integrin could also trigger additional macrophage recruitment, stimulated by CEP-mediated outside-in signaling events. Taking into consideration the differential modulation of macrophage subtypes by CEP, as reported recently,^7,8  inflamed tissue-expressed CEP might turn out to play an important modulating role in fine-tuning the transition from the initial proinflammatory phase to the subsequent resolution phase of inflammation.