Evidence from several studies suggests a role for CXCR2 receptors in brain neutrophilia and evolution of infarct volume after ischemic stroke. Studies of the CXCR1/2 inhibitor reparixin showed that receptor blockade decreased infarct volume, reduced neutrophil infiltration, and improved long-term neurological outcome in rat models of transient and permanent ischemia [
2,
3]. In another study, the CXCR1/2 antagonist G31P significantly decreased infarction when administered after transient MCAO in rats [
4]. Further, CXCR2-binding pro-inflammatory chemokines such as CXCL1 and IL-8 are markedly increased in brain and cerebrospinal fluid in inflammatory neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, and stroke, and have been proposed to be mediators of neuronal death, primarily through pro-inflammatory signaling [
16‐
19]. Mounting evidence from in vitro studies, including the results presented in this study, suggest that ligand binding by neuronal CXCR2 receptors mediates neuronal cell death directly, in addition to promoting inflammation which may potentiate long-term neurological damage.
A recent study demonstrated that binding of a rodent IL-8 functional homolog, macrophage inflammatory protein-2 (MIP-2), by CXCR2 receptors induced motor neuron death in primary cultures [
20]. In another study, treatment of cultured neurons with IL-8 resulted in the induction of MMP-2 and MMP-9 expression and activities, upregulation of pro-apoptotic proteins, and expression of cyclin D1 [
21]. These findings suggest that inflammatory chemokines such as IL-8 and its rodent functional homologs may have direct pathogenic effects in central nervous system disorders independent of their roles in the induction of leukocyte migration and activation. Our in vitro results demonstrating the neurotoxicity of ac-PGP confirm the induction of apoptosis by prolonged activation of neuronal CXCR2 receptors. Neuronal apoptosis after exposure to ac-PGP involves cleavage of caspase-3 and activation of ERK1/2 MAP kinase. While activation of neuronal ERK1/2 by growth factors, ischemic preconditioning, and hypothermia may mediate neuroprotection, activation of ERK1/2 kinase by inflammatory factors in ischemic injury has been shown to promote neuronal apoptosis [
22]. Interestingly, activation of ERK1/2 was similarly observed in neutrophils upon exposure to ac-PGP, and an inhibitor of the ERK1/2 pathway attenuated the release of MMP-9 by neutrophils stimulated with ac-PGP [
12]. In stem cells, ac-PGP activates ERK1/2 MAP kinase and upregulates CXCR1/2 receptor expression [
23]. In another study, ac-PGP was shown to induce increased vascular permeability through CXCR2-mediated induction of signaling pathways in endothelial cells [
24]. Thus, in ischemia/reperfusion injury, we hypothesize that the production of ac-PGP in brain may promote inflammation and neurodegeneration through upregulation of CXCR1/2 receptors, increased permeability of the blood-brain barrier, recruitment and activation of leukocytes, and direct neuronal injury mediated through prolonged CXCR2 receptor activation.
The presence of ac-PGP in unused culture medium and significant production of ac-PGP during normal culturing of primary neurons may have important implications for neuronal viability and in vitro studies of neuronal responses to neurotoxic stressors, such as OGD. We hypothesize that ac-PGP in unused culture medium may be of FBS origin. A previous study demonstrated high levels of MMP-2 and MMP-9 in conditioned neuronal culture medium which were unchanged following OGD [
11]. Thus, ac-PGP is likely produced in neuronal cultures by the action of MMPs on collagens or other substrates, and the lack of a significant increase in ac-PGP in neuronal cultures after OGD is not surprising. The increase in ac-PGP in brain after stroke and lack of significant change in the level of the molecule after OGD in cultured neurons may be a fundamental difference between in vitro and in vivo stroke models.