CCL2 modulates cytokine production in cultured mouse astrocytes

The chemokine CCL2 (also known as monocyte chemoattractant protein-1, or MCP-1) is upregulated in patients and rodent models of traumatic brain injury (TBI), contributing to post-traumatic neuroinflammation and degeneration by directing the infiltration of blood-derived macrophages into the injured brain. Our laboratory has previously reported that Ccl2-/- mice show reduced macrophage accumulation and tissue damage, corresponding to improved motor recovery, following experimental TBI. Surprisingly, Ccl2-deficient mice also exhibited delayed but exacerbated secretion of key proinflammatory cytokines in the injured cortex. Thus we sought to further characterise CCL2's potential ability to modulate immunoactivation of astrocytes in vitro.

Primary astrocytes were isolated from neonatal wild-type and Ccl2-deficient mice. Established astrocyte cultures were stimulated with various concentrations of lipopolysaccharide (LPS) and interleukin (IL)-1β for up to 24 hours. Separate experiments involved pre-incubation with mouse recombinant (r)CCL2 prior to IL-1β stimulation in wild-type cells. Following stimulation, cytokine secretion was measured in culture supernatant by immunoassays, whilst cytokine gene expression was quantified by real-time reverse transcriptase polymerase chain reaction.

LPS (0.1-100 μg/ml; 8 h) induced the significantly greater secretion of five key cytokines and chemokines in Ccl2-/- astrocytes compared to wild-type cells. Consistently, IL-6 mRNA levels were 2-fold higher in Ccl2-deficient cells. IL-1β (10 and 50 ng/ml; 2-24 h) also resulted in exacerbated IL-6 production from Ccl2-/- cultures. Despite this, treatment of wild-type cultures with rCCL2 alone (50-500 ng/ml) did not induce cytokine/chemokine production by astrocytes. However, pre-incubation of wild-type astrocytes with rCCL2 (250 ng/ml, 12 h) prior to stimulation with IL-1β (10 ng/ml, 8 h) significantly reduced IL-6 protein and gene expression.

Our data indicate that astrocytes are likely responsible for the exacerbated cytokine response seen in vivo post-injury in the absence of CCL2. Furthermore, evidence that CCL2 inhibits cytokine production by astrocytes following IL-1β stimulation, suggests a novel, immunomodulatory role for this chemokine in acute neuroinflammation. Further investigation is required to determine the physiological relevance of this phenomenon, which may have implications for therapeutics targeting CCL2-mediated leukocyte infiltration following TBI.