CCL2 modulates cytokine production in cultured mouse astrocytes

Abstract

BackgroundThe 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.MethodsPrimary 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.ResultsLPS (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.ConclusionsOur 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.

Generation of human pluripotent stem cell reporter lines for the isolation of and reporting on astrocytes generated from ventral midbrain and ventral spinal cord neural progenitors

“…The only drastic decline observed between treatments concerned CCL2 (also known as MCP-1), whose secretion was decreased following IL-1 treatment in both midbrain and spinal cord cultures ( Figure 6A). This finding is in line with recent work showing a down regulation of this protein in cultures composed of primary inflammatory human astrocytes (Choi et al, 2014), and whose absence exacerbates the release of several pro-inflammatory cytokines including IL-6, G-CSF, IL-1 and CXCL1, indicating a negative regulatory role of CCL2 in neuroinflammation (Semple et al, 2010). In general, no dramatic differences in cytokine and chemokine secretions were observed between midbrain and spinal cord cultures treated with IL-1.…”

CXCR3 promotes plaque formation and behavioral deficits in an Alzheimer’s disease model

“…Previous studies using murine AD models have demonstrated that chemokine receptor systems such as CCR5 (36), CCR2 (37)(38)(39), and CX3CR1 (40)(41)(42)(43) can modulate the disease course by influencing microglial function, accumulation, and clustering (37,(40)(41)(42). In addition, a positive correlation between CXCL10 concentrations in the cerebrospinal fluid and cognitive impairment in AD patients has been demonstrated (44,45).…”

CCR2 Deficiency Impairs Macrophage Infiltration and Improves Cognitive Function after Traumatic Brain Injury

“…31 Although the mechanism has yet to be defined, inhibition of CCL2 also has been observed to promote pro-inflammatory cytokine production from astrocytes. 29,32 Taken together, these data suggest that to maximize blockade of macrophage infiltration and inflammation, it may be advantageous to target the receptor, CCR2, for which drugs have recently been generated. 33 We therefore investigated TBI outcomes in CCR2-deficient (Ccr2 -/ -) mice, using a controlled cortical impact model of experimental TBI.…”