MCP-1-deficient mice show reduced neuroinflammatory responses and increased peripheral inflammatory responses to peripheral endotoxin insult

Abstract

Background: An endotoxin insult mimics a severe peripheral infection and recent evidence suggests that a single exposure can cause long-term cognitive deficits. A peripheral injection of LPS results in production of pro-inflammatory cytokines, such as IL-1beta and TNF-alpha, in the brain and periphery and these cytokines mediate many effects of the acute phase response including activation of the HPA axis. The chemokine MCP-1 is highly expressed during endotoxemia and although much is known about the importance of MCP-1 in peripheral inflammatory responses to LPS, information about MCP-1 and CNS responses to peripheral LPS is lacking.

Methods: C57Bl/6 mice were administered LPS by intraperitoneal (i.p.) injection, serum and brains were collected at several time points, and the time course of MCP-1 protein up-regulation was measured. To examine the role of MCP-1 in activation of the brain during acute systemic inflammation, we injected MCP-1 knockout (MCP-1-/-) or control C57Bl/6 (MCP-1+/+) mice with LPS i.p. and measured the levels of selected cytokines and chemokines in serum and brain extracts 6 hours later. Activated microglia were examined by CD45 immunohistochemistry, and serum corticosterone and ACTH levels were measured by enzyme immunoassay.

Results: We report that LPS injection induces a robust increase in MCP-1 protein levels in serum and brain, with peak brain levels reached at 6 hrs after LPS administration. MCP-1-/- mice injected with LPS showed higher levels of serum IL-1beta and TNF-alpha compared to LPS-treated MCP-1+/+ mice. In contrast, these MCP-1-/- mice showed significantly lower inductions of brain pro-inflammatory cytokines and chemokines, fewer activated microglia, and a reduction in serum corticosterone levels.

Conclusion: MCP-1-/- mice have decreased brain inflammation after a peripheral LPS insult, despite an exaggerated peripheral response. These data demonstrate an important role for MCP-1 in regulation of brain inflammation after peripheral endotoxemia.

Figure 1

MCP-1 is increased in a time-dependent manner in serum and brain after LPS treatment. C57Bl/6 mice were injected i.p. with LPS, and serum and brain frontal cortex were collected at 1.5, 3, 6, 9, 12, 18, and 24 hours after LPS treatment. The 0 hr time point represents saline-injected controls. MCP-1 protein levels were measured in serum (A) and brain (B) as described in Methods. Values are represented as mean ± SEM of duplicate determinations per sample and are pooled from two independent experiments, n = 2–9 mice per time point. * represents significant difference from 0 hr control, p < 0.05.

Figure 2

Pro-inflammatory cytokines IL-1β and TNF-α are increased in serum from MCP-1^-/- mice after LPS treatment. MCP-1^+/+ or MCP-1^-/- mice were injected i.p. with LPS or saline, and serum was collected 6 hours later. IL-1β (A) and TNF-α (B) levels were measured by ELISA as described in Methods. Values are represented as mean ± SEM of duplicate determinations and are pooled from two independent experiments, n = 5–11 mice per group. LPS-injected MCP-1^+/+ mice mean value is 577.6 pg/ml (IL-1β) and 573.4 pg/ml (TNF-α). ** represents significant difference between LPS-treated MCP-1^+/+ mice and LPS-treated MCP-1^-/- mice, p < 0.001; # represents significant difference between saline-treated MCP-1^+/+ mice and LPS-treated MCP-1^+/+ mice, p < 0.05; and ≠ represents significant difference between saline-treated MCP-1^-/- mice and LPS-treated MCP-1^-/- mice, p < 0.001.

Figure 3

Pro-inflammatory cytokines IL-1β and TNF-α are decreased in brains of MCP-1^-/- mice after LPS treatment. MCP-1^+/+ or MCP-1^-/- mice were injected i.p. with LPS or saline, and brains were removed and dissected 6 hours later. IL-1β (A) and TNF-α(B) levels in entorhinal cortex, frontal cortex, and hippocampus supernatants were measured by MSD as described in Methods. Values are represented as mean ± SEM of duplicate determinations and are pooled from two independent experiments, n = 5–11 mice per group. ** represents significant difference between LPS-treated MCP-1^+/+ mice and LPS-treated MCP-1^-/- mice, p < 0.001; # represents significant difference between saline-treated MCP-1^+/+ mice and LPS-treated MCP-1^+/+ mice, p < 0.001; and ≠ represents significant difference between saline-treated MCP-1^-/- mice and LPS-treated MCP-1^-/- mice, p < 0.05.

Figure 4

Brain CD45 immunoreactivity is reduced in MCP-1^-/- mice after LPS treatment. MCP-1^-/- and MCP-1^+/+ mice were injected i.p. with LPS or saline, and brains were removed 6 hours later. Brain sections (30 μm) were stained with anti-CD45 as described in Methods. Fields of view were randomly generated using StereoInvestigator for 2–3 sections per mouse and photographed at 20× magnification using a Nikon Eclipse 80 i microscope. Arrow shows example of morphologically identified leukocytes. Immunoreactive density was measured on at least 7 pictures from both the hippocampus (A) or cortex (B) per mouse and analyzed by Image J. Values for the hippocampus (C) and cortex (D) are represented as mean ± SEM, n = 5 mice per group. ** represents significant difference between LPS-treated MCP-1^+/+ mice and LPS-treated MCP-1^-/- mice, p < 0.001; # represents significant difference between saline-treated MCP-1^+/+ mice and LPS-treated MCP-1^+/+ mice, p < 0.001; and ≠ represents significant difference between saline-treated MCP-1^-/- mice and LPS-treated MCP-1^-/- mice, p < 0.01. Scale bar = 50 μm.

Figure 5

CC Chemokines are decreased in brains of MCP-1^-/- mice after LPS treatment. MCP-1^-/- and MCP-1^+/+ mice were injected i.p. with LPS or saline (MCP-1^+/+ mice only), and brains were removed and dissected 6 hours later. Entorhinal cortex was homogenized and supernatant assayed by Rules-Based Medicine for MIP-1α (A), MIP-1β (B), MIP-1γ (C), MCP-3 (D), eotaxin (E), and MIP-3β (F). Values are represented as mean ± SEM, n = 4–6 mice per group. ** or * represents significant difference between LPS-treated MCP-1^+/+ mice and LPS-treated MCP-1^-/- mice, **p < 0.001, *p < 0.01; # represents significant difference between saline-treated MCP-1^+/+ mice and LPS-treated MCP-1^+/+ mice, p < 0.001.

Figure 6

Serum corticosterone levels are reduced in MCP-1^-/- mice after LPS treatment. MCP-1^-/- and MCP-1^+/+ mice were injected i.p. with LPS or saline, and serum was collected 6 hours later. Corticosterone (A) or ACTH (B) levels were measured by EIA as described in Methods. Values are represented as mean ± SEM of singlet determinations and are pooled from two independent experiments, n = 4–11 mice per group. * represents significant difference between LPS-treated MCP-1^+/+ mice and LPS-treated MCP-1^-/- mice, p < 0.01; # represents significant difference between saline-treated MCP-1^+/+ mice and LPS-treated MCP-1^+/+ mice, p < 0.001; and ≠ represents significant difference between saline-treated MCP-1^-/- mice and LPS-treated MCP-1^-/- mice, p < 0.001.