Monocyte chemoattractant protein-1-deficiency impairs the expression of IL-6, IL-1β and G-CSF after transient focal ischemia in mice

Abstract

Monocyte chemoattractant protein-1 (MCP-1), a chemokine secreted by neurons and astrocytes following stroke is known to aggravate ischemia-related damage. Previous studies revealed that MCP-1-deficient mice develop smaller infarcts and have an improved neurological outcome, whereas mice overexpressing MCP-1 show worsened brain damage and impaired neurological function. The aim of the present study was to elucidate the molecular background of the enhanced recovery in MCP-1-deficient mice after stroke. For this purpose, we (1) performed expression analyses on crucial post-stroke related inflammatory genes in MCP-1-deficient mice compared to wildtype controls, (2) analyzed a possible impact of MCP-1 on astrocyte activation (3) investigated the cellular origin of respective inflammatory cytokines and (4) analyzed the impact of MCP-1 secretion on the migration of both neutrophil granulocytes and T-cells. Here we report that MCP-1-deficiency leads to a shift towards a less inflammatory state following experimental occlusion of the middle cerebral artery including an impaired induction of interleukin-6, interleukin-1β and granulocyte-colony stimulating factor expression as well as a subsequent diminished influx of hematogenous cells. Additionally, MCP-1-deficient mice developed smaller infarcts 36 hours after experimental stroke. Investigations revealed no differences in transcription of tumor necrosis factor-α and astrogliosis 12 and 36 hours after onset of ischemia. These novel results help to understand post ischemic, inflammatory mechanisms and might give further arguments towards therapeutical interventions by modulation of MCP-1 expression in post stroke inflammation.

Figure 1. Laser capture microdissection was performed on PEN-membrane mounted and toluidine-stained brain sections.

A: Three areas, namely cerebral cortex (cor), infarct core (inf) and contralateral striatum (cnl) were cut out of specimen and total RNA for subsequent analyses was extracted. B: Total RNA integrity of each sample was measured with an Agilent 2100 Bioanalyzer (exemplary data shown). Distinct peaks for 18S and 28S rRNA indicate intact RNA. Only total RNA with a RNA integrity number greater than or equal to 6 was used for subsequent reverse transcription and qRT-PCR. C: Real-Time PCR data showed no significant differences of IL-6, IL-1β, TNF-α and G-CSF expression between wildtype and MCP-1-deficient control animals. D: Infarct size assessed 12 and 36 hours after MCAO. Lesion volume is expressed as percentage of the ipsilateral hemisphere. Values represent mean ± SD, *p<0.05.

Figure 2. MCP-1-deficiency has no impact on astrocyte activation.

Whole hemisphere GFAP-stained images comparing the activation of astrocytes between wildtype and MCP-1-deficient animals. Analyses showed no differences with respect to immunoreactivity and morphology between wildtype and MCP-1-deficient mice. GFAP staining showed the common distribution and activation of astrocytes reacting to oxygen deprivation. Astrogliosis occurred primarily surrounding the infarcted core in both wildtype and MCP-1-deficient mice.

Figure 3. IL-6, IF-1β, TNF-α and G-CSF mRNA expression within three defined microdissected areas.

RT-PCR was performed to detect and compare mRNA levels of wildtype and MCP-1-deficient mice within the cerebral cortex, infarcted core and contralateral striatum 12 hours (A−C) and 36 hours (D−F) after focal transient ischemia. Data represent mean levels ± SD of mRNA expression in wildtype and knock-out mice compared to respective sham-operated mice. RT-PCR was repeated twice for each sample. *p<0.05; **p<0.01; ***p<0.001: statistically significant differences between wildtype versus knock-out mice (ANOVA with Bonferronis post hoc-test).

Figure 4. Immunofluorescence-staining within the ipsilateral cerebral cortex revealed astrocytes (A−C) and neurons (E−G) as the main source of interleukin-6-synthesis.

In MCP-1-deficient mice, IL-6 signal pattern also showed double positive signals colocalized with astroctyes (D) and neurons (H) but with reduced intensity indicating a diminished IL-6 concentration on the translational level. Expression of IL-1β within the ischemic core was mainly restricted to neurons (I−K) in both wildtype and MCP-1-deficient mice, the latter displaying a reduced IL-1β-fluorescence intensity confirming the results of the expression analyses (L). Shrinked cell morphology indicates apoptotic processes within the infarcted core. Double immunofluorescence-staining also showed co-localization of G-CSF-positive cells and astrocytes (M−O), microglia (Q−S) and occasionally, neurons (T). In MCP-1-deficient mice astrocytic G-CSF secretion was clearly diminished, visualized by the reduction of fluorescence signal intensity reveals (P). Scale bar = 25 µm.

Figure 5. Quantification of IL-6, IF-1β, TNF-α and G-CSF expressing cells.

IL-6, IF-1β, TNF-α and G-CSF-positive cells as well as DAPI-positive cells were counted 0, 12 and 36 hours following MCAO. Cell numbers are presented as percentage in relation to the total DAPI-positive cell number within the respective area. Data are shown as mean±SD. *p<0.05; **p<0.01; ***p<0.001: statistically significant differences between wildtype versus MCP-1-deficient animals (ANOVA followed by Bonferronis post hoc-test).

Figure 6. MCP-1-deficiency leads to an impaired influx of neutrophil granulocytes and T-cells.

(A) Quantitative assessment of migrated neutrophils within ipsilateral cortex (cor, fig.1 A) following 30 min transient middle cerebral artery occlusion. Values are obtained counting the absolute number of cortical 7/4-positive cells in 8 sections per animal. Twelve hours after MCAO, MCP-1-deficient mice showed an impaired influx of neutrophil granulocytes within the ipsilateral cortex compared to wildtype controls (*p<0.001). (B) Quantitative analysis of T-cell migration within the ipsilateral cortex of MCP-1-deficient and wildtype mice. Thirty-six hours after MCAO T-cell numbers were significantly lower in MCP-1-deficient animals. (C) Immunohistochemistry of neutrophil granulocytes within the ipsilateral cortex. Representative 7/4 stained image taken from a wildtype animal. Following transient cerebral ischemia most neutrophil granulocytes were found within the meningeal layers and the cortical tissue. Scale bar = 100 µm. (D) T-cell within the ipsilateral cortex of a wildtype mouse. Arrowheads mark the border of a blood vessel. Nuclear counterstain with DAPI (blue). Scale bar = 25 µm.