Increased circulating leukocyte numbers and altered macrophage phenotype correlate with the altered immune response to brain injury in metallothionein (MT)-I/II null mutant mice

Abstract

Background: Metallothionein-I and -II (MT-I/II) is produced by reactive astrocytes in the injured brain and has been shown to have neuroprotective effects. The neuroprotective effects of MT-I/II can be replicated in vitro which suggests that MT-I/II may act directly on injured neurons. However, MT-I/II is also known to modulate the immune system and inflammatory processes mediated by the immune system can exacerbate brain injury. The present study tests the hypothesis that MT-I/II may have an indirect neuroprotective action via modulation of the immune system.

Methods: Wild type and MT-I/II(-/-) mice were administered cryolesion brain injury and the progression of brain injury was compared by immunohistochemistry and quantitative reverse-transcriptase PCR. The levels of circulating leukocytes in the two strains were compared by flow cytometry and plasma cytokines were assayed by immunoassay.

Results: Comparison of MT-I/II(-/-) mice with wild type controls following cryolesion brain injury revealed that the MT-I/II(-/-) mice only showed increased rates of neuron death after 7 days post-injury (DPI). This coincided with increases in numbers of T cells in the injury site, increased IL-2 levels in plasma and increased circulating leukocyte numbers in MT-I/II(-/-) mice which were only significant at 7 DPI relative to wild type mice. Examination of mRNA for the marker of alternatively activated macrophages, Ym1, revealed a decreased expression level in circulating monocytes and brain of MT-I/II(-/-) mice that was independent of brain injury.

Conclusions: These results contribute to the evidence that MT-I/II(-/-) mice have altered immune system function and provide a new hypothesis that this alteration is partly responsible for the differences observed in MT-I/II(-/-) mice after brain injury relative to wild type mice.

Figure 1

Quantification of injury size and neuron death after cryolesion injury in wild type (grey bars) and MT-I/II^-/- mice (white bars). Injury size (A) was quantified by measurement of the area of the injury in sections taken from the widest point of the injury site. Neuron death identified by fluoro-jade C labelling (B). Fluorojade-C+ cells were counted in the injury site and the surrounding tissue. Counts were standardised per linear mm (width) of the injury site. Lower case letters indicate significance determined by Tukey's B post-hoc test. Time-points sharing letters indicates lack of statistically significant difference. n = 5-7, error bars = SEM. Representative images of fluoro-jade C staining in the injury site of wild type animals at 1 DPI (C), 3 DPI (D) and 7 DPI (E) with scale bars = 200 μm.

Figure 2

Leukocyte counts in sections of the injury site of MT-I/II^-/- mice (white bars) and wild type mice (grey bars) standardised to injury area. Neutrophil numbers (A) were determined by NIMP-14 immunoreactivity. Microglial and monocyte derived macrophages numbers (B) were determined by Iba1 immunoreactivity. T cell numbers (C) were determined by CD3 immunoreactivity. Lower case letters indicate significance determined by Tukey's B post-hoc test. Time-points sharing letters indicates lack of statistically significant difference. n = 5-7, error bars = SEM. Immunohistochemistry within the injury site is shown for neutrophils at 1 DPI with nuclear-fast red counter stain (D), macrophages at 7 DPI with nuclear-fast red counter stain (E) and T cells at 7 DPI without counterstain (D). Arrows indicate examples of positively stained cells, Scale bars = 100 μm.

Figure 3

MT-I and MT-II mRNA expression in wild type mice after cryolesion brain injury was measured by quantitative RT-PCR. Peak expression for both mRNAs was observed at 1 DPI with statistical significance relative to uninjured animals. For all groups n = 7 except for wild type mice at zero DPI where n = 6, error bars = SEM.

Figure 4

Circulating leukocyte counts after brain injury were obtained with the Advia 120 haemocytological analyser. Absolute cell numbers (A) show an increase at 7 DPI which was significantly different to all time points for wild type mice and from 0-3 DPI time points for MT-I/II^-/- mice as determined by Tukey's B post-hoc test. n = 4-6, error bars = SEM. Relative ratios of leukocytes (B) were compared between wild type mice (solid lines) and MT-I/II^-/- mice (dashed lines) for lymphocytes (blue circles), neutrophils (purple crosses) and monocytes (red triangles). No significant differences were found between strains for any cell type and no significant changes over time were found for any cell type. n = 4-6, error bars = SEM.

Figure 5

CD4⁺ T cell ratios after brain injury were assessed by flow cytometry for CD3 and CD4 labelled cells shown in a representative scatter plot (B). Temporal changes in CD4⁺ T cell ratios after brain injury reveal no significant differences between wild type (solid line) and MT-I/II^-/- mice (dashed line) (A), n = 6-7, error bars = SEM. The CD4⁺ cell gate revealed the ratios of CD25⁺ and FoxP3⁺ naturally occurring regulatory T cells (C). At 3 and 7 DPI no significant differences were observed between wild type mice (grey bars) and MT-I/II^-/- mice (white bars) (D), n = 7, error bars = SEM.

Figure 6

Scatter plot showing detectable plasma IL-2 concentrations in MT-I/II^-/- mice (crosses) and wild type mice (circles) after brain injury. Values below the detection limit (0.4 pg/ml) are not shown. Increases in IL-2 in plasma after injury were sporadic with few animals posting detectable concentrations. At 7 DPI only MT-I/II^-/- mice have detectable levels of plasma IL-2. n = 7 for all groups except wild type mice at zero DPI and MT-I/II^-/- mice at 7 DPI for which n = 6, error bars = SEM.

Figure 7

(A) Ym1 mRNA expression is greater in the injury site of wild type mice (solid lines) than in MT-I/II^-/- mice (dashed lines), n = 6-7, error bars = SEM. (B) Ym1 mRNA expression is significantly greater in the circulating PBMCs of wild type mice (Solid lines) than in MT-I/II^-/- mice (dashed lines), independent of time after injury, n = 6, error bars = SEM.