T cells contribute to stroke-induced lymphopenia in rats

Abstract

Stroke-induced immunodepression (SIID) results when T cell and non-T immune cells, such as B cells, NK cells and monocytes, are reduced in the peripheral blood and spleen after stroke. We investigated the hypothesis that T cells are required for the reductions in non-T cell subsets observed in SIID, and further examined a potential correlation between lymphopenia and High-mobility group protein B1 (HMGB1) release, a protein that regulates inflammation and immunodepression. Our results showed that focal ischemia resulted in similar cortical infarct sizes in both wild type (WT) Sprague Dawley (SD) rats and nude rats with a SD genetic background, which excludes the possibility of different infarct sizes affecting SIID. In addition, the numbers of CD68-positive macrophages in the ischemic brain did not differ between WT and nude rats. Numbers of total peripheral blood mononuclear cells (PBMCs) or splenocytes and lymphocyte subsets, including T cells, CD4(+) or CD8(+) T cells, B cells and monocytes in the blood and spleen, were decreased after stroke in WT rats. In nude rats, however, the total number of PBMCs and absolute numbers of NK cells, B cells and monocytes were increased in the peripheral blood after stroke; nude rats are athymic therefore they have few T cells present. Adoptive transfer of WT splenocytes into nude rats before stroke resulted in lymphopenia after stroke similar to WT rats. Moreover, in vitro T cell proliferation stimulated by Concanavalin A was significantly inhibited in WT rats as well as in nude rats receiving WT splenocyte adoptive transfer, suggesting that T cell function is indeed inhibited after stroke. Lastly, we demonstrated that stroke-induced lymphopenia is associated with a reduction in HMGB1 release in the peripheral blood. In conclusion, T cells are required for stroke-induced reductions in non-T immune cells and they are the most crucial lymphocytes for SIID.

Figure 1. PCR-RFLP assay for genotyping SD-rnu rats.

A. The PCR products were analyzed on 4% agarose gel. Lane M: DNA size marker; Lane 1, 4, 6: SD homozygote (+/+); Lane 2, 3: heterozygote (rnu/+); Lane 5, 7: nude homozygote (rnu/rnu). B. Representative appearance of WT SD rat and nude rat without hair. C. The WT SD rat has a thymus (arrow) while the nude rat is athymic.

Figure 2. Cortical infarction and inflammation in WT and nude rats after stroke.

A. Representative infarctions in the cortex are shown. Animals were euthanized 3 d after stroke. Brains were collected, coronally sectioned into 5 slices and stained with a 2% TTC solution. B. Infarct regions were measured and normalized to the contralateral, non-ischemic cortex, and expressed as percentage. The bar graphs represent average infarct sizes (n = 7–9/group). C. CD 68-positive macrophage staining in the ischemic brain. Ischemic brains were collected 72 h post-stroke and immunostained with CD 68 antibodies (green), and counterstained with DAPI (blue). CD 68 positive cells were counted from 3 sections of each animal. Representative images of CD68 positive macrophages are shown. The statistical results suggest no difference in the CD 68 positive macrophage numbers between WT and nude rats. N = 4–5/group. Scale Bar: 50 μm. D. Ischemic brain cortices were collected 3 d after stroke and total mononuclear cells were extracted for FACS analyses. Data from the non-ischemic hemispheres were used as controls. Gating strategy for T cells and subpopulations are shown on the upper panel. In the ischemic brain, numbers of T cells and CD4⁺ T cells, as well as CD8⁺ T cells were significantly increased compared with those in the control hemisphere. Note that nude rats, in principle, have no T cells, including CD4⁺ T cells, CD8⁺ T cells. * vs. corresponding controls (the contralateral, non-ischemic hemisphere), P<0.05, n = 4.

Figure 3. Gating strategy for immune cell subpopulations in the blood and spleen.

Blood was harvested from the tail vein 3 d after stroke and total PBMCs were isolated for immunostaining. The first column in each row shows the gated total PBMCs based on Forward Scatter (FSC) and Side Scatter (SSC) parameters. Further analyses after immunofluorescent staining for specific cell populations are shown in columns 2 and 3. The relative percentage of the gated population is shown at the corner. Cell populations are defined as: B cells, CD45R⁺TCR⁻; CD4⁺ T cells, CD4⁺TCR⁺; CD8⁺ T cells, CD8⁺TCR⁺; macrophages, SIRP⁺TCR⁻; NK cells, NKR⁺TCR; Tregs, CD4⁺CD25⁺FoxP3⁺. A. Representative FACS profiles after gating to detect cell types in WT rats. B. Representative FACS profiles after gating to detect cell types in nude rats.

Figure 4. Total number of PBMCs and immune cell subsets in the peripheral blood of WT and nude rats.

A. Total number of PBMCs and absolute numbers of T cells, CD4⁺ T cells, CD8⁺ T cells, Tregs, B cells, NK cells, monocytes, CD4⁺/CD8⁺ as well as the Foxp3⁺/Foxp3⁻ ratio in CD4⁺ T cells in SD WT rats (n = 5–11/group). B. Total number of PBMCs, absolute numbers of B cells, NK cells and monocytes in nude rats (n = 5–8/group), Blood was harvested from the tail 48 h or 72 h after stroke and total PBMCs were isolated for immunostaining. Relative percentages of each cell type were obtained from the FACS analyses using PBMCs. Absolute cell numbers were calculated by multiplying the percentage of each cell type with the total cell number in an analyzed sample, and adjusted to a number in 1 ml of blood. For naïve groups, no surgeries were performed. For sham groups, rats received sham surgery without stroke. *, **, *** vs. naïve, ^Δ, ^ΔΔ, ^ΔΔΔ vs. sham, p<0.05, 0.01, 0.001, respectively.

Figure 5. Spleen size, total number of splenocytes and individual subsets in WT and nude rats.

A. Spleen weight (n = 5–6/group). B. Total number of splenocytes, absolute numbers of T cells, CD4⁺ T cells, CD8⁺ T cells, Tregs, B cells, NK cells, monocytes, CD4⁺/CD8⁺ and Foxp3⁺/Foxp3⁻ ratio in CD4⁺T cells in WT SD rats (n = 5–11/group). C. Total number of splenocytes and absolute numbers of B cells, NK cells as well as monocytes in nude rats (n = 5–8/group). Spleen was collected 3 d after stroke and total splenocytes isolated for immunostaining. Relative percentages of each cell type were obtained from the FACS analyses using splenocytes. Absolute cell numbers were calculated by multiplying the percentage of each cell type with the total cell number in an analyzed sample, and adjusted to a number in 1 spleen. *, **, *** vs. stroke, p<0.05, 0.01, 0.001, respectively.

Figure 6. Infarct size and numbers of PBMCs, splenocytes, and individual cell subsets in nude rats receiving adoptive lymphocyte transplant.

Nude rats were reconstituted by intravenous injection of 1×10⁸ splenocytes from WT rats and stroke was conducted 24 h later. Infarct sizes, PBMCs and splenocytes were measured 3 days after stroke. A. Comparison of infarct sizes between vehicle and lymphocyte reconstitution group in nude rats (n = 6/group). B. Total number of PBMCs and absolute numbers of T cells, CD4⁺ T cells, CD8⁺ T cells, B cells, NK cell, as well as monocytes in the blood in reconstituted nude rats (n = 6–7/group). C. Total number of splenocytes, absolute numbers of T cells, CD4⁺ T cells, CD8⁺ T cells, B cells, NK cell and monocytes in the spleen in reconstituted nude rats (n = 6–7/group). *, ** vs. sham, p<0.05, 0.01, respectively.

Figure 7. T cell proliferative assay after stroke.

Splenocytes were prepared 72 h after stroke from naïve, sham surgery and stroke animals. Cultured splenocytes were stimulated with 0 μg/ml, 1 μg/ml Con A. The cells were incubated for 66 h at 37°C and 5% CO₂. [³H]-Thymidine was added to the culture and incubated for 18 h. Cells were harvested onto a glass fiber filter. The amount of radioactivity that integrated into divided lymphocytes was measured by a beta-plate scintillation reader. Results are expressed as counts per minute (cpm). A. Splenocytes proliferation in WT SD rats (n = 3–6/group). B. Splenocytes proliferation in T cells reconstituted nude rats (n = 3–5/group). Nude rats were reconstituted by intravenous injection of 1×10⁸ splenocytes in 100 μl RPMI 1640, and dMCAO was performed 24 h after reconstitution. *, **, *** vs. naïve, ^Δ, ^ΔΔ, ^ΔΔΔ vs. sham, p<0.05, 0.01, 0.001, respectively.

Figure 8. HMGB1 levels in the plasma and corresponding total number of PBMCs after splenectomy or treatment with Glycyrrhizin followed by stroke.

A. Western blot detected HMGB1 release in the plasma 48 h after stroke in rats with and without splenectomy. The left panel shows representative results of Western blot for HMGB1. Note that no control protein, such as β-actin, was used to show even loading, because the examined samples are plasma, for which the same amount volume (15 μl) from different animals was loaded. The bar graphs in the right panel show the average optical density of protein bands. The relative density of protein bands in rats receiving splenectomy plus stroke was set as 100%. * vs. control (stroke without splenectomy), P<0.05, n = 4/group. B. Total number of PBMCs in stroke rats with or without splenectomy. Forty-eight hours after stroke followed splenectomy, blood was harvested and total PBMCs were isolated and counted. Splenectomy significantly attenuated reduction in total number of PBMCs induced by stroke. *, ** vs. control, p<0.05, 0.01, respectively. N = 9–11/group. Ctrl: Control; Spx: splenectomy. C. Total number of PBMCs in stroke rats treated with or without glycyrrhizin. Glycyrrhizin (200 mg/kg) was injected intraperitoneally immediately before stroke and 2 h after reperfusion. The blood was harvested and total PBMCs were isolated and counted 3 d post stroke. Glycyrrhizin significantly reversed the reduction in total PBMCs induced by stroke in vehicle. ^## vs. naïve, ^ΔΔ, vs. Sham, p<0.01, * vs. vehicle, p<0.05. N = 9–11/group.