Effects of pristane alone or combined with chloroquine on macrophage activation, oxidative stress, and TH1/TH2 skewness

Abstract

We investigated the protective role of chloroquine against pristane-induced macrophage activation, oxidative stress, and Th1/Th2 skewness in C57BL/6J mice. Those mice were treated with pristane alone or combined with chloroquine. Hematological and biochemical parameters, macrophage phagocytic function, the oxidant/antioxidant index, cytokine for IFN-γ, TNF-α, IL-4, and IL-6, and the isotypes of IgG2a and IgG1 were determined. And the expression of T-bet/GATA-3 and IL-12/IL-10 mRNA in spleen were analyzed by real-time PCR. We found that pristane treatment for a period of 12 or 24 weeks triggered macrophage activation syndrome, characterized by hemophagocytosis in spleen and peripheral blood, enhanced lipid phagocytosis by peritoneal macrophages in vitro, erythropenia and leucopenia, increased anti-Smith, lactic dehydrogenase, triglyceride, and ferritin, as well as hypercytokinemia of IFN-γ, TNF-α, IL-4, and IL-6. In parallel, a significant increase in lipid peroxidation and a decrease in superoxide dismutase, glutathione, and catalase activity, as well as a skewed Th1/Th2 balance in spleen, were observed. However, chloroquine supplementation showed a remarkable amelioration of these abnormalities. Our data indicate that pristane administration induces macrophage activation, oxidative stress, and Th1/Th2 skewness, which can be attenuated by chloroquine.

Figure 1

Hemophagocytosis, extramedullary hematopoiesis, and macrophage infiltration in liver and spleen. Hemophagocytosis (arrow), extramedullary hematopoiesis (arrow head) in liver and spleen were illustrated by HE staining ((a), (b)). The distribution and expression of F4/80 in liver and spleen were depicted with immunohistochemistry ((c), (d)) and western blotting (e), respectively. ×200; inset, ×400. CQ: chloroquine. Results were expressed as mean ± SE for eight animals. *P < 0.05 versus corresponding control mice, ^# P < 0.05 versus mice exposed to pristane for 12 weeks.

Figure 2

Effects of pristane alone or combined with chloroquine on cytological characteristics in blood and bone marrow. Wright-Giemsa staining was performed in peripheral blood (a) and bone marrow (b) smears. Hemophagocytosis (arrows), poikilocytosis, polychromasia, hypochromia, and anisocytosis of erythrocytes were observed in blood smear of pristane-treated mice. Bone marrow cytology depicted hypocellularity in erythroid progenitors, poikilocytosis, and nonerythroid lineage clonal expansion following treatment with pristane for 12 weeks or 24 weeks. And the severity of poikilocytosis was attenuated in the presence of chloroquine.

Figure 3

Macrophage phagocytic function revealed by oil red O staining. Peritoneal macrophages were exposed to 500 ng/mL ox-LDL for 24 h.

Figure 4

Effects of chloroquine on oxidative stress induced by pristane. Pristane increased the content of superoxide (red, DHE stain) and MDA and decreased the levels of SOD, GSH, and CAT, which could be ameliorated by coadministration of pristane and CQ. Original magnification ×400. CQ: chloroquine; MDA: malondialdehyde; SOD: superoxide dismutase; GSH: glutathione; CAT: catalase. *P < 0.05 versus the corresponding control mice, ^# P < 0.05 versus mice exposure to pristane for 12 weeks.

Figure 5

Effects of pristane alone or in combination with chloroquine on Th1/Th2 associated gene expression by RT-PCR analysis. Relative mRNA expression was normalized to GAPDH. CQ: chloroquine. Bars represent mean ± SE of three replicate experiments, *P < 0.05 versus the corresponding control mice, ^# P < 0.05 versus mice exposure to pristane for 12 weeks.