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. 2020 Oct;33(4):237-246.
doi: 10.1293/tox.2020-0016. Epub 2020 Jul 31.

Effects of dexamethasone on hepatic macrophages in normal livers and thioacetamide-induced acute liver lesions in rats

Noa Hada  1 , Mizuki Kuramochi  1 , Takeshi Izawa  1 , Mitsuru Kuwamura  1 , Jyoji Yamate  1
Affiliations

Effects of dexamethasone on hepatic macrophages in normal livers and thioacetamide-induced acute liver lesions in rats

Noa Hada et al. J Toxicol Pathol. 2020 Oct.

Abstract

Resident and infiltrative macrophages play important roles in the development of pathological lesions. M1/M2 macrophage polarization with respective CD68 and CD163 expression remains unclear in chemically induced liver injury. This study was aimed at investigating the influence of macrophages on normal and chemically induced liver injury. For this, dexamethasone (DX), an immunosuppressive drug, was administered in normal rats and thioacetamide (TAA)-treated rats. Liver samples were collected and analyzed with immunohistochemical methods. Repeated injections of DX (0.5 or 1.0 mg/kg BW) for 3, 7 and 11 days reduced the number of CD163 positive hepatic resident macrophages (Kupffer cells) in normal livers, while increasing AST and ALT levels. In TAA (300 mg/kg BW)-treated rats injected with DX (0.5 mg/kg BW) pretreatment, the number of M1 and M2 macrophages showed a significant decrease compared with that of TAA-treated rats without DX treatment. Additionally, reparative fibrosis resulting from hepatocyte injury induced by TAA injection was suppressed by DX pretreatment. Our data suggested that macrophages could influence not only normal hepatic homeostasis (reflected by AST and ALT levels) but also chemically induced hepatic lesion development (reduced reparative fibrosis).

Keywords: M1 macrophage; M2 macrophage; dexamethasone; thioacetamide.

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Conflict of interest statement

The authors have no conflicts of interest directly relevant to the content of this article.

Figures

Fig. 1.
Fig. 1.
Histopathology of control liver and dexamethasone (DX)-treated livers at 0.5 and 1.0 mg/kg body weight. (A) Livers of control and (B, C) continuous treatments of 0.5 or 1.0 mg/kg DX do not show any histopathological changes. (D) Four-day withdrawal after 11-day continuous DX treatment does not cause histopathological changes in the liver. (E) The level of alanine transaminase (ALT) gradually increases in rats after 3, 7 and 11-day DX treatment, compared with control. Four-day withdrawal after 11-day DX treatment results in recovery of these levels close to controls. Liver samples were stained with hematoxylin and eosin. CV, central vein; bar = 100 μm; *, P<0.05, vs. control; Dunnett’s test.
Fig. 2.
Fig. 2.
Immunohistochemical analysis for CD163 as a marker of M2 macrophage. (A) CD163-positive cells (arrowheads) are seen along the sinusoids in controls, indicative of liver resident macrophages (Kupffer cells). (B) CD163-positive cells (arrowheads) are obviously decreased after DX treatment. (C) The number of CD163-positive macrophages significantly decreases in rats after 3, 7 and 11-day DX treatment. The number tends to recover after 4-day withdrawal of DX treatment. CV, central vein; bar = 50 μm. P<0.05;*, vs. control; Dunnett’s test.
Fig. 3.
Fig. 3.
Polymerase chain reaction for TNF-α mRNA using livers of control, 11-day DX of 0.5 mg/kg, and 4-day withdrawal after 0.5 mg/kg DX, as well as positive control of TAA-injected rat. TNF-α expression is not seen in control and 11-day DX treatment; however, it appears during 4-day withdrawal after continuous injection of 0.5 mg/kg DX.
Fig. 4.
Fig. 4.
Histopathology of thioacetamide-treated livers (TAA; 300 mg/kg BW) and DX pre-treated (3-day continuous DX treatment) livers followed by TAA injection: (A-D) saline injection instead of DX treatment, followed by TAA injection in liver samples (TAA group) and (E-H) DX pre-treated TAA-injected liver samples (DX + TAA group). In TAA group, (A) coagulation necrosis is seen in the centrilobular area on day 1; (B) inflammatory cells, including macrophages, are seen in the centrilobular area on day 3 (inset: high magnification); (C, D) the lesion is almost recovered on days 5 and 7, although degenerative hepatocytes are still seen on day 5. In DX + TAA group, (E) coagulation necrosis is also seen on day 1; (F) inflammatory macrophages are rarely seen in the centrilobular area on day 3; (G, H) the lesion is recovered on days 5 and 7. (J, K) The levels of hepatic deviation enzymes, such as aspartate transaminase (AST) and alanine transaminase (ALT), increase on day 1 in both TAA and DX + TAA groups; these levels begin to decrease from day 3. Liver samples were stained with hematoxylin and eosin. CV, central vein; bar = 100 μm. P<0.05; *, vs. control; Dunnett’s test.
Fig. 5.
Fig. 5.
Evaluation for collagen deposition for fibrosis with the azan-Mallory method. (A) Collagen deposition is not seen in the control liver. (B) In TAA group, along with degenerative hepatocytes, collagen deposition is observed in the centrilobular area (arrowheads) on day 5 as reparative fibrosis. (C) In DX + TAA group, the degree of collagen deposition on day 5 is lesser compared with that in TAA group. (D) The degree of collagen deposition is notably suppressed in DX + TAA groups compared with that in TAA groups. CV, central vein; bar = 100 μm. P<0.05; *, vs. control; #, vs. DX+TAA; Tukey-Kramer’s test.
Fig. 6.
Fig. 6.
Immunohistochemical analysis for CD68 for M1 macrophages in liver samples. (A) In TAA group, CD68-positive macrophages appear in the centrilobular area on day 1; (B) CD68-positive macrophages are still seen on day 3. (C, D) in DX + TAA group, CD68-positive macrophages are decreased in the centrilobular area compared with those in TAA group on days 1 and 3. (E) The numbers of CD68-positive cells in DX + TAA group are temporarily increased on day 1 compared with control group; however, they are statistically less than that in TAA group on each examination day. CV, central vein; bar = 100 μm. P<0.05; *, vs. control; #, vs. DX+TAA; Tukey-Kramer’s test.
Fig. 7.
Fig. 7.
Immunohistochemical analysis for CD163 for M2 macrophages. (A) In TAA group, CD163-positive macrophages with swollen cytoplasm are seen in the centrilobular area on day 1; (B) CD163-positive macrophages are still seen on day 3. (C) In DX+TAA group, CD163-positive macrophages also appear in the centrilobular area; (D) the appearance of CD163-positive macrophages is notable on day 3. (E) However, the numbers of CD163-positive macrophage in DX + TAA group are statistically less than that in TAA group on days 1 and 3. CV, central vein; bar = 100 μm. P<0.05; *, vs. control; #, vs. TAA; †, vs. DX+TAA; Tukey-Kramar’s test.
Fig. 8.
Fig. 8.
Immunohistochemistry for macrophage-migration inhibitory factor (MIF) in liver samples. (A) Hepatocytes in the centrilobular area strongly express MIF in TAA group on day 1. (B) The MIF expressions is weaker in DX + TAA group liver samples. CV, central vein; bar = 100 μm.
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