Cyclooxygenase-2 deficiency enhances Th2 immune responses and impairs neutrophil recruitment in hepatic ischemia/reperfusion injury

Abstract

Cyclooxygenase-2 (COX-2) is a prostanoid-synthesizing enzyme that is critically implicated in a variety of pathophysiological processes. Using a COX-2-deficient mouse model, we present data that suggest that COX-2 has an active role in liver ischemia/reperfusion (I/R) injury. We demonstrate that COX-2-deficient mice had a significant reduction in liver damage after I/R insult. The inability of COX-2(-/-) to elaborate COX-2 products favored a Th2-type response in these mice. COX-2(-/-) livers after I/R injury showed significantly decreased levels of IL-2, as well as IL-12, a cytokine known to have a central role in Th1 effector cell differentiation. Moreover, such livers expressed enhanced levels of the anti-inflammatory cytokine IL-10, shifting the balance in favor of a Th2 response in COX-2-deficient mice. The lack of COX-2 expression resulted in decreased levels of CXCL2, a neutrophil-activating chemokine, reduced infiltration of MMP-9-positive neutrophils, and impaired late macrophage activation in livers after I/R injury. Additionally, Bcl-2 and Bcl-x(L) were normally expressed in COX-2(-/-) livers after injury, whereas respective wild-type controls were almost depleted of these two inhibitors of cell death. In contrast, caspase-3 activation and TUNEL-positive cells were depressed in COX-2(-/-) livers. Therefore, our data support the concept that COX-2 is involved in the pathogenic events occurring in liver I/R injury. The data also suggest that potential valuable therapeutic approaches in liver I/R injury may result from further studies aimed at identifying specific COX-2-derived prostanoid pathways.

FIGURE 1

COX-1 and COX-2 mRNA expression in the liver of COX-2 null and WT mice after I/R injury. COX-2 mRNA expression (A) was readily detected in WT livers at 6 (lanes 2 and 3) and 24 h (lanes 6 and 7) of reperfusion following 90 min of warm ischemia. COX-2 mRNA expression was absent in COX-2^−/− livers after 6 (lanes 4 and 5) and 24 h (lanes 8 and 9) of I/R injury and in naive WT livers (lane 1). In contrast, COX-1 mRNA expression was comparable in all studied groups. The densitometric ratios COX-1/β-actin mRNA and COX-2/β-actin mRNA are shown in B. Real-time PCR of COX-1 gene expression confirms that the mRNA levels of COX-1 were similar in both COX-2^−/− and WT livers after reperfusion (C).

FIGURE 2

Prostanoid synthesis in COX-2 and WT livers after I/R injury. The concentrations of COX reaction products PGE₂, 6-keto-PGF_2α, and TXB₂ were significantly reduced in COX-2-deficient livers as compared with respective controls at 6 h after I/R injury (*, p < 0.008, **, p < 0.004, and §, p < 0.04).

FIGURE 3

Liver transaminases and histological preservation in COX-2^−/− and WT mice. sGPT and sGOT levels (IU/L) (A) were measured in the blood samples taken at 6 and 24 h after I/R injury. sGPT and sGOT levels in the COX-2^−/− mice were significantly lower than those in the respective WT control littermates at both 6 (*, p < 0.003, §, p < 0.03) and 24 h (**, p < 0.01). Representative H&E staining of livers at 6 and 24 h post-I/R injury (B). At 6 h, control WT livers were mostly characterized by elevated sinusoidal congestion (A), and COX-2^−/− livers (B) showed reduced sinusoidal congestion and some focal necrotic areas. At 24 h, whereas WT livers (C) showed extensive signs of necrosis, COX-2^−/− livers (D) had very good histological preservation (×100, H&E stain).

FIGURE 4

Apoptotic markers in COX-2^−/− and WT mice. A, Bcl-2 and Bcl-x_L were readily expressed in naive livers (lane 1) and in COX-2^−/− livers at 6 h post-I/R injury (lanes 5–7). In contrast, the levels of these inhibitors of cell death were markedly depressed in 6-h WT controls (lanes 2–4). B, Ratios of Bcl-2/β-actin and of Bcl-x_L/β-actin in WT and COX-2-deficient livers after 6 h of I/R injury. C, Caspase-3 activity was significantly depressed in COX-2^−/− livers at 6 h when compared with controls (*, p < 0.01, **, p < 0.05).

FIGURE 5

TUNEL staining in COX-2^−/− and WT mice. TUNEL-positive cells (A) were readily detected in WT livers and were significantly depressed in COX-2^−/− livers at 6 h of hepatic I/R injury (*, p < 0.05). Dual staining with TUNEL reagents and Abs to CD45 (B) showed a virtual lack of TUNEL-positive cells that co-express CD45 in 6-h WT livers. Filled arrows denote TUNEL-positive cells (green), and open arrows indicate CD45⁺ cells (red).

FIGURE 6

Intrahepatic MPO enzyme activity and Ly-6G neutrophil infiltration in COX-2^−/− and WT mice. MPO enzymatic activity (A), an index of neutrophil infiltration, was markedly reduced in the COX-2^−/− mice at 6 and 24 h of reperfusion following 90 min of warm ischemia. Additionally, Ly-6G neutrophil infiltration (B) was lower in COX-2^−/− livers as compared with controls at both 6 and 24 h post-I/R injury (*, p < 0.01). Arrows indicate Ly-6G cell labeling in liver specimens (immunostaining magnification ×200, 6 h).

FIGURE 7

T and Mac-1 leukocyte infiltration in COX-2^−/− and WT mice. Elevated infiltration of T (A) and Mac-1 (B) leukocytes in both COX-2^−/− livers and respective controls at 6 h post-I/R injury is shown. In contrast, T and Mac-1 cell infiltration was minimal in both groups at 24 h after I/R injury. Arrows indicate leukocyte labeling in liver specimens (immunostaining magnification ×200, 6 h).

FIGURE 8

Gelatin zymography and immunostaining for MMP-9 in COX-2^−/− and WT livers. MMP-9 activity (A) was reduced in COX-2^−/− livers both at 6 (lanes 4 and 5) and 24 h (lanes 8 and 9) as compared with respective controls at 6 (lanes 2 and 3) and 24 h (lanes 6 and 7) post-I/R injury. MMP-9 was virtually undetected in naive livers (lane 1). Additionally, MMP-9-positive leukocytes (B) were significantly depressed in COX-2^−/− livers at 6 h after I/R injury (*, p < 0.01).

FIGURE 9

Colocalization of MMP-9 and leukocyte markers in livers after I/R injury. All panels represent confocal optical sections of immunostained COX-2^−/− and WT livers at 6 h post-I/R injury. Ly-6G (A and D) and Mac-1 (G and J) cells were stained in green (Alexa Fluor 488), MMP-9 was labeled in red (Alexa Fluor 594) (B, E, H, and K), and cell colocalization of both Ly-6G/MMP-9 (C and F) and Mac-1/MMP-9 (I and L) markers are shown in a yellow-orange. Although MMP-9 + Ly-6G neutrophils were profoundly reduced in COX-2^−/− livers, MMP-9 + Mac-1 macrophages were present in comparable numbers in both COX-2^−/− and control livers after I/R injury.

FIGURE 10

Chemokine gene expression in COX-2^−/− and WT livers. Real-time PCR of chemokine gene expression shows that the mRNA levels of MCP-1, a monocyte chemoattractant, were up-regulated in both COX-2^−/− and WT livers after reperfusion. However, whereas the expression of CXCL1, a neutrophil chemoattractant, had little variation between COX-2^−/− and control livers, the expression of the neutrophil-activating CXCL2 chemokine was significantly reduced in COX-2^−/− livers at 6 and 24 h post-I/R injury as compared with controls. Data were normalized to actin gene expression (*, p < 0.03, **, p < 0.05).

FIGURE 11

Cytokine gene expression in COX-2^−/− and WT livers. The expression of IL-2 mRNA, a Th1-type cytokine, was profoundly depressed in COX-2^−/− livers as compared with controls at both 6 and 24 h post-I/R injury. In contrast, the anti-inflammatory IL-10 mRNA, a Th2-type cytokine, was significantly up-regulated in COX-2^−/− livers at 6 h after liver I/R injury. Moreover, IL-12 mRNA, a cytokine required for the development of adaptive Th1 responses, was also significantly depressed in the COX-2^−/− at 6 h post-I/R injury. Thus, COX-2 deficiency favored a Th2-type immune response in liver I/R injury, as indicated by the markedly decreased IL-2/IL-10 mRNA and protein ratios in the COX-2 null livers at 6 and 24 h post-I/R injury (*, p < 0.05, **, p < 0.01, and §, p < 0.001).

FIGURE 12

Cytokine expression in murine splenocytes. IL-2 and IL-10 expression were up-regulated in anti-CD3-activated splenocytes. However, the addition of NS-398, a COX-2-selective inhibitor, to the anti-CD3-activated splenocytes, significantly depressed the expression of IL-2 and increased the levels of IL-10 by these cells (*, p < 0.01, **, p < 0.002, ***, p < 0.02, and §, p < 0.03).

FIGURE 13

iNOS expression in COX-2^−/− and WT livers. iNOS expression at mRNA (A) and protein (C) levels. iNOS is virtually absent in naive livers (lane 1), and it is similarly up-regulated in WT (lanes 2 and 3) and COX-2^−/− (lanes 4 and 5) livers after 6 h of I/R injury. However, COX-2^−/− livers express almost negligible levels of iNOS at 24 h of I/R injury (lanes 8 and 9), whereas respective WT livers (lanes 6 and 7) still express elevated levels of iNOS. B and D show the ratios of iNOS/β-actin mRNA and protein, respectively (*, p < 0.01).