To clarify interleukin (IL)-6 roles in wound healing, we prepared skin excisions in wild-type (WT) and IL-6-deficient BALB/c [knockout (KO)] mice. In WT mice, the wound area was reduced to 50% of original size at 6 days after injury. Microscopically, leukocyte infiltration was evident at wound sites. Furthermore, the re-epithelialization rate was approximately 80% at 6 days after injury with increases in angiogenesis and hydroxyproline contents. The gene expression of IL-1, chemokines, adhesion molecules, transforming growth factor-beta1, and vascular endothelial growth factor was enhanced at the wound sites. In contrast, the enhanced expression of these genes was significantly reduced in KO mice. Moreover, in KO mice, the reduction of wound area was delayed with attenuated leukocyte infiltration, re-epithelialization, angiogenesis, and collagen accumulation. Finally, the administration of a neutralizing anti-IL-6 monoclonal antibody significantly delayed wound closure in WT mice. These observations suggest that IL-6 has crucial roles in wound healing, probably by regulating leukocyte infiltration, angiogenesis, and collagen accumulation.

Several lines of in vitro evidence suggest the potential role of IFN-γ in angiogenesis and collagen deposition, two crucial steps in the wound healing process. In this report, we examined the role of IFN-γ in the skin wound healing process utilizing WT and IFN-γ KO mice. In WT mice, excisional wounding induced IFN-γ mRNA and protein expression by infiltrating macrophages and T cells, with a concomitant enhancement of IL-12 and IL-18 gene expression. Compared with WT mice, IFN-γ KO mice exhibited an accelerated wound healing as evidenced by rapid wound closure and granulation tissue formation. Moreover, IFN-γ KO mice exhibited enhanced angiogenesis with augmented vascular endothelial growth factor mRNA expression in wound sites, compared with WT mice, despite a reduction in the infiltrating neutrophils, macrophages, and T cells. IFN-γ KO mice also exhibited accelerated collagen deposition with enhanced production of TGF-β1 protein in wound sites, compared with WT mice. Furthermore, the absence of IFN-γ augmented the TGF-β1-mediated signaling pathway, as evidenced by increases in the levels of total and phosphorylated Smad2 and a reciprocal decrease in the levels of Smad7. These results demonstrate that there is crosstalk between the IFN-γ/Stat1 and TGF-β1/Smad signaling pathways in the wound healing process.

Neutrophils and macrophages infiltrate after acetaminophen (APAP)-induced liver injury starts to develop. However, their precise roles still remain elusive. In untreated and control IgG-treated wild-type (WT) mice, intraperitoneal APAP administration (750 mg/kg) caused liver injury including centrilobular hepatic necrosis and infiltration of neutrophils and macrophages, with about 50% mortality within 48 h after the injection. APAP injection markedly augmented intrahepatic gene expression of inducible nitric oxide synthase (iNOS) and heme oxygenase (HO)-1. Moreover, neutrophils expressed iNOS, which is presumed to be an aggravating molecule for APAP-induced liver injury, while HO-1 was mainly expressed by macrophages. All antigranulocyte antibody-treated neutropenic WT and most CXC chemokine receptor 2 (CXCR2)-deficient mice survived the same dose of APAP, with reduced neutrophil infiltration and iNOS expression, indicating the pathogenic roles of neutrophils in APAPinduced liver injury. However, APAP caused more exaggerated liver injury in CXCR2-deficient mice with reduced macrophage infiltration and HO-1 gene expression, compared with neutropenic WT mice. An HO-1 inhibitor, tin-protoporphyrin-IX, significantly increased APAP-induced mortality, implicating HO-1 as a protective molecule for APAP-induced liver injury. Thus, CXCR2 may regulate the infiltration of both iNOS-expressing neutrophils and HO-1-expressing macrophages, and the balance between these two molecules may determine the outcome of APAP-induced liver injury. IntroductionAcetaminophen (APAP) is widely prescribed as an analgesic and antipyretic drug in clinics and is also sold as numerous over-counter preparations as a single compound or in combination with other medications [1, 2]. Although it is generally safe, an overdose of APAP can cause severe liver failure with a significant morbidity and mortality. Thus, its wide availability and severe toxicities has placed APAP overdose as the leading cause for calls to Poison Control Centers in the United States (>100 000/year). Moreover, APAP overdose accounts for more than 56 000 emergency room visits, 2600 hospitalizations, and an estimated 458 deaths due to acute liver failure each year in the United States alone [3]. The toxic response is initiated by the metabolism of APAP to a toxic metabolite, N-acetyl-p-benzoquinone imine. Because N-acetyl-p-benzoquinone imine can react rapidly with sulfhydryl groups, it first depletes glutathione in hepatocytes and then reacts with a number of intracellular proteins, thereby causing their dysfuntions [4, 5]. APAP-induced liver injury is histopathologically characterized by centrilobular hepatic necrosis with a massive infiltration of leukocytes, particularly neutrophils. The recruitment of leukocytes in the damaged tissue is a hallmark of the inflammatory process, which may contribute to the development of APAP-induced liver injury [6,7]. In line with this assumption, accumulating evidence has implicated neutrophils as the leukocyte type essentially involved i...

Cyclic ADP-ribose (cADP-ribose) 1 is synthesized from ␤-NAD ϩ , an abundant intracellular substrate, by ADP-ribosyl cyclase in sea urchin eggs and in mammalian cells (1, 2). Pharmacological studies suggest that cADP-ribose is an endogenous modulator of ryanodine-sensitive Ca 2ϩ release channels (3-10). If cADP-ribose acts as an intracellular second messenger, ADPribosyl cyclase, as an effector enzyme, should be activated or inhibited in response to stimulation by hormones or neurotransmitters, which should simultaneously be associated with a transient decrease in the intracellular NAD ϩ concentration ([NAD ϩ ] i ) and an increase in cADP-ribose concentration (11).ADP-ribosyl cyclase seems to be present in both cytosolic and membrane-bound forms (1, 2, 12). The mammalian membranebound form of ADP-ribosyl cyclase has been identified as a cellsurface antigen, CD38 (13-19) and .Recently, it has been shown that the formation of cADPribose is regulated by nitric oxide or cGMP (21-23) and that nitric oxide or cGMP is increased by stimulation with agonists (24, 25). These findings suggest the hypothesis that the regulation of the cADP-ribose level is located far downstream in the signal transduction cascade from receptors (11). An alternative hypothesis is that the cADP-ribose formation is regulated by ADP-ribosyl cyclase through the direct action of G proteins activated by receptors within the surface membrane, as already shown for the formation of cyclic AMP, inositol 1,4,5-trisphosphate, and diacylglycerol (26 -28). To test this hypothesis, we used NG108-15 neuroblastoma ϫ glioma hybrid cells (29), in which signal transduction from receptors to effectors has been extensively characterized (29,30). In particular, in NGPM1-27 cells (31), which overexpress muscarinic acetylcholine receptors (mAChRs), it has been shown that intracellular NAD ϩ or NAD ϩ metabolites are involved in signal transduction from m1 mAChRs to K ϩ channels (32,33). In this context, such neuronal cell lines have advantages for analyzing receptor-ADP-ribosyl cyclase coupling in detail.For measurement of ADP-ribosyl cyclase, high pressure liquid chromatography (HPLC) is commonly used to separate cADP-ribose-related compounds (1,2,8,14,15,17,19,34,35). However, since it takes 30 -60 min to process one sample, it is essential to develop a much more rapid method that can allow processing of multiple samples at once. There are two papers that describe ADP-ribosyl cyclase assay by TLC (21,36), in which NAD ϩ migrates faster than cADP-ribose. The methods used in those reports seem to be affected by large amounts of radiolabeled substrates. We here developed a TLC method that overcomes this problem and allows separation of cADP-ribose in up to 19 samples within 40 -50 min. Our TLC method was first tested on COS-7 cells overexpressing human CD38 and was shown to be applicable for measuring ADP-ribosyl cyclase activity. We demonstrate that crude cell membranes of NG108-15 cells possess ADP-ribosyl cyclase activity and that such activity is activated or inhibi...

Acetaminophen (APAP) causes a massive production of intrahepatic tumor necrosis factor alpha (TNF-alpha). However, it still remains elusive regarding the roles of TNF-alpha in APAP-induced liver injury. Hence, we examined pathogenic roles of the TNF-alpha-TNF receptor with a molecular weight of 55 kDa (TNF-Rp55) axis in APAP-induced hepatotoxicity using TNF-Rp55-deficient [TNF-Rp55-knockout (KO)] mice. When wild-type (WT) BALB/c and TNF-Rp55-KO mice were intraperitoneally injected with a lethal dose of APAP (750 mg/kg), the mortality of TNF-Rp55-KO mice was marginally but significantly reduced compared with WT mice. Upon treatment with a nonlethal dose (600 mg/kg), WT mice exhibited an increase in serum transaminase levels. Histopathologically, centrilobular hepatic necrosis with leukocyte infiltration was evident at 10 and 24 h after APAP challenge. Moreover, mRNA expression of adhesion molecules, several chemokines, interferon-gamma (IFN-gamma), and inducible nitric oxide synthase (iNOS) was enhanced in the liver. On the contrary, serum transaminase elevation and histopathological changes were attenuated in TNF-Rp55-KO mice injected with APAP (600 mg/kg). The gene expression of all molecules except for IFN-gamma and iNOS was significantly attenuated in TNF-Rp55-KO mice. Moreover, anti-TNF-alpha neutralizing antibodies alleviated liver injury when administered at 2 or 8 h after but not at 1 h before APAP challenge to WT mice. Collectively, the TNF-alpha-TNF-Rp55 axis has pathogenic roles in APAP-induced liver failure.