Pathogenesis of alcoholic liver disease: interactions between parenchymal and non-parenchymal cells


  • Jessica I COHEN,

    1. Departments of Pathobiology
    2. Department of Nutrition, Case Western Reserve University, Cleveland, Ohio, USA
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  • Laura E NAGY

    Corresponding author
    1. Departments of Pathobiology
    2. Gastroenterology and Hepatology, Cleveland Clinic
    3. Department of Nutrition, Case Western Reserve University, Cleveland, Ohio, USA
      Laura E NAGY, Cleveland Clinic Foundation, Lerner Research Institute/NE-40, 9500 Euclid Avenue, Cleveland, OH 44195, USA. Email:
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  • Financial support: This work was supported in part by National Institutes of Health grant RO1AA011975, RO1 AA16399 and P20AA17069 to LEN and F31AA016434 to JIC.

Laura E NAGY, Cleveland Clinic Foundation, Lerner Research Institute/NE-40, 9500 Euclid Avenue, Cleveland, OH 44195, USA. Email:


The development of alcoholic liver disease (ALD) is a complex process involving both the parenchymal and non-parenchymal cells in the liver. The impact of ethanol on hepatocytes can be characterized as a condition of organelle stress with multifactorial changes in hepatocellular function accumulating during ethanol exposure. These changes include oxidative stress, mitochondrial dysfunction, decreased methylation capacity, endoplasmic reticulum stress, impaired vesicular trafficking and altered proteasome function. Injury to hepatocytes is attributed, in part, to ethanol metabolism by the hepatocytes. Changes in the structural integrity of hepatic sinusoidal endothelial cells, as well as enhanced inflammation in the liver during ethanol exposure are also important contributors to injury. Activation of hepatic stellate cells initiates the deposition of extracellular matrix proteins characteristic of fibrosis. Kupffer cells, the resident macrophages in the liver, are particularly critical to the onset of ethanol-induced liver injury. Chronic ethanol exposure sensitizes Kupffer cells to activation by lipopolysaccharides via toll-like receptor 4. This sensitization enhances the production of inflammatory mediators, such as tumor necrosis factor-α and reactive oxygen species that contribute to hepatocyte dysfunction, necrosis and apoptosis of hepatocytes and the generation of extracellular matrix proteins leading to fibrosis. In this review we provide an overview of the complex interactions between parenchymal and non-parenchymal cells in the liver during the progression of ethanol-induced liver injury.


Alcohol abuse is regarded as major contributor to health problems worldwide and is associated with over 60 diseases including hepatitis, cirrhosis and insulin resistance. Estimates suggest that about 18 million Americans abuse alcohol and that alcoholic liver disease (ALD) affects over 10 million people in the USA. In the USA alone, $166 billion a year is spent on the medical costs associated with alcohol abuse.1 According to the National Institute of Alcohol Abuse and Alcoholism, as of 2004, approximately 28 000 US residents died of alcoholic cirrhosis and alcohol is involved in approximately 40% of all fatal car accidents. As few as two drinks per day is associated with increased mortality2 and chronic abuse of alcohol leads to alcoholic liver disease in approximately 20% of alcoholics.3


Long-term alcohol consumption leads to liver injury. Liver injury progresses in a systematic order and is characterized by distinct pathological conditions: alcoholic fatty liver, alcoholic steatohepatitis, fibrosis and cirrhosis.1 Fatty liver, also known as steatosis, is the earliest sign of liver injury and is present in about 90% of people who consume alcohol.4 Lipid accumulation in hepatocytes and increased liver size are hallmarks of steatosis. Approximately 20% of alcohol abusers will progress to the next stage of liver injury, hepatitis. This stage is marked by inflammation, hepatocyte death by both necrosis and apoptosis and increases in circulating liver enzymes such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST). The clinical spectrum of alcoholic hepatitis can range from mild transaminase elevation on laboratory testing as the only indication of disease to severe liver dysfunction with complications such as jaundice, hepatic encephalopathy, ascites, esophageal varices, coagulopathy and coma. Like steatosis, mild stages of hepatitis can be reversed if alcohol consumption is ended. In contrast, in severe alcoholic hepatitis, reported mortality ranges from 30 to 60%.5

In about half of all patients with ethanol-induced hepatitis, as injury progresses, stellate cells are activated and produce excess amounts of collagen which leads to the development of fibrosis and cirrhosis. At this stage, excess extracellular matrix is deposited resulting in a loss of hepatic sinusoids, liver hardening and, in some cases, death.6 Progression to cirrhosis occurs at a variable rate and different studies have reported this outcome in 10 to 30% of affected patients with ALD. Alcoholic cirrhosis remains among the most common indications for liver transplantation worldwide.


A two-hit hypothesis has been suggested in the progression of ethanol-induced liver injury, with the first hit being ethanol exposure resulting in hepatic steatosis.5,7 Chronic ethanol exposure has profound effects on lipid homeostasis in the liver, decreasing fatty acid oxidation and increasing fatty acid synthesis, as well as suppressing triglyceride export from the liver.8,9 These changes in lipid metabolism culminate in the development of steatosis. The second hit occurs as a result of continued ethanol exposure, for example, increased oxidative stress as a result of ethanol metabolism. Additionally, co-morbid conditions, such as obesity or viral hepatitis, can act as the second hit and exacerbate ethanol-induced liver injury.

Similar to the two-hit hypothesis, the sensitization and prime theory suggests that the alcohol consumption sensitizes the liver and makes it more susceptible to injury by a secondary risk factor.10 The impact of ethanol metabolism on hepatocyte function is a likely contributor to sensitization of the liver. ‘Prime’ refers to an early event that induces or activates a mechanism that is responsible for the damage to the liver. For example, after chronic ethanol exposure, the Kupffer cell, the resident macrophage in the liver, becomes sensitized to endotoxins, resulting in the induction of inflammatory cytokines and reactive oxygen species (ROS) leading to liver injury.11


Ethanol is primarily metabolized in the liver by the enzyme alcohol dehydrogenase (ADH). The metabolism of ethanol by ADH results in the formation of acetaldehyde and the production of reducing equivalents via the conversion of nicotinamide adenine dinucleotide (NADH) to reduced nicotinamide adenine dinucleotide.12 (Fig. 1). This pathway is responsible for most ethanol metabolism when there is a low blood alcohol concentration due to the low Km of ADH, approximately 0.2–2 mmol/L. The formation of the toxic metabolite acetaldehyde induces oxidative stress in the liver and results in impaired mitochondrial function.13 The increased production of NADH disrupts the redox balance in the liver impairing gluconeogenesis.12 Acetaldehyde is metabolized by the enzyme acetaldehyde dehydrogenase to produce acetate, a substrate in the Krebs cycle that can generate energy.13

Figure 1.

Major pathways of ethanol metabolism in the liver. NAD, nicotinamide adenine dinucleotide; NADH, reduced nicotinamide adenine dinucleotide.

The second major pathway of ethanol metabolism is the microsomal ethanol-oxidizing system, which is catalyzed by the enzyme cytochrome P450 2E1 (CYP2E1)12 (Fig. 1). Like ADH, CYP2E1 is found predominantly in the hepatocyte14 but several studies have reported increased CYP2E1 expression in Kupffer cells after chronic ethanol exposure.15,16 This pathway is responsible for the metabolism of excessive amounts of ethanol due to a high CYP2E1 Km of 10–15 mmol/L.12 CYP2E1 is increased fourfold to 10-fold in the liver of humans and rodents after chronic ethanol exposure.17 In animal and cell models, ethanol exposure increases CYP2E1 synthesis and decreases degradation both in vivo and in vitro.18,19 In addition to ethanol, CYP2E1 also metabolizes pharmaceuticals such as acetaminophen,17 anesthetics and industrial solvents.12 The metabolism of ethanol by CYP2E1 generates ROS, which react with proteins and lipids leading to oxidative damage.14

The third pathway of ethanol metabolism is non-oxidative and is mediated by fatty acid ethyl ester (FAEE) synthase, and results in the production of fatty acid ethyl esters. FAEEs are esterification products of ethanol and fatty acids and are considered cytotoxic.20 The liver and pancreas, organs commonly damaged by alcohol abuse, have the highest concentrations of FAEE and FAEE synthase.21–23 In the serum, FAEE are clinical markers of both acute and chronic ethanol consumption because of their presence after ethanol is cleared from the circulation20


Organelle stress in hepatocytes

Hepatocytes are a primary target of the toxic effects of ethanol in the liver. Chronic ethanol exposure impairs the function of a variety of metabolic and detoxification pathways in the liver and also has a profound impact on the activity of a number of organelles in the liver (Fig. 2). One critical target of ethanol toxicity in the liver is the mitochondria. Chronic ethanol exposure impairs mitochondrial function at multiple levels,24 resulting in impaired bioenergetics,25 increased ROS production,26 damage to mitochondrial DNA and inhibition of mitochondrial protein synthesis,27,28 abnormal transport of glutathione29 and increased sensitivity to mitochondrial permeability transition.30 Chronic ethanol exposure also results in endoplasmic reticulum stress31 and decreases the activity of the proteasome.32,33 Vesicular trafficking in the hepatocyte is disrupted, associated with impaired Golgi trafficking and receptor-mediated endocytosis, which is likely to be due to a disruption of the microtubule function.34–39 Finally, recent studies have demonstrated that chronic ethanol exposure also dysregulates the precise control of histone acetylation.40,41 The cumulative effects of ethanol-induced organelle stress in hepatocytes contributes to impaired metabolic and detoxification activity and sensitizes the hepatocyte to cell death via necrotic and apoptotic pathways.42

Figure 2.

Illustration of intracellular organelles in hepatocytes targeted by ethanol.

Structural changes in hepatic sinusoidal endothelial cells

Sinusoidal endothelial cells represent a specialized endothelial cell group characterized by fenestrations that facilitate the flux of macromolecules and red blood cells into the Space of Disse.43 Very little is known about the direct or indirect effects of chronic ethanol on hepatic sinusoidal endothelial cells, or both, although it is clear that ethanol exposure can result in a loss of fenestrae.44

Role of immune responses in the initiation and progression of alcoholic liver disease

The innate and adaptive immune systems are two distinct branches of the immune response, yet these two components of immunity are intimately linked at many stages of an organism's response to injury or stress. Components of the innate immune response, including nature killer (NK) and NK T-cells,45 Kupffer cells (resident hepatic macrophages)46 and the complement system,47,48 as well as T-cells and antibody-dependent adaptive immune responses,49 are involved in the hepatic response to various types of injury, including bacterial and viral infections, exposure to toxins (including ethanol), partial hepatectomy and ischemia-reperfusion. The role of Kupffer cells in response to injury is most well understood (discussed below). Chronic ethanol decreases NK cell activity.50,51 NK cells are thought to participate in the killing of hepatic stellate cells (HSC) during early activation, as well as acting as important effector cells in killing virally infected cells via the release of granules containing granzyme and perforin, as well as death ligands such as tumor necrosis factor-related apoptosis-inducing ligand, and a variety of inflammatory cytokines.52 In contrast, NK T-cells are activated early in the response to ethanol feeding and play a key role in the defensive mechanisms of the innate immune response in the liver.45

Increased activation and sensitivity of Kupffer cells, the resident macrophage in the liver

Long-term exposure to ethanol results in commensal bacterial overgrowth in the jejunum.53 The increase in microflora, coupled with a disruption of the barrier function of the small intestine in response to ethanol, increases the translocation of gram-negative bacteria into the portal circulation.54 The concentration of endotoxin is increased in the blood of both rats and mice exposed to ethanol via intragastric feeding, as well as in humans in response to alcohol consumption.55–57 In rats, when gram-negative bacteria are depleted by treatment with antibiotics the effects of ethanol on liver injury are reduced.58

The first capillary bed through which the portal blood passes carries endotoxin or lipopolysaccharide (LPS) into the liver. Kupffer cells detoxify this blood and clear LPS from circulation. LPS activates Kupffer cells, leading to the production of inflammatory mediators, including cytokines, such as tumor necrosis factor (TNF)-α, and ROS. Kupffer cells are thought to be critical to the progression of ethanol-induced liver disease. Treatment with the macrophage toxin gadolinium chloride depletes Kupffer cells and prevents ethanol-induced liver injury in rats.59 LPS-mediated signaling is also essential for the progression of ethanol-induced liver injury. LPS signaling occurs when LPS and LPS binding protein (LBP) attach to the cell surface receptor CD14. CD14, interacting with toll-like receptor 4 (TLR-4), comprise two major components of the LPS receptor complex. The activation of the LPS receptor complex stimulates a number of signal transduction cascades.60 Mice that lack LPS receptor components (CD14, TLR-4, LBP) are protected from chronic ethanol-induced liver injury after intragastric ethanol exposure61–63

TLR-4 signaling is mediated via MyD88-dependent and independent pathways.60 While the rapid LPS-stimulated expression of TNF-α is characteristic of MyD88-dependent signaling, MyD88-independent signals are more slowly activated and result in the increased expression of type I interferon (IFN) and IFN-dependent genes.60 Chronic ethanol exposure sensitizes Kupffer cells to TLR-4-MyD88-dependent responses, such as the rapid activation of mitogen-activated protein kinases and nuclear factor κB as well as TNF-α expression.46 TNF-α is elevated in the blood of rats64 and humans65 in many models of chronic ethanol exposure. TNF-α receptor I (TNFR1) is the surface receptor responsible for the signaling capabilities of TNF-α. TNFR1-deficient mice64 and mice treated with polyclonal anti-mouse TNF-α rabbit serum i.v.66 do not exhibit signs of ethanol-induced liver damage. Recent data suggest that the MyD88-independent/Toll-interleukin-1 receptor domain-containing adapter IFN-β (TRIF)-dependent pathway of TLR-4 signaling is also a key contributor to chronic ethanol-induced liver injury.67,68 However, very little is known about the impact of chronic ethanol on the regulation of MyD88-independent signaling. Taken together, these studies indicate that both LPS-TLR-4 signaling and Kupffer cell activation is critical in the progression of ethanol-induced liver injury.

Activation of HSC

Activation of HSC, the primary source of increased collagen expression in the injury liver, is a hallmark of fibrosis.69 Fibrosis, or scarring, in the liver is characterized by excessive extracellular matrix (ECM) deposition, resulting from both increased ECM protein synthesis and decreased degradation.

In normal liver HSC have an adipocyte-like phenotype containing vitamin A-rich cytoplasmic lipid droplets.70 Signals generated in response to liver injury prime quiescent HSC; subsequent signals then induce their transdifferentiation from a quiescent to an active phenotype. Pro-fibrogenic signals such as platelet-derived growth factor stimulate HSC proliferation and migration to areas of injury. Additional autocrine and paracrine signals, including transforming growth factor-β (TGF-β) and connective tissue growth factor stimulate HSC activation and fibrogenesis. The fibrotic phenotype of the activated HSC is characterized by excessive type I collagen synthesis, as well as the development of contractile capacity.70 Stellate cells are maintained in their active state both by the sustained activity of initiation signals, along with a number of additional autocrine and paracrine mediators produced by HSC and other cell types in the liver.


HSC contribute to angiogenesis in the liver, both during development and in response to injury. HSC serve as liver-specific pericytes in angiogenesis;71 however, their role as pericytes in response to liver injury may differ from that of typical pericytes.72 This difference, along with the two different types of microvasculature (the hepatic sinusoids, as well as larger vessels), gives unique characteristics to angiogenesis in the liver.72 While the role of HSC in angiogenesis has been well studied in liver regeneration, there is a growing appreciation of the critical role of HSC-mediated angiogenesis during the development of fibrosis in response to liver injury.71 Inappropriate angiogenesis is associated with impaired hepatic sinusoidal remodelling during repair, contributing to the capillarization of sinusoidal endothelial cells, as well as shunting between sinusoidal vessels.73 The formation of functional vessels involves tightly regulated cellular and molecular networks, with the cytokines vascular endothelial growth factor (VEGF), TGF-β and hepatocyte growth factor acting as critical regulators of angiogenesis. Angiopoietin-1, an angiogenic cytokine required for vascular remodelling, is secreted by HSC.74 The inhibition of angiopoetin-1 signalling ameliorates liver fibrosis in response to chronic carbon tetrachloride (CCl4) or bile duct ligation.74

Interactions between parenchymal and non-parenchymal cells in the development of ALD

The pathophysiology of fibrosis involves complex interactions between the different cell types in the liver.69 Cellular components of the innate immune system in the liver are also common elements in the development of liver fibrosis in response to a variety of insults. Kupffer cell depletion attenuates fibrosis induced by the commonly used model of CCl4-induced liver injury.75 Infiltrating neutrophils, NK T-cells, T-cells and B-cells,52 as well as hepatocytes, sinusoidal and vascular endothelial cells contribute to fibrosis.69 In contrast, NK cells suppress fibrosis in the liver.51

The production of inflammatory cytokines is a critical step in the fibrogenic response to injury. Additional mediators include reactive oxygen and nitrogen species, as well as apoptotic cell bodies.69 Each of these mediators is activated during ethanol exposure.46,76 Indeed, there is a growing appreciation that many of the critical elements in ethanol-induced inflammation in the liver are also required for fibrosis. For example, the expression of TLR-4, the receptor for LPS, is required for ethanol-induced steatosis and inflammation.46 Recent studies have also identified a role for TLR-4 signaling on HSC and in the development of fibrosis.77,78 Recent data demonstrate that ethanol also suppresses the NK function in the liver, attenuating the anti-fibrotic role of NK cells.51


Chronic alcohol abuse is one of the most common causes of liver disease and may also interact with additional environmental or genetic factors, or both, to accelerate the progression of fibrosis and liver injury. For example, alcohol consumption can accelerate the fibrotic response to hepatitis C in humans and to CCl4 in animal models.51,79,80 Neither the mechanisms for alcohol-induced fibrosis nor its ability to interact synergistically with other hepatotoxins to accelerate fibrosis are well understood. The pro-fibrotic effect of alcohol is likely to be the result, at least in part, of the increased vulnerability of hepatocytes in the alcohol-injured liver. Additional molecular mechanisms are also likely to contribute to the acceleration of fibrosis by ethanol. For example, ethanol-induced increases in LPS, via interaction with TLR-4 on Kupffer cells and HSC, stimulate inflammation and HSC activation. Ethanol-induced oxidative stress, the generation of highly reactive aldehydes or apoptosis, or both, may also accelerate fibrosis.81 Further understanding of the complex impact of ethanol on the pathogenesis of fibrosis is needed in order to facilitate the development of therapeutic interventions for patients with ALD.