HANS POPPER HEPATOPATHOLOGY SOCIETY

The Pathogenesis Of Alcoholic Liver Disease

Samuel W. French
Department of Pathology
Harbor-UCLA Medical Center
Torrance, California

It would be presumptuous to claim that the pathogenesis of alcoholic liver disease is understood. Most studies done so far have focused on the early changes in the liver observed over one or two months of alcohol feeding in rats or mice. More chronic studies utilized primates or pigs. Cirrhosis is rarely observed experimentally except in primates1 and this result has not been confirmed in other laboratories.

Studies in man using repeated liver biopsies do show positive correlation between the progression of the disease and the dose of alcohol consumed daily and the duration of alcohol consumption. Women required a lower dose of ethanol to develop cirrhosis. The type of alcoholic beverage and the type of meat consumed also influences the development of cirrhosis.2 Follow-up studied done on German veterans clearly showed a progression from the initial fatty liver stage to the stage of alcoholic hepatitis and to the final stage of cirrhosis3 depending on the dose of alcohol consumed. Of course, all three stages may co-exist. In one large VA study of 281 patients with alcoholic hepatitis 26 had fatty liver, 106 had acute alcoholic hepatitis, 39 had cirrhosis and 111 had both alcoholic hepatitis and cirrhosis.4 The histologic changes included 96.8% showed fatty change, 3.8% showed cholestasis, 85.3% showed limiting plate erosion, 76.2% showed Mallory body formation and 9.6% showed central hyaline sclerosis. At the alcoholic hepatitis stage 97% showed fatty change, 57% showed apoptosis, 78% showed Mallory bodies, 32% showed megamitochondria, 58% showed inflammation. The inflammation was predominantly PMN's in the regions of necrosis and Mallory body formation.5 Although many of these changes have been observed in experimental animal models of alcoholic hepatitis, progression to cirrhosis has been seen only rarely.6

Experimental studies
Investigations where animals are fed ethanol for 1 to several months have shown that the mechanisms of liver injury caused by ethanol ingestion are numerous and this indicates that the pathogenesis of alcoholic liver disease (ALD) is complex and, multifactorial. Liver injury is minimal if ethanol is fed for less than 15 days probably because it takes this long to induce cytochrome P450 2E1 (CYP2E1). Because of this fact it is likely that the induction of CYP2E1 is one key player in the pathogenesis of the initial liver injury (see Figure 1).

1. CYP2E1 induction
CYP2E1 is induced by ethanol primarily by stabilizing the CYP2E1 protein. CYP2E1 is turned over by the ubiquitin-proteasome pathway which is inhibited by ethanol. CYP2E1 knockout mice do not show this inhibition of the proteasome in chronic ethanol fed animals indicating that CYP2E1 is responsible for the inhibition of the proteasome.7

CYP2E1 induction by ethanol plays an important role in ALD pathogenesis because of the free radicals generated when it oxidizes ethanol to acetaldehyde. Free radicals generated include superoxide and hydroxyethyl radical. These free radicals generate reactive intermediates such as hydroxyethyl radical (HE) and products of lipid peroxidation which form adducts with proteins such as malondialdehyde (MDA) and 4-hydroxylenoneal (4HNE).8,9,10,11 The protein adducts such as HE-CYP2E1 act as neoantigens which stimulate antibody formation and immunocyte dependent liver cell damage.8 Circulating antibodies to adducts of CYP2E1 are present in alcoholics with liver disease as well as rats fed ethanol12 (Figure 1).

2. Fatty Acid Metabolism
CYP2E1 induction alters fatty acid metabolism, which leads mainly to a decrease in arachidonic acid through several mechanisms (Figure 1). Some arachidonic acid is lost through lipid peroxidation by free radicals generated by CYP2E1. Some is used by cyclooxygenase-2 (COX-2) in prostaglandin synthesis. COX-2 expression is increased by free radicals generated by CYP2E1. COX-2 gene expression is increased at low blood ethanol levels (BAL) in the rat intragastric fed rat model of ALD13 (Figure 2). Alcohol ingestion also reduces arachidonic acid synthesis by inhibiting delta 5 and 6 desaturase activity. Lastly, arachidonic acid and other fatty acids are diminished through hydroxylation by CYP2E1 and 4A.14,15 The products of hydroxylation (hydroxides and epoxides) are biologically active agents which modulate blood flow.

3. Oxidative Damage
Chronic ethanol feeding of rats increases oxidized proteins in the liver.16 This does not occur in CYP2E1 knockout mice fed ethanol.7 This indicates that the increased oxidation of cytoplasmic proteins in the liver results from free radicals generated by CYP2E1 oxidation of ethanol (Figure 1) which may overwhelm the damaged protein disposal by the 20S proteasome system which is inhibited by ethanol.17,18

4. Kupffer cell-Stellate cell activation
Endotoxin is increased in the blood after chronic ethanol ingestion. The concentration achieved is dependent on the BAL.19 Endotoxenemia activates the Kupffer cells which then express numerous cytokines, chemokines and free radicals which initiate a local inflammatory response in the liver sinusoid20 (Figure 3). The Kupffer cell is the origin of TGFb which activates the stellate cell to produce collagen21 (Figure 1). This activation is presumably the causative interaction which leads to pericellular fibrosis in alcoholic hepatitis (Figure 3). This process is initiated very late (many months) in the course of experimental liver fibrosis in rats fed ethanol.22

5. Role of Liver Hypoxia
Centrilobular fibrosis is in part the result of hypoxia which occurs in zone 3 of the liver lobule when high BAL is achieved.19 (Figure 1 and Figure 2). Centrilobular hypoxia has been documented by a variety of means including the measurement of O2 tension and measurement of liver ATP by NMR in vivo in rats and by HPLC in rats.23,24 The ischemic necrosis followed by healing leads to focal centrilobular fibrosis as in scar formation.22 Centrilobular hypoxia initiates multiple other processes through the activation of the transcription factor H1F-1a (Figure 2). These factors include increased expression of VEGF and iNOS. iNOS generates NO which reacted with O2 generated by CYP2E1 to form peroxynitrate which forms adducts with proteins at tyrosine residues. The level nitrotyrosine in the liver is increased as are liver nitrates. Hypoxia also increases serum lipopolysaccharide (LPS) which stimulates Kupffer cells to release macrophage chemoattractant protein (MCP-1) (Figure 2) and inflammatory cytokines and chemokines (Figure 2), most notably TNFa (Figure 1). The latter may induce apoptosis of hepatocytes. IL-8 which is chemotaxic for PMNs is released by neighboring hepatocytes in response to TNFa from activated Kupffer cells (Figure 3).

Other important consequences of hypoxia includes a shift in the redox state of the liver cells, which increases NADH and decreases NAD+ catalyzed reactions. Lack of O2 inhibits fatty acid b oxidation by the mitochondria which leads to fatty liver (Figure 1). When BAL falls reoxygenation injury occurs in the ischemia damaged liver (Figure 2). Mitochondria damage caused by free radical generation is partially prevented by the induction of MnSOD. However, glutathione (GSH) is reduced in the mitochondria as the result of ethanol reduction of methyl donor metabolism (methyl transferase activity reduction) and inhibition of GSH transport from the cytoplasm into the mitochondria. Cox-2 is induced which increases prostaglandin (PGE-2) synthesis (Figure 1 and Figure 2). Growth factors are upregulated which stimulate liver cell regeneration.25,26

6. Role of Immunologic Synapse
"Piecemeal" necrosis is a common feature in alcoholic hepatitis.27 T cells home to and are sequestered in the liver28,29 presumably as a consequence of activation of T cell receptors on endothelial cells in the portal tracts30 and attachment to target liver cells through the formation of immunological synapses.31 CD4 cells predominate but CD8 cells are also sequestered.32 Expression of class II MHC molecules is enhanced and this correlates with hepatocellular necrosis and Mallory body (MB) formation.33 Damage to hepatocytes by the lymphocytes that form an immunological synapse is thought to be due to endocytosis of the synapse which overtime reduces the hepatocyte to the point that it ultimately disappears.34

7. Mechanism of MB formation
MB formation is an important component of alcoholic liver disease even in the fatty liver stage. Ethanol fed mice form MBs35 if the mice are first fed a porphyrinogenic drug which transforms groups of hepatocytes. This suggests that gene expression changes must first occur before hepatocytes can form MBs. This idea is supported by the fact that MBs can be formed by hepatocellular carcinomas. It now appears that MBs form in the same way that aggresomes form in cells containing mutant or misfolded proteins.36 First, CK18 and 8 become hyperphosphorylated (Figure 1). This step can be shown using phosphatase inhibitors such as ethanol or okadaic acid. Hyperphosphorylation leads to conformational changes in the cytokeratins as shown by infrared spectra deconvolution studies. Ubiquitin covalent binding occurs in response to the misfolding of the CK proteins. A mutant ubiquitin forms as a consequence of molecular misreading at the time of transcription so that both normal ubiquitin and mutant ubiquitin coexist in the same cell. Mutant ubiquitin inhibits the deubiquitination step of proteolysis of the CK proteins by the proteasome. Consequently, the altered CK proteins accumulate and form aggresomes which move to a perinuclear region of the cells next to the centrisome. At this point the aggresome (MB) is visible and stains positive with antibodies to CK8, CK18, ubiquitin, mutant ubiquitin, transglutaminase, protreasome subunits, tubulin and HSPs 70 and 90. Transglutaminase further covalently cross-links the aggregated proteins which form the MB making the MB insoluble and resistant to proteolysis.

8. Mechanism of periportal fibrosis
Although fibrosis usually starts centrilobularly in alcoholic liver disease, sooner or later periportal fibrosis appears as a result of bile duct metaplasia at the limiting plate of hepatocytes. The mechanism of periportal fibrosis is quite different from the mechanism of centrilobular and pericellular fibrosis in this way. Since bile duct metaplasia recapitulates the embryologic mechanism of bile duct formation from the limiting plate of hepatocytes37 it's not surprising to find stellate cells surrounding and supporting the metaplastic bile ducts. The transformed liver cells involved in forming bile ductules were originally flanked by stellate cells. The stellate cells, in turn, form a collar of collagen around the metaplastic ducts (Figure 4). This provides the collagen which leads to the periportal scarring.38

Figures  (Click to enlarge)


Figure 1
Figure 2
Figure 3
Figure 4

References

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