ETIOLOGY AND PATHOGENESIS

Much remains to be clarified about the pathogenesis of FILD. Therefore the concepts proposed in this chapter should be considered hypotheses that likely will need to be refined and revised in the future as clinical experience and scientific information expand. As with many noninfectious inflammatory diseases, it is unclear if FILD is due to a single cause, such as an inflammatory response to a certain stimulus (e.g., infection, xenobiotics, or various toxins), inappropriate triggering of an immunological response, or loss of self tolerance, or if the disease is due to a combination of these factors or a progression through various stages involving these mechanisms. The hepatic parenchyma is exposed to portal blood returning from the intestines. This portal blood contains increased levels of bacterial waste products, small numbers of bacteria, endogenous and exogenous toxins, various xenobiotic compounds, and potential food-derived antigens or metabolites acting as potential haptens or neoantigens with the local immune system (e.g., gut-associated lymphoid tissues, [GALT]).

The liver plays an important role in the body’s immune response. The defense mechanisms include innate immune cells (e.g., macrophages, natural killer T cells, and CD3+ T cells), hepatocellular metabolism (i.e., detoxification), biliary excretion of waste products (e.g., xenobiotics, enterotoxins, endotoxins, and others), the flushing action of bile flow, and elimination of microorganisms by endothelial cells and Kupffer cells. To defend against potentially toxic agents effectively and quickly, and to avoid launching a harmful systemic immune response against self, the liver needs to differentiate potential threats from nutrients. The ability for a differential recognition of pathogenic bacteria, normal flora, and self-related antigenic substances is critical to prevent an inappropriate activation of an inflammatory response in the biliary system.¹⁰

A complex theory concerning the pathogenesis of immune-mediated cholangiohepatitis has been described in the human medical literature. This theory involves many factors, including genetic and individual predispositions, the biosynthesis of a variety of soluble pathogen-recognition receptors (PRRs), previously generated toll-like receptors (TLR) that guide tolerance for the enteric flora, inappropriate loss of tolerance to enteric luminal antigens, development of biliary epithelial neoantigens and subsequent activation of complement components, and chemotaxis of inflammatory cells.¹¹ Attention currently is focused on understanding how TLRs and germline-encoded PRRs activate the innate immune system and assist the process of differentiating pathogens from nonpathogens. The discovery of the toll-like receptors helped to identify the innate immune receptors responsible for tolerance towards the normal flora and self-antigens. It is interesting that TLRs seem only to be involved in the cytokine production and cellular activation of the immune system in response to microbes; they do not play a significant role in the adhesion and phagocytosis of microorganisms.¹² Activated immune cells can produce cytokines, which in turn will trigger inflammatory responses. Bacterial pathogens can be phagocytosed, digested, and processed for antigen recognition. The antigens can be presented to CD4+ T cells that release further cell signaling substances and cytokines.¹³

One of the primary theories concerning the pathogenesis of cholangitis in cats is that the disease process involves ascension of enteric bacteria into the bile ducts via the sphincter of Oddi, which serves as a valve between the duodenum on one side and the pancreatic duct and the common bile duct on the other. This in part may explain the relationship between pancreatitis, cholangitis, and inflammatory bowel disease that appears to exist in some cats. The liver’s immune system plays an important role in controlling local innate immunity. The antigen-processing cells of the liver (e.g., Kupffer cells and dendritic cells), along with T cells of the GALT, are important in the development of immunological responses. Potentially aberrant immune responses directed against intestinal epithelial mucosal cells (as can result from failure of the mechanisms involving TLR and PRR) can lead to shared or similar antigenic recognition sites with the epithelial cells lining the biliary ducts, pancreatic ducts, and the intestinal mucosa.¹³

The interplay involving the misdirected immune response, the inflammatory reaction (e.g., mediated through cytokines and inflammatory cells), regenerative effects of hepatic tissue (e.g., through the action of hepatic growth factors, cytokines, and cellular signaling mechanisms), and progression to fibrosis is very complex. Progressive disease is associated with the development of fibrosis, sinusoidal capillarization, and alterations to structure and function of the lobular terminal hepatic venules. These latter changes also result in biliary hyperplasia or atrophy, cholestasis, and portal venous hypertension. The hepatocytes can be injured by activation of pathways that lead to apoptosis (premature programmed cell death). Some authors propose that unless there is an outright cause of hepatocellular necrosis (e.g., through primary hepatocellular injury), hepatocytes are only injured indirectly and that losses of hepatocytes result mostly from apoptosis. However, if the limiting plate is breached by inflammatory cells, further damage of hepatocytes is expected. This response to inflammation in the liver leads to a harmful microenvironment for hepatocytes via retention of bile acids, cell membrane injury, relative hypoxia, mitochondrial failure, and more shunting of blood to the terminal hepatic venules, which may in turn be compromised by fibrosis.^14,15 It has been postulated that CH begins to develop with a systemic infection or ascension of microorganisms via the biliary tree, followed by a response of the immune system that leads to inflammation. One hypothesis is that there is a progression from the acute neutrophilic form of the disease to the chronic neutrophilic form. This would be followed by a decreasing neutrophilic component and an increasing lymphocytic component, which would lead to the characteristic lesions of the lymphocytic form of FILD. The apparently less pathogenic form, LPH, might represent a less pronounced response of the liver to the everyday challenges of life because older cats commonly show some degree of LPH. Although this pathogenetic model is not documented in the literature, it is an interesting hypothesis to help explain the difficulty in understanding the overlaps seen between histopathological examination and clinical findings in the described forms of FILD.

There also is an ongoing effort to define small cell lymphoma involving the portal and biliary system because this condition can be confused easily with marked lymphocytic cholangitis. Determining the clonality of the involved lymphocytes may prove helpful in separating inflammation from lymphoma.

Many authors have suggested previously that bile is not normally sterile and that some degree of ascension of bacteria from the intestinal tract can occur even in normal cats.^16,17 However, there is contrasting evidence suggesting that bile within the gall bladder of normal cats usually is sterile when cultured for aerobic and anaerobic growth.¹⁸ In one study, a group of feline and canine patients with hepatic inflammation was evaluated and seven of 30 (23.3 per cent) patients had positive bile cultures but only six of 103 (5.8 per cent) had positive hepatic cultures. Escherichia coli, Enterococcus spp., Bacteroides spp., Streptococcus spp., and Clostridium spp. were the most common isolates identified.¹⁶ In another report, in six cats with cholangitis or cholecystitis, a similar pattern of bacterial cultures was identified, with E. coli being the predominant species. In addition, a Streptococcus organism and one Salmonella enterica serovar typhimurium also were cultured.¹⁸ In this study, concurrent pathological changes involving the pancreas and intestines were found in three of the six patients. However, these data do not provide any proof whether bacteria are secondary or primary agents in the pathogenic sequence of events. More studies are needed to clarify the true role of bacteria in the pathogenesis of FILD. Newer methodologies such as fluorescent in situ hybridization (FISH) are being used to detect and localize the presence of specific bacterial deoxyribonucleic acid (DNA) sequences in hepatic specimens. Primary sclerosing cholangitis (PSC) is a severe, progressive liver disease of human beings. It is similar in histopathological appearance to more advanced cases of feline LPH, in which fibrosis is marked and cholestasis is severe. The etiology and pathogenesis of PSC in human beings remains unknown.¹⁹ Also, as it does regarding LC and LPH in cats, controversy exists as to whether PSC in human beings should be considered an autoimmune disease.^20,21 A large number of autoantibodies have been identified in patients with PSC, suggesting that it is an autoimmune disease. However, the specificity of these antibodies generally is low, and the frequencies of the presence of these antibodies in affected patients vary greatly among different studies. In general, there appears to be a reversal of the common female gender predilection of many autoimmune disorders. It has been hypothesized that the presence of autoantibodies in patients with PSC may be the result of a nonspecific dysregulation of the immune system. It is interesting that the literature also points to the possible presence of specific antibody targets, including the biliary epithelium and neutrophilic granulocytes.¹⁹ This relationship gives further support to the hypothesized pathogenic relationship among infections, the suppurative response, and the lymphocytic response directed against both the suppurative neutrophilic cells and the biliary epithelium. This hypothesis is similar to an older concept that infection-related haptens bind with innate cell membranes, leading to neoantigen formation as a mechanism for loss of self-tolerance and development of autoimmunity within the liver. Major histocompatibility complex Class II (MHC II) molecules normally are expressed by the epithelium of biliary ducts and Kupffer cells. In addition, there also are portal and bile duct intraepithelial lymphocytes (CD3+ or T lymphocytes). In one study involving immunohistochemical examination, cats who were diagnosed with progressive lymphocytic cholangitis showed increased numbers of CD3+ lymphocytes in the periportal and portal tissue.¹⁷ There were fewer B cells forming aggregates or follicles within the most affected areas of lymphocytic infiltration. Low numbers of immunoglobulin A–positive plasma cells also were seen. It is interesting that both acute and chronic types of FILD had a similar composition of infiltrates. Expression of MHC class II was increased for the vascular endothelium and fibroblasts in areas of fibrosis, supporting the role of immunological stimulation.¹⁴

Chronic infections with organisms that are difficult to culture are suspected as one possible etiological cause for inflammatory liver disease in human beings and cats. Possible organisms being investigated in cats with cholangitis/cholangiohepatitis include common enteric bacteria, Helicobacter spp., and Bartonella spp.^22,23 Five Bartonella spp. have been shown to infect cats naturally (see Chapter 4 in the fifth volume of this series). However, in most instances, infections resolve spontaneously. In one study, DNA markers were used to look for the presence of Helicobacter sequences in hepatic tissue from cats with liver disease. DNA of Helicobacter spp. was found in hepatic tissue in two of 32 cats (6.3 per cent) with cholangiohepatitis and one of 16 cats (6.3 per cent) with noninflammatory liver disease.²² These findings also suggest that Helicobacter spp. identified in the liver are distinct from those isolated normally from the stomach in cats. Although the low incidence of infection with Helicobacter spp. in cats with FILD would suggest that this organism is not a major cause of FILD, it represents an example warranting further research regarding infectious organisms and a possible and chronic stimulation of the immune system. Bartonella henselae (the main causative agent of cat scratch disease in human beings) is interesting as another example for the possible pathogenesis of inflammatory liver disease. Experimental studies have shown that Bartonella can cause chronic intraerythrocytic and vascular endothelial infections of multiple organs, including the liver.²³ It has been shown that the organism can persist in the host and cause lymphocytic hepatitis in healthy cats who were infected experimentally with B. henselae and/or B. clarridgeiae (coinfections).²⁴ In another study, Helicobacter spp. DNA was identified in the bile from four of 15 cats (27 per cent) with LPH and from eight of 51 cats (16 per cent) without LPH.²⁵ However, these results are not definitive proof of a cause-and-effect relationship; rather they provide a basis for further study of the role of infectious organisms such as Helicobacter and Bartonella spp. in the pathogenesis of chronic lymphocytic infiltration of the liver.