Serum cytokines as biomarkers of disease and clues to pathogenesis

Authors

  • Cara L. Mack

    Corresponding author
    1. Pediatric Liver Center and Liver Transplant Program, Section of Pediatric Gastroenterology, Hepatology and Nutrition, The Children's Hospital, University of Colorado at Denver Health Sciences Center, Denver, CO
    • Pediatric Liver Center and Liver Transplant Program, Section of Pediatric Gastroenterology, Hepatology and Nutrition, The Children's Hospital, University of Colorado at Denver Health Sciences Center, Denver, CO
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  • See Article on Page 180.

  • Potential conflict of interest: Nothing to report.

Cytokines are polypeptides produced mainly by cells of innate and adaptive immunity. These proteins are often pleiotropic, whereby one cytokine can have multiple effects on diverse cell types. For example, interleukin-4 (IL-4) promotes the naive T cell to differentiate into a Th2 effector cell, stimulates the B cell to produce immunoglobulin E, and inhibits the macrophage from becoming activated. Cytokines also exhibit redundancy, and many cytokines can have identical or overlapping functions. For instance, IL-2, IL-4, and IL-5 all stimulate B cell proliferation. Furthermore, cytokines may act synergistically [interferon-γ (IFN-γ) and tumor necrosis factor α (TNF-α) together enhance major histocompatibility complex class I cellular expression] or antagonistically (IFN-γ activates macrophages and IL-10 inhibits them).1 All in all, cytokines are powerful mediators of inflammation and microbial elimination. Most cytokines act in close proximity to where they are produced within the diseased target organ; however, the generation of large amounts of cytokines may lead to entrance into the circulation and detection within the serum. Measurements of serum cytokine levels can be used as biomarkers of disease severity and provide clues to the mechanisms of inflammatory mediated liver injury.

Abbreviations

BA, biliary atresia; ICAM, intercellular adhesion molecule; IFN-γ, interferon-γ; IL, interleukin; KP, Kasai portoenterostomy; PBC, primary biliary cirrhosis; sICAM, soluble intercellular adhesion molecule; TNF-α, tumor necrosis factor α.

Levels of serum cytokines as biomarkers of disease progression or severity have been analyzed in many adult chronic liver diseases. An analysis of serum cytokines in patients with a variety of chronic liver diseases revealed significant elevations in IL-1β, IL-6, and TNF-α in patients with cirrhosis versus patients without cirrhosis.2 In alcoholic steatohepatitis, serum levels of IL-1, IL-6, TNF-α, and IL-8 increased during acute decompensation of the liver disease, and patients with the highest concentration of serum cytokines had the highest rate of in-hospital mortality.3–5 A comparison of serum cytokine and chemokine levels between patients with nonalcoholic fatty liver disease and nonalcoholic steatohepatitis demonstrated significantly higher levels of TNF-α and macrophage chemoattractant protein-1 in the patients with steatohepatitis.6 A recent multicenter study of 778 chronic hepatitis C patients correlated the level of serum cytokines with the degree of hepatic inflammation and fibrosis.7 A positive correlation was found between the serum TNF-α levels and the degree of hepatic inflammation in patients without cirrhosis versus patients with cirrhosis. In addition, TNF-α induces IL-8 production, and IL-8 was found to be significantly increased in those patients with higher histological activity and continued to increase in patients with cirrhosis. This study concluded that serum cytokines are useful biomarkers of intrahepatic inflammation and could be used to screen for disease progression in chronic hepatitis C infection.

Serum cytokine levels have also been analyzed in autoimmune liver diseases. Serum IL-18 levels correlated with disease severity and progression in primary biliary cirrhosis (PBC) and autoimmune hepatitis.8 The IL-18 levels in PBC positively correlated with serum bilirubin concentrations and fell promptly after liver transplantation. In another study analyzing serum cytokines in PBC, TNF-α was significantly higher in patients with advanced disease versus patients with early disease, and TNF-α decreased substantially in response to ursodeoxycholic acid therapy.9 All the serum cytokines that were found to correlate with disease progression in these studies of chronic liver disease are components of innate immunity. Macrophages are a major cell type within innate immunity and are the principal source for the production of TNF-α, IL-1β, IL-6, and IL-18. One can conclude that the innate immune system is highly stimulated in a variety of chronic liver diseases.

The measurement of serum cytokines has also been performed to provide clues to the underlying mechanisms of inflammatory injury in chronic liver diseases. To attempt to correlate the specific serum cytokine being analyzed with disease pathogenesis, one must study the target organ, such as the liver, concurrently. For example, TGF-β serum levels were found to be elevated in active autoimmune hepatitis, and a concurrent immunohistochemical analysis of liver tissue revealed strong TGF-β expression on infiltrating immune cells and hepatocytes.10 Subsequently, both the TGF-β serum levels and tissue expression normalized during disease remission, and this suggested that TGF-β is an important mediator of liver injury in active autoimmune hepatitis. Krams et al.11 found significant increases in serum IFN-γ and IL-5 in PBC patients and had previously reported that messenger RNA transcripts for these cytokines were up-regulated in the livers of PBC patients.12 The serum IFN-γ and IL-5 levels correlated positively with increased levels of soluble CD30. CD30 is a cell surface protein induced on activated memory T cells, and these CD30+ T cells are known to produce both IFN-γ and IL-5 and to provide enhanced help for B cell immunoglobulin production. The authors speculated that CD30+ T cells might regulate autoantibody and cytokine production (IFN-γ and IL-5) that contributes to the autoimmune liver damage found in PBC.

In this issue of HEPATOLOGY, Narayanaswamy et al.13 seeks to establish the course of the inflammatory process in biliary atresia (BA) as expressed by plasma cytokine levels and to determine if these cytokines are accurate biomarkers of the severity of the disease. A thorough analysis of the plasma levels of Th1 cytokines (IL-2, IFN-γ), Th2 cytokines (IL-4, IL-10), macrophage-generated cytokines (IL-18, TNF-α), and soluble adhesion molecules [soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular cell adhesion molecule 1] from infants with BA at the time of Kasai portoenterostomy (KP) and serially thereafter for 6 months was performed. In addition, plasma from other neonatal cholestatic diseases and normal controls were available for comparison with BA at baseline (before KP). No significant differences in the baseline cytokine levels were found between BA and normal controls; however, IL-2 and IL-10 were significantly higher in comparison with other cholestatic disease controls. With respect to soluble adhesion molecules, sICAM-1 levels were substantially higher in BA plasma at baseline in comparison with normal or other cholestatic disease controls. Within the first 6 months after KP, all plasma cytokine and adhesion molecule levels increased significantly, with the exception of IL-10. The authors have surmised that the inflammatory process in BA is progressive and involves both nonpolarized T cell and macrophage immune responses that are not ameliorated by portoenterostomy.

An analysis of the immune modulators as biomarkers of disease severity led to the finding that sICAM-1 is a candidate plasma biomarker for BA severity. Intercellular adhesion molecule 1 (ICAM-1) is an adhesion molecule expressed on the surface of cells of multiple lineages at sites of inflammation and mediates leukocyte adhesion to endothelial cells, granulocyte migration through the endothelium, and antigen-induced T cell proliferation. Previous investigations have shown increased expression of ICAM within BA liver tissue14 and a significant correlation of the age at surgery with the sICAM serum levels.15 In this study, 1, 3, and 6 months after KP, the total serum bilirubin level was significantly positively correlated with the sICAM-1 level. Moreover, for those children who underwent transplantation by 1 year of age, the sICAM-1 levels were consistently elevated beginning 1 month after KP. It was determined that a cutoff level of sICAM-1 of 1779 ng/ml would predict the need for transplantation in the first year of life (87% sensitivity and 92% specificity). This work provides strong evidence for the use of plasma sICAM levels as a measure of disease progression and outcome in BA.

BA is characterized by progressive inflammation and fibrosis of both the extrahepatic and intrahepatic bile ducts. Because of the progressive nature of the disease, approximately 80% of patients with BA will require liver transplantation, accounting for half of all pediatric liver transplants.16 The cause of BA is not known, and immune-mediated mechanisms of bile duct injury have been proposed.17, 18 Numerous investigations have focused on the immune response surrounding the intrahepatic bile ducts. The portal tract infiltrates surrounding bile ducts are composed of both CD4+ and CD8+ T cells and macrophages.19–21 These lymphocytes have been shown to invade between bile duct epithelia, leading to degeneration of intrahepatic bile ducts.22 The T cells are highly activated, expressing the proliferation cell surface marker CD71 and activation markers CD25 and lymphocyte function–associated antigen 1 (a receptor for ICAM)19 and secreting Th1 cytokines (IL-2, IFN-γ, and TNF-α).21, 23 Narayanaswamy et al.13 eloquently revealed the immune response within the plasma of BA patients and identified a biomarker of disease severity (sICAM). This study did not investigate liver or bile duct remnant tissue concurrently, and therefore one is not able to make conclusions regarding the role of these immune modulators in disease pathogenesis. Future research efforts should utilize both murine models of BA24–26 and human tissue, focusing on the role of each of these cytokines in bile duct injury in order to identify potential targets for immunomodulatory therapies.

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