Mast cells and the liver aging process

Aging and the immune system

Aging is associated with many physiological changes in a variety of organs [12]. It is today recognized that many diseases observed in the elderly have an immunological basis and are associated with the decline of immune response [13]. Clinically, the consequences of impaired immune function in the elderly include an increased susceptibility to infections, malignancies and autoimmunity [14–16]. It has been demonstrated that aging leads to the replacement of unprimed virgin T-lymphocytes by primed memory T-lymphocytes subpopulations and to the accumulation of cells with signal transduction defects [17]. T-lymphocytes are more severely affected than B-lymphocytes. This is mainly due to the involution of the thymus, which is almost complete at the age of 60 [18]. In humans, the thymus is a central lymphoid organ devoted to thymocyte differentiation and maturation, and is therefore the primary source of circulating T-lymphocytes. Although its size continues to increase until it reaches its maximum absolute weight during puberty, its functional compartments and T-lymphocytes output activity diminish after the first years of life onwards. Although it continues to serve as the site of T-lymphocytes differentiation and maturation throughout adulthood, the thymus undergoes a process known as involution, which is defined as a decrease in size, weight and activity of the gland with advancing age [19,20].

Other immunological cells, including macrophages, neutrophils, natural killer (NK) cells, and NKT lymphocytes, are affected by aging [21]. In addition, it has been shown that aged dendritic cells (DCs) are less able to stimulate T and B-lymphocytes, although the results of studies in humans and rodents focusing on DCs and aging are still conflicting.

Functional defects and altered frequencies of innate and adaptive immune cells impair local responses at the site of vaccine injection, hamper the generation of primary responses to neo-antigens, prevent the effective induction of memory lymphocytes, and decrease the effect of booster vaccination. As a result, antibody responses of elderly vaccines are weaker and decline faster, and long-term protective effects of vaccination cannot be taken for granted in elderly peoples [13,22–24]. Evidences exists that immunization procedures are less effective at older age than in young adults. Influenza virus vaccines have generally proved limited in preventing morbidity and mortality among the elderly because of the lower immunological protection that they may confer on older adults compared to younger persons [25]. Additionally, the measure of anti-hepatitis virus and anti-Hepatitis B surface antibodies (anti-HBs) in elderly people after a combined hepatitis virus A and B vaccination has underlined the decreased response to vaccination with increasing age [25].

It is now accepted that aging is characterized by a peculiar state of chronic inflammation (the so-called “inflamm-aging”) under genetic control, which seems to be a consequence of a person’s life-long antigenic load [26–30] and leads to a long-term tissue-related risk of all-cause mortality in older people [31,32]. A number of studies have highlighted that chronic inflammation is the underlying biological mechanism responsible for the decline in physical function observed in the elderly [33].

It has also been shown that aging is accompanied by a 2-4-fold increase in the plasma levels of inflammatory mediators, cytokines and acute-phase proteins [34–37]. Additionally, human aging is associated with a deregulated cytokine response following stimulation: chemotaxis and phagocytosis, as well as antigen processing and presentation, are all depressed in the elderly, whereas cell activation and the secretion of pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6 and Tumor-Necrosis Factor (TNF)-α are markedly increased. Recently, it has been reported that inflammation in excess is detrimental, and that excessive production and secretion of cytokines may lead to pathology [25]. Kundu and Surh evidenced that a low-grade systemic inflammation characterizes aging and that inflammatory markers are significant predictors of mortality in old humans [38]. It is widely accepted that many aging-related diseases, including cardiovascular diseases, atherosclerosis, Alzheimer’s disease and diabetes, share a common inflammatory background.

Aging and the liver

The incidence of liver disease increases with age while the ability to withstand a hepatic insult falls with each decade [39]. It is indubitable that interest in the role of aging in the sphere of hepatology has increased, especially with the recent recognition of the critical importance of age in determining the clinical outcome in chronic hepatitis C virus (HCV) infection and the influence of donor age on graft survival after liver transplantation [39]. Human and experimental studies suggest that, in comparison with other organs, the liver ages fairly well [40]. Aging is however associated with a variety of morphological changes in the liver [40,41], but their underlying mechanisms are still unclear. The liver progressively shrinks by 20-40% during the course of a human life [42–44], and there is a concomitant age-related decrease in liver volume [43,45]. The classic gross appearance of the liver in the elderly is known as “brown atrophy”, and the brown is due to an accumulation of highly oxidized insoluble proteins, known as lipofuscin, stored into hepatocytes. These accumulations of highly cross-linked protein are thought to relate to chronic oxidative stress and a failure to degrade damaged and denatured proteins [39]. Increasing evidence suggests that lipofuscin interferes with complex cellular pathways due to its ability to trap metallic cations and facilitate further free radical formation. Other sub-cellular hepatocyte changes with age involves a marked decline in smooth endoplasmic reticulum surface, which correlates with decreased hepatic microsomal protein concentrations and enzymatic activity including glucose-6-phosphatase; furthermore, the human liver tends to develop macro and polyploid hepatocytes with increased nuclei and nucleoli during aging [46]. Kudryavtsev et al. reported that after the age of 85, around 27% of human hepatocytes demonstrate polyploidy compared to around 6% for individuals in their twenties [47].

One of the most important age-related changes in liver function observed in animal models is a significant decrease in regenerative capacity of the liver, but not in the capacity to restore the organ to its original volume [48]. Liver regeneration in both young and old animals was complete by day 7 after 70% hepatectomy, but at day 1 younger animal had significantly increased liver mass and increased intrahepatic mitotic activity. It has also been shown that aging is associated with multiple changes in [49–53]. Elderly humans secrete less bile acid, have increased biliary cholesterol levels, and show an increased oxidative stress that is mainly attributable to a reduced capacity to eliminate metabolically generated superoxide radicals as efficiently as before [41]. The reduction in hepatic blood flow during aging reduces the metabolism of rapidly cleared drugs [54]. Aging of the liver is also associated with impaired metabolism of drugs, adverse drug interactions, and susceptibility to toxins [53,55]. Additionally, it is now accepted that drug [53] and pharmacodynamics may be altered in the elderly. An important contribution is made by decreased renal function, but biotransformation in the liver may also play a fundamental role [56,57]. Serum bilirubin and classical enzymes unchanged with aging [58]. However, sensitive tests of liver function have revealed a small decline with aging. This has been confirmed in experimental studies that have demonstrated changes in mRNA in the genes involved in cell stress and fibrosis [59]. Albumin synthesis decreased slightly with age, but this seems to be secondary to increased cytokine production owing to the levels of chronic inflammation [60].

Older people have a higher incidence of acute liver failure and a higher mortality with acute hepatitis A [61,62]. Along with the considerable growth in Western societies of relatively healthy elderly populations, it is inevitable that there will also be an increase in the number of elderly people with chronic liver diseases (mainly hepatitis C virus (HCV)-related cirrhosis, alcoholic cirrhosis and HCC).

Poynard et al. showed that age at HCV infection was a main risk factor for fibrosis [63,64]. The rate at which fibrosis progressed was low in individuals infected when younger than 20 years, intermediate in those infected at age 21 to 40 years, increased in those infected at age 40 to 50 years, and highest in those infected at 50 years of age or older. The reason for more rapid progression of liver fibrosis in older patients affected by HCV is still unknown, but seems to be related to the decline in immune function with age. Hartmann et al. showed that telomere shortening (the telomere length of somatic cells becomes shorter with aging because of the “end replication problem”) represents a causal factor impairing liver regeneration and accelerating cirrhosis formation in response to chronic liver disease [65].

Age is an independent risk factor for poor outcome in primary biliary cirrhosis (PBC) in addition to the presence of portal hypertension and impaired liver function [66–68]. It has been hypothesized that the increased susceptibility of aging people to neoplastic diseases are due to a decrease in basic immune defense functions [39,69].

It is well recognized that the incidence of HCC in patients with HCV correlates with progression of liver fibrosis. However, there is little information on the impact of aging on hepatocarcinogenesis. Furthermore, it is not known whether the putative etiologic factors and clinical and pathological features of HCC differ between young adults and older patients [70]. The elderly patients developed HCC more often, despite their lower grade of fibrosis, compared with the younger patients. Youssef et al. reported that the hepatocarcinogenic effect of Peroxisome Proliferator-Activated Receptor Alpha (PPAR alpha) agonists is enhanced by aging [71]. Exposure to these chemicals produces a five- to seven-fold higher yield of grossly visible hepatic tumors in old relative to young animals [71]. Newer evidence indicates an active age-related change in the regulation of malignant hepatocyte proliferation. Substantial attention has focused on the telomere/telomerase system as a mediator of replicative capacity. Telomeres are repeating hexanucleotide sequences that function to protect chromosomes against chromosomal end-end fusion and non-reciprocal translocations. Telomerase is a reverse transcriptase consisting of enzymatic (TERT), RNA template (TERC) and several other protein components including heat-shock protein 90 (hsp90) and dyskerin [39,72]. Telomerase can maintain telomere length by adding TTAGGG repeats, but its expression is controlled in differentiated and stem-cell populations [73–75]. Short human telomeres have been linked to the subsequent onset of disease and death in healthy older persons [76]. Kitada et al. [77] and Urabe et al. [78] showed that subjects with chronic viral hepatitis had shorter hepatic telomeres than healthy controls and that increasing fibrosis was associated with shorter telomere lengths. The role of the telomere/telomerase system in the pathogenesis of HCC have led some to suggest that therapeutic manipulation may hold the promise for future therapies [39]. Telomerase inhibitors are currently in phase II clinical trials and would represent a rational therapy for HCC. However, the potential for inducing fatal liver failure, given the results from mouse models, may limit their safety and efficacy.

Mast cells: a heterogeneous innate immune cell population

Mast cells (MCs) have a rather unique position among cells of the immune response. Paul Ehrlich first described MCs in his 1878 doctoral thesis: he called them “mastzellen” (maestung – a root of the English word mastication; the active form “measten” is still in use) because of their characteristic staining of proteoglycan and protease-rich cytoplasmic granules. Ehrlich also noted the tendency of MCs to be associated with blood vessels, nerves, and glandular ducts.

It has been estimated that human MCs contain 2.4 to 7.8 μg heparin per 10⁶ cells [79]. This observation, along with the knowledge that heparin is a negatively charged molecule helps explain why MC granules are preferentially stained with cationic dyes [80]. In their traditional role, MCs are key players in immunoglobulin E (IgE)-associated immune responses via aggregation of the high-affinity IgE receptor, FcεRI, that is expressed on MCs as a heterotetrameric receptor with subunits that initiate specific signaling events [81]. Both positive and negative effects are elicited by FcεRI, which includes degranulation, gene transcription and eicasanoid production. More recently, it has been noted that MCs are not regulated solely by IgE-dependent mechanisms. New reports show that MCs express other surface receptor binding sites such as Toll-like receptors (TLRs), β2-integrins, intercellular adhesion molecule-1 (ICAM-1), androgen receptors, purinergic P2X receptors (P2X1, P2X4 and P2X7) and the serotonin receptor, 5-HT1A [81].

It has long been recognized that MCs elicit allergic symptoms [82], but it is now widely accepted that they are multifunctional effector cells of the immune system, although the various phases of their differentiation are still only partially known. It was initially suggested that they derive from T lymphocytes, fibroblasts or macrophages, but the current general consensus suggests that they originate from pluripotent hemopoietic stem cells in bone marrow, from where they are released into the blood as progenitors before they undergo terminal differentiation by invading connective or mucosal tissue as morphologically unidentifiable MC precursors and then differentiating into phenotypically identifiable MCs [83] (Figure  ). It has been demonstrated that MC are long-lived cells [83].

MCs are found in almost all of the major organs of the human body, and in several body sites that come into contact with the external environment, including the skin, respiratory system and digestive tract. These main accumulations in sites where foreign material attempts host invasion suggest that MCs are one of the first cell populations to initiate defense mechanisms.

The ability of the lineage to generate individual MC populations with different biochemical and functional properties gives them greater diversity and flexibility in meeting the requirements of the physiological, immunological, inflammatory or other biological responses in which they may be involved [84–88].

A number of Authors have identified a local or systemic increase in the number of MCs in various pathological conditions, including interstitial pneumonia, ulcerative colitis, intestinal helminthosis and ectodermal parasitosis, as well as skin disorders such as atopic dermatitis, psoriasis, scleroderma and wound healing, and various neoplastic diseases [89–92].

Tryptases and chymases are the major proteases components of MC secretory granules. There are two well-known human MC classes that are distinguished by enzymatic immunostaining for mast cell proteases, tryptase (M_CT) alone or tryptase and chymase together (MC_TC). M_CT are found mainly in mucosal sites whereas MC_TC[93]. These two populations of MCs exhibit functional differences [84]. TNF was the first cytokine clearly associated with normal MCs in 1990 [87,92]. Other MC products include interleukins (IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-18, IL-2), chemokines (macrophage inflammatory protein alpha [MIP-1]), hematopoietic factors (granulocyte macrophage colony stimulating factor [GM-CSF]), stem cell factor (SCF), growth factors (transforming growth factor beta [TGF-β], vascular endothelial growth factor [VEGF], nerve growth factor [NGF]), several metalloproteinases, heparin, histamine, chondroitin sulfates, cathepsin, carboxypeptidases and peroxidase [84,87].

These products may be released when MCs are activated via IgE- or IgG-dependent mechanisms, and may also be produced under other circumstances such as in response to stimulation by bacterial products through Toll-like receptors (TLRs) [94].

MCs and IgE have long been associated with the pathogenesis of the acute manifestations of the immediate hypersensitivity reaction, the pathophysiologic hallmark of allergic rhinitis, allergic asthma, and anaphylaxis. The central role of MCs in these disorders is now widely accepted. Additionally, MCs are considered to be critical effectors in many human inflammatory diseases, and the core of an immediate hypersensitive reaction: they have been incriminated in different diseases including allergy, asthma, rheumatoid arthritis, arteriosclerosis, chronic graft-versus-host disease, fibrotic disease, ischemic heart disease and malignancy, and contribute to the progression of chronic diseases [95–99]. Any alteration in cell programs that determines a requirement for MC degranulation may therefore have a considerable impact on disease severity. Nguyen et al. have shown the ability of Prostaglandin E2 (PGE₂) to initiate MCs degranulation changes in the aging animal, thus suggesting that aging induces reprogramming of MC degranulation [100].

The parenchyma of the liver is a complicated structure with numerous cell types having different functions in normal liver function and disease response. These include: Kupffer cells, hepatic stellate cells, sinusoidal endothelial cells, vascular endothelial cells, fibroblasts and pit cells. The majorities of these cells play definitive roles in liver pathophysiology and interact with hepatic MCs (Figure  ). MC quantity has been shown to increase in PBC by staining for either toluidine blue or immunohistochemical staining for tryptase and chymase, evidence that these cells are presumably MC_TC. Increased plasma histamine levels were also found in patients with PBC, in contrast to healthy patients, raising the possibility of a role for in vivo mast cell activation and mediator release in these diseases [101]. Hepatic MC expression and quantity have been also shown to increase in patients with primary sclerosing cholangitis (PSC) compared to healthy controls. Increased MC infiltration has been noted in bile ducts in patients with early stage PSC with a diffuse staining in the fibrous septa in the late stage of PSC [102].