Journal of Hepatology
Volume 41, Issue 6 , Pages 1045-1049, December 2004

Enhanced TH1 cytokine production in hepatitis C virus-infected patients with mixed cryoglobulinemia: understanding the pathological issues

Department of Internal Medicine and Center for Research, Transfer and High Education MCIDNENT, University of Florence, Viale Morgagni, 85, 50134 Florence, Italy

published online 01 November 2004.

See Article, pages 1031–1037

Article Outline

 

Mixed cryoglobulinemia (MC) is a distinct syndrome clinically characterized by purpura, weakness, arthralgia, and involvement of one or more organ systems, including membranoproliferative glomerulonephritis, peripheral neuropathy, skin ulcers, liver damage and diffuse vasculitis. Cryoprecipitable immunocomplexes, namely mixed (IgG–IgM) cryoglobulins, are the serological hallmark of the disease: IgG is the autoantigen, IgM, with rheumatoid factor (RF) activity, the autoantibody. Mixed cryoglobulinemia is classified in type II and type III according to the presence of polyclonal or oligo-monoclonal IgMs. Because expansion of RF-producing B-cells is the underlying disorder of MC, this condition is considered a ‘benign’ B-cell lymphoproliferative disease (LPD) [1], [2], [3].

The mechanism(s) responsible for the lymphoproliferation surrounding MC remain unknown. Due to geographical heterogeneity in prevalence of MC in HCV-infected patients, it is conceivable that unknown genetic and/or environmental factors may influence the development of this syndrome [4]. Several data are consistent with the possibility that chronic stimulation of B-cells by viral epitopes could play an important role [5], [6], [7]. Furthermore, it is possible to hypothesize that some pathogenically important B-cell subpopulations may be selected because of persistent antigenic stimulation. In this context, analogies may exist between the mechanisms by which HCV leads to MC or other LPDs, and the pathogenetic mechanisms of Helicobacter pylori lymphomagenesis. The latter is in fact considered a multi-step process evolving from a physiological polyclonal immune response, to an oligoclonal and finally monoclonal one, due to the acquisition of genetic mutations that gradually make lymphoproliferation less and less antigen-dependent [8].

A wide body of evidence, in addition, strongly suggests that a key factor in the pathogenesis of HCV-related MC is represented by the inhibition of the apoptosis of B-cells, leading to their progressive accumulation. First, this is suggested by the histopathological characteristics of liver and/or bone-marrow lymphocyte infiltrates in MC patients [8], as well as by the high prevalence of bcl-2 rearrangement (t(14;18) translocation) in patients with MC, with regression of translocated B-cell clones after successful antiviral therapy [3], [9], [10]. Although the high frequency of this translocation—which is considered an error during physiological V-D-J rearrangement processes in B-cells—may be favoured by strong and sustained B-cell activation, recent data provide evidence that in HCV infected patients, in analogy with NHL patients, the frequency of JH6 gene involvement in t(14;18) is significantly higher than in controls. Therefore, the increased prevalence of t(14;18) translocation in HCV+ patients may not be fully explained by an unbiased enhancement of processes governing the possible creation of t(14;18)+B cells in normal subjects [11]. Moreover, it has been suggested that in t(14;18)-negative cases, other genetic aberrations may also account for the strong bcl-2 expression [12]. In addition, recent in vitro data suggest that HCV may act as a potent mutagen [13].

Overall, research regarding the pathogenesis of HCV-related MC is now in an extremely fervent phase, since a number of apparently different data exists suggesting a very complex, multi-step mechanism based on a common pathogenetic factor (HCV infection), as well as a constitutional predisposition, and which may develop through multiple hits, amid a combination of ‘systemic’ as well as ‘local’ factors.

In this context, the study by Saadoun et al. [14] in this issue of the Journal is focused on the definition of a potentially critical aspect, the local immune response in the liver, which is considered the principal site for immune reactions involved in MC pathogenesis. In that study, the cytokine profile of liver-infiltrating T lymphocytes from HCV infected patients with and without MC (of type II) were compared. By using intracellular staining and flow cytometry, Saadoun et al. evaluated the ability of freshly isolated liver T cells to produce IFN-γ, TNF-α, IL-2, IL-4 and IL-10 in response to stimulation with phorbol miristate acetate (PMA) and ionomycin, and showed that, although no differences were found in the proportion of CD4+, CD8+ liver T cells, the ability of freshly isolated liver T cells to produce type-1 cytokines in response to stimulation with PMA and ionomycin for 6h was significantly higher in patients with MC than in HCV-infected controls without MC, whereas production of type-2 cytokines by these cells was similar (IL-4) or reduced (IL-10).

Cytokines are regulatory molecules that play an important role in many physiological/pathophysiological processes. CD4 T cells, which are central to the induction of anti-viral responses, have been subdivided according to two predominant cytokine secretion profiles originally described in mouse T-cell clones [15]. T helper (Th) 1 cells produce cytokines such as IL-2 and IFN-γ, which are important factors promoting cell-mediated immune response. In contrast, Th2 cells produce cytokines involved in the humoral response, such as IL-4 and IL-10. However, it is now recognized that these cytokines can be produced by cells other than CD4+T cells and therefore these polarized cytokine responses better referred to as Th1-like or type 1 and Th2-like or type 2 response [16].

Briefly, this study represents the first observation of an increased production of type-1 cytokines by liver T cells of HCV+ patients with type II MC. This agrees with previous data obtained in peripheral blood mononuclear cells [17], ruling out the possibility of a discrepancy between the response of peripheral and liver T cells, that could indeed occur because of compartimentalization of T cells, which may be due to sequestration of HCV-specific T cells in the liver or homing from the periphery to the liver [18], [19]. Interestingly, in both studies—by Lofreda et al. and Saadoun et al.—a reduced expression of IL-10 (a strong inhibitor of IFN γ production) [20] is demonstrated regardless of the different sources. The observation that the evolution of HCV infection toward MC is characterized by a strong Th1 response is also notable. In fact, it has been hypothesised that CD4+-dependent B-cell growth as well as persistence of HCV infection with persistent B-cell stimulation may be favoured in a Th2 microenvironment, with a predisposition to LPD development as a consequence [21].

Further studies should assess whether the higher number of cytokine producing cells actually corresponds to the increased production of these cytokines. Furthermore, in addition to the cytokine pattern, analysis of cytotoxic activity, including granzyme and perforin production or pro-apoptotic activity of Th1 cells could also be carried out [22], [23].

Several issues can be drawn from this study.

First, there is a need to determine whether a similar immune response may play a key role in the pathogenesis of MC. Indeed, this study suggests that chronic activation of liver infiltrating T cells by intrahepatic antigen recognition and induction of a strong immune reaction dominated by Th1 helper cells takes place in MC patients. These data agree with the observation that the major immunoglobulinin isotypes in HCV-associated type II and type III MC are IgM (RF) and IgG, mainly of IgG1 and IgG3 subclasses [4].

It is also conceivable that the antigens—or antigen products—which induce this immune reaction are of viral origin; further studies utilizing HCV recombinant proteins, in order to evaluate intrahepatic HCV-specific T cell responses in MC, and possibly showing different reactivity against different HCV epitopes [24], would be helpful in better characterizing the potential role of viral infection and pathogenetic mechanisms.

A more controversial issue regards the possible consequences of Th1 polarization of the T-cell response on the severity of liver damage in MC. Indeed, the only two published studies on this aspect show concordant data with respect to the strong Th1 response (the present study and [17]), but suggest opposite (yet acceptable) interpretations of the possible effects of such a polarization on MC-associated liver disease.

In consideration of the complexity of this topic, the principal issue consists of the disagreement still existing regarding the epidemiological association between HCV-positive MC and a severe liver damage. In fact, in light of data showing a significant association with cirrhosis [25], [26], in different reports, generally from Southern Europe, the prevalence of minimal and mild hepatitis is greater and the prevalence of cirrhosis is decreased in MC+ patients compared with patients selected for chronic hepatitis C without MC [27], [28], [29]. In this context, the effort of Kayali et al. to perform a metanalysis of data from the literature appears meritorious [26]. However, some negative studies are not included in the metanalysis because either they did not fit the rigorous inclusion criteria used or simply they were unavailable at that time (i.e. [27], [29]).

In light of differing geographical distribution of MC prevalence, with a South-North gradient, one could also hypothesize that the differing association of HCV+ MC with liver cirrhosis may be related to some still unknown genetic/environmental factors making the development of MC more difficult and time-requiring, thereby increasing the probability to find advanced liver damage in patients with MC. If this is the case, the difficulty in specifying the exact time of infection (frequently estimated through history of exposure to risk factors and/or first detection of abnormal ALT or positive HCV markers), would make an exact evaluation very hard. Interestingly, in the study by Saadoun et al, in which no differences were found between MC+ and MC− patients in terms of the length of infection, clinical and laboratory findings of liver damage (Metavir score, ALT levels, HCV viraemia) were similar.

Thus, the question whether diagnosis of MC in HCV-positive patients may actually be or not be considered of prognostic value with respect to liver damage may still be considered open.

From a more general point of view, apart from the specific case of HCV+ MC, the effects of the host immune response in determining hepatitis C pathogenesis and progression of HCV-related liver fibrosis, and, in particular, the problem of the association between Th1 polarization of the intrahepatic immune response and the severity of liver damage are not fully resolved.

Studies of mice showed that immune deviation to a Th1 response results in decreased liver fibrosis, whereas enhancement of Th2 cytokine production promotes an increase in hepatic fibrosis [30], [31]. In addition, the analysis of the relationship of Th1 versus Th2-like cytokines in chronic infections, such as human immunodeficiency virus, leprosy, and leishmaniasis, suggests that a decrease in cell-mediated immunity, i.e. a Th2-type profile, is associated with increased pathogen load and progressive disease [16], [32], [33].

The question is: where does HCV infection in respect to this paradigm lie?

Some studies have shown that the spontaneous resolution of acute hepatitis is associated with vigorous and persistent HCV-specific CD4 Th1 and CD8 cytotoxic T cell response [24], [34], [35]. However, the role of these cellular immune responses, particularly in the liver, once chronic infection is established, is less clear since the natural history of HCV is highly variable and occurs over the course of decades.

In one study, the production of IFN-γ, IL-4, and IL-5 by a wide series of T-cell clones derived from the liver of patients with chronic hepatitis C and generated by limiting dilution from freshly isolated mononuclear cells was analyzed. The majority of liver-infiltrating T cells were Th1 cells [36]. In another study, through a PCR method to identify intrahepatic mRNA expression of Th1-like versus Th2-like cytokines, patients with chronic HCV infection had an increase in mRNA expression of Th1-like cytokines (IFN-γ and IL-2) which correlated with disease severity and liver damage [33]. More recently, Kamal et al., in a prospective longitudinal study, characterized the HCV-specific CD4+T cell responses in the liver and peripheral blood at an early phase of chronic HCV infection (6–10 months after acute HCV infection) and then more than 8 years later, in subjects with and without coinfection with HCV and Schistosoma mansoni, a parasite that induces increased rates of chronic evolution and accelerated progression of liver disease. Alterations of cytokine patterns in the form of insufficient intrahepatic Th1 response or augmented Th2 response enhanced progression of fibrosis [24].

In consideration of the inconstant and late occurrence of MC following HCV infection, it is reasonable to wonder whether a similar longitudinal study comparing patients who develop MC or not may ever be possible.

Finally, the present study raises the important question of whether the identification of a strong Th1-oriented immune response may have therapeutic applications.

In this respect, it should be noted that the gold-standard therapy of chronic HCV infection, including cases with MC, is currently represented by IFN α and ribavirin (RBV). Both IFN α and RBV have been shown to enhance Th1 response [37]. In particular, IFN α seems to favour Th1 development by enhancing IL-12 secretion by macrophages and/or by maintaining the expression of functional IL-12 receptors on Th cells. In consideration of these data, the conclusion of the present study, which suggests an association between Th1 polarization and high severity of liver damage, should not discourage the use of these drugs. In fact, available studies regarding HCV+ patients treated with IFN α± RBV indicate that, even in the case of no virological response, liver biopsies taken after treatment did not show liver damage progression, but, on the contrary, histological improvement [38]. The use of immunoregulatory cytokines to tilt the balance away from Th1 dominance, should be taken with caution at the moment. In fact, in mice, activation of Th1 responses induce and, conversely, enhancement of Th2 responses lower anti-tumor immunity. This may suggest that switching to Th2-like response in chronic HCV infection may increase the risk of developing tumours such as hepatocellular carcinoma and/or lymphoma [20], [38]. On the contrary, enhancement of Th1 response may protect against this risk. The complexity of the argument and the limited amount of available data in humans make any practical application of the presented data premature at the moment, even if, in theory, the clarification of Th1/Th2 differentiation and its pathological correlates is expected to be critical in influencing the evolution of HCV-related damage and potentially important in the development of new therapies and vaccines.

In conclusion, this study is the first step in characterizing intrahepatic T-cell response in HCV+ MC patients by showing an increased production of type-1 cytokines by liver T cells of HCV-positive patients with MC when compared with HCV+, MC-negative subjects, and raises important, still unresolved issues.

Further investigations are needed, aimed at clarifying the physiopathological implications of these findings, including both the evolution of MC syndrome and liver damage as well as the possible improvement of therapeutic and preventive measures.

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Acknowledgements 

Supported by the ‘Italian Liver Foundation’, AIRC and European Commission; Grant Number: QLK-2-CT-2002-00954 (Hepac-resist).

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PII: S0168-8278(04)00466-0

doi:10.1016/j.jhep.2004.10.007

Journal of Hepatology
Volume 41, Issue 6 , Pages 1045-1049, December 2004

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