Clinical Rheumatology

, Volume 28, Issue 10, pp 1179–1185

High interleukin-6 and tumor necrosis factor-alpha serum levels in hepatitis C infection associated or not with mixed cryoglobulinemia

Authors

    • Department of Internal MedicineUniversity of Pisa, School of Medicine
  • Clodoveo Ferri
    • Chair and Rheumatology UnitUniversity of Modena and Reggio E. School of Medicine
  • Silvia Martina Ferrari
    • Department of Internal MedicineUniversity of Pisa, School of Medicine
  • Emiliano Ghiri
    • Department of Internal MedicineUniversity of Pisa, School of Medicine
  • Santino Marchi
    • Department of Internal MedicineUniversity of Pisa, School of Medicine
  • Michele Colaci
    • Chair and Rheumatology UnitUniversity of Modena and Reggio E. School of Medicine
  • Fabrizio Bruschi
    • Department of Experimental Pathology, Medical Biotechnology, Infectivology and EpidemiologyUniversity of Pisa, School of Medicine
  • Poupak Fallahi
    • Department of Internal MedicineUniversity of Pisa, School of Medicine
Original Article

DOI: 10.1007/s10067-009-1218-8

Cite this article as:
Antonelli, A., Ferri, C., Ferrari, S.M. et al. Clin Rheumatol (2009) 28: 1179. doi:10.1007/s10067-009-1218-8

Abstract

The objective of this study is to evaluate serum levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-a) in a series of patients with hepatitis C virus (HCV)-related mixed cryoglobulinemia (HCV-MC) and to correlate these parameters with the clinical features of the disease. Serum IL-6 and TNF-a were assayed in 61 patients with HCV-MC, in 61 sex- and age-matched patients with HCV chronic hepatitis without cryoglobulinemia (HCV+), and in 61 sex- and age-matched healthy controls. HCV-MC patients showed significantly higher mean IL-6 levels than controls (p=0.005) or HCV+ patients (p = 0.02). Moreover, IL-6 was increased in cryoglobulinemic patients with active vasculitis, even if the statistical significance was not reached (p=0.056). Serum TNF-a levels were significantly higher in HCV-MC than in HCV+ or in controls (p<0.01). The study demonstrates high IL-6 and TNF-a serum levels in HCV-MC patients; moreover, IL-6 levels tended to be higher in HCV-MC patients in presence of active vasculitis.

Keywords

CryoglobulinemiaHepatitis CIL-6TNF-alpha

Introduction

Mixed cryoglobulinemia (MC) is a systemic leukocytoclastic vasculitis involving small and medium-sized vessels. The preeminent role of hepatitis C virus (HCV) in the etiology of MC was clearly established; moreover, an involvement of the immune system in the pathogenesis of MC has been shown [1, 2].

Cytokines may be of particular relevance in this context, playing a central role in the immune response to viral agents [3, 4].

We have recently shown markedly high serum levels of CXCL10 and tumor necrosis factor-alpha (TNF-a) in patients with HCV-related MC (HCV-MC) compared to HCV+ and healthy controls; moreover, in HCV-MC patients, increased CXCL10 levels were significantly associated with the presence of active vasculitis [5].

Interleukin-6 (IL-6) is a multi-functional protein produced by lymphoid and non-lymphoid cells, and by normal and transformed cells, including T-cells, monocyte/macrophages, fibroblasts, hepatocytes, and vascular endothelial cells [6, 7]. The production of IL-6 is regulated, either positively or negatively, by a variety of signals including mitogens, antigenic stimulations, lipopolysaccharides, IL-1, TNF-a, platelet-derived growth factor, and viruses. On the basis of its various activities, IL-6 has also been called interferon-2 (IFN-2), 26 kDa protein, and B-cell stimulatory factor-2 [6, 7].

The effects of IL-6 on different cells are several and varied. The effects on B-cells are the stimulation of differentiation and the antibody secretion [8, 9]. IL-6 represents also a co-stimulant of T-cells, with sub-optimal concentrations of phytohemagglutinin or concanavalin A, to stimulate IL-2 production and IL-2 receptor expression. IL-6 exhibits growth factor activity for mature thymic or peripheral T-cells and reportedly enhances the differentiation of cytotoxic T-cells in presence of IL-2 or IFN-gamma. Finally, it stimulates production of acute phase proteins by hepatocytes and presents colony-stimulating activity on hematopoietic stem cells [8, 9].

The various activities of IL-6 described above suggest that this factor will have a major role in the mediation of the inflammatory and the immune responses triggered by infection or injury. Although the exact functions of IL-6 in vivo are not known, elevated IL-6 levels have been reported to be associated with a variety of diseases, including autoimmune disorders such as rheumatoid arthritis, Castleman’s disease, mesangial proliferative glomerulonephritis, psoriasis, and inflammatory bowel diseases [8, 9].

Until now, a few studies evaluated the importance of IL-6 in patients with MC. A preliminary work showed that IL-6 serum concentrations were increased in HCV-infected patients with or without MC [10]. An increase in the circulating levels of IL-6 was also observed in HCV-MC patients by Falasca et al. [11].

To our knowledge, no study evaluated serum levels of IL-6 in a large series of patients with HCV-MC in relation to the clinical picture. The aim of this study is to evaluate serum levels of IL-6 in a series of HCV-MC patients and to correlate this parameter with the clinical features of the disease and with the circulating levels of TNF-a.

Materials and methods

Patients

Sixty-one HCV-MC patients (43 females and 18 males; mean age 62±10 SD years; mean disease duration 12±10 SD years), consecutively referred to our Rheumatology Unit, were recruited for the study between 2001 and 2006. The diagnosis of HCV-MC was based according to the preliminary criteria proposed by the Italian Group for the Study of Cryoglobulinemias in 1989 [2]; in particular, we considered the presence of serum mixed (IgG-IgM) cryoglobulins and the classical clinical triad—purpura, weakness, and arthralgias—excluding other well-known systemic disorders, such as immune-rheumatic and neoplastic diseases [1, 2, 1214].

The study included only patients with HCV-MC, without liver cirrhosis or hepatocellular carcinoma (by histology, laboratory evidence of liver failure, and/or ultrasound-proven portal hypertension) [14, 15]. After a thyroid screening (clinical history, physical examination, thyrotropin, free triiodothyronine, free thyroxin, anti-thyroglobulin and anti-thyroid peroxidase antibodies measurements, and ultrasonography), we excluded the patients with thyroid autoimmune disorders, a well-known cause of high serum CXCL10 [1618], in the purpose to eliminate another possible cause of a T-helper 1 response.

Forty-six out of 61 (75%) HCV-MC patients underwent liver biopsy for diagnostic purposes; liver histology activity index (grade) or stage of liver fibrosis was evaluated according to Ishak et al. [19]. The mean activity index (grade) in HCV-MC patients was 5.2±1.1, and the stage was 2.1±0.7. Main demographic and clinico-serological features of HCV-MC patients are reported in Table 1.
Table 1

Demographic and clinico-serological features of 61 HCV-MC patients

Features

Values

Age (years)

62 ± 11

Men/women

18/43

Disease duration with MC (years)

12 ± 10

Purpura

87%

Active vasculitis

41%

Weakness

92%

Arthralgias

90%

Arthritis

15%

Raynaud’s phenomenon

52%

Sjogren’s syndrome

46%

Peripheral neuropathy

67%

Renal involvementa

13%

Aminotransferases elevation and/or histologic activity

70%

Cryocrit (%)

4.3 ± 9.1

CH50 (normal, 160–220 units)

115 ± 36

C3 (normal, 60–130 mg/dl)

81 ± 38

C4 (normal, 20–55 mg/dl)

12 ± 10

Autoantibodiesb

26%

aSerum creatinine >1.5 mg/dl and/or proteinuria >0.5 gr/24 h

bPresence of anti-nuclear autoantibodies

Among them, 22 had been previously treated with IFN-a for an average of 7 months (range 1 to 15), at a mean dosage of 10.1 MU/week; the time elapsed from the last course of IFN-a treatment ranged from 6 to 69 months (mean 40±19). No statistically significant differences were observed in the main demographic and clinico-serological features of HCV-MC patients treated or untreated with IFN-a.

At the time of study, 43 HCV-MC patients were taking low doses of corticosteroids (5 mg of prednisone a day), nine had previously been on corticosteroids, and nine had never been treated with corticosteroids. No HCV-MC patient had had plasma-exchange treatment in the last year before the study. In both patients and controls, a careful medical history was collected, in particular, with regard to family history of thyroid disease, smoking habits, and drugs. The presence of Raynaud’s phenomenon, sicca syndrome, skin ulcers, peripheral neuropathy, and renal and liver involvement in HCV-MC patients was evaluated. Routine blood chemistry, including alanine aminotransferase (ALT), was carried out by standard methods.

Controls

Each of the 61 HCV-MC patients eligible for the study was matched, by sex and age, one-to-one with a control group of healthy subjects of the general population from the same geographic area (North-West Tuscany). This control group was extracted from a larger sample of 1,640 subjects in a population-based survey of thyroid disorders; only HCV-negative subjects without clinical and laboratory evidences of thyroid and liver disorders and autoimmune diseases and not treated with immune-modulators were included.

Another control group consisted of 61 sex- and age-matched patients with “chronic type C hepatitis” (HCV+) extracted from a larger cohort of 491 chronic hepatitis referrals without cryoglobulinemic syndrome; subjects with other liver infection, liver cirrhosis (by histology, laboratory evidence of liver failure, and/or ultrasound-proven portal hypertension) [14, 15], and hepatocellular carcinoma were excluded.

Forty-three out of 61 (70%) HCV+ patients underwent liver biopsy for diagnostic purposes; the mean activity index (grade) was 5.1±1.2, and the stage was 2.0±0.5.

Extraction of the control group from the original populations was performed by finding the closest age-match (±2 years) to each case within either gender. When more than one age-match was available per case, the choice was made at random.

The study protocol was approved by the local Ethics Committee. All subjects gave their informed consent to enter the study.

Immunological studies

Cryocrit was measured as the percentage of packed cryoglobulins after cold centrifugation of the serum stored at +4°C for 7 days; cryoglobulins composition was determined by detecting the presence in cryoprecipitates of monoclonal or polyclonal IgM-rheumatoid factor (MC type II or MC type III, respectively). Anti-nuclear, anti-smooth muscle, and anti-mitochondrial autoantibodies were detected by current techniques, to exclude other autoimmune disorders. Sera with a titer >1:40 were considered positive. Anti-extractable nuclear antigen antibodies, including anti-Scl70, -Sm, -RNP, -SSA/SSB, -PCNA, and -Jo1 specificities, were detected by counter-immune-electrophoresis according to Bunn et al. [20].

Virological studies

Anti-HCV antibodies and HCV-RNA were determined on serum clotted and centrifuged at 37°C and stored at −70°C. Antibodies against HCV (anti-HCV) and HCV-RNA (polymerase chain reaction technique) in the serum were investigated as previously described [21].

Cytokines, chemokines, and analytical assays

Serum IL-6 levels were assayed by a quantitative sandwich immunoassay using a commercially available kit (R&D Systems, Minneapolis, MN, USA), with a sensitivity of 0.7 pg/ml. The intra- and inter-assay coefficients of variation were 2.5% and 3.0%.

Serum TNF-a concentrations were measured using commercially available kits (R&D Systems, Minneapolis, MN, USA). The mean minimum detectable dose was 0.12 pg/ml for TNF-a; the intra- and inter-assay coefficients of variation were 5.8% and 10.2%. Samples were assayed in duplicate. Quality control pools of low, normal, or high concentration for all parameters were included in each assay.

Data analysis

Values are given as mean ± SD for normally distributed variables or as median ± interquartile range for not normally distributed variables. Group values were compared by univariate analysis of variance, for normally distributed variables, or by Kruskal–Wallis (≥3 groups) or Mann–Whitney U (two groups) tests for not normally distributed ones. Proportions were compared by the chi-square test. Post hoc comparisons on normally distributed variables were carried out using the Bonferroni–Dunn test. Univariate analysis was performed by simple regression. A multivariate logistic regression analysis was performed in HCV-MC patients, considering age, gender, ALT, TNF-a, presence or absence of high levels of IL-6 as dependent variable, and presence or absence of active vasculitis as independent variables. We have considered “active vasculitis” the presence of palpable purpura and/or vasculitic skin ulcers.

Results

Patients with HCV-MC showed significantly higher mean IL-6 serum levels (mean 82±164 SD ng/L; median 13.6 ng/L, range 0.7–783) than controls (mean 8±11 SD ng/L; median 0.9 ng/L, range 0.6–43; p=0.005), or HCV + patients (mean 42±64 SD ng/L; median 9.6 ng/L, range 0.6–452; p=0.02; Fig. 1). HCV+ patients showed slightly but not significantly higher mean IL-6 serum levels than controls (Mann–Whitney U test).
https://static-content.springer.com/image/art%3A10.1007%2Fs10067-009-1218-8/MediaObjects/10067_2009_1218_Fig1_HTML.gif
Fig. 1

Patients with HCV-MC showed significantly higher mean (±SE) IL-6 serum levels than controls (p = 0.005) or HCV + patients (p = 0.02) (Mann–Whitney U test). HCV+ patients showed slightly but not significantly higher mean IL-6 serum levels than controls (Mann–Whitney U test)

By defining high IL-6 level as a value higher than the 95th percentile of the control group (>2.8 pg/ml), 47/61 (77%) of patients with HCV-MC, 3/61 (5%) of the control subjects, and 19/61 (31%) of HCV+ patients had high IL-6 (p<0.002, HCV-MC vs controls; p=0.005, HCV-MC vs HCV+ ; p=0.05, HCV+ vs controls; chi-square).

In order to better define the role of increased serum IL-6 in HCV-MC, mean levels of this interleukin were separately evaluated among HCV-MC patients’ subgroups defined according to main demographic and clinical features (age >55 years; gender; disease duration >10 years; presence or absence of purpura, active vasculitis, weakness, arthralgias, arthritis, Raynaud’s phenomenon, sicca syndrome, peripheral neuropathy, renal involvement, ALT elevation, and/or histological activity in the liver). The levels of IL-6 observed in 25 patients with active vasculitis (palpable purpura and/or vasculitic skin ulcers) at the time of the present study were higher even if not significantly (p=0.056) in comparison to those without (18.9 (35.4) vs 7.6 (18.5); median and (interquartile range); Mann–Whitney U test).

No significant correlations were observed between IL-6 levels and other clinical features or serological findings of HCV-MC (i.e., levels of cryocrit or complement) or previous/ongoing treatments.

Serum TNF-a was detectable in 54/61 (89%) of controls and in all HCV-MC and HCV+ patients; mean levels were significantly higher in HCV-MC (mean 44±86 SD ng/L; median 12.1 ng/L, range 2.1–369) or HCV+ (mean 9.4±23 SD ng/L; median 8.2 ng/L, range 1.3–122) than in controls (mean 2±2 SD ng/L; median 1.2 ng/L, range 0.6–8.4; p<0.0001 for both; median and range; Mann–Whitney U test; Fig. 2). Furthermore, TNF-a levels were higher in HCV-MC than in HCV+ (p=0.005; Mann–Whitney U test). No correlation was found between serum TNF-a and IL-6, ALT, liver histology activity index, stage of liver fibrosis, presence of active vasculitis, or the other demographic, serological, and clinical features of MC.
https://static-content.springer.com/image/art%3A10.1007%2Fs10067-009-1218-8/MediaObjects/10067_2009_1218_Fig2_HTML.gif
Fig. 2

TNF-a mean (±SE) levels were significantly higher in HCV-MC or HCV+ than in controls (p < 0.0001 for both; Mann–Whitney U test), furthermore, TNF-a levels were higher in HCV-MC than in HCV+ (p = 0.005; Mann–Whitney U test)

HCV+ and HCV-MC patients had, obviously, raised ALT enzymes (p<0.0001), in comparison with controls; however, there was no significant difference in serum ALT between HCV+ and HCV-MC patients. No association was observed between IL-6 and ALT levels both in HCV+ and HCV-MC patients. The grade and the stage of hepatitis in HCV+ and in HCV-MC patients were not significantly different.

Discussion

Our study demonstrates significantly higher serum levels of IL-6 in patients with HCV-MC compared to healthy controls and HCV+ patients. Interestingly, the levels of IL-6 observed in patients with active vasculitis were higher even if not significantly in comparison to those without. Furthermore, our study confirms significantly high serum levels of TNF-a in patients with HCV-MC or HCV+ compared to healthy controls [5].

IL-6 is a typical pleiotropic cytokine that plays a central role in inflammation. In the blood circulation, the IL-6 level stays low in healthy young individuals but increases with aging. By contrast, IL-6 production is rapidly induced in the course of acute inflammatory reactions. Not only immune cells but also a variety of other cell types, such as muscle cells, adipocytes, and hepatocytes produce IL-6 [22]. The IL-6 receptor component, gp130, is broadly distributed in the body. It has been hypothesized that a deregulated, high-level production of IL-6 could induce an undesired inflammatory state in many organs. In these lights, a number of reports implicate IL-6 in the pathogenesis of many human disorders such as inflammatory bowel disease, multiple myeloma, rheumatoid arthritis, Sjogren’s syndrome, systemic lupus erythematosus, and others. Evidence from in vitro studies indicates that IL-6 has an important assisting effect on the functions of a variety of immune cells. However, IL-6 appears to be essential for the progression of experimentally induced-immunological disorders in animals, making IL-6 an attractive therapeutic target [23].

TNF-a has both growth stimulating and inhibitory properties, is secreted by monocytes/macrophages, and is considered a pivotal cytokine for inflammation: in particular, it stimulates the acute phase reactant proteins in the liver, activates neutrophils, T- and B-cells, and participates to endothelial activation [24]. Furthermore, in the last 10 years, TNF-a represents one of the most important targets in the therapy of autoimmune diseases, such as rheumatoid and psoriatic arthritis.

Our results, showing high levels of circulating IL-6 in HCV-MC, are in agreement with two other previous studies.

In the first work [10], peripheral blood mononuclear cells of HCV patients associated with non-Hodgkin lymphoma B (n=12), MC (n=14), uncomplicated hepatitis C (n=12), and healthy volunteers (n=12) were incubated with the recombinant HCV proteins E2, core, and secreted non-structural protein 3 to study induction of cytokine production. HCV core was the only studied protein, which induced production of IL-6 and IL-8 in CD14(+) cells. Furthermore, IL-6 serum concentrations were increased in HCV-infected patients with or without MC [10].

In the second study [11], serum levels of IL-6 were evaluated in HCV+ patients with (n=30) and without (n=30) MC. IL-6 serum levels were higher in the MC+ group than the MC− group, according to our study. Interestingly, the IL-6 levels observed (8.7 pg/mL) were similar to those observed in our HCV-MC patients without active vasculitis (7.6 pg/ml). However, we have found an increase of IL-6 in HCV-MC with active vasculitis. This discrepancy may be due to different factors: (a) the numerosity of our series of HCV-MC patients is larger (61 vs 30), (b) the different patient selection criteria, and (c) the inclusion of HCV-MC patients with active vasculitis.

The fact that, among HCV-MC subjects, the IL-6 levels showed a trend to be higher in patients with signs of active vasculitis is in agreement with the findings of an up-regulation of the expression of IL-6 in other vasculitis, such as in active Behcet’s disease [2527] and in giant-cell arteritis [28, 29].

Change of serum chemokine levels in the course of other autoimmune disorders has been demonstrated. Recent experimental evidences have shown that CXC chemokines, particularly CXCL10 (T-helper 1 response), play an important pathophysiological role in the initial phases of autoimmune thyroid disorders [3034]. In our study, we have not found any relation between serum IL-6 levels and the duration of HCV-MC, probably because the disease is characterized by a relapsing clinical course; in fact, vasculitic manifestations may appear at any time during the follow-up, possibly triggered by multiple pathogenetic co-factors [2].

On the other hand, the increase of IL-6 in the active phase of the disease is in agreement with findings arisen from previous reports in which serum CXCL10 has been found high in the active phase of a number of diseases, such as multiple sclerosis and Graves’ disease.

According to these data, Sansonno D. et al. showed that increased levels of CXCL13 strongly correlated with active cutaneous vasculitis in HCV-MC patients compared to healthy controls and HCV-infected patients without MC [35].

In the pathophysiology of HCV-MC, B-cell proliferation leads to immune-complexes production, mainly HCV-containing cryoglobulins [12], which are responsible for immune-complex-mediated vasculitis [1, 2, 21]; secondly, high serum IL-6 levels due to HCV-related vascular injury strongly amplify the inflammatory process through co-stimulation of T and B-cell proliferation.

Longitudinal studies evaluating serum IL-6 levels in large HCV-MC patients’ series will be necessary to evaluate if serum IL-6 measurement could represent an easily detectable prognostic marker for clinical management of HCV-MC patients.

Our study confirms that serum TNF-a resulted high in HCV+ accordingly to other studies in hepatitis C patients [3638]. Kaplanski G. et al. found increased levels of soluble TNF-a receptors in HCV-MC [39]. Other works have shown an increased production of TNF-a by lymphocytes of HCV-MC patients [40, 41], suggesting a predominance of T-helper 1 response in the liver of HCV-MC. It is unlikely that the increase of TNF-a may be due to a more aggressive liver disease in HCV-MC compared to HCV+; in fact, no correlation was found between TNF-a levels and ALT or degree of liver inflammation in the present paper. Although the importance of TNF-a in the pathophysiology of MC has been shown, up to date, the results obtained with infliximab (anti-TNF-a antibody) in HCV-MC are not satisfying [42].

In conclusion, our study demonstrates significantly high serum levels of IL-6 in patients with HCV-MC compared to healthy controls and HCV+ patients, overall, in patients with signs of active vasculitis. Furthermore, significantly high serum levels of TNF-a in patients with HCV-MC compared to healthy controls have been confirmed. Future prospective studies in larger patients’ series will be needed to evaluate the relevance of serum IL-6 and TNF-a determination as clinico-prognostic markers of HCV-MC; moreover, being the availability of monoclonal antibodies directed to these cytokines, their study could be useful in the therapeutic approach to these patients.

Guarantors of the article

Antonelli A. and Ferri C.

Specific author contributions

Alessandro Antonelli and Clodoveo Ferri were the principal investigators. All other authors participated in the design and analysis of the study and in writing the manuscript.

Financial support

None.

Disclosures

None.

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© Clinical Rheumatology 2009