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Abstract

Objective:

To study the clinical significance of intercellular interleukin (IL)-33 in hepatocellular carcinoma (HCC).

Methods:

Using immunohisto - chemistry, this prospective study compared IL-33 protein levels in samples of HCC tissue and normal tissue adjacent to the tumour in 60 patients with HCC, and in normal liver tissue from six healthy controls. Interferon (IFN)-α, IFN-γ and IL-33 serum levels were also analysed by enzyme-linked immunosorbent assay in HCC 30 patients and 10 healthy controls. The level of IL-33 immunohistochemical staining was compared with the rate of lymph node metastasis in HCC patients.

Results:

IL-33 was strongly positive in the cytoplasm of hepatocytes. The median percentage of IL-33-positive tissue was higher in HCC than in normal liver tissue samples (adjacent to the tumour or fromcontrols). Serum IFN-α, IFN-γ and IL-33 levels were higher in pre- and postoperative samples from HCC patients than in control samples, and in patients with metastasis compared with those without metastasis.

Conclusions:

Increased IL-33 protein levels were observed in serum and liver tissue from HCC patients; IL-33 may be a useful biological marker for monitoring HCC growth and metastasis.

Keywords

Hepatocellular carcinoma Immunohistochemical analysis Interferon-α Interferon-γ Interleukin-33

Introduction

The production of immunomodulatory factors by hepatocellular carcinoma (HCC) cells, and their subsequent suppression of the immune system, are involved in the impairment of liver immune function in patients with HCC.¹ The evolution of HCC is very complex due to the large number of pathogenic mechanisms involved;² thus, the study of inflammatory cytokines in HCC is becoming increasingly important. Resection of cancerous liver tissue can decrease the tumour burden by reducing or eliminating the source of a variety of factors that lead to immunosuppression, thereby restoring the normal immune balance.³

Interleukin (IL)-33 is a pleiotropic cytokine that can regulate T helper 2 (Th2) immune responses, stimulate mast cells to produce proinflammatory factors, and act as a nuclear factor (NF) to inhibit cell transcription.⁴ Research has demonstrated that low levels of IL-33 levels are detected in the liver;⁵ however, there have been no relevant studies to assess IL-33 levels in liver tissue or blood from patients with HCC.

The aetiology of HCC is unclear due to the complexity of its pathogenesis, although interferon (IFN)-α, IFN-γ and other cytokines are known to play important roles.⁶ The present study explored the role of cytokines in HCC pathogenesis and their potential clinical significance. Levels of IL-33 protein in HCC tumour and normal liver tissue samples, and serum levels of IFN-α, IFN-γ, and IL-33 in HCC patients and healthy control subjects, were examined.

Patients and methods

Study Population

This prospective study included liver tissue specimens from consecutive HCC patients admitted for liver resection to the Department of General Surgery, Norman Bethune First Hospital, Jilin University, Changchun, China, between January 2010 and October 2010. HCC was diagnosed according to guidelines of the American Association for the Study of Liver Diseases;⁷ the differentiation of HCC was diagnosed by liver biopsy. Tumour metastasis was identified postoperatively, by pathological examination. Healthy individuals from Changchun, undergoing routine physical examination at the same institute, served as controls.

Written informed consent was obtained from each participant. The study protocol followed the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Human Research Committee of Jilin University.

Sample Collection

For each case of HCC, liver tissue specimens were collected from the central tumour area and from nontumour tissue 2 – 5 cm away from the central tumour area. Liver tissue specimens were collected from HCC patients at the time of surgical resection, and from healthy control subjects who voluntarily underwent liver biopsy puncture. Tissue samples were divided into two parts: one part was immediately snap frozen, embedded in Optimal Cutting Temperature compound (Tissue-Tek^® Miles Laboratories, Elkhart, IN, USA), and stored at –70 °C; the second part was fixed in 10% neutral buffered formalin overnight at room temperature, followed by routine paraffin embedding and processing using an automated processor (Tissue-Tek^® VIP^® Sakura Finetek, Torrance, CA, USA).

Peripheral venous blood (5 ml) was collected from HCC patients and healthy subjects after a 12-h fast. In HCC patients, samples were taken 10 days pre- and 10 days postsurgery. Serum was separated from blood at 300 g for 10 min at 4°C within 1 h of collection and stored at –80 °C until analysis.

Histopathological Staining

Paraffin sections were treated with xylene twice for 10 min followed by dehydration in 100% ethanol twice for 5 min. Sections were then stained with filtered 0.1% Mayers haematoxylin for 10 min, rinsed in double-distilled water for 5 min, dipped in eosin (0.5% in 95% ethanol) 12 times and rinsed again in ethanol until the eosin stopped streaking. Sections were then dehydrated in 50%, 70%, 95% and 100% ethanol, followed by xylene, several times before being mounted.

Immunohistochemistical Analysis of Il-33 Protein

Levels of IL-33 protein in HCC tumour tissue and normal liver tissue were assessed by immunohistochemistry. Four sections, cut from frozen tissue blocks, were assessed for each sample. Antigen retrieval was carried out by heating tissue sections in target retrieval solution, pH 6.1 (Dako Cytomation, Carpinteria, CA) in a pressure cooker for 20 min, then in a microwave oven for 10 min (maximum power, 500 W). Sections were rinsed under running water for 2 min, treated with 3% H₂O₂/methanol solution for 5 min to quench endogenous peroxidases and blocked with 5% bovine serum albumin (BSA) for 1 min. Primary antibodies, mouse antihuman IL-33 antibody and mouse IgG (diluted 1:200 in 0.01 mM PBS, pH 7.0, containing 5% BSA; both from R&D systems, Minneapolis, MN, USA) were applied to the tissue sections in a humid chamber at 37°C and irradiated intermittently for 10 min (250W, 4 s on, 3 s off). After three washes for 1 min with 0.05 M Tris-buffered saline, pH 7.6, containing 1% Tween (TBST), peroxidase-conjugated rabbit antimouse antibody (1:50 dilution in 0.01 mM PBS, pH 7.0, containing 5% BSA) from an EnVision™+ Kit (Westang Biotechnology, Shanghai, China) was applied in a humid chamber according to the manufacturer's instructions. Irradiation was then performed intermittently for 10 min as described above. After washing five times with TBST, sections were immersed in diaminobenzidine solution (Sigma-Aldrich, St Louis, MO, USA) with 0.01% H₂O₂ and counterstained with haematoxylin (Dako Cytomation).

Immunohistochemical staining was identified as granular brown deposits in the cytoplasm. Four photographs for each tissue section were taken using a × 200 objective, involving a Leica DFC420 camera system (Leica Microsystems Imaging Solutions, Cambridge, UK). The signal was quantitated using Image Pro^® Plus analysis software, version 5.0 (Media Cybernetics, Bethesda, MD, USA). The optical density of each core point examined represented the degree of protein staining. The median percentage of tissue positive for IL-33 was calculated for all samples.

Analysis of Serum Cytokine Levels

Levels of IL-33, IFN-α, IFN-γ in serum from HCC patients and healthy controls were detected by enzyme-linked immunosorbent assay (ELISA) kits for human IL-33, IFN-α and IFN-γ (all from R & D Systems) according to the manufacturer's instructions.

Statistical Analyses

Statistical analyses were carried out using the SPSS^® statistical package, version 19.0 (SPSS Inc., Chicago, IL, USA) for Windows^®. Data are shown as median (25^th – 75^th percentile) values. Differences in the percentages of tissue stained positive for IL-33 protein between the three groups were compared using Fisher's exact test. Differences in serum IFN-α, IFN-γ and IL-33 levels were compared using a two-tailed unpaired Mann–Whitney U-test. A P-value of < 0.05 was considered to be statistically significant.

Results

In total, 60 HCC patients (43 males and 17 females; mean age 47 years, range 23 – 71 years) and 20 healthy volunteers (13 males and seven females; mean age 37.5 years, range 25 – 68 years) were recruited into the study. Immunohistochemical analyses were carried out in tumour tissue and normal adjacent tissue samples from the 60 HCC patients, and in normal liver tissue samples from the only six control subjects who agreed to undergo liver biopsy. Analyses of serum cytokine levels were carried out in 30 HCC patients for whom both pre- and postoperative samples were available (15 with, and 15 without, metastasis), and in 10 control subjects (only 10 volunteers agreed to provide blood samples).

Representative samples of IL-33 immunohistochemical staining are shown in Fig. 1. IL-33 was mainly localized in the cytoplasm of liver cells for all tissue types. Quantitative analysis of the degree of IL-33 staining showed that the median percentage of tissue positive for IL-33 was significantly higher in HCC tumour tissue (P = 0.0002) and in normal adjacent tissue from HCC patients (P = 0.009) compared with normal liver tissue from healthy controls (Table 1).

FIGURE 1:

Increased levels of interleukin (IL)-33 protein in hepatocellular carcinoma (HCC) tumour tissue and normal adjacent liver tissue from HCC patients, compared with normal liver tissue from healthy control subjects: (A) immuno histochemical staining of IL-33 in tumour tissue from a HCC patient; (B) haematoxylin and eosin staining in tumour tissue from a HCC patient; (C) immunohistochemical staining of IL-33 in normal adjacent liver tissue from an HCC patient; (D) Immunohistochemical staining of IL-33 in normal liver tissue from a healthy control subject. Brown cytoplasmic staining indicates samples positive for IL-33 protein

TABLE 1:

Median percentages of tissue immunopositive for interleukin (IL)-33 protein in tumour tissue from hepatocellular carcinoma (HCC) carcinoma patients, normal adjacent liver tissue from HCC patients and normal liver tissue from healthy controls

Tissue sample	n	Tissue positive for IL-33 Median (range) %	Statistical significance^a
HCC carcinoma tissue	60	85.33 (38.45 – 99.86)	P = 0.0002
Normal adjacent liver tissue from HCC patients	60	30.56 (9.87 – 38.97)	P = 0.009
Normal liver tissue from healthy controls	6	9.78 (2.56 – 12.34)

Statistically significantly different versus healthy controls; Fisher's exact test.

Serum levels of IFN-α, IFN-γ and IL-33 were significantly higher in pre- and postoperative serum samples from HCC patients compared with normal healthy controls (P < 0.05 for all comparisons; Table 2). Serum levels of IFN-α, IFN-γ and IL-33 in the metastatic HCC group were significantly higher than those in the nonmetastatic HCC group (P < 0.001 for all comparisons; Table 2)

TABLE 2:

Serum interferon (IFN)-α, IFN-γ and interleukin (IL)-33 levels in preoperative and postoperative hepatocellular carcinoma (HCC) I

Serum sample	n	IFN-α, pg/ml	Statistical significance	IFN-γ, pg/ml	Statistical significance	IL-33, pg/ml	Statistical significance
Preoperative HCC	30	193.45 (79.77-233.02)	P=0.0024^a	1935.74 (338.84-2318.38)	P<0.0001^a	116.14 (16.38-137.02)	P<0.0001^a
Postoperative HCC	30	24.38 (9.32-215.90)	P=0.0383^a	723.43 (123.61 -2313.90)	P<0.0001^a	27.86 (8.25-130.66)	P<0.0001^a
Healthy controls	10	8.89 (7.21 -9.89)	—	9.66 (9.33-12.63)	—	4.32 (3.25-5.20)
Metastatic HCC	15	223.02 (214.79-226.21)	P=0.0005^b	2352.94 (2242.24-2422.34)	P=0.0003^b	137.53 (128.02-146.53)	P=0.0002^b
Nonmetastatic HCC	15	50.73 (6.43-87.5)	—	454.51 (98.72-1344.72)	—	8.66 (7.50-26.12)

Data are median (25^th - 75^th percentiles).

Statistically significantly different versus

healthy controls or

nonmetastatic HCC group; two-tailed unpaired Mann-Whitney U-test.

Discussion

Interferon-α is a lymphokine with a wide range of biological effects. HCC has a high prevalence in China;⁸ however, long-term high-dose IFN-α treatment dose-dependently inhibited metastasis, tumour recurrence and growth of human HCC implanted in the nude mice model, LCI–D20.⁹ Randomized, controlled clinical trials have confirmed that IFN-α can increase the tumour-free survival rate by 10% following liver resection, as reviewed by Qin et al. 2002.¹⁰ IFN-α treatment was found to inhibit tumour angiogenesis significantly in a model of corneal neovascularization, where LCI-D20 tumour tissue was implanted to corneas of nude mice.¹¹ IFN-α can also effectively inhibit vascular endothelial cell proliferation and migration.¹² These results suggest that antiangiogenesis is the main target of IFN-α therapy in HCC.

Interferon-γ is a cytokine with a variety of important physiological functions, particularly the promotion of the innate and adaptive immune responses, as noted in reviews by Bach et al. and Boehm et al.^13,14 In higher biological species, the loss of IFN-γ or its associated signalling response may lead to fatal pathogenic infection.¹³ Research suggests that IFN-γ may also inhibit tumour formation.¹⁴ Loss of sensitivity to IFN-γ may also increase susceptibility to cancer: immunogenic fibrosarcomas grew faster in animals treated with neutralizing IFN-γ antibodies than in normal mice.¹³ The ability of lipopolysaccharide to decrease tumour formation was inhibited following administration of neutralizing IFN-γ antibodies.¹³ Similarly, in primary mouse tumour models induced by chemical carcinogens, IFN-γ was shown to have a protective effect against tumour invasion.¹⁵

Interferon-α and IFN-γ are important in the prevention of primary hepatocellular carcinoma development,^16,17 and IFN-γ may also be used as an early indicator of liver cancer severity.¹⁷ Serum concentrations of both cytokines in patients with HCC in the present study were significantly higher those observed in healthy controls.

Interleukin-33 (also known as IL-1F11), is a cytokine that interacts with the ST2 receptor,^18,19 first described by Schmitz et al.²⁰ when comparing the homology of the IL-33 protein with that of IL-1. Sequence analysis confirmed that the C terminal of the molecule is similar to that of the IL-1 family of cytokines, therefore it was named IL-33 according to the naming conventions for interleukins. Coimmuno precipitation experiments demonstrated that IL-33 binds to ST2, thereby activating NF-κB and other downstream molecules, which may explain why IL-33 and IL-1 share similar receptor signalling pathways.^21,22

Interleukin-33 can recruit the downstream signalling molecules IL-1-associated protein kinase 1, IL-1-related protein kinase 4 and tumour necrosis factor receptor-associated factor 6. Following binding to ST2, this can activate NF and mitogen-activated protein kinase, and induce cytokine transcription and subsequent Th2 regulatory biological functions.⁵ IL-33 can also stimulate IL-6, IL-1β and IFN-α secretion from mouse bone marrow-derived primary mast cells.²³,²⁴ IL-1β and IFN-α stimulation of primary lung and skin fibroblasts can induce keratinocytes to secrete IL-33, suggesting that IFN-α and IL-33 are likely to be part of a positive feedback loop, and that IL-33 in liver cancer may play a similar proinflammatory role with IFN-α. Foot or joint injection of IL-33 in mice can induce nociception in a time- and dose-dependent manner, whereas treatment with soluble ST2 or morphine can reduce injury²⁵ Injection of methylated BSA can induce tissue injury and high levels of IL-33, which can be can alleviated by soluble ST2 in a dose-dependent manner.⁵ Based on the above, we speculate that IL-33 is likely to play a similar proinflammatory role with IFN-α in HCC.

In summary, the present study findings suggest that high levels of IL-33 protein in serum and HCC liver tumour tissue may reflect the degree of disease development and metastasis. Higher serum levels of IL-33, IFN-α and IFN-γ were observed in HCC patients compared with healthy controls, and in HCC patients with metastatic disease compared with those with no metastasis, indicating that IL-33 is activated in early HCC. Thus, IL-33 may be useful as a new diagnostic indicator for HCC.

Footnotes

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (No. 30972610) and Jilin Province Science and Technology Agency (No. 200705128). In addition, it was a component of Chinese Medical Science and Technology Projects in Administration of Chinese Medicine of Jilin Province (08sys-086), Health Department Research Projects in Jilin Province (2009Z054) and Cutting-edge Science and Interdisciplinary Innovation Projects of Jilin University.

The authors had no conflicts of interest to declare in relation to this article.

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Detection of Interleukin-33 in Serum and Carcinoma Tissue from Patients with Hepatocellular Carcinoma and its Clinical Implications