Abstract
Background
The present study determined type I interferon (IFN) receptor (interferon-α/β receptor (IFNAR)) and its predicable role in interferon-α2b treatment in chronic hepatitis B (CHB) patients.
Methods
Expression of IFN-α/βR-1 and IFN-α/βR-2 in peripheral blood mononuclear cells and in liver tissue was measured by flow cytometry, immunofluorescence, immunohistochemistry and RT-PCR.
Results
IFN-α/βR-1 and IFN-α/βR-2 in monocytes and lymphocytes increased in CHB patients. Expression of IFNAR-1 and IFNAR-2 in liver had positive correlation with HBV-DNA in liver tissue. Expression of IFN-α/βR in lymphocytes and monocytes increased in the first month, but then decreased during the subsequent interferon-α2b treatment, patients who had higher levels of IFN-α/βR-2 in monocytes prior to therapy showed better viral response than those with lower levels.
Conclusions
Expression of IFN-α/βR-2 in monocytes can be used as a predictable parameter to evaluate the effect of IFN-α treatment in CHB patients.
Keywords: Interferon-α/β receptor, chronic hepatitis B, interferon-alpha, peripheral blood mononuclear cells (PBMCs)
Introduction
Patients with chronic hepatitis B (CHB) are at increased risk of developing hepatic decompensation, cirrhosis, and hepatocellular carcinoma [1, 2]. Interferon (IFN)-α, the first drug approved for use against CHB by the Food and Drug Administration (FDA) in the USA, has been shown to be effective in suppressing hepatitis B virus (HBV) replication [3] as well as in induction remission of liver disease. However, its efficacy is limited to a small percentage of highly selected patients.
It is now known that all type I IFNs bind to the same dimeric receptor, the interferon-α/β receptor (IFNAR). It is a heteromeric receptor composed of one chain with two subunits, referred to as interferon-α/β receptor 1 (IFNAR-1) and interferon-α/β receptor 2 (IFNAR-2) [4]. IFNAR-2 binds to type I IFNs with different affinities, mostly in the nanomolar range, and its low-affinity partner, IFNAR-1, which binds to IFNs with micromolar affinities, also differentially recognizes the type I IFNs [5, 6]. IFNs bind initially to IFNAR-2, and this complex then recruits IFNAR-1 [7, 8].
The expression of the type I IFN receptor in liver tissues may be directly involved in the pathogenesis of viral hepatitis and the response to IFN therapy. After a sequence of IFN-a receptor subunit genes was identified [9, 10], several investigators assessed the amounts of IFNAR-1 and IFNAR-2 expression in patients with hepatitis C by polymerase chain reaction (PCR) assay [11]. mRNA levels of IFNAR-1 and IFNAR-2 were significantly higher in patients with sustained virological and biochemical response to interferon therapy compared with those with nonsustained response. Moreover, mRNA expression ratio of IFNAR-1 to IFNAR-2 was also significantly high in patients with sustained virological and biochemical response to IFN therapy. The expression level of IFNAR-2 in the liver was related to that in peripheral blood mononuclear cells (PBMCs). These results suggest that hepatitis C virus infection may up-regulate the expression of the type I interferon receptor and that the measurement of IFNAR-2 expression in PBMCs may be useful for monitoring its expression in liver.
The efficacy of IFN-α is limited to a small percentage of highly selected patients, only 20% to 40% have a complete, sustained inhibition of viral replication [12–14]. One possibility might be the difference in interferon-α/β receptor (IFNAR) expression in those patients, based on the observation that monocytes and B cells express the highest levels of IFNAR among peripheral hematopoietic cells [15]. These results prompted us to determine which parameter could be used for predicting response to IFN-α therapy; we detected IFNAR expression in CHB patients before and after application of interferon-α2b.
To gain more insights into the alteration of IFNARs in CHB patients, we measured mRNA expression of IFNAR-1, IFNAR-2, interleukin (IL)-6, IL-8, tumor growth factor (TGF)-β1 and tumor necrosis factor (TNF)-α both in PBMCs and liver tissues by real-time reverse-transcription polymerase chain reaction (RT-PCR). In addition, we evaluated the expression of IFN-α/βR-1 and IFN-α/βR-2 on peripheral lymphocytes and monocytes by flow cytometry, and performed immunofluorescence histochemistry to detect the expression of IFN-α/βR-1 and IFN-α/βR-2 in liver.
Patients and Methods
Patients
From March 2007 to June 2010, 84 patients (54 men and 30 women, age 21–51 years), who met the criteria for chronic hepatitis B according to the AASLD practice guideline and Chinese Society of Hepatology guideline [1, 16], and ten healthy people were enrolled in Qilu Hospital, Shandong University. All the patients were grouped into two groups: group A (56 patients): HBsAg+ (hepatitis B surface antigen), HBeAg+ (hepatitis B e antigen), Group B (28 patients): HBsAg+, HBeAg−. Of all the patients, 25 patients accepted liver biopsy. Twenty-five patients who met the criteria for IFN-α treatment [16] were treated with recombinant human interferon-α2b (Yuan Ce Pharm, Beijing China) for at least 3 months (administered three million unit thrice a week), and blood samples were collected every 4 weeks to monitor the alteration of biochemical parameters. The effect of interferon-α2b was verified by biochemical response (BR): decrease in serum ALT to within the normal range; virologic response (VR): decrease in serum HBV-DNA to undetectable levels by PCR assays, and loss of HBeAg in patients who were initially HBeAg positive; and early virologic response (EVR): decrease in serum HBV-DNA more than 2 log10 copies/mL in 3 months.
Clinical characteristics were showed in Table I. All patients gave written informed consent for research testing under protocols approved by the local Research and Ethics Committee at Qilu Hospital of Shandong University in accordance with the guidelines of the 1975 Declaration of Helsinki.
Table I.
Clinic characteristics of chronic hepatitis B patients and control health group
| Group | HBV-DNA≥105 | HBV-DNA<105 | Control group |
|---|---|---|---|
| Case number (gender) | 43 (M31/F12) | 41 (M13/F18) | 10 (M6/F4) |
| Age (year) | 33±10 | 41 ±13 | 26±5 |
| ALT (IU/L) | 256.7±186.5 | 151.4± 167.2 | 25.8+15.2** |
| AST (IU/L) | 221.3±145.2 | 121.5± 152.3 | 28.8±23.2** |
| ALP (IU/L) | 102.5±56.8 | 121.4±87.8 | 102.3±76.4*** |
| GGT (IU/L) | 102.3± 119.3 | 123.5± 121.3 | 93.6±98.7*** |
| TP (G/L) | 73.9±9.3 | 71.3±8.6 | 72.7±8.7*** |
| ALB (G/L) | 38.6±8.9 | 39.2±9.1 | 38.8±7.8*** |
| TBIL (umol/L) | 18.8±20.2 | 17.6± 17.0 | 15.2± 18.3*** |
| DBIL (umol/L) | 10.5±12.3 | 11.5± 10.4 | 16.2±9.4* |
| Cho (mmol/L) | 3.24±1.05 | 2.98± 1.32 | 3.12±2.05*** |
| TG (mmol/L) | 0.81 ±0.21 | 0.87±0.32 | 0.78±0.32*** |
| GLU (mmol/L) | 5.04±0.81 | 4.97±0.78 | 4.56±0.76*** |
| BUN (mmol/L) | 4.87±1.01 | 4.88± 1.03 | 4.84±0.89*** |
| Ccr (mmol/L) | 72.4±16.9 | 74.3± 15.7 | 68.2±17.3*** |
| HBV-DNA in serum (copies/mL) | (12.1±16.5)×105 | (13.2±13.1)×103 | <103** |
| HBV-DNA in liver (copies/mL) | (10.1±6.5)×105 | (11.4±13.5)×105 | NC |
| n=13 | n=12 | ||
| Grade of inflammatory (number of patients) | |||
| G0 | 0 | 0 | |
| G1 | 3 | 4 | |
| G2 | 5 | 5 | |
| G3 | 4 | 3 | |
| G4 | 1 | 0 | |
| Stage of fibrosis (number of patients) | |||
| S0 | 4 | 4 | |
| S1 | 5 | 4 | |
| S2 | 3 | 4 | |
| S3 | 1 | 0 | |
| S4 | 0 | 0 |
All the data are presented as means±standard error
ALT alanine aminotransferase, AST aspartate transaminase, ALP alkaline phosphatase, GGT r-glutamyletransferace, TP total protein, ALB albumin, TBIL total bilirubin, DBIL direct bilirubin, Cho total cholesterol, TG triglycerides, GLU glucose, BUN blood urea nitrogen, Ccr endogenous creatinine clearance
p<0.05;
p<0.01;
p>0.05
Cell Isolation
PBMCs were isolated from whole blood by means of Ficoll-Paque Plus (GE Healthcare Piscataway, NY, USA) density gradient centrifugation [17]; 10-mL heparinized whole blood diluted 1:1 with phosphate-buffered saline (PBS), were layered on top of 5-mL Ficoll and centrifuged without a brake at 500×g (20°C) for 30 min and the PBMC ring was collected, and diluted with GIBCO RPMI 1640 (Invitrogen, Carlsbab, CA, USA) to a final volume of 50 mL, and centrifuged at 500×g for 15 min at room temperature. The pellet was washed again with RPMI 1640 for the measurement. CD14+ monocytes and CD14− lymphocytes were purified from PBMCs by positive or negative selection using microbeads MACS system (Miltenyi Biotech, Bergisch-Gladbach, Germany) according to the manufacturer’s instructions.
Isolation of Human PBMCs and Quantitative RT-PCR
Total RNA was extracted and purified from PBMCs using RNeasy Mini Kit (Qiagen, Valencia, CA, USA). RNA was isolated from human liver tissue by homogenization and purification using TRIzol (Invitrogen) followed by RNeasy clean-up (Qiagen); 2 µg RNA was used as a template for cDNA synthesis according to the handbook of High Capacity cDNA Reverse-Transcription Kit (Applied Biosystem, Foster City, CA, USA). Quantitative RT-PCR was performed with the SYBR Green RT-PCR kit (Bio-Rad, Hercules, CA, USA) on the ABI PRISM 7000 (Applied Biosystems). The primers of IFNAR-1, IFNAR-2, IL-6, IL-8, TGF-β and TNF-α were designed using Primer Express software, version 2.0 (Applied Biosystems) (Table II). GAPDH was used to normalize the samples in each PCR reaction. The absence of nonspecific primer-dimer products was verified by melting-curve and gel-migration analyses. Quantification was performed by comparing the Ct values of each sample with a standard curve and normalization to GAPDH [18, 19].
Table II.
RT-PCR primers
| Gene | RefSeq ID | Forward | Reverse |
|---|---|---|---|
| h IFNAR-1 | NM_000629 | 5′-TGACCAGAAATGAACTGTGTCA-3′ | 5′-TTTAAATAGTTAAGAGCTTGCCCG-3′ |
| h IFNAR-2 | NM_000874 | 5′-GAAGGTGGTTAAGAACTGTGC-3′ | 5′-CCCGCTGAATCCTTCTAGGACGG-3′ |
| h IL-6 | NM_000600 | 5′-GTCAGGGGTGGTTATTGCAT-3′ | 5′-AGTGAGGAACAAGCCAGAGC-3′ |
| h IL-8 | NM_000584 | 5′-AAATTTGGGGTGGAAAGGTT-3′ | 5′-TCCTGATTTCTGCAGCTCTGT-3′ |
| h TGF-β1 | NM_000660 | 5′-CTTCCAGCCGAGGTCCTT-3′ | 5′-CCCTGGACACCAACTATTGC-3′ |
| h TNF-α | NM_000594 | 5′-AGATGATCTGACTGCCTGGG-3′ | 5′-CTGCTGCACTTTGGAGTGAT-3′ |
| h GAPDH | NM_002046 | 5′-AATGAAGGGGTCATTGATGG-3′ | 5′-AAGGTGAAGGTCGGAGTCAA-3′ |
Flow Cytometry
IFN-α/βR-1 and IFN-α/βR-2 expression was measured by flow-cytometric analysis [20]. Briefly, 2.0×105 cells were incubated with 10 µl (25 µg/mL) primary antibodies (goat monoclonal anti-human IFN-α/βR-1 antibody or rabbit polyclonal anti-human IFN-α/βR-2: R&D Systems, Minneapolis, MN, USA) for 1 h, followed by incubation with 1% fluorescein phycoerythrin (PE red)-conjugated donkey anti-goat IgG (Santa Cruz Biotech, Santa Cruz, CA, USA) or PerCP-cyanine dye 5.5 (Cy5.5 blue)-conjugated mouse anti-rabbit IgG (Santa Cruz Biotech) as the secondary antibodies. Background staining was determined using matched isotype controls, and all staining was done in the presence of 1% human serum to block nonspecific binding to Fcg receptors. Mean fluorescence intensity (MFI) of the background stain was subtracted from IFN-α/βR-1 and IFN-α/βR-2 MFIs. The cells were analyzed using a FACSCalibur (Becton Dickinson, San Diego, CA, USA) to measure the forward and sideward light scatter and PE and Cy5.5 fluorescence [21].
Liver Histology and Immunofluorescence Histochemistry
All liver biopsies were obtained through percutaneous techniques, and histological examination was performed according to the Batts–Ludwig scoring system at a single pathology laboratory [22, 23]. Immunofluorescence double staining was carried out to detect IFN-α/βR-1 and IFN-α/βR-2 in liver biopsy tissue. After deparaffinization and dehydration, liver sections were microwaved to get antigen retrieval, blocked in the Dako protein block serum free (Dako, Carpinteria, CA, USA), and then incubated in a mixture of goat monoclonal anti-human anti-IFN-α/βR-1 (1:200) and rabbit polyclonal anti-human anti-IFN-α/βR-2 (1:200; R&D Systems.). One percent PE red-conjugated donkey anti-goat IgG (Santa Cruz Biotech, Santa Cruz, CA, USA) and Cy5.5 blue-conjugated mouse anti-rabbit IgG (Santa Cruz Biotech) as the secondary antibodies. After staining, the sections were mounted with Dako mounting medium (Dako.) and observed with a fluorescence microscope (Olympus 1X71, Japan). The positive area was measured in six high-power (*200) fields on each slide and quantified using NIH recommended software Image J.
Immunohistochemistry
Paraffin sections were deparaffinized by xylene and dehydrated in graded alcohol. Endogenous peroxide was inactivated using 3% cold hydrogen peroxide (Sigma-Aldrich St. Louis, MO) in methanol for 15 min. Slides were microwaved in citrate buffer for 20 min to get antigen retrieval, followed by blocking in the Dako protein block serum free (Dako) for 30 min, and an overnight incubation at 4°C in anti-IFN-α/βR-2 (1:200; R&D Systems.) in PBS. After washing, goat anti-rabbit IgG (horseradish peroxidase conjugated; Santa Cruz Biotech, Santa Cruz, CA, USA) was used as secondary antibody for 1 h at room temperature and then the signal was detected using an ABC Elite kit (Santa Cruz Biotech.). For negative control, sections were incubated with secondary antibodies only.
Statistical Analysis
Results were expressed as the mean±standard deviation. Statistical significance between CHB patients and the control group were evaluated by the unpaired Student’s t test. Differences among the patients groups were analyzed by Student’s t test. The relationships between variations were analyzed by nonlinear (curve fit) and linear regression. A p value of <0.05 was considered statistically significant. All analysis was performed by Graph Pad Prism 5.0 (GraphPad Software, San Diego, CA, USA).
Results
Expression of IFN-α/βR in PBMCs Increased Significantly in CHB Patients
In concordance with a previous study [20], lymphocytes and monocytes from CHB patients showed up-regulated expression of IFN-α/βR-1 and IFN-α/βR-2 (Table III). IFN-α/βR-1 and IFN-α/βR-2 were predominantly expressed in the control group by lymphocytes, but not monocytes (IFN-α/βR-1, 26.65±2.15 vs. 16.11±1.96; IFN-α/βR-2, 25.25±2.65 vs. 15.35±3.98; Figure 1a). Both lymphocytes and monocytes from CHB patients expressed similar levels of IFN-α/βR-1 and IFN-α/βR-2 (IFN-α/βR-1, 39.27±2.10 vs. 40.44±1.49; IFN-α/βR-2, 38.40±2.67 vs. 45.67±3.59. Figure 1b).The MFI of IFN-α/βR-1 and IFN-α/βR-2 in peripheral lymphocytes was increased by Δ11.62±2.34 and Δ14.15±3.94, respectively. Moreover, IFN-α/βR-1 and IFN-α/βR-2 expression in monocytes from CHB patients was increased much higher than in lymphocytes (Δ25.33±1.65 and Δ27.32±2.56, respectively). Monocytes showed much more IFN-α/βR-1 and IFN-α/βR-2 expression increasement than lymphocytes in response to CHB virus (p<0.0001).
Table III.
Expression of IFN-α/βR in control group and CHB patients (By FACS)
| Control group | CHB patients | Increasement | |
|---|---|---|---|
| IFN-α/βR-1 in monocytes (MFI) | 16.11±1.96 | 40.44±1.49* | Δ25.33±1.65 |
| IFN-α/βR-2 in monocytes (MFI) | 15.35±3.98 | 45.67±3.59* | Δ27.32±2.56 |
| IFN-α/βR-1 in lymphocytes (MFI) | 26.65±2.15 | 39.27±2.10* | Δ11.62±2.34 |
| IFN-α/βR-2 in lymphocytes (MFI) | 25.25±2.65 | 38.40±2.67* | Δ14.15±3.94 |
All the data are presented as means±standard error
MFI mean fluorescence intensity
p<0.01
Fig. 1.
Expression of IFN-α/βR-1 and IFN-α/βR-2 in peripheral lymphocytes and monocytes increased in CHB patients. a IFN-α/βR-1 and IFN-α/βR-2 increased both in peripheral lymphocytes and monocytes in CHB patients. b In control group, IFN-α/βR-1 and IFN-α/βR-2 expression in lymphocytes was much higher than in monocytes, while in chronic HBV patients the levels of two kinds of receptor showed no difference between lymphocytes and monocytes. *p<0.05; **p<0.01; #p>0.05
Relationship Between IFNAR and IL-6, IL-8, TGF-β1 and TNF-α in mRNA Levels
We found out that expression of IFNAR-1, IFNAR-2, IL-6, IL-8, TGF-β1 and TNF-α in PBMCs and livers significantly increased in patients with CHB (Fig. 2a). While, mRNA levels of IFNAR-1 and IFNAR-2 in PBMCs had no correlation with the severity of viral infection in all the patients (data not shown). There was no difference in IFNAR-1 and IFNAR-2 as well as TGF-β1, TNF-α, IL-6 and IL-8 mRNA levels between the two groups of patients (data not shown). Elevated hepatic mRNA expression of IFNAR-1 and IFNAR-2 was strongly correlated with HBV-DNA content in liver biopsy samples from CHB patients (Fig. 2b). The mRNA expression of IFNAR correlated with all the inflammatory cytokines such as IL-6, IL-8, TGF-β1 and TNF-α detected in livers (Fig. 3).
Fig. 2.
mRNA expression of IFNAR-1, IFNAR-2, IL-6, IL-8, TGF-β1 and TNF-α in PBMCs and liver increased in CHB patients. a All cytokines (IL-6, IL-8, TGF-β1 and TNF-α) and receptors (IFNAR-1, IFNAR-2) mRNA increased significantly in CHB patients, especially in the liver (*p<0.01). b IFNAR-1 and IFNAR-2 in liver displayed a positive correlation with HBV-DNA in liver tissues
Fig. 3.
Correlations between IFNAR and IL-6, IL-8, TGF-β1 and TNF-α in liver mRNA levels. a, b Correlations between mRNA levels of IFNAR-1 and IL-6, IL-8, TGF-β1, TNF-α in liver
Taken together, our data indicate that mRNA levels of hepatic IFNAR-1 and IFNAR-2 correlate with the HBV-DNA content in liver tissue of CHB patients.
Expression of IFN-α/βR-1 and IFN-α/βR-2 in Liver Tissues Increased Significantly in CHB Patient
Immunofluorescence histochemistry of liver tissues were double stained with IFN-α/βR-1 and IFN-α/βR-2 antibodies and showed increased expression compared with control liver tissues (Fig. 4a). Immunohistochemistry of liver tissues stained with IFN-α/βR-2 antibody had the same result (Fig. 4b). However, there was no relationship between IFN-α/βR-1 and IFN-α/βR-2 expression and inflammatory grade or fibrosis stage (data not shown).
Fig. 4.
Expression of IFN-α/βR-1 and IFN-α/βR-2 in liver went up in CHB patients. a Liver tissues double stained with IFN-α/βR-1 and IFN-α/βR-2 antibodies were more positive than the normal control. b Immunohistochemistry of liver tissues stained with IFN-α/βR-2 antibody exhibited the same result
Alterations of IFNAR Expression in Response to Interferon-α2b Therapy
Among the 25 chronic hepatitis B patients treated with interferon-α2b, 12 patients reached BR, only one patients achieved VR, while nine patients had EVR (Table IV).
Table IV.
Clinic parameters of interferon-α2b–treated patients
| Group | Before treatment | 1 month | 2 months | 3 months |
|---|---|---|---|---|
| Case number (gender) | 25 (M16/F9) | 25 (M16/F9) | 25 (M16/F9) | 25(M16/F9) |
| Age (year) | 32±12 | 32±12 | 32±12 | 32±12 |
| ALT (IU/L) | 267.7±176.5 | 165.8±147.6 | 151.4±137.2 | 87.2±52.3** |
| AST (IU/L) | 196.7±185.4 | 213.3±167.2 | 132.5±107.3 | 97.3±67.6** |
| AKP (IU/L) | 96.5±48.6 | 101.3±87.6 | 123.4±78.9 | 101.4±75.2*** |
| GGT (IU/L) | 94.3±109.2 | 100.4±79.2 | 103.5±97.3 | 110.8±78.4*** |
| TP (G/L) | 74.1±8.9 | 73.1±7.5 | 73.2±6.9 | 71.4±8.9*** |
| ALB (G/L) | 37.6±7.9 | 37.2±6.7 | 38.2±8.4 | 37.8±7.6*** |
| TBIL (umol/L) | 35.8±10.2 | 26.9±9.1 | 25.6± 10.1 | 24.8±9.4*** |
| DBIL (umol/L) | 15.5±12.3 | 16.4± 10.4 | 15.5±8.4 | 13.4±8.1*** |
| HBsAg | 3,387.6±1,434.3 | 3,476.8±1,132.4 | 2,987.6±1,034.5 | 3,076.5±1,213.4*** |
| HBeAg | 1,209.2±978.6 | 1,023.6±765.4 | 874.5±654.7 | 543.6±467.9** |
| HBV-DNA in serum (copies/mL) | (12.1±16.5)×105 | (11.2±13.6)×105 | (14.2±6.1)×104 | (10.2±5.8)×104* |
| Biochemical respond (BR) | 1/25 | 8/25 | 12/25 | |
| Virologic responds (VR) | 0/25 | 0/25 | 1/25 | |
| Early virologic responds (EVR) | 2/25 | 4/25 | 9/25 |
All the data are presented as means±standard error
p<0.05;
p<0.01;
p>0.05
During interferon-α2b treatment, IFN-α/βR-1 and IFN-α/βR-2 in lymphocytes and monocytes in all the 25 patients treated with interferon-α2b increased in the first month, but then decreased in the following 2 months (Fig. 5b). The mRNA levels of IFNAR-1 and IFNAR-2 in PBMCs showed similar patterns (data not shown). After we analyzed the difference between responders (patients who achieved EVR, BR or VR. Some patients could achieve BR, EVR and VR at the same time, so the total number of responders was 12.) and nonresponders (NR, n=13), we found out that before interferon-α2b treatment, the expression of IFN-α/βR-1 and IFN-α/βR-2 in lymphocytes and monocytes magnified almost no difference between the two groups. But the expression of IFN-α/βR-2 in monocytes in responders was higher than that in nonresponders (Fig. 6d) before the application of interferon-α2b. During the 3-month treatment, the expression of IFN-α/βR-1 and IFN-α/βR-2 in lymphocytes and monocytes was much higher in responders than non-responders (Fig. 6a – d).
Fig. 5.
Alteration of IFN-α/βR in lympocytes and monocytes in the 25 patients treated with interferon-α2b. a IFN-α/βR-1 and IFN-α/βR-2 in lympocytes and monocytes significant increased after interferon-α2b treatment in the first month, while in the following 2 months, both IFN-α/βR-1 and IFN-α/βR-2 decreased dramatically. b In all the 25 patients treated with interferon-α2b, IFN-α/βR-1 and IFN-α/βR-2 in lympocytes and monocytes first increased in the first month, but then decrease in the following 2 months (*p<0.05; **p<0.01)
Fig. 6.
Comparisons between responders (n=12) and nonresponders (n=13) at 3 months of treatment. a, b IFN-α/βR-1 and IFN-α/βR-2 in lympocytes were almost at the same levels before interferon-α2b treatment both in responders and nonresponders. Expression of IFN-α/βR-1 and IFN-α/βR-2 in lympocytes in the two groups demonstrated the same trend of alteration (increased in the first month and then deceased dramatically in the following 2 months). Expression of IFN-α/βR-1 and IFN-α/βR-2 in lymphocytes in responders was much higher than nonresponders. c, d IFN-α/βR-1 and IFN-α/βR-2 in monocytes showed almost the same results, but IFN-α/βR-2 expression in responders was much higher than nonresponders (**p<0.01)
Along with interferon-α2b treatment, the mRNA levels of IL-6, IL-8, TGF-β and TNF-α, all decreased (Fig. 7a), implying that recombinant IFN-α can significantly suppress the inflammatory cytokines induced by hepatitis B virus. Expression of IFN-α/βR-2 in monocytes in the response patients was significantly higher than the NR patients (Fig. 7b).
Fig. 7.
Alteration of IL-6, IL-8, TGF-β1 and TNF-α in PBMCs during interferon-α2b treatment. a The mRNA levels of IL-6, IL-8, TGF-β1 and TNF-α in PBMCs continuously decreased according to interferon-α2b treatment in all the 25 treated patients. b Compared the expression of IFN-α/βR in PBMCs in responders (total, 12 of 25) and nonresponders (total, 13 of 25) before treatment, the expression of IFN-α/βR-2 in monocytes in responders was significantly higher than the nonresponders (43.60±5.69 vs. 29.53±6.67). *p<0.05; **p<0.01; #p>0.05
Discussion
IFN-α is a critical mediator of immune response to HBV infection. IFN-α can stimulate the immune responses, which target infected hepatocytes leading to a decrease of hepatitis B virus. IFNs bind initially to IFN-α/βR-2, and this complex then recruits IFN-α/βR-1. The extracellular ligand-binding domain of IFN-α/βR-2, by virtue of its smaller size and higher affinity for IFNs than IFN-α/βR-1, is a more tractable target for mutagenesis and structural studies [24].
Sarah L. Pogue demonstrated that expression levels of IFNAR-1 and IFNAR-2 were highest on the monocyte population, followed by B cells. Neutrophils and NK cells expressed approximately one third the level of receptor as B cells, and the T cell subsets expressed low but detectable levels of the receptor [15]. Shiro Tochizawa’s results indicated that all leukocyte subsets possessed IFNAR-2, and the expression was higher in monocytes and granulocytes than in lymphocytes [20]. We found that IFN-α/βR-1 and IFN-α/βR-2 were expressed ubiquitously in leukocytes, including lymphocytes, monocytes and neutrophils, while the expression of IFN-α/βR-1 and IFN-α/βR-2 in neutrophils shows no difference between CHB patients and healthy controls. To observe IFNAR’s fluctuation during IFN-α treatment, we focused on IFN-α/βR-1 and IFN-α/βR-2 in lymphocytes and monocytes. Lau JY first reported the expression and regulation of IFNAR in chronic hepatitis B virus infection in 1991 [25]. Their data indicated that IFNARs are highly expressed and regulated normally in chronic hepatitis B virus infection by interferon-α therapy. Their results are limited by enrollment (only 20 HBV patients) and the fact that they did not measure the two subunits of IFNAR. Shiro Tochizawa also examined the effects of IFN-α on IFNAR-2 expression using heparin-treated blood. A decrease in IFNAR-2 expression in lymphocytes, monocytes, and granulocytes by 2-h treatment with IFN-α was observed in a concentration-dependent manner [20]. But he got the results only in vitro cultured leukocytes, there were no more data available about the effect of IFN-α on IFNAR expression in CHB patients.
From 2002 to 2007, three groups of researchers from Japan worked on the relationship between type I IFN receptors in response to interferon-α treatment in hepatitis C and gastrointestinal cancers. These results suggest that hepatitis C virus infection may up-regulate the expression of type I interferon receptor and that the measurement of IFN-α/βR-2 expression in PBMCs may be useful for monitoring its expression in liver [26–28]. Our data showed that in chronic hepatitis B patients, IFN-α/βR-1 and IFN-α/βR-2 in PBMCs and liver were elevated significantly after HBV infection. Moreover, IFNAR-1 and IFNAR-2 in livers have a positive relationship with the level of HBV-DNA in livers.
Our study revealed that the expression of IFN-α/βR-1 and IFN-α/βR-2 were higher in lymphocytes than in monocytes in the control group, while it is not the case in the CHB patients. During interferon-α2b treatment, the levels of IFN-α/βR-1 and IFN-α/βR-2 on lymphocytes and monocytes increased in the first month, but then decreased in the following 2 months. The pattern of alteration of IFNAR expression caused by interferon-α2b in CHB patients is not the same as Shiro Tochizawa reported in cultured leukocytes. There are several possible explanations: (a) application of interferon-α2b in the first month can induce the duplication and production of IFNAR in mRNA and protein levels in lymphocytes and monocytes, while continuous stimulation of interferon-α2b can suppress the induction of IFNAR; (b) remaining IFNAR has a higher binding affinity, therefore partially compensating for the decrease in receptor number; (c) the intracellular effects are cumulative, so that with long-term treatment, less interferon-α2b binding is required to sustain the same biological effects; and (d) the maximum biological response requires the occupation of only a small proportion of the accessible receptors. Our study only focused on the expression of IFNAR in PBMCs and in liver tissues, to gain better explanation to such alteration of IFNAR needs further studies.
The IFN molecule itself has no antiviral effect, the binding of IFN to its receptor is an essential first step required to exhibit antiviral activity. However, whether upregulation of the type I IFN receptor results in enhanced antiviral activity by IFN therapy is not confirmed. Patients who achieved EVR or BR had higher levels of IFN-α/βR-2 before interferon a2b treatment in monocytes than the no-response patients. Perhaps the expression of IFN-α/βR-2 in monocytes can be used as a predictive parameter for the efficacy of interferon-α in the treatment of chronic hepatitis B. Perhaps this could explain the clinic manifestation that if a patient could not achieve early virus response in the first 3-month interferon-α2b application, simply extending the course of IFN-α has no use in improving the rate of virus response. During the application of interferon-α2b, the responders exhibited higher expression of IFN-α/βR-1 and IFN-α/βR-2 both in lymphocytes and monocytes than the nonresponders. These results presented a concept to us that higher IFNα/βR expression in PBMCs was a predictive factor associated with sustained virologic response to interferon-α2b therapy in chronic hepatitis B patients.
In summary, we demonstrated that IFNAR expression in CHB patients is higher than in healthy controls, especially in peripheral monocytes. The expression of IFN-α/βR-2 on monocytes before interferon treatment may be used as a predictive parameter. Higher expression of IFNα/βR in lymphocytes and moncytes during interferon-α2b treatment maybe predictive for response to IFN therapy.
Acknowledgments
We would like to thank all the patients who donated blood and liver samples. We thank Dr. Zhaoxia Qi, Dr. Longyan Chen, Dr. Yuchen Fan and clinic nurses in the Department of Hepatology, Qilu Hospital for recruiting patients for this study. We acknowledge Prof. Xun Qu for her excellent technical assistance. We really appreciate Dr. Karin Diggle for her critical reading. This study was supported by Shandong Province Science and Technology Plan (grant: 2006GG2202042) and Key Project of Chinese Ministry of Science and Technology (grant: 2008ZX10002-007). This work was presented in part at the 19th Conference of the Asian Pacific Association for the Study of Liver (APASL) in Hong Kong February 2009 and won the Travel Grant (Auth Code: ATH0900153).
Abbreviations
- HBV
Hepatitis B virus
- CHB
Chronic hepatitis B
- HBsAg
Hepatitis B surface antigen
- HBeAg
Hepatitis B e antigen
- IFN-α
Interferon-α
- IFNAR
Interferon-α/β receptor
- RT-PCR
Real-time reverse-transcription polymerase chain reaction
- GAPDH
Glyceraldehydes-3-phosphate dehydrogenase
- BR
Biochemical response: decrease in serum ALT to within the normal range
- VR
Virologic response: decrease in serum HBV-DNA to undetectable levels by PCR assays, and loss of HBeAg in patients who were initially HBeAg positive
- EVR
Early virologic response: decrease in serum HBV-DNA more than 2 log10 copies/mL in 3 months
Footnotes
The Authors declare that they do not have anything to disclose regarding funding from industries or conflicts of interest with respect to this manuscript.
Contributor Information
Fanli Meng, Department of Hepatology, Qilu Hospital, Shandong University, 107 Wenhua Xi Road, 250012, Jinan, People’s Republic of China.
Jiefei Wang, Department of Hepatology, Shanghai Public Health Clinical Center, Fudan University, 2901 Cao Lang Road, Jinshan District, 201508, Shanghai, People’s Republic of China.
Jian Ge, Department of Hepatology, Qilu Hospital, Shandong University, 107 Wenhua Xi Road, 250012, Jinan, People’s Republic of China.
Xiaopeng Fan, Department of Hepatology, Qilu Hospital, Shandong University, 107 Wenhua Xi Road, 250012, Jinan, People’s Republic of China.
Bing Wang, Department of Hepatology, Qilu Hospital, Shandong University, 107 Wenhua Xi Road, 250012, Jinan, People’s Republic of China.
Liyan Han, Department of Hepatology, Qilu Hospital, Shandong University, 107 Wenhua Xi Road, 250012, Jinan, People’s Republic of China.
Tatiana Kisseleva, Department of Medicine, San Diego, School of Medicine, University of California, 9500 Gilman Drive, La Jolla, CA 92093-0702, USA.
YongHan Paik, Department of Medicine, San Diego, School of Medicine, University of California, 9500 Gilman Drive, La Jolla, CA 92093-0702, USA.
David A. Brenner, Department of Medicine, San Diego, School of Medicine, University of California, 9500 Gilman Drive, La Jolla, CA 92093-0702, USA
Kai Wang, Email: wangdoc876@yahoo.com.cn, Department of Hepatology, Qilu Hospital, Shandong University, 107 Wenhua Xi Road, 250012, Jinan, People’s Republic of China.
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