Altered Expression of IFN-λ2 in Allergic Airway Disorders and Identification of Its Cell Origins

This study investigated the expression levels of interferon- (IFN-) λ2 in peripheral blood and tissues. The results showed that the levels of IFN-λ2 were elevated by 17.9% and 14.2% in the plasma of allergic rhinitis (AR) and combined rhinitis with asthma (AR + AS), which was positively correlated with the level of tryptase but negatively correlated with the level of IL-10. IFN-λ2 was predominately expressed in the CD16+ cells and CD14+ cells in healthy control subjects (HC) but upregulated only in CD8+ cells of AR and in eosinophils of asthma. It was observed that approximately 6.6% and 7.0% dispersed tonsil cells and 5.8% and 0.44% dispersed lung cells are IFN-λ2+ mast cells and macrophages. Moreover, tryptase and agonist peptides of PAR-2 induced enhanced IFN-λ2 mRNA expression in A549 cells. In conclusion, the elevated levels of IFN-λ2 in the plasma of AR and AR + AS indicate that IFN-λ2 is likely to contribute to the pathogenesis of allergic airway disorders. The potential origins of the elevated plasma IFN-λ2 include mast cells, macrophages, and epithelial cells in tissues, neutrophils, monocytes, CD8+ T cells, and eosinophils in peripheral blood. Development of IFN-λ2 related therapy may help to treat or prevent allergic airway disorders.


Introduction
Human IFN-2 (IL-28A) is a relatively new cytokine, in which the genomic structure resembles that of the IL-10 family, but the protein structure is more closely related to type I IFN than to interleukin-(IL-) 10 [1,2]. IFN-2 has been discovered to play a role in innate immunity. For example, it can induce antiviral activity in cell lines, though the potency is weaker than other IFNs [3], and has the potential antitumor effect against human lung cancer cells [4].
It has also been discovered that IFN-2 is capable of exacerbating T-cell-mediated autoimmune diseases such as uveitis [5]. Treatment with IFN-2 completely halts and reverses the development of collagen-induced arthritis, dramatically reduces the numbers of proinflammatory IL-17producing Th17 and T cells in the joints and inguinal lymph nodes, and restricts recruitment of IL-1b-expressing neutrophils [6]. However, IFN-2 seems not effective in inducing Tr1 cells [7] and cannot induce proliferation of regulatory T cells from cord blood CD4(+) T cells [8].
Recently, it was found that the expression level of IFN-2 mRNA was significantly increased during naturally occurring respiratory viral infections in children with asthma [9] and that IFN-2 modulates lung dendritic cells (DC) function to promote Th1 immune skewing and suppresses allergic airway disease [10]. These suggest that IFN-2 is not only involved in autoimmune diseases but also associated with allergic airway disorders. We therefore investigated the potential involvement of IFN-2 in allergic airway diseases in the present study.
To our surprise, information on the IFN-2 expressing cells is very limited. It was found that IFN-2 expressed in tracheobronchial tissue cells from the patients with COPD [11]. DC express moderate quantity of IFN-2 when using 2 Mediators of Inflammation lipopolysaccharide (LPS) as the maturation stimulus [12], and vitiligo patient skin and/or peripheral blood mononuclear cells express IFN-2 mRNA [13]. In order to understand the role of IFN-2, we examined the cell origins of IFN-2 in the present study.
The aim of the study is to investigate the expression of IFN-2 in peripheral blood of allergic airway disorders, its correlation with cytokines and tryptase, and its potential cell location. We found that the levels of IFN-2 were elevated in the plasma of AR and AR + AS and that several cell types express IFN-2.

Patients and Samples.
A total of 33 allergic rhinitis (AR), 26 asthma, 12 combined rhinitis with asthma (AR + AS), and 20 healthy control subjects (HC) were recruited in the study. The diagnosing criterion of asthma was conformed to the Global Initiative for Asthma [14], and diagnosis for allergic rhinitis was based on Allergic Rhinitis and its Impact on Asthma (ARIA) [15]. All patients were asked to stop antiallergy medication for at least 2 weeks prior to attending the study (those who could not stop antiallergy drugs were excluded). The recruited patients did not have any airway infection more than one month. The informed consent from each volunteer according to the Declaration of Helsinki and agreement with the Ethical Committee of the First Affiliated Hospital of Liaoning Medical University and General Hospital of Shenyang Military Area Command were obtained. The general characteristics of the patients and control subjects were summarized in Table 1. Peripheral venous blood sample (10 mL) was collected from each patient or HC and was immediately processed to collect cells and plasma for analysis. Specimens of human tissues for immunohistochemistry and flow cytometry analysis were collected from the Department of Pathology, The First Affiliated Hospital of Liaoning Medical University. Macroscopically normal lung tissue was removed at lobectomy from patients with carcinoma. Tonsillar tissue was removed at tonsillectomy. Nasal polyps were collected from AR patients. The protocol for ethical use of human tissue in research was according to the Declaration of Helsinki (2000) and approved by the Committees of the First Affiliated Hospital of Liaoning Medical University.

Flow Cytometry Examination of Expression of IFN-2 in
Peripheral Blood Cells from Allergic Patients. To detect IFN-2 expression on leukocytes excluding T cells, the following antibodies were added to different testing tubes: (1) to detect IFN-2 expression in basophils: FITC-anti-human CD123 and PerCP-anti-human HLA-DR; (2) to detect IFN-2 expression in CD16+ polynucleated cells, CD16− polynucleated cells, and CD14+ cells and CD19+ cells: PerCP-antihuman CD16, PE/Cy7-anti-human CD14, and APC-antihuman CD19 before 200 L of whole blood being added at room temperature for 15 min in dark. Following ligation of red blood cells, white blood cells were fixed and permeabilized by using Cytofix/Cytoperm Fixation/Permeabilization Kit according to the manufacturer's instructions. Following washing with BD washing buffer, the cell pellets were resuspended and rabbit anti-human IFN-2 followed by PE or FITC conjugated goat anti-rabbit IgG antibodies were added at 4 ∘ C for 30 min. Finally, cells were resuspended in fluorescence-activated cell sorting-(FACS-) Flow solution and analyzed with FACSVerse flow cytometer (BD Biosciences, San Jose, CA). A total of 10,000 events were analyzed per population for each sample. Data were analyzed with CellQuest software (BD Immunocytometry systems).  Median (range) data are shown for the number of subjects indicated. All patients stop using long-acting corticosteroids for at least two weeks and any other antiallergic drugs for one week before skin prick being taken. na = not applicable.

Dispersing Cells from Tissues and Flow Cytometry Analysis of IFN-2 Expression.
The procedure for dispersing tissue cells was mainly adopted from the procedure described previously [16]. Cells were then incubated with each labeled monoclonal antibody including (1) PE/Cy7 conjugated mouse anti-human tryptase, anti-human chymase antibody CC4 (IgM subtype), PE conjugated rat anti-mouse IgM, rabbit anti-human IFN-2, and FITC conjugated goat anti-rabbit IgG antibodies to detect mast cells; (2) PE/Cy7-anti-human CD14, APC-anti-human CD19, rabbit anti-human IFN-2, and FITC conjugated goat anti-rabbit IgG antibodies to detect macrophages and B cells at 4 ∘ C for 30 min in dark. After washing, the cell pellets were resuspended in FACS-Flow solution and analyzed with FACSVerse flow cytometer. A total of 10,000 events were analyzed for each sample. Data were analyzed with CellQuest software.

Immunocytochemical Staining of IFN-2 in Human
Tissues. Tissues were fixed in Carnoy's fixative, dehydrated, and embedded in paraffin wax. Sections (4 m) were dewaxed, rehydrated, and incubated for 10 min with 0.5% H 2 O 2 in methanol followed by 0.1% sodium azide for 10 min in order to inhibit endogenous peroxidase activity. PBS containing 5% BSA was added for 1 h and the same solution was employed as the diluent for the antibodies added subsequently. Sequential sections of tonsil, lung, or nasal polyps were incubated with biotinylated rabbit anti-human IFN-2 for 2 h. After washing with PBST, ExtrAvidin-peroxidase conjugate was applied to sections for 1 h. Staining was developed over 4 min by using DAB chromogen system before being counterstained with Mayer's haematoxylin and mounted in AquaMount. For each section, the number of positively stained cells was counted in at least 30 fields (the area of each field equals 0.19 mm 2 ). A single-blind method was used to examine the slides.
For challenge experiments, cells were detached from culture flasks using trypsin, seeded into 12-well cell culture plates, and grown to about 80% confluence. The cells were then cultured with the serum-free basal medium for an additional 16 h before challenge. Cells were exposed to tryptase (2 g/mL, 1 g/mL = 7.4 nM) with or without its inhibitor leupeptin (3 g/mL), 100 M of SLIGKV-NH 2 with or without PAR-2 antagonist FSLLRN-NH 2 (400 M) and its reverse peptide VKGILS-NH 2 , and 100 M of tc-LIGRLO-NH 2 with or without PAR-2 antagonist FSLLRN-NH 2 (400 M) and its reverse peptide tc-OLRGIL-NH 2 , respectively. Cells (1.5 × 10 6 per well) were collected at 2 h or 6 h, centrifuged at 4 ∘ C, and stored at −80 ∘ C until use.

Quantitative Real-Time PCR (qPCR) Analysis of IFN-2 mRNA Expression in A549
Cells. The expression of IFN-2 mRNA in A549 cells was determined by qPCR following the manufacture's protocol. Briefly, after synthesizing cDNA from total RNA by using Superscript first strand synthesis system for RT-PCR and oligo-dT primers, real-time PCR was performed by using SYBR Premix Ex Taq kit on the ABI Prism 7700 Sequence Detection System (Perkin Elmer Applied Systems, Foster City, CA, USA). Sequence-specific standard curves were generated using 10-fold serial dilutions of plasmid DNA, and the values for the initial concentrations of unknown samples were calculated by using the software (version 1.7) provided with the ABI 7700 system. IFN-2 mRNA expression in each sample was finally determined after correction with -actin expression. Each measurement of a sample was conducted in duplicate. The primers for IFN-2 were forward: 5 -CACCCTGCACCATATCCTCT-3 , reverse: 5 -GGAGGGTCAGACACACAGGT-3 and foractin were forward: 5 -AGAGCTACGAGCTGCCTGAC-3 , reverse: 5 -AGCACTGTGTTGGCGTACAG-3 .

Determination of Levels of Tryptase and Cytokines.
Levels of tryptase, IL-4, IL-10, IL-12, and IFN-2 in the plasma of AR, asthma, AR + AS, and HC were measured by using ELISA kits according to the manufacturer's instructions.
2.9. Statistical Analysis. Statistical analyses were performed by using SPSS software (Version 13.0). Data were expressed as mean ± SEM. Where analysis of variance indicated significant differences between groups with ANOVA, Student's -test was applied. Data for allergic patients are presented as scatter plot. Where Kruskal-Wallis analysis indicated significant differences between groups, for the preplanned comparisons of interest, the paired Mann-Whitney test was employed.
Correlations were determined using Spearman rank correlation. For all analyses, < 0.05 was taken as significant.

Levels of IFN-2 in the Patients with Allergic Rhinitis and Asthma and Its Correlation with Tryptase and Cytokines.
In order to evaluate the potential role of IFN-2 in allergic airway disorders, the most direct evidence is to examine the changes of its levels in clinical specimen. We therefore examined the levels of IFN-2 in the plasma and its cellular location in blood of the patients with AR and asthma. The results showed that the levels of IFN-2 were elevated by 17.9% and 14.2% in the plasma of AR and combined rhinitis with asthma (AR + AS), but not of asthma (Figure 1(a)). The plasma levels of tryptase were increased by 34.7% and 38.3% in the patients with AR and asthma, but not AR + AS (Figure 1(b)). The plasma levels of IL-4 were increased by 21.1% in the patients with asthma but decreased by 55.3% and 26.3% in AR and AR + AS (Figure 1(c)). The plasma levels of IL-10 ( Figure 1(d)) and IL-12 (Figure 1(e)) were decreased by 29.8% and 100% in the patients with AR, by 54.3% and 100% in the patients with asthma, and by 100% and 100% in the patients with AR + AS, respectively. There were positive correlation between IFN-2 and tryptase and negative correlation between IFN-2 and IL-10 in the plasma of AR. Similarly, plasma IFN-2 positively correlates with tryptase, and IL-10 positively correlates with IL-12 in asthma ( Table 2).

Immunohistochemical Staining of IFN-2 in Tissue Cells.
In order to further investigate the potential source of IFN-2, we examined the expression of IFN-2 in cells of various tissue origins by using immunohistochemical staining technique. The results showed that IFN-2 clearly expresses in glandular epithelial cells and some large cells (most likely mast cells or macrophages) in tonsillar tissue (Figure 3    and in some large cells in lung tissue (Figure 3(d)) and nasal polyps (Figure 3(f)) as compared with the negative control tissues (Figures 3(a), 3(c), and 3(f)). dispersed cells are IFN-2+ MC T mast cells, MC TC mast cells, macrophages, and B cells (Figure 4).

Induction of the Expression of IFN-2 mRNA in A549
Cells. Positive correlation of IFN-2 with tryptase implicated that the increased level of IFN-2 in the plasma of patients with AR and AR + AS may be elicited by mast cell tryptase. To confirm this anticipation, we examined the effect of tryptase and agonist peptides of PAR-2 on IFN-2 mRNA expression in A549 cells. It was found that the expression of IFN-2 mRNA over baseline control was increased by approximately 1.4-and 0.5-fold when the cells were incubated with tryptase at 2 g/mL for 2 and 6 h ( Figure 5). Similarly, SLIGKV-NH 2 and tc-LIGRLO-NH 2 induced approximately 1.4-and 0.9fold increase in expression of IFN-2 mRNA over baseline control, respectively, when they were incubated with A549 cells for 2 h (Figure 5). At 6 h following incubation with SLIGKV-NH 2 and tc-LIGRLO-NH 2 , the expression of IFN-2 mRNA was enhanced by approximately 0.6-and 1.0-fold, respectively ( Figure 5). The reverse peptides VKGILS-NH 2   and tc-OLRGIL-NH 2 showed little effect on the expression of IFN-2 mRNA in A549 cells following 2 and 6 h incubation periods ( Figure 5).
Since FSLLRN-NH 2 and leupeptin were able to inhibit tryptase induced upregulation of expression of IFN-2 mRNA and FSLLRN-NH 2 suppressed SLIGKV-NH 2 and tc-LIGRLO-NH 2 induced upregulation of IFN-2 mRNA expression ( Figure 5), the action of tryptase is likely to be mediated by PAR-2 and requires its enzymatic activity.

Discussion
We have demonstrated for the first time that the levels of IFN-2 are elevated in plasma of the patients with AR and AR + AS, but not with asthma, which provides the first hard evidence for proving that IFN-2 may participate in adoptive immune response such as allergic airway reactions. The recent reports that the expression level of IFN-2 mRNA was significantly increased during naturally occurring respiratory viral infections in children with asthma [9] and that IFN-2 was capable of exacerbating a T-cell-mediated autoimmune disease [5] may support our observation.
It is difficult to evaluate the role of IFN-2 in allergic airway inflammation at this stage as we do not know if the increased serum level of IFN-2 is a causative or resulting factor in the pathogenesis of the allergic airway disorders. Our observation that elevated IFN-2 levels were positively correlated to tryptase level in the plasma of AR suggests that these two compounds are likely released from the same source. Since tryptase is a relatively selective marker of mast cell degranulation and the most abundant secretory product from mast cells [17], it is likely that IFN-2 is also released from mast cells upon degranulation. Indeed, we have demonstrated in the present study that large numbers of tonsil and lung MC T and MC TC subtypes of mast cells express IFN-2, confirming that mast cells are the major source of IFN-2. Our previous report that IFN-1 (IL-29) highly expressed in mast cells [18] may support our current observation.
However, unlike tryptase acting as a potent proinflammatory mediator which is capable of provoking microvascular leakage in the skin of guinea pigs [19], stimulating the release of histamine from dispersed human tonsil mast cells [16], and inducing accumulation of eosinophils and neutrophil in the peritoneum of mice [20], IFN-2 appears to act as a suppressor of allergic airway diseases. For example, IFN-2 can modulate lung DC function to promote Th1 immune skewing and suppress allergic airway disease [10]. Since the information on the role of IFN-2 in allergy is very limited, the study that treatment with IFN-2 completely halts and reverses the development of collagen-induced arthritis, dramatically reduces numbers of proinflammatory IL-17-producing Th17 and T cells in the joints and inguinal lymph nodes, and restricts recruitment of IL-1b-expressing neutrophils [6] may support the anticipation that IFN-2 may play an inhibitory role in allergic airway diseases.
Since a large population of macrophages express IFN-2, it is likely one of major sources of IFN-2, considering huge numbers of macrophages in lung and tonsil. Epithelial  cells could be another source of IFN-2 as tonsil glandular epithelial cells express IFN-2, and A549 cells express IFN-2 mRNA. Our observation that tryptase induced upregulation of expression of IFN-2 mRNA in A549 cells is mediated by PAR-2 and requires tryptase enzymatic activity implicates that tryptase may provoke IFN-2 production in lung epithelial cells through activation of PAR-2, and released IFN-2 could contribute to the elevated plasma level of IFN-2 in allergic airway disorders. Obviously, further work is required to prove this speculation. Since little is known of the relationship between PARs and IFN-s, our previous report that the actions of thrombin on A549 cells are most likely carried out through hydrolytic cleavage of N-terminal of PAR-1 [21] may help to understand our observation above.

Mediators of Inflammation
We have also observed the declined plasma levels of IL-10 and IL-12 in the allergic patients. Since the correlation between IL-12 and IL-10 levels in serum has been reported in the patients with atopic dermatitis [22], and diminished IL-12 levels were previously found in the serum of allergic patients [23], our observation may further suggest that reduced IL-10 and IL-12 production may contribute to the pathogenesis of the airway allergic disorders. The negative correlation between IFN-2 and IL-10 in the plasma of AR suggested they are not likely to be released from same sources, which means that if mast cells are major source of IFN-2, they should not be the major source for IL-10 in AR.
In order to identify the potential source of increased IFN-2, we investigated the expression of IFN-2 in peripheral blood leukocytes. Our data showed that IFN-2 expression was downregulated in AR, in asthma, and in AR + AS in monocytes and neutrophils. Since neutrophils and monocytes are predominant IFN-2-expressing cells in blood of HC, the decreased expression of IFN-2 in these 2 cell types could contribute to diminished level of IFN-2 in the plasma of asthma, even though IFN-2 expression appeared to be upregulated in blood cytotoxic T cells and eosinophils in asthma as cytotoxic T cells only weakly express and eosinophils do not express IFN-2 in HC. Downregulation of expression of IFN-2 in peripheral blood monocytes and neutrophils of AR and AR + AS seemed to conflict with the observation of increased level of IFN-2 in the plasma of AR and AR + AS, which suggests that there must be some other sources to generate large amount of IFN-2 apart from blood leukocytes. Moreover since helper T cells including regulatory T cells do not express IFN-2, they are one of the major sources of IL-10, which may at least partially explain the negative correlation between IFN-2 and IL-10 in the plasma of AR.

Conclusion
In conclusion, the elevated levels of IFN-2 in the plasma of AR and AR + AS and positive correlations of plasma IFN-2 with tryptase in AR and asthma indicate that IFN-2 is likely to contribute to the pathogenesis of allergic airway disorders. Mast cells, macrophages, and epithelial cells in human tonsil and lung tissues express IFN-2, and upregulated IFN-2 expression was observed in CD8+ T cells and eosinophils of allergic airway disorders indicate that they are the potential sources of IFN-2. Development of IFN-2 related therapy may help to treat or prevent allergic airway disorders.