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* Graduate Institute of Biochemistry and
Graduate Institute of Microbiology and Immunology Medical College, National Cheng Kung University, Tainan, Taiwan; and
Chi-Mei Medical Center, Tainan, Taiwan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and IL-6, it is a normal endogenous feedback factor for the control of immune responses and inflammation (1, 2). Autoimmune models of rheumatoid arthritis, thyroiditis, and collagen-induced arthritis and a model of herpetic stromal keratitis all suggest negative regulatory roles for IL-10 in limiting inflammation and immunopathology (2). However, IL-10 is also a stimulatory factor for mast cells, B cells, and thymocytes (3, 4, 5), is pleiotropic, and acts on many other cell types, including monocytes, macrophages, T cells, NK cells, neutrophils, endothelial cells, and PBMC (6, 7).
IL-19 belongs to the IL-10 family, which includes IL-19, IL-20, IL-22, MDA-7 (IL-24), and AK155 (IL-26). IL-19 induces IL-6 and TNF- production in monocytes (8). It also induces cell apoptosis and reactive oxygen species production in monocytes (8). The IL-19 gene is up-regulated by LPS and GM-CSF (9).
Asthma is characterized by airway hyperreactivity to a variety of specific and nonspecific stimuli, by chronic airway hypersecretion, and by increased IgE levels. The pathology in asthma occurs as a consequence of excessive production of IL-4, IL-5, and IL-13 by Th2 cells. IL-4 is critical for Th2 differentiation and IgE Ab switching (10, 11, 12). IL-5 is a key factor for eosinophil maturation and egress from bone marrow (13, 14, 15). IL-13 contributes to mucus hypersecretion, induces airway smooth muscle hyperresponsiveness, and activates airway stromal cells to produce chemokines such as eotaxin (16, 17). Although the immunological mechanisms that induces asthma and allergy are relatively well characterized, the specific mechanisms that transpire in vivo to down-regulate Th2 cell-mediated allergic inflammatory response are not yet clear.
T cells secreting IL-10 in the respiratory mucosa can regulate Th2-induced airway hyperreactivity and inflammation, and it has been suggested that IL-10 plays an important inhibitory role in allergic asthma (18). Our aim, therefore, was to determine whether IL-19, a member of IL-10 family, is also associated with the pathogenesis of asthma.
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Materials and Methods |
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One hundred asthmatic patients (range, 3–12 years old), treated at the pediatric allergy clinic of National Cheng Kung University Hospital and Chi-Mei Hospital, were included in this study. The asthma severity of these patients was classified as severe-persistent, moderate-persistent, or mild-persistent, according to the guidelines of the Global Initiative for Asthma (April 2002). These patients had taken no oral glucocorticosteroids for at least 1 wk before the blood sampling.
Five milliliters of heparinized venous blood were collected from each patient. The blood samples were centrifuged, and the plasma was collected and stored in aliquots at –80°C before analysis. The control serum from healthy age-matched children and adults was similarly prepared.
Expression and purification of IL-19 recombinant protein
A cDNA clone coded for human or mouse IL-19 sequence from leucine to leucine (aa 25–176) was inserted into the expression vector of Pichia pastoris (pPICZ-; Invitrogen, San Diego, CA). IL-19 was expressed and purified from the culture medium of the yeast cells by affinity chromatography. This protein was used in biological function analysis in vitro and for the generation of polyclonal and mAbs as described below.
Generation of IL-19 mAb
BALB/cJ mice were immunized s.c. weekly for 4 wk with recombinant human IL-19 protein (100 µg/mouse) emulsified with an equal volume of Freund’s complete/incomplete adjuvant. Three days before fusion, three mice were boosted by i.v. injection of the Ag without adjuvant. Spleen cells (1.2 x 108) from immunized mice were fused with X63-Ag8-6.5.3 myeloma cells (1.5 x 107) in the presence of PEG 4000 (Merck, Whitehouse Station, NJ). After fusion, the cells were distributed into 24-well plates and cultured in hypoxanthine/aminopterin/thymidine medium for 14 days. Via ELISA, culture supernatant was tested for the presence of Ab reacting with human IL-19 (hIL-19).3 For the cloning of the selected hybridoma cell, the limiting dilution was conducted twice. The hybridoma cells were cultured in DMEM (Invitrogen Life Technologies) containing 15% FCS, 1% penicillin/streptomycin, 2% L-glutamine, and 1% adjusted NaHCO3 solution. The isotype of the selected Ab was IgG as determined by isotyping ELISA. The Ab was purified from ascites using protein A chromatography.
Generation of anti-hIL-19 polyclonal Ab
Human IL-19 polyclonal Ab was generated by injection of hIL-19 recombinant protein into a rabbit, following the standard procedure. Serum samples were collected, and the Ab was purified using protein A chromatography.
Detection of IL-19 protein in human serum
Human serum IL-19 was detected by ELISA, using anti-hIL-19 polyclonal Ab as the capture Ab and anti-hIL-19 mAb as the detecting Ab.
Detection of IL-4, IL-5, IL-10, and IL-13 cytokines in serum and culture medium
The levels of cytokines in serum and culture medium were detected by ELISA, following the manufacturer’s protocol (R & D Systems, Minneapolis, MN).
Detection of IL-19 protein in murine sera
Mice were bled from the retro-orbital plexus. The blood was centrifuged at 5000 rpm for 10 min, and serum was collected for an ELISA to detect the murine IL-19 level.
Asthmatic mouse model
The allergen Dermatophagoides farinae (Der f; 1 g of lyophilized whole body extract in ether; Allergon, Engelholm, Sweden) (19) was dissolved in pyrogenic-free isotonic saline, filtered through a 0.22-µm pore size filter, and stored at –70°C before use. LPS concentration of the Der f preparations was <0.96 endotoxin U/mg Der f (Limulus amebocyte lysate test, E-Toxate; Sigma-Aldrich, St. Louis, MO). Groups of specific pathogen-free, female, 6- to 8-wk-old BALB/cJ mice (Laboratory Animal Center, National Cheng Kung University, Tainan, Taiwan) were intratracheally inoculated with five doses of Der f (0.5 mg/ml, 50 µl) at 1-wk-intervals according to the method previously described (19). Blood samples were collected via the orbital sinus after the fourth inoculation every other day for 14 days. Control mice were inoculated with saline instead of Der f. The lungs from control mice and asthmatic mice were removed. Total RNA was isolated from the lung for real time PCR analysis.
Electroporation i.m. of IL-19 plasmid
Full-length mouse IL-19 plasmid DNA with a 6-histidine tag at the 3' end was cloned in an expression vector of pcDNA 3.1 (Invitrogen). Mice were anesthetized with 2 ml/kg acepromazine maleate i.p. (Tech America, Elwood, KS). Fifty micrograms of murine IL-19 plasmid DNA was injected, using a 27-gauge needle, into the bilateral quadriceps muscle of each animal. Immediately after injection, transcutaneous electric pulses were applied on each side of the leg with a pair of stainless steel needle electrodes (Genetrode, model 508(S); BTX, San Diego, CA) 5 mm long and 0.4 mm in diameter with a 5-mm fixed distance between them. Electrical contact with the leg skin was ensured by shaving each leg. Electrical pulses were delivered with an electrical pulse generator (Electro Square Porator ECM 830; BTX). The shape of the pulse was a square wave; i.e., the voltage remained constant during the pulse. Six pulses of the indicated voltage (100 V or 900 V) were administered to each injection site at a rate of 1 pulse/s, with each pulse 100 µs in duration (20). Electroporation was performed every other day for 8 days on the muscle of alternate tibias, for a total of four administrations per experiment.
Real time PCR analysis of the transcripts of cytokines
To amplify the IL-13 transcripts, real time PCR was performed using the LightCycler-Fast Start DNA Master SYBR Green I kit (Roche, Indianapolis, IN) according to the manufacturer’s instructions. Sense (5'-CTTGCTTGCCTTGGTGGT-3') and antisense (5'-TGGTCTTGTGTGATGTTGCTC-3') primers were used in the real time PCR of the transcripts. A LightCycler was used for real time PCR. cDNA was diluted (1/50) with nuclease-free water, and 2 µl of the solution were used for the LightCycler SYBR-Green mastermix: 0.5 µM primers, 3 mM magnesium chloride, and 2 µl of Master SYBR-Green in nuclease-free water in a final volume of 20 µl. Individual PCR products were analyzed by melting point analysis. Samples were heated from 50°C to 95°C, and the decline in fluorescent signals of each individual sample was assessed. Melting point characteristics differed between individual PCR products. The fluorescence/time-dependent generation of signals was assessed by the manufacturer’s software program, and the melting point of each product was matched with its individual melting temperature. GAPDH was used as an internal control gene to normalize for RNA amounts. Real time PCR was analyzed using the comparative Ct method according to the manufacturer’s instructions. In brief, sample variation was corrected by subtracting GAPDH Ct values from the Ct values (=
Ct) of the obtained cytokines.
In vitro activity of IL-19 on CD4+ T cells
Single-cell suspensions were prepared from splenocytes depleted of RBCs. CD4+ T cells were subsequently isolated to >97% purity with positive selection using anti-CD4 (L3T4) magnetic beads (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instruction. For in vitro differentiation assays, CD4+ T cells were stimulated for 2 days with 1 µg/ml plate-bound anti-CD3 and 1 µg/ml plate-bound anti-CD28 mAbs in the presence of IL-2 (20 U/ml) alone for Th0 cells. IL-2 (20 U/ml), 5 ng/ml IL-12, and 10 µg/ml anti-IL-4 mAb were added to the T cells for Th1 differentiation, whereas 20 U/ml IL-2, 10 ng/ml IL-4 (10 ng/ml), 1 µg/ml anti-IL-12 mAb, and 10 µg/ml anti-IFN-
mAb were added for Th2 differentiation. All the cytokines and Abs used were purchased from R & D Systems. Three days after primary stimulation, cells were washed and then further cultured in the presence of IL-2 (20 U/ml), IL-12, anti-IL-4 mAb (Th1 polarized) or IL-2 (20 U/ml), IL-4, anti-IL-12 mAb, and anti-IFN- mAb (Th2 polarized) for 7 days. Fresh cytokines and Ab were added every 2 days. On day 7, cell culture supernatants were collected for ELISA of Th1 and Th2 cytokines production.
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Results |
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To explore whether IL-19 was associated with asthma, we compared the average serum level of IL-19 in 100 asthmatic patients with the average levels in 50 healthy adults and 50 healthy age-matched children. A majority of asthmatic patients are younger than 15 years old. Therefore, we chose nonasthmatic adults as well as nonasthmatic children as controls to rule out any age effect. Asthmatic patients had twice the average level of serum IL-19 as controls (Fig. 1).
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Because IL-19 levels in asthmatic patients were higher than those of healthy controls, we analyzed IL-19 expression in an established murine model of asthma induced by Der f allergen. Serum IL-19 level was twice higher in asthmatic mice than in healthy controls (Fig. 3A). In addition, real time PCR analysis showed that 80% more IL-19 transcript was expressed in the lungs of asthmatic mice than in healthy mice (Fig. 3B).
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Because IL-19 was up-regulated in asthmatic patients and asthmatic mice, and because its level was correlated with those of IL-4 and IL-13, we speculated that IL-19 might up-regulate Th2 cytokines. To test this hypothesis, we injected IL-19 cDNA in the expression vector pcDNA3.1 into mice using i.m. electroporation and then monitored the serum levels of IL-4, IL-5, IL-10, and IL-13. Empty pcDNA3.1 vector was injected into the negative-control mice. Three days after the first injection, anti-histidine Ab against recombinant IL-19 detected the expression of exogenous IL-19 in the serum of the experimental mice, and the IL-19 level lasted for >14 days (data not shown). IL-19 up-regulated serum levels of IL-4, IL-5, and IL-10 in the experimental mice compared with the negative controls (Fig. 4). Elevated IL-13 levels, however, were not detected in the serum of the experimental mice (data not shown).
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IL-19 induced IL-4, IL-5, and IL-10, but not IL-13, production in healthy mice. In contrast, both asthmatic patients and asthmatic mice had higher levels of IL-19 and IL-13. We therefore speculated that IL-19 might induce IL-13 only in asthmatic patients or mice. To prove this hypothesis, we injected IL-19 cDNA into asthmatic mice using i.m. electroporation. IL-13 levels were analyzed by real time PCR amplification of lung tissue. Compared with levels in empty vector-treated asthmatic mice, IL-13 transcripts increased in the asthmatic mice (Fig. 5). This result demonstrated that IL-19 induced IL-13 only in asthmatic mice and suggested that IL-19 may up-regulate IL-13 only in activated T cells.
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IgE was elevated in the asthmatic mice. To analyze whether IL-19 would up-regulate IgE production, we also used i.m. electroporation to inject IL-19 into asthmatic mice and monitored their serum IgE levels. Compared with serum IgE levels in healthy control mice, serum IgE levels were 2.43 times higher in asthmatic mice untreated with IL-19 but were 3.17 times higher in IL-19-treated asthmatic mice (Fig. 6). IL-19 up-regulated IgE production in asthmatic mice.
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To further determine whether IL-19 could induce the production of Th2 cytokines in vitro, we isolated CD4+ T cells from the spleens of mice. The CD4+ T cells were incubated on a plate coated with anti-CD3 and anti-CD28 mAbs and stimulated with IL-2 for 2 days. IL-19 was added, and the CD4+ T cells were incubated with IL-19 and IL-2 for another 3 or 7 days. The conditioned medium was collected on day 3 or 7, and the secretion of IL-4, IL-5, IL-10, and IL-13 was analyzed by ELISA, which showed that IL-19 up-regulated production of all these cytokines (Fig. 7). In contrast, IFN- production was not detected (data not shown).
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mAb were added to direct differentiation of Th2 cells. IL-19 was added to these two groups of Th cells at the same time. Conditioned medium was collected on day 7 from the control and IL-19-treated Th cells. We used ELISA to analyze levels of IL-4, IL-5, IL-10, IL-13, and IFN- to monitor their response to IL-19 treatment. IL-19 induced production of IL-4, IL-5, IL-10, and IL-13 in Th2 cells but inhibited production of IFN- in Th1 cells (Fig. 8).
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IL-19 induced IL-13 production on activated T cells, and the in vivo induction of IL-13 occurs only in asthmatic mice. To provide further evidence that this effect occurred directly on activated T cells and was not attributable to any indirect effect of, for example, trace contaminated B cells or monocytes in the primary culture of T cell populations, we treated the activated and nonactivated Jurkat T cells with IL-19 and analyzed IL-13 transcript. Treatment of Jurkat T cells with Con A-PMA increased IL-13 transcript 2-fold (Fig. 9). IL-19 alone did not induce IL-13 transcript. When Jurkat T cells were treated with Con A-PMA followed by IL-19, IL-13 was up-regulated 4-fold. In contrast, if Jurkat T cells were treated with IL-19 followed by Con A-PMA or incubated with IL-19 and stimulant (Con A-PMA) at the same time, induction of IL-13 transcript was similar to that of Con A-PMA alone. These results demonstrated that IL-19 induced IL-13 expression only on activated T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-5 is a key factor for eosinophil maturation and egress from bone marrow. Induction of IL-5 in vivo by IL-19 suggests that IL-19 may also play some role in the maturation of eosinophil in asthmatic patients. Whether IL-19 induces eosinophil maturation and egress from bone marrow in vivo awaits further study.
The immunostimulatory CpG oligonucleotide sequences present in most plasmid DNAs of vector are potent inducers of cytokines (24). Therefore, we used empty vector without IL-19 insert as negative controls in all of our studies of in vivo delivery of IL-19 cDNA into mice by i.m. electroporation. Induction of cytokine protein or up-regulation of cytokine transcript by IL-19 were compared with this negative control to rule out the effect of CpG oligonucleotides. The slight induction of IL-4, IL-5, and IL-10, shown in Fig. 4, by the empty vector may have been due to the CpG sequences in the vector.
In addition to the induction of Th cytokine in vivo, we demonstrated that IL-19 could induce Th cytokine production in vitro on activated CD4+ T cells. To further differentiate which Th cells subset was affected by IL-19, we tested the effect of IL-19 on cytokine production of Th1- and Th2-polarized T cells. IL-19 induced IL-4, -5, -10, and -13 production by Th2 cells but inhibited IFN- production by Th1cells. Our result is similar to the observation by Gallagher et al. (25), who also demonstrated that IL-19 up-regulated IL-4 and down-regulated IFN- in whole PBMC culture. Furthermore, at the end of 7 days of incubation with IL-19, if Th2 cells were collected and washed twice with fresh medium and recultured on the plate without anti-CD3 and anti-CD28 mAbs and incubated with IL-19 alone, Th2 cytokine were found to be up-regulated by IL-19, except for IL-13, which was slightly down-regulated by IL-19 in Th2 cells (data not shown). These data further supported the hypothesis that IL-19 induces IL-13 production only on activated T cells. Studies with the Jurkat T cell line confirmed that the activation of T cells is required for IL-19 to induce IL-13 production from T cells. This result is also consistent with the observation that IL-19 up-regulated IL-13 only in the asthmatic mouse model in which the T cells have been activated. IL-13 is a crucial molecule in the pathogenesis of asthma. The data presented here demonstrate that IL-19 was up-regulated in asthma patients and induced IL-13 production. Therefore, it may play an important role in the pathogenesis of asthma.
In summary, we demonstrated that IL-19, another member of the IL-10 family, was associated with asthma by its modulatory effect on Th2 cytokines, including IL-4, IL-5, and IL-13. Regulation of IL-13, a critical molecule in the pathogenesis of asthma, was dependent on the prior activation of Th cells. The discovery of a new contributor to asthma provides further understanding of the complex mechanism of this disease.
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Footnotes |
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1 This work was supported by a grant from Chi-Mei Medical Center, Tainan, Taiwan. 
2 Address correspondence and reprint requests to Professor Ming-shi Chang, Graduate Institute of Biochemistry, Medical College, National Cheng Kung University, 1 Dahsueh Road, Tainan 701, Taiwan. E-mail address: mschang{at}mail.ncku.edu.tw
3 Abbreviation used in this paper: hIL-19, human IL-19.
Received for publication July 30, 2004. Accepted for publication September 8, 2004.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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hIL-19, Jurkat T cells stimulated with Con A-PMA for 8 h first followed by IL-19 for another 8 h; hIL-19