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INTERLEUKIN-12 INHIBITS EOTAXIN SECRETION OF CULTURED PRIMARY LUNG CELLS AND ALLEVIATES AIRWAY INFLAMMATION IN VIVO
doi:10.1006/cyto.2002.1950, available online at http://www.idealibrary.com on
INTERLEUKIN-12 INHIBITS EOTAXIN SECRETION OF CULTURED PRIMARY LUNG CELLS AND ALLEVIATES AIRWAY INFLAMMATION IN VIVO Yi-Ling Ye,1 Wan-Ching Huang,2 Yueh-Lun Lee,2 Bor-Luen Chiang,1,2 The mechanisms that cause the inflammation of airway and lung tissue in asthma have been studied extensively. It is noted that type 1 T helper cell (Th1)-related cytokines could decrease the accumulation of eosinophils in lung tissue and relieve airway constriction. But the therapeutic mechanisms of Th1 cytokines remain unclear. In this study, interleukin-12 (IL-12) DNA plasmid as a therapeutic reagent was delivered intravenously. Bronchoalveolar lavage (BAL) fluids were collected from IL-12 treated and control mice, and analyzed for cell composition and eotaxin level. The results showed that IL-12 DNA plasmid could effectively inhibit eosinophilia and airway inflammation in vivo. The level of eotaxin in BAL fluid also decreased. To further investigate the effect of Th1-related cytokines such as IL-12 or interferon- (IFN-) on the eotaxin level produced by lung cells, primary lung cell culture was established. The results demonstrated that both IL-12 and IFN- could suppress eotaxin secretion from IL-13 or IL-4 stimulated primary lung cell culture. Moreover, the inhibitory effect of IL-12 could not be reversed by the administration of anti-IFN- antibody. All the evidences suggested that IL-12 could regulate airway inflammation by suppressing the eotaxin secretion of lung tissue through an IFN- independent mechanism. 2002 Elsevier Science Ltd. All rights reserved.
Interleukin-12, also known as natural killer cell stimulatory factor (NKSF) and cytotoxic lymphocyte maturation factor, was originally identified and purified from the conditioned medium of an EBVtransformed human lymphoblastoid cell line.1 IL-12 is a 75 KDa heterodimer glycoprotein composed of two covalently linked proteins of p40 and p35 subunits. In general, IL-12 can enhance the IFN- production of NK and T cells, stimulate a number of cell surface molecules such as IL-2 receptor (CD25), inhibit IgE secretion, and act as a synergistic factor for hematopoietic stem cells.2–5 It also enhances type 1 T helper From the Departments of1Graduate Institute of Immunology, 2 Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China Correspondence to: Dr Bor-Luen Chiang, Department of Medical Research, National Taiwan University Hospital, No. 1, Chang-Teh Street, Taipei, Taiwan, Republic of China. Tel: 886-2-2312-3456 ext. 7299 or 7302; Fax: 886-2-2397-2031; E-mail: [email protected] Received 28 January 2002; received in revised form 22 May 2002; accepted for publication 6 June 2002 1043–4666/02/$-see front matter 2002 Elsevier Science Ltd. All rights reserved. KEY WORDS: Eotaxin/Interferon-/Interleukin-12/Lung cell culture This study was supported by a grant, DOH 90-TD-1030, from the Department of Health of the Republic of China. 76
but not type 2 T helper cell proliferations. Based on these potent immunologic effects, IL-12 has been applied in several disease models such as parasitic infection, malignancies and allergic asthma.6–9 Evidence exists that Th2 type immune response plays a major role in allergic disorders. Using cloning techniques or in situ hybridization, cells from local infiltrates, BAL fluid, or biopsies of bronchial mucosa show predominately Th2 cytokine expression, including IL-4, IL-5 and IL-13.10 After being challenged with allergen, activated mast cells secrete various inflammatory mediators that are responsible of subsequent airway inflammation. Then infiltrating cells, including neutrophils, lymphocytes, and eosinophils, accumulated locally in the lungs. Chemokines, such as eotaxin, RANTES, MCP-1, MCP-3 and MIP-1 also increased.11 Eosinophils are so important due to the toxic products in its granules that were proven to directly damage lung tissue.12 Eotaxin can chemoattract CCR3 positive cells, including eosinophils and Th2 cells.13,14 There is strong evidence regarding the therapeutic effect of Th1 cytokine administration. Using Th1related cytokine proteins,9,15–16 constructed cytokine plasmids,17–21 and adenovirus vectors expressing CYTOKINE, Vol. 19, No. 2 (21 July), 2002: pp 76–84
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cytokine genes,22,23 airway inflammation could be decreased. However, the mechanisms responsible for the therapeutic effect of Th1 cytokine have not yet been clarified. Bottomly et al. reported24 cotransferring T cell receptor transgenic Th1 and Th2 cells could reduce the recruitment of eosinophils. But the effect of these Th1 cells is not by regulating Th2 cell activity because Th1 and Th2 cytokines coexist in the lung. Der p1 (Dermatophagoides pteronyssinus) is a major component of the allergic immune response in house dust mite (HDM) asthmatic patient. Its cysteine protease activity has been indicated to cleave the surface marker CD23 from B cells25 and CD25 from T cells.26 It can also catalytically inactivate 1-antitrypsin.27 This unique character is believed to intensify IgE secretion and airway inflammation.26 Previously, we have demonstrated that administration of IL-12 protein to Der p1 sensitized mice, in which Th2-associated response were established, had a significant effect on abolishing recruitment of eosinophils.28,29 It is reported that the eotaxin producing cells include lung epithelial cells, endothelial cells, and lung fibroblast. Eotaxin production by these cells is induced by certain cytokines such as IL-4, IL-13, IL-1 or TNF-.30–35 However, the kinds of cytokine and chemokine-secreting pattern of different lung cell lines are controversial. We utilize primary lung cell culture system to evaluate the effect of Th1 cytokine on the eotaxin level produced by the lung cells in vitro. The therapeutic role of IL-12 and its mechanism in asthma is also discussed.
RESULTS
Figure 1.
Gene expression in lung tissue after 24 h and 48 h.
C57BL/6 mice (n =4 per group) were intravenously injected with DNA and lipofectAMINE 200 l per mouse and were sacrificed at 24 h or 48 h after injection. sIL-12 mRNA expression was measured by RT-PCR after isolating the mRNA by oligotex mRNA kit. The size of sIL-12 is 1.64 Kb.-actin, 540 bp, expression in each mouse organ is measured also (A). Luciferase activity was determined as the concentration of luciferase activity (pg) within lung tissue protein (per mg) (B). These data was the one result of three repeated experiments respectively.
Intravenous injection with plasmid liposome complex To evaluate the expression of mRNA and protein of the DNA plasmid after intravenous injection, the levels of sIL-12 mRNA (single chain IL-12) expression or reporter luciferase were assayed. sIL-12 mRNA expression could be detected in mouse lung organ that received intravenous injection with IL-12 plasmid after 24 h or 48 h (Fig. 1A). In contrast, mice that injected with vector plasmid could not detect the 1.64 Kb specific sIL-12 band. The luciferase activity in the lung tissues 24 h after injection was detected also (Fig. 1B). However, the luciferase activity decreased after 48 h. This data is similar to that reported from several other studies that characterize the expression of cationic liposome-mediated gene transfer in vivo by intravenous administration.36–38 These results suggest that the method of mixing plasmid with lipofectAMINE can induce a detectable but transient expression of protein derived from plasmid in lung organ.
IL-12 exerted the therapeutic effect on the animal model of asthma After intravenous injection with IL-12 DNA plasmid into Der p1 sensitized mice, the mice were sacrificed 24 h after inhalation of crude mite. Analysis of the cells populations in BAL fluid showed that IL-12 can specifically inhibit the infiltration of eosinophils (Fig. 2A). In addition, eotaxin levels in BAL fluid (bronchoalveolar lavage fluids) also decreased in IL-12 treated groups (Fig. 2B). The data showed that the eotaxin level correlates with the reduction of eosinophils in BAL fluid. Histopathologically, the damage and infiltrative cells were less severe in the IL-12 plasmid treated group (Fig. 3C); in the contrast, many cells infiltrated around the bronchial and lung alveoli in both the control and vector treated groups (Fig. 3A, 3B). These results demonstrated that intravenous injection with IL-12 DNA plasmid could specifically inhibit
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Figure 2. The effect of IL-12 DNA plasmid on inflammative cell population (A) and eotaxin levels (B) in BAL fluid from mice after aerosol challenge. On day 56, mice were challenged with inhaled D. pteronyssinus and BAL fluid samples were taken after 24 h. The BAL fluids from these mice were measured by using a specific ELISA. Cells from BAL fluids were cytospined and stained with Liu’s stain. A minimum of 200 cells were counted and classified as monocytes, lymphocytes, neutrophils, or eosinophils. Data represented the meanSEM of 5–7 mice per group. *P<0.05, IL-12p versus vector control group, #P<0.1, IL-12p versus positive control group. The data was the one result of two repeated experiments.
the infiltration of cells and reduce the pathological damage within the lung in this mouse model. Furthermore, we measured the extent of airway constriction of mice using the Buxco system. The Penh (pause of enhance) increased as the concentration of methacholine increased. The application of IL-12 plasmid can suppress airway hyperresponsiveness (Fig. 4). The relative percentage increased of Penh in IL-12 treated mice was inhibited compared to the positive control group (P<0.05) when made to inhale with 100 mg/ml methacholine. The mice that were immunized with 1 PBS and challenged with crude mite could not express detectable eotaxin and the cell population in BAL fluid is mostly monocytes (data not shown).
Figure 3. Histological study of the lungs of immunized mice with or without IL-12 treatment. The data showed extensive cellular infiltration of the periairway region from positive control group mice (A) and vector DNA treated mice (B). In contrast, lung tissue from IL-12 plasmid treated mice demonstrated a much less serious histological inflammation (C).
Eotaxin secretion of primary cells after rIL-4 or rIL-13 stimulation Many studies have reported that rIL-4 (recombinant interleukin 4) and rIL-13 (recombinant interleukin 13) could stimulate lung cells to secrete eotaxin.30,31,34,35 We establish a primary culture system to assay the stimulatory effect of rIL-4 or rIL-13. After two weeks of culture, the lung cells that were derived from normal B6 mice could secrete detectable eotaxin in a dose dependent pattern after 24 h and increases after 48 h with rIL-4 (Fig. 5A) or rIL-13 (Fig. 5B) stimulation.
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Figure 4. IL-12 plasmid treatment can alleviate airway hyperresponsiveness (AHR) when compared with that of the positive control mice. After being intravenously treated with IL-12 plasmid or vector plasmid and challenged with crude mite extract, each mouse was analysed using the Buxco system. The data represents the meanSEM. Compared to the positive group, the relative percentage increase of Penh in IL-12 treated mice was inhibited (*P<0.05) when stimulated with 100 mg/ml methacholine.
IL-12 and IFN- could decrease eotaxin level in primary lung cell culture In order to investigate the role of rIL-12 (recombinant interleukin 12) on the lung cells, the primary cells were cultured for two weeks and treated with Th1 cytokine for 2 h before the stimulation of rIL-4 or rIL-13. Th1 cytokine, IFN- as a therapeutic agent, could inhibit eotaxin secretion stimulated by rIL-4 (Fig. 6A) or rIL-13 (Fig. 6B) administration. Although it is not significant, low concentration of IFN- could slightly enhance the eotaxin expression after rIL-4 or rIL-13 stimulation. In addition, rIL-12 could also suppress eotaxin expression even with subsequent stimulation of rIL-4 (Fig. 7A) or rIL-13 (Fig. 7B). The eotaxin level produced by the primary lung cells at 24 h was much lower and the inhibition pattern of IL-12 or IFN- was similar to that of 48 h (data not shown). Because of the IFN- inducing activity of IL-12, we measured the level of IFN- within the supernatant of IL-12 treated cell culture. In addition, we added the anti-IFN- Ab to neutralize IFN- to avoid any possible indirect inhibitory effect of IFN-. The levels of IFN- were undetectable (or lower than 31.3 pg/ml) in rIL-12 treated primary cell cultures. Furthermore, the inhibitory effect of IL-12 was not suppressed by the addition of various concentration of anti-IFN- antibody (Fig. 7C). As a whole, these data demonstrated that IL-12 has a therapeutic effect on the treatment of asthma and this effect is not through IFN- directly.
Figure 5. Primary lung cells secreted eotaxin after IL-4 (A) or IL-13 (B) stimulation. Lung organs derived from B6 mice were cultured and stimulated with different concentrations of IL-4 or IL-13 cytokine. The supernatant was collected and measured after 48-h incubation using the R&D ELISA kit for eotaxin. The data represents the meanSEM and was one representative result of two repeated experiments.
DISSCUSSIONS It has been well documented that the worldwide prevalence of allergic diseases such as bronchial asthma is increasing every year.39 Allergic diseases are characterized by the presence of Th2 cells and related cytokines such as interleukin-4 (IL-4) and interleukin-5 (IL-5), with the subsequent development of eosinophils infiltration and chronic inflammation. By contrast, allergen-specific T cells isolated from non-allergic individuals show Th1 activity.40 Both IFN- protein and gene therapy have been successfully applied in a murine model of asthma.16,17,21 Interleukin-12 has been shown to have the ability to direct the development of Th1 cells and its therapeutic effects have been assessed in several disease models including infectious diseases, tumors and allergic diseases.6–9 To establish
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Figure 6. Eotaxin level from the supernatant of primary cells that were stimulated with IL-4 (A) or IL-13 (B) treatment with IFN-. The lung cells were cultured with 300 units/ml IL-4 or 10 ng/ml IL-13 two h after being treated with IFN-. After 48 h, the supernatant was collected and measured using the ELISA kit for mouse eotaxin. The data represents the meanSEM and *P<0.05 means each condition when compared to the IL-4 or IL-13 only. The data here was one representative result of four repeated experiments.
an animal model that closely resembles human bronchial asthma, mite Der p1 allergen was used in the study. Previous studies have demonstrated that IL-12 protein could decrease allergen-specific IgE and eosinophils infiltration in the mouse model of airway inflammation.9,15,18–20,22 Furthermore, the effect of IL-12 on allergen-induced eosinophils infiltration largely involves the promotion of Th1 development but could not completely suppress the development of Th2 cells after multiple antigen challenge in the absence of IL-12.28,29 These data suggest that IL-12 might be as useful an immunotherapeutic agent as a vaccine adjuvant in the treatment of bronchial asthma. Several studies have shown that intravenous injection of DNA plasmid mixed with liposome achieved the highest protein expression in the lungs.36–38 The results of the present study also support the finding that intravenous injection of IL-12 encoding DNA plasmid can dramatically decrease eosinophils infiltration in a murine model of airway inflammation. The
Figure 7. Eotaxin level from the supernatant of primary cells that were stimulated with IL-4 (A) or IL-13 (B) treatment with IL-12. The lung cells were cultured with 300 units/ml IL-4 or 10 ng/ml IL-13 2 h after treatment with IL-12. After 48 h, the supernatant was collected and measured using ELISA kit for mouse eotaxin. The supernatant was analyzed after 48 h as mentioned above. (C) The inhibitory effect of IL-12 on eotaxin secretion was not influenced by anti-IFN- Ab. IL-12 10 ng/ml was added with different concentration of anti-IFN- Ab simultaneously. The data represents the meanSEM and *P<0.05 means each condition when compared to the IL-4 or IL-13 only. The data here was one representative result of four repeated experiments.
toxicity of IL-12 that some studies have mentioned before41,42 did not affect our study because of the short-term expression. Actually, only the IL-12 plasmid with lipofetAMINE injection did not induce any inflammatory cells such as neutrophils, lymphocytes, and eosinophils to infiltrate lung tissue. These data suggest that the mechanism of IL-12 on the inhibition of eosinophils recruitment and inflammation may not occur totally through an antigen-specific pathway, since a single injection of IL-12 plasmid 24 h before the inhalation challenge is probably not long enough to
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achieve a good Th 1 antigen-specific immune response. The therapeutic effect of IL-12 may occur through the influence on the secretion or bio-function of eotaxin. Actually, we found the level of eotaxin in BAL fluid was decreased significantly. Eotaxin, secreted by lung epithelial cells, is a specific chemokine for the recruitment of eosinophils.11 It is interesting to note if IL-12 plays any role in the modulation of chemokine or cytokine expression in lung tissue and if there are subsequent diminution in airway inflammation. IL-12 has been reported to inhibit the production of IL-4 and IL-10 expression in human CD4 positive T cells of allergic individuals when restimulated with allergen for 12 days. However, the effect on the IL-4 production by activated CD4 positive T cells is not very obvious.43 In our data, the short period of IL-12 expression could reduce the eotaxin expression even the splenic Th2 cytokine profile and IgE Ab titer show no difference with or without IL-12 treatment. Based on these findings, it is hypothesized that IL-12 might exert a direct effect on the eotaxin production of lung cells. Many reports indicate that administration of IL-4,30,44,45 IL-13,30,35 TNF-31,44,45 or IL-1 31 can induce the chemokine expression by lung related cell line or primary lung cell culture. Although the human epithelial cell line A549 system has proven that IFN- can enhance the mRNA expression of eotaxin31 when stimulated with IL-1 or TNF-, the other cell line BEAS-2B, shown the opposite effect of IFN- when added with TNF- plus IL-4 or plus IL-13.45 We have established a primary lung cell culture system derived from normal B6 mice. This system allows us to evaluate the relationship between cytokines and chemokines in these cells that form the first line of defense against allergen stimulation. In this study, Th1 cytokines could suppress the eotaxin secretion of lung cells stimulated by IL-4 or IL-13. IFN- could slightly enhance the eotaxin level when treated at low concentration. The similar effect of low dose IFN- (10 unit/ ml) on eotaxin levels of human bronchial epithelial cell line (BEAS-2B) has been reported by the other group.45 However, this phenomenon was not found on IL-12 treated lung cells in our study. The reason is still unclear, but the signal pathways of these two Th1 cytokines might be different. For example, IFN- alone could induce lung cells to secrete RANTES in a concentration dependent manner but not IL-12 (data not shown). It still needs further investigation to analyze the further detailed mechanisms. Collectively, these data suggests the role of Th1 cytokine in allergic immunotherapy is not only through immune regulation but also through the other mechanisms. Nutku et al.46 have observed that PMAactivated human peripheral blood eosinophils increased cell apoptosis by dose dependent hIL-12 treatment, which can be blocked by the addition of
IL-5. In the other hand, Wang et al.47 reported that IL-12 knockout mice have less eosinophilia after challenging with OVA compared to that of control mice. The eotaxin level is no difference but IL-12 KO mice showed deficiency in VCAM-1 expression on lung endothelium. Interestingly, the report from Zhao et al.48 is opposite. IL-12 KO mice that they manipulated could enhance BAL eotaxin level and display a more serious eosinophilia. Endogenous deficiency of IL-12 may directly or indirectly influence many other unknown factors, which make the phenotype of IL-12 KO mice more complicated. Recently, more and more reports have shown that the lung epithelium system is not only a barrier for passive protection but also plays an important role through its ability to secrete many kinds of cytokines, chemokines, growth factors and neuropeptides. It can also express MHC II, B7.1, B7.2 or adhesion molecules49,50 with proper stimulation. Our data have demonstrated that IL-12 could decrease the eotaxin level both in vitro and in vivo and subsequently alleviate airway inflammation. In conclusion, the therapeutic effects of Th1 cytokines on airway inflammation might be through more complicated mechanisms and need to take the airway environment into consideration in the future study.
MATERIAL AND METHODS Mice C57BL/6 mice between four to six weeks of age were obtained from and maintained in the Animal Center of the College of Medicine of National Taiwan University. The animal room was kept on a 12-h light and dark cycle with constant temperature (25C2C) and humidity maintained. Animal care and handling conformed to the NIH Guide for the Care and Use of Laboratory Animals.
Establishment of the animal model of airway inflammation The immunization protocol used was similar to that described previously.28,29 Briefly, the mice were immunized with an intraperitoneal injection of 10 g Der p 1 (Dermatophagoides pteronyssinus, group 1 protein) and 2 mg Alum, using 400 ng pertussis toxin (List Biological Lab. Inc., Campbell, CA, USA) as an adjuvant. On day 0, 14, 28, and 42, the mice were boosted with the same dose of Der p1 and PT adjuvant. The allergen Der p 1 was isolated by affinity column from spent mite media, which was kindly provided by Dr K.-Y. Chua (The National University of Singapore). To examine the therapeutic effects of murine IL-12 DNA plasmid, mice received intravenous injection of 200 l IL-12 DNA plasmid liposome one day before the inhalation challenge. Instead, vector control mice were injected with pCMV vector. Positive group mice that were immunized with Der p1 and pertussis toxin did not receive this treatment. Negative control mice were given intraperitoneally injection of
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1PBS on each immunization. All mice were exposed to aerosolized crude mite extract, (Dermatophagoides pteronyssinus, Angelholm, Sweden) over a 20-min period by placing them in a chamber which could contain six to eight mice at a time. The aerosols were generated and conducted into the chamber using an ultrasonic nebulizer (DeVilbiss, PA, USA). The output of the nebulizer was 0.3 ml/min and the particles produced were 0.5–5 m in size. The concentration of crude mite extract in the nebulizer was 0.1 % (W/V).
Preparation of DNA plasmids and measurement of single chain IL-12 mRNA expression Luciferase plasmid or mouse IL-12 plasmid in 1 X PBS solution at a concentration of 1 g/l was mixed with lipofectAMINE (Life Technologies Pacific Ltd., Tsuen Wan, Hong Kong) in a 1:1 ratio. Each mouse received a single intravenous injection of 200 l volume of plasmid liposome through the tail vein. The IL-12 plasmid which we have constructed51,52 was delivered into mouse and picked up the lungs after 24 h or 48 h. In order to avoiding contamination of plasmid DNA, the Oligotex mRNA spin-column (Qiagen, CA, USA) was used to purify mRNA. The mRNA product then react with M-MuLV reverse transcriptase (BioLab, MA, USA) and cDNA was amplified by Pro Taq DNA polymerase (Protech Technology, Taiwan, ROC).
Analysis of the luciferase protein expression In determining luciferase activity, the mice were given lethal doses of ether, their lungs removed, and the luciferase was assayed using a commercial kit (Promega, WI, USA). The lung organ was removed from the mice at 24 h or 48 h after injection. The tissues were washed and then frozen in liquid nitrogen.36 These were then individually pulverized into a fine powder by hand grinding with liquid nitrogen using a porcelain mortar and pestle. Lysis buffer was added at a volume to weight ratio of 4 l per mg of collected lung organ. The mixture was then vigorously vortexed for 15 min at 4C and centrifuged at 10000 g for 10 min at room temperature. The supernatants were collected and luciferase activity was assayed by luminometer (Turner Designs, CA, USA). The protein concentration of each sample was determined through BCA protein reagent assay (Pierce, IL, USA). The luciferase activity from serially diluted concentrations of recombinant luciferase was the standard curve used. The activity for each lung was recorded in luciferase activity (pg) per mg of tissue protein.
Bronchoalveolar lavage and cell differential counts After all of the groups of mice were anesthetized, they were bled from the retro-orbital venous plexus and then sacrificed. Using a cannula, their lungs were immediately lavaged through the trachea with 31 ml of 1HBSS, which is free of ionized calcium and magnesium. The lavaged fluid was centrifuged at 400 g for 10 min at 4C. After the wash, the cells were resuspended in 1 ml 1HBSS and the total cells counts were determined using a hemocytometer. Cytocentrifuged preparations were stained with Liu’s stain for differential cell counts. Based on standard morphologic
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criteria, a minimum of 200 cells were counted and classified as macrophages, lymphocytes, neutrophils or eosinophils.
Histopathological study of lung organs In order to evaluate the effects of IL-12 treatment, the lungs were immediately removed after lavaging and fixed in a solution of 3% v/v formalin (in 0.01M phosphate buffer pH 7.2). The tissues were subsequently embedded in paraffin and cut into 5 m thick sections. These sections were stained with hematoxylin-eosin and examined by light microscopy for histopathological changes.
Airway hyperresponsiveness In order to investigate the effects of IL-12 on airway hyperresponsiveness (AHR), the Buxco system (Biosystem XA; Buxco Electronics Inc., CT, USA) was used to evaluate the extent of airway constriction in different groups of mice following the protocol mentioned previously.53,54 This system is non-invasive and can measure the respiratory changes in animals that are awake, excluding both anesthetic effect and surgical artifact. The value of Penh is calculated by collecting data information derived from transducer (differential pressure transducer; Buxco) and preamplifier (MAX II, Buxco). Penh=PausePIF/PEF; Pause=(Te-Tr)/Tr, (PIF; peak inspiratory flow, PEF; peak expiratory flow, Te; expiratory time, Tr; relaxation time). In this experiment, the mice were challenged three times with crude mite extract and injected intravenously with DNA plasmid-lipofectAMINE 24 h before each crude mite inhalation. One day after final inhalation, the mice were given aerosolized normal saline, 12.5 mg/ml, 25 mg/ml, 50 mg/ml or 100 mg/ml methacholine (Sigma; MO, USA) serially. The mice inhaled normal saline or increasing concentrations of methacholine for 3 min each. The Penh value for each minute was recorded and after the third recorded value, the average Penh value was divided by the Penh of normal saline and was presented as relative percentage increase of Penh.
Primary mouse lung cell culture Four to six weeks B6 female mice were sacrificed by dislocation in order to avoid influence of ether or pentobarbital. The lungs were removed and washed with 1PBS buffer until the blood has been removed. The connective tissue and blood vessels were removed while the lung tissues were cut to small pieces. These were centrifuged and the cell precipitate was collected while the supernatant was discarded after. The lung cells were cultured with MEM (alpha minimum essential medium, Life Technologies) complete medium including 10% fetal bovine serum (FBS), 4 mM L-glutamine, 25 mM HEPES (pH 7.2), 5105 M 2-mercaptoethanol, 100 U/ml penicillin, 100 ug/ml streptomycin and 0.25 mg/ml amphotericin. After 10–14 days, the primary cell population can reach into 80% confluent. The cell culture dish was then washed with 1PBS, treated with 1 trypsin—EDTA, and incubated at 37C, 5% incubator for 3–5 min to detach cells. 1PBS was added and after flushing with a pippette, cell suspension were collected in 50 ml centritubes. These were centrifuged at 400 g at 4C for 5 min. After the supernatant was discarded, the cells were seeded with 1105 cells per well in 48-well plates. These
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were cultured in 37C, 5% CO2 incubator until the cells get 80% confluence. Then replaced with MEM serum-free medium. After 16–18 h, cells were treated with different kinds of cytokines. Recombinant mouse IL-12 (R&D) was added 2 h before rIL-13 (R&D) or rIL-4 (R&D) stimulation. In neutralization assay, anti-IFN- Ab (BD PharMingen; CA, USA) was added simultaneously with rIL-12 2 h before rIL-13 stimulation. After 48 h of incubation, the supernatant of each well was collected and frozen in 20C before further analysis was made.
Eotaxin level in culture supernatant or bronchoalveolar lavage The eotaxin was assayed with ELISA kit (R&D) used according to the manufacturer’s instructions. Briefly, the culture supernatant or bronchoalveolar lavage of each condition was added to wells precoated over night at 4C with anti-eotaxin Ab. After 2 h of incubation, the plates were washed and biotin-conjugated Ab was added. After two more hours at room temperature, HRP-avidin was added to each well. The substrate tetramethylbenzidine was then added and the OD (at 450 nm) values were converted to concentrations of chemokine in the suppernatant. The sensitivity of this assay was 1.9 pg/ml for eotaxin.
Statistical analysis Individual experimental values were compared by the student’s t test. Differences between two groups were considered statistically significant at exact P-values, P<0.05.
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hyperresponsiveness, inflammation, and Th2 cytokine expression in mice. J Exp Med 182:1527–1536. 10. Piccinni MP, Maggi E, Romagnani S (2000) Environmental factors favoring the allergen-specific Th2 response in allergic subjects. Ann NY Acad Sci 917:844–852. 11. Homey B, Zlotnik A (1999) Chemokines in allergy. Curr Opin Immunol 11:626–634. 12. Gleich GJ (1990) The eosinophil and bronchial asthma: current understanding. J Allergy Clin Immunol 85:422–436. 13. Griffiths-Johnson DA, Collins PD, Rossi AG, Jose PJ, Williams TJ (1993) The chemokine, eotaxin, activates guinea-pig eosinophils in vitro and causes their accumulation into the lung in vivo. Biochem Biophys Res Commun 197:1167–1172. 14. Meeusen EN, Premier RR, Brandon MR (1996) Tissuespecific migration of lymphocytes: a key role for Th1 and Th2 cells? Immunol Today 17:421–424. 15. Hofstra CL, Van Ark I, Hofman G, Kool M, Nijkamp FP, Van Oosterhout A J (1998) Prevention of Th2-like cell responses by coadministration of IL-12 and IL-18 is associated with inhibition of antigen-induced airway hyperresponsiveness, eosinophilia, and serum IgE levels. J Immunol 161:5054–5060. 16. Brusselle GG, Kips JC, Peleman RA, Joos GF, Devos RR, Tavernier JH, Pauwels RA (1997) Role of IFN- in the inhibition of allergic airway inflammation caused by IL-12. Am J Respir Cell Mol Biol 17:767–771. 17. Li XM, Chopra RK, Chou TY, Schofield BH, Wills-Karp M, Huang SK (1996) Mucosal IFN- gene transfer inhibits pulmonary allergic responses in mice. J Immunol 157:3216–3219. 18. Iwamoto I, Kumano K, Kurasawa K, Nakao A (1996) Interleukin-12 prevents antigen-induced eosinophil recruitment into mouse airways. Am J Respir Crit Care Med 154:1257–1260. 19. Schwarze J, Hamelmann E, Cieslewicz G, Tomkinson A, Joetham A, Bradley K, Gelfand EW (1998) Local treatment with IL-12 is an effective inhibitor of airway hyperresponsiveness and lung eosinophilia after airway challenge in sensitized mice. J Allergy Clin Immunol 102:86–93. 20. Hogan SP, Foster PS, Tan X, Ramsay AJ (1998) Mucosal IL-12 gene delivery inhibits allergic airways disease and restores local antiviral immunity. Eur J Immunol 28:413–423. 21. Dow SW, Schwarze J, Heath TD, Potter TA, Gelfand EW (1999) Systemic and local interferon gene delivery to the lungs for treatment of allergen-induced airway hyperresponsiveness in mice. Human Gene Ther 10:1905–1914. 22. Stampfli MR, Neigh GS, Wiley RE, Cwiartka M, Ritz SA, Hitt MM, Xing Z, Jordana M (1999) Regulation of allergic mucosal sensitization by interleukin-12 gene transfer to the airway. Am J Respir Cell Mol Biol 21:317–326. 23. Walter DM, Wong CP, DeKruyff RH, Berry GJ, Levy S, Umetsu DT (2001) IL-18 gene transfer by adenovirus prevents the development of and reverses established allergen-induced airway hyperreactivity. J Immunol 15; 166:6392–6398. 24. Cohn L, Homer RJ, Niu N, Bottomly K (1999) T helper1 cells and interferon regulate allergic airway inflammation and mucus production. J Exp Med 189:1309–1317. 25. Schulz O, Sutton BJ, Beavil RL, Shi J, Sewell HF, Gould HJ, Laing P, Shakib F (1997) Cleavage of the low-affinity receptor for human IgE (CD23) by a mite cysteine protease: nature of the cleaved fragment in relation to the structure and function of CD23. Eur J Immunol 27:584–588. 26. Shakib F, Schulz O, Sewell H (1998) A mite subversive: cleavage of CD23 and CD25 by Der p1 enhances allergenicity. Immunol Today 19:313–316. 27. Kalsheker NA, Deam S, Chambers L, Sreedharan S, Brocklehurst K, Lomas DA (1996) The house dust mite allergen Der p 1 catalytically inactivates 1-antitrypsin by specific reactive center loop cleavage: a mechanism that promotes airway inflammation and asthma. Biochem Biophys Res Commun 221:59–61. 28. Lee YL, Fu CL, Ye YL, Chiang BL (1999) Administration of IL-12 prevents mite Der p 1 allergen-IgE antibody production and eosinophils infiltration in animal model of airway inflammation. Scand J Immunol 49:229–336. 29. Lee YL, Fu CL, Chiang BL (1999) Administration of IL-12 exerts therapeutic instead of long-term preventive effect on
84 / Ye et al. Der p 1 allergen-Induced animal model of airway inflammation. Immunology 97:232–240. 30. Doucet C, Brouty-Boye D, Pottin-Clemenceau C, Canonica GW, Jasmin C, Azzarone B (1998) Interleukin (IL)4 and IL-13 act on human lung fibroblasts. J Clin Invest 101:2129–2139. 31. Lilly CM, Nakamura H, Kesselman H, Nagler-Anderson C, Asano K, Garcia-Zepeda EA, Garcia-Zepeda EA, Rothenberg ME, Drazen JM, Luster AD (1997) Expression of eotaxin by human lung epithelial cells. Introduction by chemokines and inhibition by glucocorticoids. J Clin Invest 99:1767–1773. 32. Gonzalo JA, Jia, GQ, Aguirre V, Friend D, Coyle AJ, Jenkins NA, Lin GS, Katz H, Lichtman A, Copeland N, Kopf M, Gutierrez-Ramos JC (1996) Mouse eotaxin expression parallels eosinophil accumulation during lung allergic inflammation but it is not restricted to a Th2-type response. Immunity 4:1–14. 33. Ying S, Robinson DS, Meng Q, Rottman J, Kennedy R, Ringler DJ, Mackay CR, Daugherty BL, Springer MS, Durham SR, Williams TJ, Kay AB (1997) Enhanced expression of eotaxin and CCR3 mRNA and protein in atopic asthma. Association with airway hyperresponsiveness and predominant co-localization of eotaxin mRNA to bronchial epithelial and endothelial cells. Eur J Immunol 27:3507–3516. 34. Zhu Z, Homer RJ, Wang Z, Chen Q, Geba GP, Wang J, Zhang Y, Elias JA (1999) Pulmonary expression of interleukin-13 causes inflammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production. J Clin Invest 103:779–788. 35. Li L, Xia Y, Nguyen A, Lai YH, Feng L, Mosmann TR, Lo David (1999) Effects of Th2 cytokines on chemokine expression in the lung: IL-13 potently induces eotaxin expression by airway epithelial cells. J Immunol 162:2477–2487. 36. Song YK, Liu F, Chu S, Liu D (1997) Characterization of cationic liposome-mediated gene transfer in vivo by intravenous administration. Human Gene Ther 8:1585–1594. 37. Zhu N, Liggitt D, Liu Y, Debs R (1993) Systemic gene expression after intravenous DNA delivery into adult mice. Science 261:209–211. 38. Li S, Rizzo MA, Bhattacharya S, Huang L (1998) Characterization of cationic lipid-protamine-DNA (LPD) complexes for intravenous gene delivery. Gene Ther 5:930–937. 39. Woolcock AJ, Peat JK (1997) Evidence for the increase in asthma worldwide. Ciba Found Symp 206:122–134. 40. Imada M, Simons FE, Jay FT, Hayglass K (1995) Allergenstimulated interleukin-4 and interferon -production in primary culture: responses of subjects with allergic rhinitis and normal controls. Immunology 85:373–380. 41. Hendrzak JA, Brunda MJ (1995) Biology of disease: Interleukin-12, biology activity, therapeutic utility, and role in disease. Lab Invest 72:619–637. 42. Car BD, Eng VM, Schnyder B, LeHir M, Shakhov AN, Woerly G, Huang S, Aguet M, Anderson TD, Ryffel B (1995) Role
CYTOKINE, Vol. 19, No. 2 (21 July, 2002: 76–84) of interferon- in interleukin 12-induced pathology in mice. Am J Pathol 147:1693–1707. 43. Marshall JD, Secrist H, DeKruyff RH, Wolf SF, Umetsu DT (1995) IL-12 inhibits the production of IL-4 and IL-10 in allergen-specific human CD4+ T lymphocytes. J Immunol 155: 111–117. 44. Teran LM, Mochizuki M, Bartels J, Valencia EL, Nakajima T, Hirai K, Schroder JM (1999) Th1 and Th2-type cytokines regulate the expression and production of eotaxin and RANTES by human lung fibroblasts. Am J Respir Cell Mol Biol 20:777–786. 45. Fujisawa T, Kato Y, Atsuta J, Terada A, Iguchi K, Kamiya H, Yamada H, Nakajima T, Miyamasu M, Hirai K (2000) Chemokine production by the BEAS-2B human brochial epithelial cells: differential regulation of eotaxin, IL-8, and RANTES by Th2- and Th1-derived cytokines. J Allergy Clin Immunol 105:126–133. 46. Nutku E, Zhuang Q, Soussi-Gounni A, Aris F, Mazer BD, Hamid Q (2001) Functional expression of IL-12 receptor by human eosinophils: IL-12 promotes eosinophil apoptosis. J Immunol 167:1039–1046. 47. Wang S, Fan Y, Han X, Yang J, Bilenki L, Yang X (2001) IL-12-dependent vascular cell adhesion molecule-1 expression contributes to airway eosinophilic inflammation in a mouse model of asthma-like reaction. J Immunol 166:2741–2749. 48. Zhao LL, Linden A, Sjostrand M, Cui ZH, Lotvall J (2000) IL-12 regulates bone marrow eosinophilia and airway eotaxin levels induced by airway allergen exposure. Allergy 55:749–756. 49. Cunningham R, Mahon BP (2001) The immunological role of respiratory tract epithelium. Mod Asp Immunobiol 1(5): 204–209. 50.Salik E, Tyorkin M, Mohan S, George I, Becker K, Oei E, Kalb T, Sperber K (1999) Antigen trafficking and accessory cell function in respiratory epithelial cells. Am J Respir Cell Mol Biol 21:365–379. 51. Lee YL, Tao MH, Chow YH, Chiang BL (1998) Construction of vectors expressing bioactive heterodimeric and single-chain murine interleukin-12 for genomic therapy. Human Gene Ther 9:457–465. 52. Lee YL, Ye YL, Yu CI, Wu YL, Lai YL, Ku PH, Hong RL, Chiang BL (2001) Construction of single-chain interleukin-12 DNA plasmid to treat airway hyperresponsiveness in an animal model of asthma. Human Gene Ther 12:2065–2079. 53. Hamelmann E, Gelfand EW (1999) Animal models of airway sensitization. Coligan JE, Kruisbeek AM, Margulies DH, Shevach EM, Strober W (eds) Current Protocols in Immunology, Wiley Press, New York, pp. 5.18.1–15.18.13. 54. Cieslewicz G, Tomkinson A, Adler A, Duez C, Schwarze J, Takeda K, Larson KA, Lee JJ, Irvin CG, Gelfand EW (1999) The late, but not early, asthmatic response is dependent on IL-5 and correlates with eosinophil infiltration. J Clin Invest 104: 301–308.
INTERLEUKIN-12 INHIBITS EOTAXIN SECRETION OF CULTURED PRIMARY LUNG CELLS AND ALLEVIATES AIRWAY INFLAMMATION IN VIVO Yi-Ling Ye,1 Wan-Ching Huang,2 Yueh-Lun Lee,2 Bor-Luen Chiang,1,2 The mechanisms that cause the inflammation of airway and lung tissue in asthma have been studied extensively. It is noted that type 1 T helper cell (Th1)-related cytokines could decrease the accumulation of eosinophils in lung tissue and relieve airway constriction. But the therapeutic mechanisms of Th1 cytokines remain unclear. In this study, interleukin-12 (IL-12) DNA plasmid as a therapeutic reagent was delivered intravenously. Bronchoalveolar lavage (BAL) fluids were collected from IL-12 treated and control mice, and analyzed for cell composition and eotaxin level. The results showed that IL-12 DNA plasmid could effectively inhibit eosinophilia and airway inflammation in vivo. The level of eotaxin in BAL fluid also decreased. To further investigate the effect of Th1-related cytokines such as IL-12 or interferon- (IFN-) on the eotaxin level produced by lung cells, primary lung cell culture was established. The results demonstrated that both IL-12 and IFN- could suppress eotaxin secretion from IL-13 or IL-4 stimulated primary lung cell culture. Moreover, the inhibitory effect of IL-12 could not be reversed by the administration of anti-IFN- antibody. All the evidences suggested that IL-12 could regulate airway inflammation by suppressing the eotaxin secretion of lung tissue through an IFN- independent mechanism. 2002 Elsevier Science Ltd. All rights reserved.
Interleukin-12, also known as natural killer cell stimulatory factor (NKSF) and cytotoxic lymphocyte maturation factor, was originally identified and purified from the conditioned medium of an EBVtransformed human lymphoblastoid cell line.1 IL-12 is a 75 KDa heterodimer glycoprotein composed of two covalently linked proteins of p40 and p35 subunits. In general, IL-12 can enhance the IFN- production of NK and T cells, stimulate a number of cell surface molecules such as IL-2 receptor (CD25), inhibit IgE secretion, and act as a synergistic factor for hematopoietic stem cells.2–5 It also enhances type 1 T helper From the Departments of1Graduate Institute of Immunology, 2 Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China Correspondence to: Dr Bor-Luen Chiang, Department of Medical Research, National Taiwan University Hospital, No. 1, Chang-Teh Street, Taipei, Taiwan, Republic of China. Tel: 886-2-2312-3456 ext. 7299 or 7302; Fax: 886-2-2397-2031; E-mail: [email protected] Received 28 January 2002; received in revised form 22 May 2002; accepted for publication 6 June 2002 1043–4666/02/$-see front matter 2002 Elsevier Science Ltd. All rights reserved. KEY WORDS: Eotaxin/Interferon-/Interleukin-12/Lung cell culture This study was supported by a grant, DOH 90-TD-1030, from the Department of Health of the Republic of China. 76
but not type 2 T helper cell proliferations. Based on these potent immunologic effects, IL-12 has been applied in several disease models such as parasitic infection, malignancies and allergic asthma.6–9 Evidence exists that Th2 type immune response plays a major role in allergic disorders. Using cloning techniques or in situ hybridization, cells from local infiltrates, BAL fluid, or biopsies of bronchial mucosa show predominately Th2 cytokine expression, including IL-4, IL-5 and IL-13.10 After being challenged with allergen, activated mast cells secrete various inflammatory mediators that are responsible of subsequent airway inflammation. Then infiltrating cells, including neutrophils, lymphocytes, and eosinophils, accumulated locally in the lungs. Chemokines, such as eotaxin, RANTES, MCP-1, MCP-3 and MIP-1 also increased.11 Eosinophils are so important due to the toxic products in its granules that were proven to directly damage lung tissue.12 Eotaxin can chemoattract CCR3 positive cells, including eosinophils and Th2 cells.13,14 There is strong evidence regarding the therapeutic effect of Th1 cytokine administration. Using Th1related cytokine proteins,9,15–16 constructed cytokine plasmids,17–21 and adenovirus vectors expressing CYTOKINE, Vol. 19, No. 2 (21 July), 2002: pp 76–84
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cytokine genes,22,23 airway inflammation could be decreased. However, the mechanisms responsible for the therapeutic effect of Th1 cytokine have not yet been clarified. Bottomly et al. reported24 cotransferring T cell receptor transgenic Th1 and Th2 cells could reduce the recruitment of eosinophils. But the effect of these Th1 cells is not by regulating Th2 cell activity because Th1 and Th2 cytokines coexist in the lung. Der p1 (Dermatophagoides pteronyssinus) is a major component of the allergic immune response in house dust mite (HDM) asthmatic patient. Its cysteine protease activity has been indicated to cleave the surface marker CD23 from B cells25 and CD25 from T cells.26 It can also catalytically inactivate 1-antitrypsin.27 This unique character is believed to intensify IgE secretion and airway inflammation.26 Previously, we have demonstrated that administration of IL-12 protein to Der p1 sensitized mice, in which Th2-associated response were established, had a significant effect on abolishing recruitment of eosinophils.28,29 It is reported that the eotaxin producing cells include lung epithelial cells, endothelial cells, and lung fibroblast. Eotaxin production by these cells is induced by certain cytokines such as IL-4, IL-13, IL-1 or TNF-.30–35 However, the kinds of cytokine and chemokine-secreting pattern of different lung cell lines are controversial. We utilize primary lung cell culture system to evaluate the effect of Th1 cytokine on the eotaxin level produced by the lung cells in vitro. The therapeutic role of IL-12 and its mechanism in asthma is also discussed.
RESULTS
Figure 1.
Gene expression in lung tissue after 24 h and 48 h.
C57BL/6 mice (n =4 per group) were intravenously injected with DNA and lipofectAMINE 200 l per mouse and were sacrificed at 24 h or 48 h after injection. sIL-12 mRNA expression was measured by RT-PCR after isolating the mRNA by oligotex mRNA kit. The size of sIL-12 is 1.64 Kb.-actin, 540 bp, expression in each mouse organ is measured also (A). Luciferase activity was determined as the concentration of luciferase activity (pg) within lung tissue protein (per mg) (B). These data was the one result of three repeated experiments respectively.
Intravenous injection with plasmid liposome complex To evaluate the expression of mRNA and protein of the DNA plasmid after intravenous injection, the levels of sIL-12 mRNA (single chain IL-12) expression or reporter luciferase were assayed. sIL-12 mRNA expression could be detected in mouse lung organ that received intravenous injection with IL-12 plasmid after 24 h or 48 h (Fig. 1A). In contrast, mice that injected with vector plasmid could not detect the 1.64 Kb specific sIL-12 band. The luciferase activity in the lung tissues 24 h after injection was detected also (Fig. 1B). However, the luciferase activity decreased after 48 h. This data is similar to that reported from several other studies that characterize the expression of cationic liposome-mediated gene transfer in vivo by intravenous administration.36–38 These results suggest that the method of mixing plasmid with lipofectAMINE can induce a detectable but transient expression of protein derived from plasmid in lung organ.
IL-12 exerted the therapeutic effect on the animal model of asthma After intravenous injection with IL-12 DNA plasmid into Der p1 sensitized mice, the mice were sacrificed 24 h after inhalation of crude mite. Analysis of the cells populations in BAL fluid showed that IL-12 can specifically inhibit the infiltration of eosinophils (Fig. 2A). In addition, eotaxin levels in BAL fluid (bronchoalveolar lavage fluids) also decreased in IL-12 treated groups (Fig. 2B). The data showed that the eotaxin level correlates with the reduction of eosinophils in BAL fluid. Histopathologically, the damage and infiltrative cells were less severe in the IL-12 plasmid treated group (Fig. 3C); in the contrast, many cells infiltrated around the bronchial and lung alveoli in both the control and vector treated groups (Fig. 3A, 3B). These results demonstrated that intravenous injection with IL-12 DNA plasmid could specifically inhibit
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Figure 2. The effect of IL-12 DNA plasmid on inflammative cell population (A) and eotaxin levels (B) in BAL fluid from mice after aerosol challenge. On day 56, mice were challenged with inhaled D. pteronyssinus and BAL fluid samples were taken after 24 h. The BAL fluids from these mice were measured by using a specific ELISA. Cells from BAL fluids were cytospined and stained with Liu’s stain. A minimum of 200 cells were counted and classified as monocytes, lymphocytes, neutrophils, or eosinophils. Data represented the meanSEM of 5–7 mice per group. *P<0.05, IL-12p versus vector control group, #P<0.1, IL-12p versus positive control group. The data was the one result of two repeated experiments.
the infiltration of cells and reduce the pathological damage within the lung in this mouse model. Furthermore, we measured the extent of airway constriction of mice using the Buxco system. The Penh (pause of enhance) increased as the concentration of methacholine increased. The application of IL-12 plasmid can suppress airway hyperresponsiveness (Fig. 4). The relative percentage increased of Penh in IL-12 treated mice was inhibited compared to the positive control group (P<0.05) when made to inhale with 100 mg/ml methacholine. The mice that were immunized with 1 PBS and challenged with crude mite could not express detectable eotaxin and the cell population in BAL fluid is mostly monocytes (data not shown).
Figure 3. Histological study of the lungs of immunized mice with or without IL-12 treatment. The data showed extensive cellular infiltration of the periairway region from positive control group mice (A) and vector DNA treated mice (B). In contrast, lung tissue from IL-12 plasmid treated mice demonstrated a much less serious histological inflammation (C).
Eotaxin secretion of primary cells after rIL-4 or rIL-13 stimulation Many studies have reported that rIL-4 (recombinant interleukin 4) and rIL-13 (recombinant interleukin 13) could stimulate lung cells to secrete eotaxin.30,31,34,35 We establish a primary culture system to assay the stimulatory effect of rIL-4 or rIL-13. After two weeks of culture, the lung cells that were derived from normal B6 mice could secrete detectable eotaxin in a dose dependent pattern after 24 h and increases after 48 h with rIL-4 (Fig. 5A) or rIL-13 (Fig. 5B) stimulation.
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Figure 4. IL-12 plasmid treatment can alleviate airway hyperresponsiveness (AHR) when compared with that of the positive control mice. After being intravenously treated with IL-12 plasmid or vector plasmid and challenged with crude mite extract, each mouse was analysed using the Buxco system. The data represents the meanSEM. Compared to the positive group, the relative percentage increase of Penh in IL-12 treated mice was inhibited (*P<0.05) when stimulated with 100 mg/ml methacholine.
IL-12 and IFN- could decrease eotaxin level in primary lung cell culture In order to investigate the role of rIL-12 (recombinant interleukin 12) on the lung cells, the primary cells were cultured for two weeks and treated with Th1 cytokine for 2 h before the stimulation of rIL-4 or rIL-13. Th1 cytokine, IFN- as a therapeutic agent, could inhibit eotaxin secretion stimulated by rIL-4 (Fig. 6A) or rIL-13 (Fig. 6B) administration. Although it is not significant, low concentration of IFN- could slightly enhance the eotaxin expression after rIL-4 or rIL-13 stimulation. In addition, rIL-12 could also suppress eotaxin expression even with subsequent stimulation of rIL-4 (Fig. 7A) or rIL-13 (Fig. 7B). The eotaxin level produced by the primary lung cells at 24 h was much lower and the inhibition pattern of IL-12 or IFN- was similar to that of 48 h (data not shown). Because of the IFN- inducing activity of IL-12, we measured the level of IFN- within the supernatant of IL-12 treated cell culture. In addition, we added the anti-IFN- Ab to neutralize IFN- to avoid any possible indirect inhibitory effect of IFN-. The levels of IFN- were undetectable (or lower than 31.3 pg/ml) in rIL-12 treated primary cell cultures. Furthermore, the inhibitory effect of IL-12 was not suppressed by the addition of various concentration of anti-IFN- antibody (Fig. 7C). As a whole, these data demonstrated that IL-12 has a therapeutic effect on the treatment of asthma and this effect is not through IFN- directly.
Figure 5. Primary lung cells secreted eotaxin after IL-4 (A) or IL-13 (B) stimulation. Lung organs derived from B6 mice were cultured and stimulated with different concentrations of IL-4 or IL-13 cytokine. The supernatant was collected and measured after 48-h incubation using the R&D ELISA kit for eotaxin. The data represents the meanSEM and was one representative result of two repeated experiments.
DISSCUSSIONS It has been well documented that the worldwide prevalence of allergic diseases such as bronchial asthma is increasing every year.39 Allergic diseases are characterized by the presence of Th2 cells and related cytokines such as interleukin-4 (IL-4) and interleukin-5 (IL-5), with the subsequent development of eosinophils infiltration and chronic inflammation. By contrast, allergen-specific T cells isolated from non-allergic individuals show Th1 activity.40 Both IFN- protein and gene therapy have been successfully applied in a murine model of asthma.16,17,21 Interleukin-12 has been shown to have the ability to direct the development of Th1 cells and its therapeutic effects have been assessed in several disease models including infectious diseases, tumors and allergic diseases.6–9 To establish
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Figure 6. Eotaxin level from the supernatant of primary cells that were stimulated with IL-4 (A) or IL-13 (B) treatment with IFN-. The lung cells were cultured with 300 units/ml IL-4 or 10 ng/ml IL-13 two h after being treated with IFN-. After 48 h, the supernatant was collected and measured using the ELISA kit for mouse eotaxin. The data represents the meanSEM and *P<0.05 means each condition when compared to the IL-4 or IL-13 only. The data here was one representative result of four repeated experiments.
an animal model that closely resembles human bronchial asthma, mite Der p1 allergen was used in the study. Previous studies have demonstrated that IL-12 protein could decrease allergen-specific IgE and eosinophils infiltration in the mouse model of airway inflammation.9,15,18–20,22 Furthermore, the effect of IL-12 on allergen-induced eosinophils infiltration largely involves the promotion of Th1 development but could not completely suppress the development of Th2 cells after multiple antigen challenge in the absence of IL-12.28,29 These data suggest that IL-12 might be as useful an immunotherapeutic agent as a vaccine adjuvant in the treatment of bronchial asthma. Several studies have shown that intravenous injection of DNA plasmid mixed with liposome achieved the highest protein expression in the lungs.36–38 The results of the present study also support the finding that intravenous injection of IL-12 encoding DNA plasmid can dramatically decrease eosinophils infiltration in a murine model of airway inflammation. The
Figure 7. Eotaxin level from the supernatant of primary cells that were stimulated with IL-4 (A) or IL-13 (B) treatment with IL-12. The lung cells were cultured with 300 units/ml IL-4 or 10 ng/ml IL-13 2 h after treatment with IL-12. After 48 h, the supernatant was collected and measured using ELISA kit for mouse eotaxin. The supernatant was analyzed after 48 h as mentioned above. (C) The inhibitory effect of IL-12 on eotaxin secretion was not influenced by anti-IFN- Ab. IL-12 10 ng/ml was added with different concentration of anti-IFN- Ab simultaneously. The data represents the meanSEM and *P<0.05 means each condition when compared to the IL-4 or IL-13 only. The data here was one representative result of four repeated experiments.
toxicity of IL-12 that some studies have mentioned before41,42 did not affect our study because of the short-term expression. Actually, only the IL-12 plasmid with lipofetAMINE injection did not induce any inflammatory cells such as neutrophils, lymphocytes, and eosinophils to infiltrate lung tissue. These data suggest that the mechanism of IL-12 on the inhibition of eosinophils recruitment and inflammation may not occur totally through an antigen-specific pathway, since a single injection of IL-12 plasmid 24 h before the inhalation challenge is probably not long enough to
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achieve a good Th 1 antigen-specific immune response. The therapeutic effect of IL-12 may occur through the influence on the secretion or bio-function of eotaxin. Actually, we found the level of eotaxin in BAL fluid was decreased significantly. Eotaxin, secreted by lung epithelial cells, is a specific chemokine for the recruitment of eosinophils.11 It is interesting to note if IL-12 plays any role in the modulation of chemokine or cytokine expression in lung tissue and if there are subsequent diminution in airway inflammation. IL-12 has been reported to inhibit the production of IL-4 and IL-10 expression in human CD4 positive T cells of allergic individuals when restimulated with allergen for 12 days. However, the effect on the IL-4 production by activated CD4 positive T cells is not very obvious.43 In our data, the short period of IL-12 expression could reduce the eotaxin expression even the splenic Th2 cytokine profile and IgE Ab titer show no difference with or without IL-12 treatment. Based on these findings, it is hypothesized that IL-12 might exert a direct effect on the eotaxin production of lung cells. Many reports indicate that administration of IL-4,30,44,45 IL-13,30,35 TNF-31,44,45 or IL-1 31 can induce the chemokine expression by lung related cell line or primary lung cell culture. Although the human epithelial cell line A549 system has proven that IFN- can enhance the mRNA expression of eotaxin31 when stimulated with IL-1 or TNF-, the other cell line BEAS-2B, shown the opposite effect of IFN- when added with TNF- plus IL-4 or plus IL-13.45 We have established a primary lung cell culture system derived from normal B6 mice. This system allows us to evaluate the relationship between cytokines and chemokines in these cells that form the first line of defense against allergen stimulation. In this study, Th1 cytokines could suppress the eotaxin secretion of lung cells stimulated by IL-4 or IL-13. IFN- could slightly enhance the eotaxin level when treated at low concentration. The similar effect of low dose IFN- (10 unit/ ml) on eotaxin levels of human bronchial epithelial cell line (BEAS-2B) has been reported by the other group.45 However, this phenomenon was not found on IL-12 treated lung cells in our study. The reason is still unclear, but the signal pathways of these two Th1 cytokines might be different. For example, IFN- alone could induce lung cells to secrete RANTES in a concentration dependent manner but not IL-12 (data not shown). It still needs further investigation to analyze the further detailed mechanisms. Collectively, these data suggests the role of Th1 cytokine in allergic immunotherapy is not only through immune regulation but also through the other mechanisms. Nutku et al.46 have observed that PMAactivated human peripheral blood eosinophils increased cell apoptosis by dose dependent hIL-12 treatment, which can be blocked by the addition of
IL-5. In the other hand, Wang et al.47 reported that IL-12 knockout mice have less eosinophilia after challenging with OVA compared to that of control mice. The eotaxin level is no difference but IL-12 KO mice showed deficiency in VCAM-1 expression on lung endothelium. Interestingly, the report from Zhao et al.48 is opposite. IL-12 KO mice that they manipulated could enhance BAL eotaxin level and display a more serious eosinophilia. Endogenous deficiency of IL-12 may directly or indirectly influence many other unknown factors, which make the phenotype of IL-12 KO mice more complicated. Recently, more and more reports have shown that the lung epithelium system is not only a barrier for passive protection but also plays an important role through its ability to secrete many kinds of cytokines, chemokines, growth factors and neuropeptides. It can also express MHC II, B7.1, B7.2 or adhesion molecules49,50 with proper stimulation. Our data have demonstrated that IL-12 could decrease the eotaxin level both in vitro and in vivo and subsequently alleviate airway inflammation. In conclusion, the therapeutic effects of Th1 cytokines on airway inflammation might be through more complicated mechanisms and need to take the airway environment into consideration in the future study.
MATERIAL AND METHODS Mice C57BL/6 mice between four to six weeks of age were obtained from and maintained in the Animal Center of the College of Medicine of National Taiwan University. The animal room was kept on a 12-h light and dark cycle with constant temperature (25C2C) and humidity maintained. Animal care and handling conformed to the NIH Guide for the Care and Use of Laboratory Animals.
Establishment of the animal model of airway inflammation The immunization protocol used was similar to that described previously.28,29 Briefly, the mice were immunized with an intraperitoneal injection of 10 g Der p 1 (Dermatophagoides pteronyssinus, group 1 protein) and 2 mg Alum, using 400 ng pertussis toxin (List Biological Lab. Inc., Campbell, CA, USA) as an adjuvant. On day 0, 14, 28, and 42, the mice were boosted with the same dose of Der p1 and PT adjuvant. The allergen Der p 1 was isolated by affinity column from spent mite media, which was kindly provided by Dr K.-Y. Chua (The National University of Singapore). To examine the therapeutic effects of murine IL-12 DNA plasmid, mice received intravenous injection of 200 l IL-12 DNA plasmid liposome one day before the inhalation challenge. Instead, vector control mice were injected with pCMV vector. Positive group mice that were immunized with Der p1 and pertussis toxin did not receive this treatment. Negative control mice were given intraperitoneally injection of
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1PBS on each immunization. All mice were exposed to aerosolized crude mite extract, (Dermatophagoides pteronyssinus, Angelholm, Sweden) over a 20-min period by placing them in a chamber which could contain six to eight mice at a time. The aerosols were generated and conducted into the chamber using an ultrasonic nebulizer (DeVilbiss, PA, USA). The output of the nebulizer was 0.3 ml/min and the particles produced were 0.5–5 m in size. The concentration of crude mite extract in the nebulizer was 0.1 % (W/V).
Preparation of DNA plasmids and measurement of single chain IL-12 mRNA expression Luciferase plasmid or mouse IL-12 plasmid in 1 X PBS solution at a concentration of 1 g/l was mixed with lipofectAMINE (Life Technologies Pacific Ltd., Tsuen Wan, Hong Kong) in a 1:1 ratio. Each mouse received a single intravenous injection of 200 l volume of plasmid liposome through the tail vein. The IL-12 plasmid which we have constructed51,52 was delivered into mouse and picked up the lungs after 24 h or 48 h. In order to avoiding contamination of plasmid DNA, the Oligotex mRNA spin-column (Qiagen, CA, USA) was used to purify mRNA. The mRNA product then react with M-MuLV reverse transcriptase (BioLab, MA, USA) and cDNA was amplified by Pro Taq DNA polymerase (Protech Technology, Taiwan, ROC).
Analysis of the luciferase protein expression In determining luciferase activity, the mice were given lethal doses of ether, their lungs removed, and the luciferase was assayed using a commercial kit (Promega, WI, USA). The lung organ was removed from the mice at 24 h or 48 h after injection. The tissues were washed and then frozen in liquid nitrogen.36 These were then individually pulverized into a fine powder by hand grinding with liquid nitrogen using a porcelain mortar and pestle. Lysis buffer was added at a volume to weight ratio of 4 l per mg of collected lung organ. The mixture was then vigorously vortexed for 15 min at 4C and centrifuged at 10000 g for 10 min at room temperature. The supernatants were collected and luciferase activity was assayed by luminometer (Turner Designs, CA, USA). The protein concentration of each sample was determined through BCA protein reagent assay (Pierce, IL, USA). The luciferase activity from serially diluted concentrations of recombinant luciferase was the standard curve used. The activity for each lung was recorded in luciferase activity (pg) per mg of tissue protein.
Bronchoalveolar lavage and cell differential counts After all of the groups of mice were anesthetized, they were bled from the retro-orbital venous plexus and then sacrificed. Using a cannula, their lungs were immediately lavaged through the trachea with 31 ml of 1HBSS, which is free of ionized calcium and magnesium. The lavaged fluid was centrifuged at 400 g for 10 min at 4C. After the wash, the cells were resuspended in 1 ml 1HBSS and the total cells counts were determined using a hemocytometer. Cytocentrifuged preparations were stained with Liu’s stain for differential cell counts. Based on standard morphologic
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criteria, a minimum of 200 cells were counted and classified as macrophages, lymphocytes, neutrophils or eosinophils.
Histopathological study of lung organs In order to evaluate the effects of IL-12 treatment, the lungs were immediately removed after lavaging and fixed in a solution of 3% v/v formalin (in 0.01M phosphate buffer pH 7.2). The tissues were subsequently embedded in paraffin and cut into 5 m thick sections. These sections were stained with hematoxylin-eosin and examined by light microscopy for histopathological changes.
Airway hyperresponsiveness In order to investigate the effects of IL-12 on airway hyperresponsiveness (AHR), the Buxco system (Biosystem XA; Buxco Electronics Inc., CT, USA) was used to evaluate the extent of airway constriction in different groups of mice following the protocol mentioned previously.53,54 This system is non-invasive and can measure the respiratory changes in animals that are awake, excluding both anesthetic effect and surgical artifact. The value of Penh is calculated by collecting data information derived from transducer (differential pressure transducer; Buxco) and preamplifier (MAX II, Buxco). Penh=PausePIF/PEF; Pause=(Te-Tr)/Tr, (PIF; peak inspiratory flow, PEF; peak expiratory flow, Te; expiratory time, Tr; relaxation time). In this experiment, the mice were challenged three times with crude mite extract and injected intravenously with DNA plasmid-lipofectAMINE 24 h before each crude mite inhalation. One day after final inhalation, the mice were given aerosolized normal saline, 12.5 mg/ml, 25 mg/ml, 50 mg/ml or 100 mg/ml methacholine (Sigma; MO, USA) serially. The mice inhaled normal saline or increasing concentrations of methacholine for 3 min each. The Penh value for each minute was recorded and after the third recorded value, the average Penh value was divided by the Penh of normal saline and was presented as relative percentage increase of Penh.
Primary mouse lung cell culture Four to six weeks B6 female mice were sacrificed by dislocation in order to avoid influence of ether or pentobarbital. The lungs were removed and washed with 1PBS buffer until the blood has been removed. The connective tissue and blood vessels were removed while the lung tissues were cut to small pieces. These were centrifuged and the cell precipitate was collected while the supernatant was discarded after. The lung cells were cultured with MEM (alpha minimum essential medium, Life Technologies) complete medium including 10% fetal bovine serum (FBS), 4 mM L-glutamine, 25 mM HEPES (pH 7.2), 5105 M 2-mercaptoethanol, 100 U/ml penicillin, 100 ug/ml streptomycin and 0.25 mg/ml amphotericin. After 10–14 days, the primary cell population can reach into 80% confluent. The cell culture dish was then washed with 1PBS, treated with 1 trypsin—EDTA, and incubated at 37C, 5% incubator for 3–5 min to detach cells. 1PBS was added and after flushing with a pippette, cell suspension were collected in 50 ml centritubes. These were centrifuged at 400 g at 4C for 5 min. After the supernatant was discarded, the cells were seeded with 1105 cells per well in 48-well plates. These
Interleukin-12 inhibits eotaxin secretion / 83
were cultured in 37C, 5% CO2 incubator until the cells get 80% confluence. Then replaced with MEM serum-free medium. After 16–18 h, cells were treated with different kinds of cytokines. Recombinant mouse IL-12 (R&D) was added 2 h before rIL-13 (R&D) or rIL-4 (R&D) stimulation. In neutralization assay, anti-IFN- Ab (BD PharMingen; CA, USA) was added simultaneously with rIL-12 2 h before rIL-13 stimulation. After 48 h of incubation, the supernatant of each well was collected and frozen in 20C before further analysis was made.
Eotaxin level in culture supernatant or bronchoalveolar lavage The eotaxin was assayed with ELISA kit (R&D) used according to the manufacturer’s instructions. Briefly, the culture supernatant or bronchoalveolar lavage of each condition was added to wells precoated over night at 4C with anti-eotaxin Ab. After 2 h of incubation, the plates were washed and biotin-conjugated Ab was added. After two more hours at room temperature, HRP-avidin was added to each well. The substrate tetramethylbenzidine was then added and the OD (at 450 nm) values were converted to concentrations of chemokine in the suppernatant. The sensitivity of this assay was 1.9 pg/ml for eotaxin.
Statistical analysis Individual experimental values were compared by the student’s t test. Differences between two groups were considered statistically significant at exact P-values, P<0.05.
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