Role of Interleukin-12 and Stat-4 in the Regulation of Airway Inflammation and Hyperreactivity in Respiratory Syncytial Virus Infection
2001; Elsevier BV; Volume: 159; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)61734-8
ISSN1525-2191
AutoresKim K. Tekkanat, Hussein Maassab, Aaron A. Berlin, P Lincoln, Holly Evanoff, Mark H. Kaplan, Nicholas W. Lukacs,
Tópico(s)Neonatal Respiratory Health Research
ResumoRespiratory syncytial virus (RSV) is a respiratory pathogen that can cause significant morbidity in infants and young children. Interestingly, the majority of children who acquire a RSV infection do not exhibit severe symptoms. Development of a Th1 response has been associated with resolution of symptoms in viral infections and may explain mild RSV illness. The current study investigated the cytokine response observed in mild disease in C57BL/6 mice that had low airway resistance and mucus production with little pulmonary inflammation. RSV infection in these mice was accompanied by a fourfold increase in interleukin-12(IL-12). Treatment of RSV-infected mice with anti-IL-12 resulted in an increase in airway hyperreactivity, mucus production, and airway inflammation (eosinophilia). Since IL-12 activation is dependent on Stat-4-mediated intracellular signal transduction, similar experiments were performed in Stat-4 deficient mice and demonstrated similar results to those obtained from anti-IL-12 treated mice. Again, there was an increase in airway hyperreactivity and mucus production, and goblet cell hypertrophy. These studies support the importance of IL-12 in the immune response to RSV infection resulting in resolution of disease and protection from inappropriate inflammatory responses. Respiratory syncytial virus (RSV) is a respiratory pathogen that can cause significant morbidity in infants and young children. Interestingly, the majority of children who acquire a RSV infection do not exhibit severe symptoms. Development of a Th1 response has been associated with resolution of symptoms in viral infections and may explain mild RSV illness. The current study investigated the cytokine response observed in mild disease in C57BL/6 mice that had low airway resistance and mucus production with little pulmonary inflammation. RSV infection in these mice was accompanied by a fourfold increase in interleukin-12(IL-12). Treatment of RSV-infected mice with anti-IL-12 resulted in an increase in airway hyperreactivity, mucus production, and airway inflammation (eosinophilia). Since IL-12 activation is dependent on Stat-4-mediated intracellular signal transduction, similar experiments were performed in Stat-4 deficient mice and demonstrated similar results to those obtained from anti-IL-12 treated mice. Again, there was an increase in airway hyperreactivity and mucus production, and goblet cell hypertrophy. These studies support the importance of IL-12 in the immune response to RSV infection resulting in resolution of disease and protection from inappropriate inflammatory responses. RSV is an important viral pathogen that causes annual epidemics worldwide.1Eigen H The RSV-asthma link: the emerging story. Introduction.J Pediatr. 1999; 135: 1-11Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar It is responsible for a large proportion of hospitalizations of infants in the winter months in the United States and consumption of millions of health care dollars. There can be significant morbidity associated with RSV bronchiolitis, both acutely and chronically, as a link between RSV bronchiolitis and later development of asthma has been proposed.1Eigen H The RSV-asthma link: the emerging story. 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The present studies examined the role of IL-12 in the immune response to RSV infection in a mouse model using C57BL/6 mice. IL-12 is important in the initial phase of bacterial, parasitic, and viral infections, and for the development of the T helper type 1 (Th1) response.13Trinchieri G Scott P The role of interleukin12 in the immune response, disease and therapy.Immunol Today. 1994; 15: 460-463Abstract Full Text PDF PubMed Scopus (0) Google Scholar, 14Trinchieri G Biological properties and therapeutic applications of interleukin-12.Eur Cytokine Netw. 1997; 8: 305-307PubMed Google Scholar, 15Wills-Karp M Interleukin-12 as a target for modulation of the inflammatory response in asthma.Allergy. 1998; 53: 113-119Crossref PubMed Scopus (33) Google Scholar, 16Trinchieri G Interleukin-12: a cytokine at the interface of inflammation and immunity.Adv Immunol. 1998; 70: 83-243Crossref PubMed Google Scholar, 17Adorini L Aloisi F Galbiati F Gately MK Gregori S Penna G Ria F Smiroldo S Trembleau S Targeting IL-12, the key cytokine driving Th1-mediated autoimmune diseases.Chem Immunol. 1997; 68: 175-197Crossref PubMed Scopus (29) Google Scholar Production of IL-12 is thought to favor differentiation and function of (Th1) T cells while inhibiting the differentiation of Th2 cells. In many viral infections, IL-12 promotes viral clearance and host recovery from infection.18Romani L Puccetti P Bistoni F Interleukin-12 in infectious diseases.Clin Microbiol Rev. 1997; 10: 611-636Crossref PubMed Google Scholar, 19McDyer JF Wu CY Seder RA The regulation of IL-12: its role in infectious, autoimmune, and allergic diseases.J Allergy Clin Immunol. 1998; 102: 11-15Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 20Haraguchi S Day NK Nelson Jr, RP Emmanuel P Duplantier JE Christodoulou CS Good RA Interleukin 12 deficiency associated with recurrent infections.Proc Natl Acad Sci USA. 1998; 95: 13125-13129Crossref PubMed Scopus (87) Google Scholar, 21Masihi KN Highlights of the international symposium on immunotherapeutic prospects of infectious diseases.Nat Immun Cell Growth Regul. 1991; 10: 185-195PubMed Google Scholar The role of IL-12 in RSV infection in relation to airway hyperreactivity and mucus production has not been specifically addressed, but other studies have speculated on its importance in acute bronchiolitis in humans.22Blanco-Quiros A Gonzalez H Arranz E Lapena S Decreased interleukin-12 levels in umbilical cord blood in children who developed acute bronchiolitis.Pediatr Pulmonol. 1999; 28: 175-180Crossref PubMed Scopus (51) Google Scholar This study focused on the physiological and immune response to RSV infection in the C57BL/6 mouse strain, a strain that appears to have a favorable response to RSV. Specific pathogen-free C57BL/6 mice (H-2b) and DBA/J mice (H-2d) were purchased from Jackson Laboratories (Bar Harbor, ME) and housed in University of Michigan animal facilities under pathogen-free conditions. Stat-4 deficient mice were grown and maintained by Dr. Mark Kaplan at Indiana University. RSV A2 was grown and harvested in Dr. Maassab's lab at the University of Michigan School of Public Health. The virus had been through 48 passages in Hep-2 cells (human epidermoid carcinoma cells from the larynx). It was subsequently passed multiple times in Vero cells (green monkey kidney cells), MRC-5 cells (human lung cells), and Hep-2 cells for growth and amplification of the virus. Pathogen-free mice were infected intratracheally with 3 × 105 plaque- forming units (PFU) in 30 μl media. Control mice received 30 μl of vehicle intratracheally. Mice were anesthetized with sodium pentobarbital (50 mg/kg) and ketamine (40 mg/kg) injected intraperitoneally. A tracheostomy was performed and RSV was injected directly into the trachea with a Hamilton syringe (Reno, NV). Following infection, the incision was closed with surgical staples and mice were allowed to recover. No mice died from this level of viral infection or from anesthesia. Airway hyperreactivity was measured using a Buxco mouse plethysmograph (Buxco, Troy, NY) which is specifically designed for low tidal volumes as previously described.23Campbell E Hogaboam C Lincoln P Lukacs NW Stem cell factor-induced airway hyperreactivity in allergic and normal mice.Am J Pathol. 1999; 154: 1259-1265Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 24Hogaboam CM Bone-Larson CL Lipinski S Lukacs NW Chensue SW Strieter RM Kunkel SL Differential monocyte chemoattractant protein-1 and chemokine receptor 2 expression by murine lung fibroblasts derived from Th1- and Th2-type pulmonary granuloma models.J Immunol. 1999; 163: 2193-2201Crossref PubMed Google Scholar Briefly, the mouse to be tested was anesthetized as described above, and intubated via cannulation of the trachea with an 18-gauge metal tube. The mouse was placed on a Harvard pump ventilator (Harvard, Holliston, MA) (tidal volume 0.4 ml, frequency 120 breaths/min, positive end-expiratory pressure 2.5–3.0 cm H2O) and was ventilated for 5 minutes before the methacholine challenge. The plethysmograph was sealed and readings monitored by computer. Since the box is a closed system, a change in lung volume was represented by a change in box pressure (Pbox) that was measured by a differential transducer. The system was calibrated with a syringe that delivered a known volume of 2 ml. A second transducer was used to measure the pressure swings at the opening of the trachea tube (Paw), referenced to the body box (ie, pleural pressure), and to provide a measure of transpulmonary pressure (Ptp) (Ptp = Paw − Pbox). The tracheal transducer was calibrated at a constant pressure of 20 cm H2O. Resistance is calculated by the Buxco software by dividing the change in pressure (Ptp) by the change in flow (F) (δPtp/δF; units = cmH2O/ml/s) at two time points from the volume curve based on a percentage of the inspiratory volume. Once baseline levels were stabilized and initial readings were taken, a methacholine challenge was given intravenously via cannulation of one of the tail veins with a 27-gauge needle. A dose-response curve (0.01 to 0.5 mg/kg) was performed and an optimal dose of 0.1 mg/kg of methacholine was obtained. This dose was used throughout the rest of the experiments in this study. After the methacholine challenge, the response was monitored and the peak airway resistance was recorded as a measure of airway hyperreactivity. The right upper lobe from each mouse was flash-frozen in liquid nitrogen and kept frozen at −80°C. Just before running the ELISA assays, the samples were weighed and homogenized in 1 ml of homogenization buffer containing 1 Complete tablet (Boehringer Mannheim, Germany) and 0.1% Triton-X in 50 ml of phosphate-buffered saline (PBS). Cytokines were quantitated from homogenized PBS lung aqueous extracts using a double ligand ELISA system. The murine ELISAs have been developed in our laboratories using a previously described method.25Hogaboam CM Gallinat CS Taub DD Strieter RM Kunkel SL Lukacs NW Immunomodulatory role of C10 chemokine in a murine model of allergic bronchopulmonary aspergillosis.J Immunol. 1999; 162: 6071-6079Crossref PubMed Google Scholar ELISAs were conducted as follows: flat-bottomed 96-well microtiter plates (Nunc Immuno-Plate I 96-F, Lincolnshire, IL) were coated with capture antibody diluted to 3.2 μg/ml in coating buffer (borate-buffered saline, pH 8.6) and incubated overnight at 4°C. Nonspecific binding sites were blocked with 2% bovine serum albumin (BSA) in PBS and incubated for 1 hour at 37°C. Plates were washed and specimens added in triplicate followed by incubation at 37°C and washing. Biotinylated detection antibody was added and the plates incubated at 37°C for 1 hour. Plates were washed and conjugated streptavidin-peroxidase was added, followed by washing and the addition of chromogen substrate (OPD). Finally plates were incubated at room temperature, the reaction terminated with 3 mol/L H2SO4 and read at 490 nm in an ELISA reader. The individual polypeptides were standardized to total protein (ng/μg total protein). Our ELISAs routinely detect protein at concentrations above 50 pg/ml. These ELISAs are specific and do not cross-react to any other chemokine or cytokine. Rabbit anti-murine IL-12 antibodies were prepared by multiple-site immunization of New Zealand White rabbits with recombinant murine IL-12 (R&D, Rochester, MN) in Complete Freund's Adjuvant. Polyclonal antibodies were titered by direct ELISA and specifically verified by the failure to cross-react to mIL-3, mIL-1α, mTNF, mIL-4, hIL-12, mIL-10, mMIP-1α, IL-6, mJE, mMIP-1β, hMCP-1, hIL-8, hRANTES, hMIP-1α, hTNF, and hMIP-1α. The IgG portion of the serum was purified over a protein A column and used in a sandwich ELISA. Whole serum (0.5 ml) was used in vivo to block IL-12 during the RSV infection. Neutralization of IL-12 was carried out using a polyclonal rabbit anti-murine IL-12 antibody developed in our laboratory as above. The anti-IL-12 or control antibody was administered intraperitoneally 1 hour before infection and every 2 days postinfection until day 14. The in vivo half-life of the antibody was ∼30 hours. Bronchoaveolar lavage samples (BAL) samples were collected from each mouse just after airway hyperreactivity data were collected and after cervical dislocation. 1 ml of sterile 0.9 N saline was instilled intratracheally and was suctioned out after a few seconds. Samples were placed in Eppendorf tubes and centrifuged for 1500 rpm for 5 minutes. Supernatant was removed and cells were resuspended in PBS and cytospin-fixed and stained with Diff-Quick (Dade Behring Ag., Dudingen, Switzerland). Mouse lungs from each time point were preserved in 1 ml of 4% paraformaldehyde. The fixed lungs were embedded in paraffin, and multiple 50-μm sections were differentially stained with hematoxylin-eosin for the identification of eosinophils. Eosinophils were quantified by counting 100 hpf/lung using multiple step sections of the lung. The eosinophils counted were only in the peribronchial region to assure enumeration of only those eosinophils within or immediately adjacent to the airway. Periodic acid-Schiff (PAS)-stained lung sections (prepared as above) were used to assess goblet cells in the small-to-moderate sized airways. Goblet cell hypertrophy was assessed qualitatively and quantitatively (counting numbers per airway). Numerical results were expressed as means ± SEM. Analysis of variance was used to determine the level of difference between groups. Pairs of groups were compared by unpaired two-tailed Student's t-test. Analysis of variance was used to compare changes between different strains with same treatment and significance was determined with P values <0.05. The hypothesis that host genetic difference affects response to RSV was tested by comparing airway hyperreactivity (AHR) responses and BALs in C57BL/6 (H-2b) mice to those in DBA/2 mice (H-2d) during primary RSV infection. Pathophysiological response to RSV was determined by measuring change in airway resistance after a methacholine challenge. Comparison of the strains showed that the BALB/c and DBA/2 mice had significantly higher airway hyperreactivity over almost the entire time course after RSV exposure when compared to the C57BL/6 mice (Figure 1). Increased hyperreactivity was apparent by day 8 in DBA/2 mice, continued to increase to day 12, and then declined to baseline by later time points postinfection. The C57BL/6 mice had minimal change in hyperreactivity over the same postinfection time course. Interestingly, C57BL/6 and DBA-2 mice had similar levels of virus on day 4 of infection with no evidence of productive virus at the time of peak airway hyperreactivity at day 12 (data not shown). Examination of BALs from RSV-infected mice showed minimal epithelial sloughing and little mucus production in the C57BL/6 mice with a prominent increase in DBA/2 mice (data not shown). Epithelial cell sloughing was most prominent early postinfection and increasing mucus production correlated with increasing hyperreactivity. Lung histology revealed similar findings with more inflammation observed in the DBA/2 mice (data not shown). Thus, the C57BL/6 mice appeared to respond more appropriately to the RSV infection with lower pathophysiologic abnormalities. The pulmonary physiological response observed in the different mouse strains may be due to the cytokine environment established at the site of infection. In these studies, we were interested in examining the basis for the mild pathophysiological response to RSV seen in the C57Bl/6 mouse strain. Cytokine profiles were determined by running ELISA assays on whole lung homogenates. Cytokines examined included interferon-γ, IL-4, IL-12, and IL-13. The production of IFN-γ showed an increase early in the response while IL-12 production was up-regulated at later time points; specifically, at day 8 through day 14 of infection (Figure 2). IL-13 and IL-4 showed slight decreases over the same time period (data not shown). Increased IL-12 levels appeared to correlate with mild response to RSV infection suggesting that it may be an important cytokine involved in the immune response to RSV in the C57BL/6 mice. Accordingly, IL-12 production in lungs of infected DBA/2 mice did not increase over the same period of infection over background levels, ranging from 0.65 to 0.85 ng/lung on the various days (Figure 2). IL-12 is known to be an important immunoregulatory cytokine favoring differentiation and function of Th1 cells while inhibiting differentiation of Th2 cells.14Trinchieri G Biological properties and therapeutic applications of interleukin-12.Eur Cytokine Netw. 1997; 8: 305-307PubMed Google Scholar, 26Trinchieri G Scott P The role of interleukin-12 in the immune response, disease and therapy.Immunol Today. 1994; 15: 460-467Abstract Full Text PDF PubMed Scopus (26) Google Scholar Although there is not a great deal of information available on the importance of IL-12 in RSV infection, several investigators have suggested that it may have an immunomodulatory role.10Byrd LG Prince GA Animal models of respiratory syncytial virus infection.Clin Infect Dis. 1997; 25: 1363-1368Crossref PubMed Scopus (103) Google Scholar, 22Blanco-Quiros A Gonzalez H Arranz E Lapena S Decreased interleukin-12 levels in umbilical cord blood in children who developed acute bronchiolitis.Pediatr Pulmonol. 1999; 28: 175-180Crossref PubMed Scopus (51) Google Scholar, 27Tang YW Graham BS Interleukin-12 treatment during immunization elicits a T helper cell type 1-like immune response in mice challenged with respiratory syncytial virus and improves vaccine immunogenicity.J Infect Dis. 1995; 172: 734-738Crossref PubMed Scopus (67) Google Scholar To confirm that IL-12 was in fact the cytokine responsible for the mild response to RSV, antibodies specific for IL-12 were used to neutralize the cytokine in vivo. Neutralizing anti-IL-12 or control antibodies were given intraperitoneally 1 hour before intratracheal RSV (3 × 105 PFU) and every other day thereafter until day 12 of infection. The mice were then examined for changes in airway hyperreactivity at specific time points after infection (Figure 3). The mice treated with neutralizing antibodies against IL-12 had a significant increase in airway hyperreactivity, whereas untreated mice and mice treated with control antibodies had minimal airway hyperreactivity, comparable to previous experiments. IL-12 levels were reduced to baseline values in neutralized animals (data not shown) while IL-13 levels showed a slow increase with peak levels observed on day 12 (Figure 4). In control, uninfected mice, anti-IL-12 had no effect on airway hyperreactivity (data not shown). Microscopic examination of bronchoalveolar lavage fluid demonstrated minimal mucus production in control serum-treated mice and untreated mice, with a large amount of mucus production seen in the anti-IL-12-treated animals at days 8 and 12 (Figure 5). Histologically, the anti-IL-12-treated mice had increased inflammation (Figure 6) with a significantly greater number of pulmonary eosinophils (Figure 6). PAS staining revealed increased numbers of mucus-producing goblet cells in anti-IL-12 treated mice compared to RSV/control mice (Figure 7). Thus, production of IL-12 during RSV infection appears to be an important component in protecting the host from detrimental pathophysiologic responses.Figure 4IL-13 production in control C57BL/6 mice versus anti-IL-12 treated mice. RSV control mice were injected intratracheally with RSV and intraperitoneally with control serum. Anti-IL-12 treated mice were injected IT with RSV and IP with anti-IL-12 antibody. Each time point represents mean ± SEM for 8 mice with experiment repeated three times with similar results obtained for each. * Significant differences (P < 0.05) between normal serum control/RSV mice and anti-IL-12 treated mice.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Light microscopy of BAL fluid from control C57BL/6 mice and anti-IL-12 treated mice after RSV infection. BALs were collected and processed as previously described using Diff-Quick stain. Magnification, ×400.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6a: Hematoxylin and eosin stain of lung sections from control C57BL/6 mice and anti-IL-12 treated mice on days 8 and 12 after RSV infection. Magnification, ×400. b: Peribronchial eosinophil numbers in control (IT vehicle only, no treatment) versus RSV control versus RSV/anti-IL-12 treated mice.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7PAS stain of lung sections from control C57BL/6 mice and anti-IL-12 treated mice on day 12 after RSV infection.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Th1 and Th2 lymphocytes are thought to be activated by specific cytokines resulting in a differentiated immune response. Binding of the cytokine to its receptor results in activation of its associated signal transduction pathways resulting in a unique response. The signal transducer and activator of transcription (Stat) family of proteins are involved in one of these intracellular signal transduction pathways. IL-12 activates T helper cells via a Stat-4-induced signaling pathway. To confirm the importance of IL-12 in abrogating severe airway hyperreactivity responses in RSV infection, we performed similar RSV infection experiments in Stat-4-deficient mice. Figure 8 shows that Stat-4−/− mice on a C57BL/6 background had significantly higher airway hyperreactivity responses compared to Stat 4+/+ mice. This pronounced effect was present as soon as day 4 after infection and persisted at day 12 which is similar to the effects observed in anti-IL-12-treated mice. Histologically, Stat 4 −/− mice had increased numbers of eosinophils and goblet cell hypertrophy when compared to RSV controls but not to the extent observed in anti-IL-12-treated C57BL/6 mice (data not shown). RSV causes airway epithelial cell damage, peribronchial inflammation, hyperreactivity, and increased mucus production leading to respiratory distress, hospitalization, and even death in those infants and small children most severely affected. Yet, most children who are infected develop only mild symptoms. Others have described cytokine mediators involved in the severe response,28Srikiatkhachorn A Chang W Braciale TJ Induction of Th1 and Th2 responses by respiratory syncytial virus attachment glycoprotein is epitope and major histocompatibility complex independent.J Virol. 1999; 73: 6590-6597Crossref PubMed Google Scholar, 29Schwarze J Hamelmann E Bradley KL Takeda K Gelfand EW Respiratory syncytial virus infection results in airway hyperresponsiveness and enhanced airway sensitization to allergen.J Clin Invest. 1997; 100: 226-233Crossref PubMed Scopus (294) Google Scholar, 30Roman M Calhoun WJ Hinton KL Avendano LF Simon V Escobar AM Gaggero A Diaz PV Respiratory syncytial virus infection in infants is associated with predominant Th-2-like response.Am J Respir Crit Care Med. 1997; 156: 190-195Crossref PubMed Scopus (267) Google Scholar but there is little information on mediators involved in an asymptomatic or mildly symptomatic infection. The current study was designed to study the cytokine(s) that were important in mild disease. Because IL-12 was the cytokine produced in the largest amount in these mildly “symptomatic” C57Bl/6 mice after RSV infection, this study focused on its importance in modifying the pathophysiology. 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