recent evidence supports important functions of the cytokine il-33 in both innate and adaptive immune responses . il-33 was first described as a nuclear factor from small endothelial venules [ 1 , 2 ] and later by schmitz et al .   as an extracellular ligand for st2 , which in turn had previously been described as an orphan receptor . il-33 showed ability to induce t helper 2 cytokines and subsequent work has further characterized il-33 biology and roles in homeostasis and disease including bacterial and helminthic infection , allergy and chronic inflammation of lung , joint , skin , cns , and cardiovascular tissues ( as reviewed by others [ 58 ] ) . the extracellular soluble form of il-33 interacts with its receptor complex composed of st2 and il-1rap   and induces the nf - kappa b and map kinase cell signaling pathways . il-33 is typically not secreted by living cells , where it is localized primarily to the nucleus , but it is released upon cell death / necrosis similar to other alarmins such as hmgb1 and il-1 [ 5 , 6 ] . marked effects of excess soluble il-33 in mice are indicated by lethal inflammation and autoimmunity in transgenic mice with high levels of soluble il-33 bioavailability . less is known about the roles of il-33 within the nucleus ; however , cellular il-33 is associated with heterochromatin   and can modulate nf - kappa b signaling . elevated levels of il-33 are found in mouse models of allergic airway disease and in chronic human lung conditions including severe asthma [ 58 ] . il-33 influences th2-skewed immune functions in allergic responses , where engagement of il-33 receptor complexes on th2 cells enhances th2 responses . il-33 induces eosinophil activation , exacerbates the ova - induced airway inflammation model , and is required for maximal effects in models of hdm - induced airway inflammation . however , il-33 is also critical in innate immune responses such as lps - induced systemic inflammatory responses and dextran - induced colitis with t cell - independent epithelial damage . in addition , studies by luzina et al .   indicate elevated expression of il-33 in tissue from idiopathic pulmonary fibrosis ( ipf ) patients , and other studies have implicated il-33 in mouse models of lung fibrosis . exogenous il-33 increased severity in bleomycin - induced lung fibrosis , whereas inhibition of il-33 pathways reduced lung fibrosis , as assessed by anti - il-33 treatment in the bleomycin model or in st2 ko mouse responses to bleomycin . human il-33 was initially described in endothelium of lymph nodes   and subsequent work showed endothelial cell expression in many sites including chronically inflamed tissue . constitutive il-33 gene expression is evident in nuclei of endothelial and epithelial cells [ 11 , 19 ] and mouse keratinocytes . myeloid cells can also express il-33 upon induction by lps / nf - kappa b activation . among pulmonary tissue cells examined , airway smooth muscle cells [ 17 , 22 ] and type 2 alveolar epithelial cells show il-33 expression [ 7 , 14 ] . published studies on regulation of il-33 by cytokines and growth factors indicate that tnf , il-1 , or gmcsf can induce il-33 , whereas ifn - gamma appeared to repress il-33 levels depending on the cell type . il-33 expression may be regulated in homeostasis and disease by other growth factors and cytokines . while the role of gp130 cytokines ( including il-6 , lif , oncostatin m ( osm ) , il-11 , and il-31 ) in regulation of il-33 expression in lung cells is not yet known , we and others have previously shown that the gp130 cytokine osm has homeostatic and proinflammatory activities and can modify extracellular matrix ( ecm ) [ 2427 ] . given the function of osm in activating stromal cells and elevation of osm levels in ipf and severe asthma [ 28 , 29 ] , putative regulation of il-33 may be of importance in the modulation of innate immunity , chronic inflammation , and tissue repair in lung disease . in this study , we examined functions of oncostatin m in modulating il-33 expression in mouse lung in vivo and in mouse epithelial cells in vitro . osm showed prominent regulation of il-33 mrna and full length il-33 protein in vivo and induced cellular expression of full length il-33 in mouse lung epithelial cells in vitro which was repressed with stat3 inhibition . the results support a newly identified osm - il-33 axis in lung tissue cells and suggest an additional pathway in innate immune responses involving il-33 . cell lines were cultured under standard conditions in medium containing 10% fetal bovine serum supplemented with 1% penicillin / streptomycin and 1% l - glutamine . the c10 type 2 alveolar epithelial cell line was cultured in rpmi as previously indicated   ( gift from dr . mouse recombinant cytokines including osm , il-6 , lif , il-31 , tnf , tgf , il-4 , il-13 , and il-17a and mature il-33 were purchased from r&d systems ( minn . pharmacological inhibitors were added at the following final concentrations prior to osm stimulation : the stat3 inhibitor stattic at 5  m ( 30  min prior ) ( abcam biochemical , toronto , canada ) , the p38 inhibitor sb203580 at 5  m ( 1  hr prior ) ( calbiochem , san diego , ca ) , the erk inhibitor pd98059 at 25  m ( 2  hr prior ) ( calbiochem ) , and the akt inhibitor aktx at 5  m ( 1  hr prior ) ( calbiochem ) . adenovirus encoding mouse osm ( adosm ) or empty virus ( addel70 ) has been previously described and was used to transiently express virus encoded genes in lungs by endotracheal administration as previously indicated [ 30 , 31 ] . at day 7 after administration of vectors , each at 5  10  pfu with n = 5 mice per treatment , the mice were culled , bronchoalveolar lavage was completed , and the right lung lobes were extracted and snap - frozen . portions were later processed either for rna analysis or for protein analysis as previously described . the left lung lobe was excised , inflated / fixed with formaldehyde , and subsequently processed for histology and immunohistochemistry . ihc for il-33 was completed similarly to that as previously described   and as follows : paraffin embedded sections of left lungs of mice treated with ad vectors as indicated were deparaffinized , hydrated sequentially with absolute , 95% , and 70% alcohol , and then washed in 3% hydrogen peroxide ( to block endogenous peroxide ) and then in tbs / tween/0.1% saponin . the sections were then blocked with horse serum as part of the immpress system ( vector labs ) in 0.1% saponin , washed ( tbs / tween/0.1% saponin ) , and then incubated with goat anti - mouse il-33 primary antibody ( cat # af3626 , r&d systems ) at varying concentrations ( 2.5  g / ml , 1.2  g / ml , and 0.6  g / ml ) for 1  hr . after washing , immpress secondary ab ( horse anti - goat igg ) was added for 30  min and then washed and antigen detected with dab reagent ( dako ) . for prosurfactant protein c ( spc ) , methods were as described previously   using 1  :  2000 anti - spc polyclonal ab ( millipore ) and envision + rabbit ( dako ) . for cd31 stain , antigen retrieval was used ( dako target retrieval ) before rat anti - mouse cd31 ( 1  :  50 dilution , overnight rt ) and biotinylated rabbit anti - rat ( mouse absorbed ) at 1  :  100 ( vector labs ) and then revealed with envision - plus - rabbit - tru ( dako k4003 ) . for quantification , il-33 cells were enumerated per high power field ( 400x ) with 5 fields counted per left lung from at least 3 separate mice per ad vector treatment . 1520  g of protein was loaded on 10% or 12% sds - page gels and following separation proteins were transferred to a nitrocellulose membrane by standard methods   and blocked for one hour at room temperature using li - cor odyssey blocking buffer ( mandel ) . blueye molecular weight protein ladder ( froggabio , toronto , canada ) was run on all protein gels . blots were then probed using the following antibodies at 4c overnight : actin i-19 and phospho - p38 ( santa cruz biotechnology ) , diluted 1  :  2000 ; mouse il-33 ( r&d systems ) , diluted 1  :  1000 ; phosphorylated stat3 ( cell signaling technology ) , diluted 1  :  2000 ; total stat3 ( cell signaling technology ) , diluted 1  :  1000 ; and smad1 , diluted 1  :  1000 ( cell signaling technology ) . phospho - smad1/5/8 ( 1  :  1000 dilution ) was purchased from millipore . primary antibodies were detected using li - cor anti - rabbit or anti - goat irdye infrared secondary antibodies at 1  :  5000 or 1  :  10000 dilution , respectively , and imaged using the li - cor odyssey infrared scanner . blots were stripped using li - cor newblot nitro stripping buffer , as per manufacturer 's instructions , and then blocked and reprobed for different proteins . densitometry analysis of immunoblot band intensity was performed using image studio lite software ( li - cor ) . cell lysates were compared relative to untreated control samples to quantify fold changes in signal , and lung homogenate samples were compared relative to lung homogenate samples from mice treated with addel70 . for assessing soluble il-33 in cell culture supernatants , duoset antibody pairs for mouse il-33 were purchased from r&d systems inc . and used as per manufacturer 's protocols . in assessing mrna levels , rna was prepared following manufacturer 's protocol using purelink rna mini kits and analyzed by real - time quantitative pcr . predetermined assay reagents ( pdar probes ) were purchased for il-33 and st2/il1rl1 ( thermo fisher ) . graphpad prism version 5.0 was used to generate graphs to display the results and for statistical analysis . the one - way analysis of variance ( anova ) test was used to evaluate statistical differences using tukey 's post hoc multiple comparisons test at a significance level of 0.05 . we have previously shown that transient overexpression of osm using ad vector expressing mouse osm ( adosm ) induces inflammatory responses and increases extracellular matrix in lungs of mice [ 30 , 31 ] . to determine whether il-33 is expressed and/or regulated in this system , we examined both protein and mrna levels in mice treated with control addel70 vector or adosm vector ( figure 1 ) . the addel70 vector is without the mosm cdna insert , but otherwise it has the identical backbone and is used as the control vector as previously indicated [ 30 , 31 ] . immunoblots of total lung protein extracts showed consistent elevation of il-33 signal by adosm and this correlated with pstat3 activation ( figure 1(a ) ) . addel70 alone did not upregulate il-33 at day 7 , as compared to nave uninfected mice ( data not shown ) . quantification of il-33 ( corrected to actin ) and pstat3 ( corrected to total stat3 ) showed significant induction of il-33 levels in both balb / c ( figures 1(a)1(c ) ) and c57bl/6 ( figures 1(b ) and 1(c ) and supplemental figure  1 in supplementary material available online at http://dx.doi.org/10.1155/2016/9858374 ) total lung extracts . the antibody detected prominent bands corresponding to full length il-33 ( migrated at approximately 35  kd ) , whereas mature il-33 signal ( recombinant mature il-33 migrated at approximately 18 - 19  kd ) was absent / low in mouse lung extracts of both addel70- and adosm - treated balb / c mice ( supplemental figure  2 ) . to assess expression of il-33 and il-33 receptor chain ( il-33r / st2 ) mrna levels , total lung homogenates the data showed induction of il-33 mrna in both balb / c ( > 20-fold ) and c57bl/6 ( > 10-fold ) mouse strains . in contrast , mrna levels of st2 were not altered in balb / c mice and minimally elevated ( 2-fold ) in c57bl/6 mice at the time point examined . to determine which cells in the lung express il-33 upon transient osm overexpression , we examined lung sections by specific il-33 immunohistochemistry . figure 2(a ) showed some il-33 staining in addel70-treated balb / c mice which was limited to the nucleus of il-33-positive cells throughout the parenchyma . in contrast , adosm - treated mice showed lungs with marked increases in number and intensity of il-33-stained cells , localized predominantly to the nucleus of cells with morphology of type 2 alveolar epithelial cells ( figures 2(a ) and 2(b ) ) . quantification of il-33-nuclear positive cells per 400x magnification is shown in figure 2(c ) and indicated significant increases in numbers of cells . we also stained for prosurfactant protein c ( spc ) to identify type 2 alveolar epithelial cells ( figure 2(e ) ) . a similar distribution of il-33 and spc staining was observed , consistent with expression of il-33 by type 2 alveolar epithelial cells , as shown previously in other systems of mouse lung inflammation . since it has been shown that endothelial cells express il-33 in other systems , we examined mouse lung sections stained for cd31 , a typical vascular endothelial cell marker . cd31 staining was evident on the luminal surface of blood vessels ; however , these cells were not positive for il-33 in serial sections ( figure 2(d ) ) . to confirm that adosm induced the same effects in balb / c mouse lung as previously described in this system , inflammatory cell accumulation in bronchoalveolar lavage ( bal ) fluid and histopathology of lung tissue sections are shown in supplementary figure  4 . the data indicated that mice showed the expected elevation of cells in bal as well as increases in epithelial cell hyperplasia , thicker alveolar walls , and inflammatory cell accumulation in tissue sections , in response to adosm . since the data indicated type 2 epithelial cells were strongly responsive to adosm in vivo , we further explored expression of il-33 in vitro . we first tested whether osm could directly regulate il-33 in mouse type 2 alveolar epithelial cells using the c10 cell line derived from balb / c . c10 cells stimulated with osm resulted in time - dependent increases ( evident at 4 and 6 hours and more prominently at 24 hours ) and dose - dependent increases ( evident with 0.5  ng / ml osm stimulus and markedly with 5  ng / ml osm at 24  hr ) in il-33 signal detected by immunoblots of c10 cell lysates ( figures 3(a ) and 3(b ) ) . we could not detect presence of mature il-33 ( approximately 18 - 19  kd bands , as indicated by inclusion of recombinant mature il-33 in supplemental figure  2 ) in unstimulated or osm - stimulated c10 cell lysates . similar results were observed using a monoclonal anti - il-33 antibody ( nessy-1 antibody from abcam , data not shown ) . figure 3(b ) shows a quantitative 2-fold , 4-fold , and 12-fold increase in il-33 signal at 4  hr , 6  hr , and 24  hr , respectively , upon stimulation with 5  ng / ml osm ( figure 3(b ) ) . other gp130 cytokines including il-6 , lif , or il-31 did not stimulate il-33 detectably in c10 cell lysates in comparison to osm ( figures 3(a ) and 3(b ) ) nor did tnf. activation of stat3 , as assessed by pstat3 immunoblots , was evident in lysates of osm - stimulated cells in dose - dependent fashion , whereas il-6 , lif , il-31 , or tnf did not elevate pstat3 ( figures 3(a ) and 3(c ) ) . when supernatants of cells from any of these conditions were tested for il-33 by specific elisa , no detectable soluble il-33 was evident ( not shown ) at the level of sensitivity of the elisa ( 15  pg / ml , r&d systems ) . ihc staining for il-33 in c10 cell cultures in vitro showed detectable il-33 in nuclei in unstimulated cells and a marked increase in nuclear staining upon osm stimulation ( supplemental figure  3 ) consistent with the il-33 staining in whole lung ( figure 2 ) . to determine whether osm regulated il-33 or st2 expression at the mrna level in vitro , c10 cellular extracts were assessed by qrt - pcr and showed approximately 30-fold and 40-fold increases in il-33 mrna at 6 hours or at 72 hours , respectively , after osm stimulation ( figure 3(d ) ) , whereas the il-33 receptor chain st2 mrna levels did not change ( figure 3(e ) ) . since induction of il-33 protein levels was associated with activation of stat3 both in vivo and in vitro ( figures 1 and 3 ) , we then assessed whether pharmacological inhibition of stat3 affected il-33 induction in c10 cells . in figure 4(a ) ( immunoblots ) and figure 4(b ) ( quantitative densitometry ) , data showed that preincubation of cells with the stat3 inhibitor stattic , or the p38 inhibitor sb203580 , reduced the signal of il-33 compared to vehicle alone at 6  hr of osm stimulation . as compared to vehicle alone , the erk inhibitor pd908059 , and the akt inhibitor aktx , stattic inhibited early osm - induced pstat3 levels ( 20-minute time point ) in dose - dependent fashion but did not affect the osm - induced levels of phospho - p38 ( figure 4(c ) ) . absence of pstat3 activation corresponded to reduced levels of il-33 protein expression ( figure 4(a ) ) . this supports a role of stat3 activation in the regulation of il-33 levels by osm in c10 cells . we then compared osm induction of il-33 to the activity of th2 cytokines il-4 and il-13 , as well as the th17 cytokine il-17a , in their ability to regulate il-33 in c10 cell cultures . figure 5(a ) shows that osm and to a lesser extent il-17a stimulation elevated il-33 signal , whereas il-4 , il-13 , and tgf alone did not elevate il-33 . in combination , il-4 or il-13 did not detectably affect osm - induced il-33 , whereas il-17a and osm combination resulted in higher levels of il-33 signal compared to either cytokine alone . il-33 has been implicated in exacerbation of ecm accumulation in mouse lungs as shown by others [ 17 , 18 ] . since osm , bmp-2 , and tgf are cytokines that can induce ecm in various systems , we tested each in their ability to regulate il-33 in the mouse epithelial c10 cells ( figure 5(b ) ) in vitro . results indicate that stimulation by osm , but not bmp-2 or tgf , increased il-33 cellular protein in c10 cell lysates . bmp-2 activated its canonical signaling pathway as indicated by elevated psmad1/5/8 , and tgf activated its canonical pathway as indicated by elevated psmad2 in the same c10 cell lysates . this indicates that , despite functional receptors and signaling in these cells , neither tgf nor bmp2 alone could induce il-33 expression . when the cytokines were added in combination , tgf inhibited il-33 induction by osm in c10 cell cultures . to determine whether similar osm responses were also evident in a second mouse epithelial cell line , we assessed mouse la-4 in similar fashion . although the level of il-33 signal was generally lower in la-4 cells than in c10 cells , figure 6(a ) shows that osm induced il-33 protein signal in la-4 lysates , whereas tgf alone did not , and tgf again reduced il-33 signal when added in combination with osm . assessment of mrna levels of il-33 in la-4 cells ( figure 6(b ) ) showed results consistent with the protein analysis , where osm induced il-33 mrna levels , tgf alone did not , and tgf reduced il-33 mrna when added in combination with osm . our results show that transient gene expression of osm in lungs of mice induces il-33 mrna and protein levels in whole lung extracts in vivo . alveolar type 2 cells but not vascular endothelial cells expressed markedly elevated il-33 in vivo , and osm directly induced il-33 in mouse alveolar epithelial type 2 c10 cells in vitro . this is the first identification of osm as a participant in il-33 regulation in lung and suggests additional mechanisms of il-33 regulation in pulmonary inflammation and pathology . the results of ihc analysis of lung sections and c10 epithelial cells in vitro indicated that type 2 alveolar cells were prominent responders to osm in il-33 expression . other stromal cells such as fibroblasts , endothelial cells , and smooth muscle cells have been shown to express il-33 in other systems and may also contribute to il-33 levels in total lung , although here we focused on alveolar type 2 cells due to their robust response in this system . others have also shown il-33 induction in alveolar type 2 cells in lung inflammatory models including the hdm model of allergic airway inflammation in balb / c mice   and in lung alveoli upon papain - induced allergic airway inflammation in c57bl/6 mice . recent work has also shown that systemic expression of osm in mice , using the same adosm vector used here but administered intramuscularly , induced il-33 expression in liver endothelial cells in vivo . whether lung tissue il-33 expression can be modified by systemic osm elevation is not yet known . in the present work we could not detect il-33 upregulation in lung endothelial cells with local pulmonary osm overexpression ( figure 2 ) . have recently shown that il-33 is not constitutively expressed in mouse lung endothelium ( il-33 immunostaining ) but is expressed to some extent in simple cuboidal cells in lung epithelium of c57bl/6 mice . our studies indicated low / no staining of cuboidal epithelial cells in balb / c mice by ihc , and this was minimally altered upon overexpression of osm ( figure 2 ) . this may reflect technical differences in detection systems used or possibly differences in balb / c and c57bl/6 mouse strains in cell expression of il-33 protein . has also suggested significant species differences in il-33 expression between mouse and human cells / tissues . we observed induction of il-33 protein levels by osm but not by il-6 , lif , or il-31 ( figure 3 ) in vitro and this was consistent with relative activation of stat3 by each of these cytokines in the same cell lysates . osm induces a number of signaling pathways in connective tissue cells including stats 1 and 3 , mapk pi3k , and pkc delta , depending on the cell systems examined [ 26 , 27 ] . pronounced and prolonged stat3 activation is characteristic of osm responses both in vitro and in vivo [ 31 , 35 ] . in the systems examined here , other works have shown that osm can elevate il-33 in osteoblast cell cultures and may be involved in the anabolic activity of osm in bone formation   in musculoskeletal tissue ; however , the involvement of stat3 was not explored . a sequence search of the murine il-33 promoter in the ncbi nucleotide database reveals multiple stat binding sites , suggesting that stat3 activation can directly regulate transcription of the il-33 gene , although confirmation of this mechanism requires further study . upon testing th2 cytokines ( il-4 and il-13 ) or il-17a , il-17a enhanced il-33 alone and additively when used in combination with osm to stimulate c10 cells . on the other hand , il-4 or il-13 had no detectable activity . whether similar effects are evident in other lung cell types remains to be established , and regulations by osm and il-17a in lung fibroblasts or lung smooth muscle cells are currently being investigated to determine whether osm / il-17a regulation of il-33 is a more general effect . conversely , tgf ameliorated il-33 levels in c10 as well as la-4 alveolar epithelial cells in vitro ( figures 5 and 6 ) . although the mechanism by which tgf suppresses il-33 protein levels in lung cells is unclear ( we did not observe modulation of osm - induced stat3 signals ) , suppression of il-33 expression could indicate another function of tgf in modulating functions in immunity . a previous study showed tgf inhibition resulted in increased il-33 macrophages and increased pathology in colon of dss treated mice ; however , whether stromal cell expression of il-33 was modulated in that study was not indicated . collectively , our results indicate that osm action on il-33 is not shared by th2 cytokines or other gp130 cytokines in mouse lung epithelial cells . others have shown that osm potentiated responses to il-4 and il-13 in regulation of chemokines such as eotaxin-1 in other cell types . however , our results here indicate that the regulation of il-33 appears to be independent of il-4/il-13 and stat6 signaling in mouse lung epithelial cells . il-33 is expressed as a full length protein ( migrates at approximately 35  kd in immunoblots ) which can be cleaved to yield smaller isoforms . extracellular full length il-33 is bioactive at its receptor complex as are mature forms cleaved by elastase and cathepsin g which yield smaller and more active mature il-33 . il-33 can also be processed during apoptosis by caspase 3 and 8 to yield cleaved il-33 ; however , such isoforms are not biologically active . immunoblots for il-33 enabled us to simultaneously assess isoforms of il-33 present as well as signaling molecules in cell / tissue extracts . the data indicate that , in cell lysates of c10 cells , and in whole lung homogenates of mice treated in vivo , full length il-33 is the major form elevated due to osm . we could not detect il-33 species in lung homogenates or c10 cell samples by western blot which corresponded to the size of recombinant bioactive mature 18 - 19  kd form of il-33 ( supplemental figure  2 ) . upon prolonged exposure of detection by immunoblots , very minor bands lower than 35  kd were observed in total lung extracts ( supplemental figure  2 ) ; however , it is not clear if these are cleaved forms of il-33 or nonspecific protein species . such bands were not evident in cell culture lysates , and clearly the full length form is the major il-33 species induced in this system . we could not detect increases in soluble il-33 in bronchoalveolar lavage ( bal ) of the adosm- or addel70-treated balb / c mice or in supernatants of c10 type 2 alveolar epithelial cells as measured by commercially available elisa ( not shown ) . thus , in this system using balb / c mice in vivo and epithelial cells derived from balb / c mice in vitro , mature il-33 was not detectable . has shown that overexpression of mature il-33 using adenovirus vectors in c57bl/6 mouse lung mediates st2-dependent eosinophil accumulation , goblet cell increases , and higher il-4 , il-5 , and il-13 levels , whereas ad vector encoding full length il-33 did not . our previous results show that balb / c mice treated with adosm do not show increased levels of eosinophil accumulation or th2 cytokines ( il-4 and il-13 ) in bal , consistent with a lack of soluble full length or mature active il-33 presence and/or activity in balb / c mice in our system . on the other hand , we and others show that osm induces th2 cytokine skewing and pulmonary eosinophilia in c57bl/6 mice [ 29 , 31 ] . it is possible that such effects in c57bl/6 mice are mediated in part by elevated soluble il-33 in the c57bl/6 mouse strain , but this needs further study . in addition to functions upon release and action at the st2/il-1rap receptor complex , il-33 has been shown to have roles as a nuclear factor that can bind chromatin and regulate nf - kb function [ 11 , 12 ] . the increase of nuclear il-33 upon osm stimulation may thus contribute to gene regulation , and future work using il-33 deficient cells ( either gene knockout or sirna knockdown ) could assess this function . our results also lead us to suggest that osm presence in combination with necrotic cell death / damage will lead to increased release of extracellular il-33 . others have shown that influenza virus infection of mouse lung results in increased levels of soluble il-33 . have recently shown that infection of mouse epithelial tc1 cells with influenza a or rsv induces release of immunodetectable il-33 . in chronic lung conditions , where osm is elevated ( such as ipf and severe asthma ) [ 28 , 29 ] , il-33 is also upregulated [ 17 , 22 ] . we postulate that elevated osm enhances the cellular pool of il-33 , which in combination with epithelial cell damage / death results in increased release of soluble il-33 and its subsequent actions as an alarmin . collectively , our results suggest an osm - il-33 axis exists which engages stromal cells and contributes to innate and inflammatory mechanisms of disease . the enhancement of innate immune functions that release il-33 may also influence ongoing and/or subsequent adaptive immune responses .
il-33 modulates both innate and adaptive immune responses at tissue sites including lung and may play critical roles in inflammatory lung disease . although il-33 expression can be altered upon nf - kappa b activation , here we examine regulation by oncostatin m , a gp130 cytokine family member , in mouse lung tissue . 
 responses were assessed in balb / c mouse lung at day 7 of transient overexpression using endotracheally administered adenovirus encoding osm ( adosm ) or empty vector ( addel70 ) . 
 whole lung extracts showed induction of il-33 mrna ( > 20-fold ) and protein ( 10-fold increase in immunoblots ) by adosm relative to addel70 . 
 immunohistochemistry for il-33 indicated a marked induction of nuclear staining in alveolar epithelial cells in vivo . 
 oncostatin m stimulated il-33 mrna and il-33 full length protein in c10 mouse type 2 alveolar epithelial cells in culture in time - dependent and dose - dependent fashion , whereas il-6 , lif , il-31 , il-4 , or il-13 did not , and tgf repressed il-33 . 
 il-33 induction was associated with activation of stat3 , and pharmacological inhibition of stat3 ameliorated il-33 levels . 
 these results indicate oncostatin m as a potent inducer of il-33 in mouse lung epithelial cells and suggest that an osm / il-33 axis may participate in innate immunity and inflammatory conditions in lung .