the mucosal surfaces lining the respiratory tract are continuously exposed to potentially infectious agents , as well as environmental toxics and antigens .
the bronchial epithelium acts as a physicochemical barrier of the airway mucosa and is able to initiate inflammatory responses by releasing mediators upon exposure to stimuli such as cigarette smoke- or pathogen - derived molecules .
one first - line mechanism of the airway immune defense against inhaled antigens and microorganisms consists of secretory immunoglobulin a ( siga ) , the predominant immunoglobulin isotype in mucosal secretions [ 1 , 2 ] . in the lamina propria , mucosal plasma cells produce iga molecules mainly as polymers , linked through a joining chain .
polymeric iga ( piga ) then binds to the polymeric ig receptor ( pigr ) , a transmembrane glycoprotein selectively expressed on the basolateral surface of epithelial cells .
the piga - pigr complexes are transported across epithelial cells up to the apical membrane where a proteolytic cleavage releases the main part of the extracellular domain of pigr , known as secretory component ( sc ) , remaining bound to piga to form siga   which assumes protective , antiinfectious functions within the mucosal lumen .
chronic obstructive pulmonary disease ( copd ) represents the fourth leading cause of death worldwide and results in large consumption of health care resources .
this disorder characterized by progressive and mostly irreversible limitation of expiratory airflow   is mainly caused by cigarette smoking , whereas only some ( 1520% ) smokers will develop the disease . among features of copd
are epithelial changes ( mucous hyperplasia ) and chronic inflammation with recruitment / activation of leukocytes including neutrophils , macrophages , and cd8 t cells . however , the links between airway neutrophilic inflammation and impairment of epithelial functions remain poorly understood in copd .
we previously showed that expression of pigr / sc was strongly decreased in the bronchial epithelium from patients with severe copd , and that this epithelial defect correlated with airflow limitation and with infiltration of submucosal glands by neutrophils .  
neutrophils can be involved in various lung disorders notably through the release of oxidants , proteinases ( including serine proteinases ) and defensins , which have important effects on several epithelial functions such as mucus secretion , cytokine release , or receptor expression .
more specifically , we next showed that activated neutrophils can induce in vitro a cleavage of epithelial pigr / sc through the release of serine proteinases ( particularly neutrophil elastase ) , whereas a stimulatory effect on pigr / sc production by cultured bronchial epithelial cells was also observed .
however , the mechanisms regulating this opposite effect of neutrophils on pigr / sc expression in airway epithelial cells remain unclear , in particular factors responsible for increased pigr / sc synthesis upon stimulation by neutrophils are not identified .
the aim of the present work was therefore to evaluate the molecular determinants of the  dual effect  of neutrophils on pigr / sc expression in human bronchial epithelial cells .
more particularly , the involvement of soluble mediators in the epithelial pigr / sc upregulation by neutrophils was investigated , as well as the role of proteinase / antiproteinase and redox balances and of intracellular mapkinase signaling pathways .

 calu-3 , a human ( adenocarcinoma ) bronchial epithelial cell line , was obtained from the atcc ( rockville , md ) , and cultured in rpmi-1640 supplemented with 10% decomplemented fetal bovine serum , 100  iu / ml penicillin , 100  g / ml streptomycin and 2  mm l - glutamine ( cambrex , rockland , me ) and referred to as  complete medium  .
96-well plates were precoated for 2  hrs at 37c , with 3.1  g / ml vitrogen ( cohesion , palo alto , ca ) , 10 g / ml fibronectin , and 50  g / ml bovine serum albumin in rpmi and followed by two washes with rpmi . to obtain a confluent bronchial epithelial layer ,
50,000 calu-3 cells were seeded in this 96-well plate in 200  l of complete medium and cultured for 57 days until confluence . 

polymorphonuclear neutrophils were obtained from whole heparinized blood from healthy donors by a one - step density gradient method ( 40  min centrifugation , 800    g , room temperature ) using polymorphprep ( axis shield , oslo , norway ) following manufacturer 's protocol .
after washing , neutrophils were resuspended in rpmi-1640 and activated by 20 ng / ml il-8 ( r&d system , minneapolis , mn ) for 5 min followed by 1  nm formyl - methionyl - leucyl - phenylalanine ( fmlp ) ( sigma aldrich , saint louis , mo ) for 30  min at 37c . in all experiments , il-8/fmlp activated neutrophils were represented by  activated neutrophils  . 

activated neutrophils alone were incubated ( from 1.25 to 5  10 cells ) in complete medium for 48  hrs to determine the production of cytokines including tgf-1 in supernatants .  

confluent calu-3 bronchial epithelial cells were cultured for 48  hrs , with or without tgf-1 ( 20  ng / ml , r&d system ) . to evaluate the dose response to tgf-1
, cells were incubated   with increasing concentration from 0.2 to 80  ng / ml .
after culture , pigr / sc production was measured in supernatants and cell lysates . 

response effect of neutrophils on sc production by bronchial epithelial cells , confluent monolayers of calu-3 cells were incubated for 48  hrs with increasing numbers of activated neutrophils ( from 0.3 to 15  10 cells ) in the presence or not of 2.5  m human secretory leukocyte protease inhibitor ( slpi , r&d system ) .  
kinetics ( from 2 to 72  hrs ) of tgf-1 production by calu-3 epithelial monolayers alone , activated neutrophils ( 5  10 cells ) alone or bronchial epithelial cells cocultured with neutrophils was also analyzed .
similarly , kinetics ( from 1 to 48  hrs ) of sc secretion by calu-3 epithelial layers was measured after incubation with tgf--1 ( 20  ng / ml ) or activated neutrophils ( 5  10 cells ) . at the end of each culture , the required volume of supernatant was taken to measure elastase enzymatic activity , and 2  mm phenylmethylsulfonyl fluoride ( pmsf , sigma aldrich ) was then added to inhibit further serine proteinase activity .
epithelial cells were washed twice in pbs and lysed with 100  l triton ( sigma aldrich ) 0.1% w / v in pbs for 15  min on ice .
cell viability and activity of mitochondrial electron transport chain , as indicator for cytotoxicity , was determined by the capacity of cells to reduce mtt ( 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ) to formazan .
confluent calu-3 epithelial cells were incubated with increasing number of neutrophils ( from 0.3 to 15  10 ) , with increasing concentrations of tgf- ( from 0.2 to 80  ng / ml ) , with cse ( from 0.1% to 10% ) and with mapkinases inhibitors ( pd , sp , and sb ) for 48  hrs .
100  l of pbs containing 1  mg / ml mtt was added to each well and the plate was returned to 37c .
to solubilize intracellular formazan produced by active mitochondria in living cells , 100  l of dimethylsulfoxide ( dmso ) was added per well .
the absorbance at 550  nm was measured on a spectrophotometric plate reader and used for the calculation of relative cell viability where untreated cells represented 100% viability . 

sc concentration was determined in culture supernatants and cell lysates from epithelial cells by sandwich elisa , as previously reported , by using affinity purified goat antihuman sc polyclonal antibody ( developed in our laboratory , recognizing both soluble sc and membrane pigr / sc ) as capture and detection ( biotinylated ) antibody .
the reaction was revealed by streptavidine - hrp followed by tetramethylbenzidine ( tmb ) in the presence of hydrogen peroxide .
sensitivity of the immunoassay was ~0.2  ng / ml .  
 tgf- production was measured in supernatants from calu-3 cells , neutrophils or calu-3/neutrophils cocultures by elisa .
a step of sample extraction ( acidification ) was carried out to allow for the release of tgf-1 from latent complexes , making it accessible for measurement in the immunoassay .
a monoclonal antibody specific for tgf-1 had been coated onto the wells and extracted samples and standards were put into these wells followed by addition of biotinylated secondary antibody ( tgf- immunoassay kit , biosource , nivelles , belgium ) .
n - methoxysuccinyl - ala - ala - pro - val - p - nitroanilide ( sigma chemical ) was used as specific substrate , diluted at 1  mm in 0.1  m hepes buffer , ph 7.5 , with 0.5  m nacl and 10% v / v dimethylsulfoxide .
supernatants ( 50  l ) were incubated with 100  l of substrate solution at 37c in 96-well plates , and od increase was monitored at 415  nm using a microplate reader ( biorad ) .
results were compared with standard curves obtained with serial dilutions of purified neutrophil elastase ( elastin products co. , owensville , ma ) , and expressed in nmole / l .
confluent bronchial epithelial layers were incubated for 48  hrs with tgf-1 ( 20  ng / ml ) or with pmn ( 5  10 ) in the presence of the antioxidants n - acetylcystein or glutathione ( sigma ) at final concentration of 10  mm . the production of sc was measured in supernatants .
after 30  min of stimulation by tgf- of a confluent monolayer in a 12-well plate , calu-3 cells were lyzed in 150  l of ripa buffer [ 16  mm tris base , 150  mm nacl , 6  mm edta , 10  g nonidet ( np-40 ) , 12  mm sodium deoxycholate , 3  mm sds , and ph 7.4 ] with 10  g / ml of protease cocktail inhibitor ( roche ) , 1  mm pmsf , 1  mm naf and 1  mm na3vo4 .
40  l of protein lysate are diluted in 40  l of laemmli 's sample buffer , heated at 100c for 5  min , loaded in a 12% sds - page gel and run at 180  v for 1  h. cell proteins were transferred onto a nitrocellulose membrane ( hybond - c , amersham biosciences , uk ) at 70  ma for 1.5  h at room temperature .
the membrane was blocked with 5% bsa in tris - buffered saline with 0.1% tween 20 ( tbst ) for 1  h at room temperature , washed , and incubated with the primary ab diluted 1/1000 in tbst - bsa 1% ( anti - phospho erk , anti - phospho p38 or anti - phospho jnk ; cell signalling technology , beverly , ma ) overnight at 4c .
membranes were then incubated for 1  h at room temperature with a secondary ab diluted 1/2000 in tbst - bsa 1% ( anti - rabbit igg / hrp , cell signalling technology ) .
immunoreactive bands were developed using chemiluminescence ( ecl , amersham biosciences , uk ) and detected with the chemidoc xrs apparatus ( bio - rad laboratories , hercules , ca ) .
calu-3 epithelial cells were pretreated for 1  hr with 50  m pd98059 , 50  m sb203580 or 50  m sp600125 as selective inhibitors of extracellular signal - regulated kinase-1/2 ( erk ) mitogen - activated protein kinase ( mapk ) , p38 mapk , and c - jun - n - terminal kinase ( jnk ) mapk , respectively ( new england biolabs , beverly , ma ) .
calu-3 cells were then incubated for 48  hrs with tgf-1 ( 20  ng / ml ) or neutrophils ( 5  10 cells ) , and production of sc in supernatants was determined by elisa .
the differences between the groups were analyzed as appropriate by means of the mann whitney test , using graphpad prism 4.0 ( graphpad prism , san diego , ca ) .
to set up a model to examine effects of neutrophils on sc production by bronchial epithelial cells , confluent epithelial layers of calu-3 cells were incubated for 48  hrs with increasing numbers of activated neutrophils ( figure 1(a ) ) , with evaluation of cytotoxicity .
 10 did not significantly affect epithelial cell viability according to mtt assay ( viability  80% compared to control ) .
however cytotoxicity was observed for 10  10 neutrophils ( cell viability decreased by 20% ) .  
sc production increased when epithelial cells were incubated with neutrophils , starting at 1.25  10 and with a maximal increase at 2.5  10 neutrophils ( p < .01 , figure 1(a ) ) . at further increased neutrophil numbers ( > 5  10 ) ,
sc production was downregulated and tended to decrease at 15  10 neutrophils as compared to control ( p = .08 , figure 1(a ) ) , consistent with cytotoxicity ( mtt assay ) and/or proteolytic cleavage .
elastase activity evaluated in supernatants from the same calu-3/neutrophils cocultures increased as expected with the number of neutrophils ( figure 1(b ) ) .
these results indicated that this system allowed to address the two opposite effects of neutrophils on epithelial sc , and experiments were carried out to explore the mechanisms of sc upregulation by  nontoxic  numbers of neutrophils releasing elastase in a concentration range that could be observed in chronic airway neutrophilic diseases , for example , during exacerbations . to evaluate the role of serine proteinases on the regulation of sc production by neutrophils ,
calu-3 cells were cultured with increasing neutrophil numbers in the presence or not of secretory leukocyte protease inhibitor ( slpi ) , a natural inhibitor of neutrophil elastase , at a concentration ( 2.5  m ) almost completely suppressing elastase activity ( > 90% inhibition ) .
sc production was measured in both epithelial supernatants and cell lysates ( figure 2 ) . in the presence of exogenous slpi ,
sc upregulation observed in epithelial supernatants at intermediate neutrophil numbers ( p < .05 at 2.5  10 neutrophils ) was further increased .
in addition , the presence of slpi promoted an increase in pigr / sc in epithelial lysates ( p < .05 at 0.6  10 neutrophils ) , while no effect was observed in this cell compartment in the absence of slpi ( figure 2 ) or with slpi alone , without neutrophils ( data not shown ) . besides the effect of proteinases / antiproteinases on sc upregulation , involvement of other neutrophil ( and/or epithelial)-derived soluble factors was assessed .
cytokines known to induce pigr transcription such as tnf- or il-1 were not detected in coculture supernatants ( data not shown ) .  
in contrast , tgf- , which was detected in resting calu-3 cell supernatants at a similar level as in complete ( serum - supplemented ) medium , was increased upon neutrophil coculture ( 3-fold increase at 5  10 neutrophils , p < .05 ; figure 3(a ) ) .   in order to determine the respective role of epithelial cells and neutrophils in tgf- production , a time course of tgf- production by epithelial cells alone , activated neutrophils alone or epithelial cell
/ neutrophil cocultures was carried out ( figure 3(b ) ) . a trend for tgf- increase
was observed in neutrophil and coculture supernatants as early as after 2  hrs , and further increased at later time points ( p < .05 at 48 and 72  hrs , figure 3(b ) ) in cocultures .
in contrast , epithelial cells began to secrete tgf- between 4872  hrs , with a 3-fold increase as compared to 2  hrs ( p < .05 ) .
at each time point , tgf- in neutrophil supernatants was higher than the epithelial production , except at 72  hrs .
these results indicated that neutrophils represented the main source of tgf- up to 48  hrs of coculture , with a modest ( and late ) contribution from epithelial cells .
tgf- had no significant cytotoxic effect on epithelial cell viability up to 80  ng / ml , with even increased mtt reduction at 80  ng / ml ( not shown ) .
addition of recombinant human tgf-1 significantly increased pigr / sc production , in cell lysates ( p < .05 ; figure 4(a ) ) , and a trend was also observed for sc secretion in supernatants . in dose - response experiments ,
sc production upon tgf- stimulation increased from 1  ng / ml to reach a plateau ~20  ng / ml ( figure 4(b ) ) .
the kinetics of sc production by bronchial epithelial cells incubated with tgf- or neutrophils , the main source of tgf- , was assessed ( figure 4(c ) ) . from 1  hr to 14  hrs of incubation , no difference was observed between the three conditions of stimulation , constitutive sc production starting to significantly increase from 24  hrs .
effects of tgf- and neutrophils had similar kinetics on sc production , with upregulation observed at 48  hrs ( figure 4(c ) ) .
we examined whether the redox balance could affect tgf-- and neutrophil - induced sc upregulation .
calu-3 cells were cultured with tgf- or neutrophils in the presence or not of two antioxidants , n - acetylcysteine and glutathione . in the presence of these antioxidants , effects of neutrophils
n - acetylcysteine and glutathione had no effect on tgf- production , whereas they did slightly reduce elastase activity presumably by reducing the ph ( data not shown ) .
the link between p38 mapk activation and redox balance in epithelial cells cocultured with activated neutrophils was assessed .
activation of p38 and erk was assayed in calu-3 cells cocultured with neutrophils in the presence or not of n - acetylcysteine ( figure 5(b ) ) . upon neutrophil coculture erk
was activated at 60  min , while p38 was activated as soon as after 5  min and also peaked at 60  min . in the presence of the antioxidant ,
p38 mapk activation was inhibited , whereas that of erk was not affected or even promoted .
to analyse the intracellular mechanisms involved in sc upregulation and engaged in bronchial epithelial cells activated by tgf- or neutrophils , activation of erk , p38 , and jnk mapkinase was assessed , as well as effects of their selective inhibition . for each inhibitor ,
specificity and absence of significant cytotoxicity was confirmed by western blot and mtt assay , respectively ( data not shown ) .
activation of erk and p38 mapk was confirmed in calu-3 stimulated by tgf- , with a maximal effect at 5  min both for erk and p38 ( figure 6(a ) ) .
the pharmacologic inhibitors did not significantly affect baseline sc secretion ( figure 6(b ) ) . while no significant effects were observed with erk or jnk pathway inhibitors ( pd98059 and sp600125 , resp . ) , the p38 mapk inhibitor ( sb203580 ) suppressed neutrophil- and tgf--driven sc upregulation ( p < .05 , figure 6(b ) ) .
epithelial pigr / sc is the critical receptor for the transcellular routing of iga involved in frontline defense mechanisms at mucosal surfaces , including the airways .
we previously observed that the bronchial epithelium of severe copd displays a strongly reduced pigr / sc expression , which correlated with airway obstruction and neutrophil infiltration .
our previous in vitro study showed that neutrophils could induce on one hand pigr / sc cleavage and on the other hand upregulation of sc production in epithelial cells , suggesting a  dual effect  of neutrophils on pigr / sc .
this study shows that , in an in vitro coculture system set up to address the mechanisms of pigr / sc dual regulation by neutrophils , increased epithelial sc production was observed at intermediate numbers of neutrophils ( corresponding to 10  nm elastase released ) , while further increasing neutrophils downregulated sc , presumably due to proteolytic cleavage ( > 20  nm elastase ; ) and/or cytotoxic effects . in addition ,
sc upregulation was favoured in the presence of slpi , a natural elastase inhibitor , and associated with tgf- production by neutrophils .
moreover , we show for the first time that tgf- is able to stimulate epithelial pigr / sc production , in a dose - dependent manner .
we also demonstrate that tgf-- and neutrophil - driven sc upregulation depends on the redox balance ( cigarette smoke extracts and antioxidants both suppressing the effect ) and that p38 map kinase represents a downstream , intracellular signalling pathway targeted by tgf- to induce pigr / sc production upon stimulation by neutrophils .
neutrophils are able to secrete a large range of soluble factors including proteinases and oxidants , as well as cytokines / chemokines and growth factors , involved in inflammatory responses and tissue remodelling . among these factors produced by neutrophils ,
our data indicate that elastase and tgf- represent the major candidates to underlie , respectively , the down- and upregulation of pigr / sc in epithelial cells exposed to activated neutrophils .
a robust production of tgf- was detected in our coculture supernatants , mainly arising from neutrophils .
in addition , a dose - dependent increase in sc expression was observed upon exogenous tgf- stimulation .
tgf- produced in cocultures ranged from 0.5 to 1  ng / ml ( figure 3 ) , a concentration close to levels effectively upregulating sc production ( figure 4(b ) ) .
in addition , maximal sc production was reached at 48  hrs of stimulation , both for tgf- and neutrophils . moreover
, functional experiments pointed to common molecular pathways ( redox system , p38 mapk ) , further supporting that neutrophils could induce pigr / sc expression mainly by producing tgf-.   while elastase is the major serine proteinase released by neutrophils involved in lung tissue breakdown due to cigarette smoking ( know as  emphysema  ) , tgf- represents a multifunctional growth factor .
it modulates cell proliferation and differentiation and induces synthesis of extracellular matrix proteins [ 10 , 11 ] and is thereby involved in the development of tissue fibrosis .
tgf- has also been shown as chemotactic for neutrophils , while exerting prominent immunosuppressive functions .
in addition , it has a unique role in mucosal tissues in promoting class switch recombination of mucosal plasma cells to iga , a critical ig for prevention of microbial infection and for control of the commensal flora [ 13 , 14 ] . here
, we show that tgf- not only triggers iga synthesis but also promotes its transport across the epithelium , further supporting the view of this cytokine as a critical factor in mucosal homeostasis .
bronchial epithelial cells can also produce tgf- either constitutively or upon stimulation such as by cigarette smoke [ 15 , 16 ] , whereas ifn- inhibits epithelial tgf- release whilst promoting pigr / sc expression .
intriguingly , it was shown that tgf- expression is increased in small airway epithelium from smokers and patients with copd [ 17 , 18 ] , and was suggested as a factor downregulating slpi expression in these patients . a reduction in slpi secretion
can also be induced by elastase , as recently reported . of note , no production of slpi ( or elafin )
thus , it remains to investigate whether relationships between tgf- signalling and epithelial functions  including production of pigr / sc and slpi  are affected in copd . in healthy subjects
, a balance between oxidants and antioxidants keeps the extracellular environment in a reduced state .
cigarette smoke , which contains massive amounts of oxidants , is the most important risk factor for the development of copd .
our results showed that glutathione or n - acetylcysteine , a glutathione precursor , inhibited the epithelial sc production induced by tgf- or by neutrophils .
tgf- is produced in an inactive form , complexed to the latency - associated peptide ( lap ) , and this complex needs to be dissociated to release active tgf-. reactive oxygen species ( ros ) are able to oxidize lap and to release active tgf- , this effect being prevented by oxidant scavenging .
the main cellular sources of ros in the lung not only include activated phagocytes such as neutrophils but also structural cells such as endothelial cells , alveolar and bronchial epithelial cells [ 23 , 24 ] .
accordingly , it is plausible that in our study gsh and nac prevented tgf- activation by neutrophil ( or epithelial cell)derived oxidants , thereby inhibiting tgf-induced sc production .
in addition , antioxidants could also affect epithelial intracellular signalling , including p38 mapk activation [ 25 , 26 ] .
similarly , the natural antiproteinase slpi might modulate tgf-- and neutrophil - driven epithelial cell activation , such as nfb pathway .
however , our data support that slpi further promotes sc upregulation upon neutrophil coculture not by affecting epithelial cell signalling but rather by inhibiting elastase activity .  
intracellular kinases play a crucial role in the control of activation of ( anti)inflammatory genes in the airways , in t - cell function and tissue remodelling .
map kinases control in airway epithelial cells the transcription of several genes involved in copd pathogenesis such as mucins and inflammatory mediators ( e.g. , tnf- , il-8 , and matrix metalloproteinases ) .
neutrophil- and tgf--driven sc upregulation in epithelial cells was almost completely abolished by the inhibitor sb203580 , indicating the requirement for p38 mapk activation , in contrast to erk and jnk pathways . in human bronchial epithelial cells ,
exposure to oxidants ( peroxide ) activates mapk cascades , particularly stress responsive p38 mapk [ 25 , our data ] .
thus , oxidative stress , caused by inflammatory cells and/or by cigarette smoke derivatives may upregulate epithelial pigr / sc production by activating p38 mapk
. increased numbers of p38 positive cells were previously observed in alveolar walls of patients with copd , as compared to smokers and nonsmoking controls . moreover , p38 inhibitors are effective in several animal models of inflammatory disorders , including copd .
for instance , the inhibitor sb2439063 reduced neutrophilia and expression of inflammatory mediators in a rat model of copd . however , it has been observed that cytokine - induced synthesis of elafin , another natural epithelial - derived neutrophil elastase inhibitor , is also dependent on p38 mapk [ 32 , 33 ] .
thus , activation of p38 mapk pathway might be critical in the airways to both inflammatory responses and transactivation of protective epithelial genes , including local anti - proteinases and pigr / sc .   in conclusion
, this paper demonstrates that activated neutrophils trigger increased epithelial pigr / sc expression through redox- and p38 mapk - dependent mechanisms .
it also highlights a novel mucosal function for tgf- , upregulating not only iga synthesis but also its receptor - mediated transepithelial transport , and further suggests that the redox balance plays an important role in modulating epithelial responses to neutrophils .
thus , this paper provides a molecular basis for the dual effect of neutrophils on mucosal / secretory immunity which consists of on the one hand on proteolytic cleavage of pigr / sc ( and iga ) by elastase and proteinase-3 , and on the other hand on tgf--mediated activation of pigr / sc synthesis via oxidant- and p38-dependent pathways .
these findings are relevant to airway cell biology and neutrophilic airway diseases , suggesting that imbalance between the two identified opposite regulatory mechanisms of pigr / sc may lead to downregulation of secretory iga immunity , as observed in smokers who developed severe copd .
the cellular and molecular determinants of this ( im)balance and fine tuning of pigr expression will be further investigated in primary cultures of airway epithelial cells from copd patients .
 neutrophils have a dual affect on epithelial pigr / sc , the critical receptor for transcellular routing of mucosal iga , but mechanisms of pigr / sc upregulation remain elusive 
 . 
 requirements of cytokine , redox , and signalling pathways for pigr / sc production were assessed in human bronchial epithelial ( calu-3 ) cells cocultured with increasing numbers of blood neutrophils . 
 
 increased sc production was observed after incubation for 48  hrs with intermediate neutrophil numbers ( 1.25 to 2.5  106 ) , was favoured by the elastase inhibitor slpi , and correlated with increased tgf- production . 
 exogenous tgf- stimulated sc production with a maximal effect at 48  hrs and both tgf-- and neutrophil - driven sc upregulation were dependent on redox balance and p38 map - kinase activation . 
 
 this paper shows that activated neutrophils could upregulate epithelial pigr / sc production through tgf--mediated activation of a redox - sensitive and p38 mapk - dependent pathway . 
 an imbalance between the two neutrophil - driven opposite mechanisms ( sc upregulation and sc degradation ) could lead to downregulation of pigr / sc , as observed in severe copd . 