aspirin - exacerbated respiratory disease ( aerd ) is a syndrome in which chronic asthma is accompanied by nonallergic hypersensitivity to nonsteroidal anti - inflammatory drugs ( nsaids ) , leading to acute bronchoconstriction and exacerbation of other lower and upper airway symptoms [ 1 , 2 ] . the ability of classical nsaids to inhibit prostaglandin synthesis by cyclooxygenase ( cox ) isozymes , particularly cox-1 , is implicated in these acute exacerbations , but it is not known how they activate mast cells , eosinophils , macrophages , epithelium , and other cells to release a range of inflammatory mediators in susceptible subjects .  
prominent among these mediators are the cysteinyl - leukotrienes ( cys - lts ) , which are potent bronchoconstrictor lipids synthesised by the 5-lipoxygenase ( 5-lo)/ltc4 synthase pathway .
compared to nsaid - tolerant asthmatics , aerd patients have chronically elevated production of cys - lts , as demonstrated in bronchoalveolar lavage ( bal ) fluid , induced sputum , exhaled breath condensate , and urine .
together with the clinical efficacy of antileukotriene drugs , this suggests a key role of cys - lts in chronic aerd , even when nsaids are entirely avoided .
exposure to nsaids is postulated to shunt the shared substrate arachidonic acid from the prostaglandin ( cyclooxygenase ) pathway to the leukotriene ( 5-lipoxygenase ) pathway , or to reduce production of a prostaglandin , putatively pge2 , that normally suppresses leukotriene synthesis via an ep receptor mechanism , possibly via phosphorylation of ltc4 synthase . increased cys - lt production is prominent in the acute bronchoconstriction that results , but it is not understood why nsaids trigger the acute surge in cys - lt levels only in aerd subjects . in 1998 , we described with our collaborators a marked overexpression of ltc4 synthase , the terminal enzyme for the cellular biosynthesis of the first of the cys - lts , ltc4 , in the bronchial mucosa of aerd patients .
ltc4 synthase overexpression was also described in nasal polyps from aspirin - sensitive rhinitic patients . a model for nsaid sensitivity
was proposed in which enhanced expression of ltc4 synthase in airway macrophages , mast cells , and eosinophils provides the enzymatic capacity for constitutive overproduction of cys - lts in aerd [ 7 , 9 ] .
indeed , total numbers of ltc4 synthase - positive cells in the bronchial mucosa correlate strikingly with increased cys - lt levels in bal fluid in aerd subjects .
overexpression of ltc4 synthase may also explain why only aerd subjects respond adversely to nsaid challenge , as suggested by the unique relationship between bronchial ltc4 synthase and airway hyperresponsiveness to aspirin challenge .  
the view that ltc4 synthase overexpression is a central anomaly in aerd is supported by recent work in ltc4 synthase transgenic mice , although the potential mechanisms involved remain unclear .
ltc4 synthase can be upregulated by cytokines , including interleukin ( il)-3 and il-5 in maturing eosinophils   and il-4 and il-13 in human cord - blood mast cells , while tnf downregulates ltc4 synthase in monocytes .
immunoexpression of il-5 , but not il-3 or gmcsf , is increased in aerd biopsies relative to aspirin - tolerant subjects , but there are no studies of il-13 in aerd lung .
macrophages from resected human lung may represent a useful cellular model for aerd as they are primary cells that express both the 5-lo / ltc4 synthase and cox biosynthetic pathway enzymes , and also receptors for il-13 that mediate increased expression of the cyslt1 receptor .
we hypothesised that culture of human lung macrophages with il-13 would increase ltc4 synthase gene transcription and cys - lt synthesis compared to control macrophages .
we also hypothesised that the nsaid , indomethacin , would trigger a further release of cys - lts only in the il-13-treated cells .
taqman universal master mix , -actin and ltc4s gene expression assays , and microamp optical adhesive film were purchased from applied biosystems ( warrington , uk ) .
the improm - ii reverse transcription system was purchased from promega ( southampton , uk ) .
the cysteinyl - leukotriene enzyme immunoassay ( eia ) kit was from cayman chemical europe ( tallinn , estonia ) .
calcium ionophore calcimycin ( a23187 ) , indomethacin , trypan blue solution ( 0.4% ) , and dimethylsulphoxide ( dmso ) were from sigma chemical company ( poole , uk ) .
samples of lung tissue ( wet weight 1.835.2  g ) were collected from male and female patients undergoing bullectomy or lobectomy for lung cancer at the southampton general hospital , in accordance with ethical approval ( 08/ho502/32 ) from the southampton and south - west hampshire research ethics committee .
tissue samples were dissected into 3  mm fragments and suspended in dulbecco 's phosphate - buffered saline ( pbs ) containing 0.1  m nacl , 2.7  mm kcl , 1.8  mm kh2po4 , and 10  mm na2po4 ( ph 7.4 ) . the suspension was centrifuged ( 80  g , 20c ) for 5  min , the supernatant was discarded , and the pellet was resuspended in 30  ml of lysis buffer ( 155  mm nh4cl , 10  mm khco3 , 0.1  mm edta ; ph 7.4 ) .
after 5  min at room temperature , the suspension was filtered ( 70  m filter ) and centrifuged ( 80  g , 20c , 5  min ) to remove erythrocyte fragments and other debris .
the pellet was resuspended in rpmi medium supplemented with antibiotics ( penicillin 55  u / ml , streptomycin 5  mg / ml , and gentamycin 10  mg / ml ) .
the resulting cell population was > 90% pure macrophages by morphology and had viability > 90% as determined by exclusion of trypan blue dye .
macrophage aliquots were cultured in rpmi ( 0.5  10  cells / ml ) at 37c for 16  h overnight with or without il-13 ( 10  ng / ml ) in a 5% co2 humidified atmosphere .   for cys - lt assays , il-13-treated and untreated cells were incubated for a further 30  min in fresh rpmi medium ( 0.5  10 cells in 1  ml ) in a 37c water bath with no addition ( control ) , with indomethacin ( 100  m ) , with calcimycin ( a23187 , 1  m ) , or with both indomethacin and a23187 at the same concentrations .
calcimycin ( a23187 ) is a calcium ionophore that liberates arachidonic acid from membrane phospholipids and activates 5-lo to initiate leukotriene synthesis .
indomethacin is a classical nsaid which inhibits both cox-1 and cox-2 at the concentration used .
these reagents were diluted from stock solutions in dimethylsulfoxide ( dmso ) such that final dmso concentration in the cell incubations was always < 0.2% . at the end of the incubation , tubes were removed onto ice and centrifuged ( 240  g , 4c ) for 5 minutes to pellet the cells for rna analyses .
the supernatants were treated with two volumes of ethanol to precipitate protein , which was removed by centrifugation .
ethanolic supernatants were then evaporated to dryness in vacuo in a gyrovap rotary evaporator and stored at 20c before cys - lt immunoassays .   for ltc4s gene transcription assays , aliquots ( 10  10 cells ) of il-13-treated and untreated cell pellets were mixed with 0.5  ml rnalater and kept at 4c for 24  h , then stored at 20c before rna extraction and reverse - transcriptase quantitative polymerase chain reaction ( rt - qpcr ) assays for ltc4s and beta - actin mrnas .
pbs ( 1  ml ) was added , and eppendorfs were vortexed and centrifuged ( 2800  g , 20c ) for 5  min to decrease the viscosity of the rnalater and allow the macrophages to form a pellet .
rnalater was then removed and the rna extracted using trizol ( invitrogen , paisley , uk ) by the manufacturer 's protocol . residual genomic dna was digested using ambion dna - free ( applied biosystems , warrington , uk ) . for each rna sample ,
hemel hempstead , uk ) was > 1.8 , indicating an adequate level of purity , and rna was stored at 80c .
to generate cdna , 1  g of rna was reverse transcribed ( improm - ii rt system , promega , southampton , uk ) using random hexamer primers and stored at 20c .  
rt - qpcr was performed on the lightcycler 480 ( roche diagnostics , uk ) in 384-well reaction plates using ~100  ng of template cdna in quadruplicate 20  l reactions using taqman gene expression assays for ltc4s ( hs00168529_m1 ) with actb ( beta - actin , hs99999903_m1 ) as a reference gene , both using fam - labelled hydrolysis probes and taqman universal mastermix ii for 40 cycles , as in the manufacturer 's protocol ( applied biosystems ) .
gene expression relative to actb was calculated using the 2 method . paired statistical comparison between il-13-treated and untreated cells from n
= 12 donors was performed by wilcoxon signed rank test for nonparametric data . evaporated supernatants from 30 min incubations of il-13-treated and untreated macrophages with and without indomethacin and calcimycin
were resuspended in appropriate volumes of pbs buffer and aliquots taken in duplicate for eia quantification of released cys - lts .
the total cys - lt eia kits ( cayman europe ) use a monoclonal primary antibody with 100% specificity for ltc4 and ltd4 and 79% specificity for lte4 .
cross - reactivity to ltb4 , various hetes , and arachidonate is less than 4% and the assay has high sensitivity ( 34  pg / ml ) for cys - lts .
the assay is based on competition with a standard ltc4-acetylcholinesterace tracer with ellman 's reagent as substrate .
cys - lts were assayed in duplicate , and concentrations are expressed as nanograms of ltc4 released per million viable macrophages .
data are presented as mean  sem for n = 8 tissue donors , and comparisons between mean values were made by two - tailed paired student 's t - tests , with p < 0.05 considered significant .
for ltc4s gene transcription assays , human lung macrophages from 12 donors ( 9 male , three female ; mean age 64 years , range 4978 years ) were cultured overnight for 16 hours with or without il-13 ( 10  ng / ml ) .
ltc4s mrna expression in each cell sample was detected in quadruplicate using rt - qpcr , with -actin as the housekeeping gene .
the mean expression of ltc4s mrna at 16 hours was not significantly different in il-13-treated macrophages compared to their untreated cells ( mean log  cq value = 0.49 , p = 0.33 , n = 12 ) ( figure 1 ) .
the comparison remained nonsignificant when the single outlying value , caused by an anomalously low control ( -actin ) value in the untreated cells from one donor , which was not apparent in the il-13-treated cells from the same donor , was excluded from the analysis .  
following 16-hour cultures with or without il-13 , macrophages from representative donors ( 8 males , mean age 67 years , range 5676 ) underwent 30-minute incubations for detection of total cys - lt release ( figure 2 ) .
il-13 pretreatment significantly increased spontaneous cys - lt release from 544  215  pg / million cells to 825  292  pg / million cells ( p = 0.02 ) , a mean increase of 52  17% .
incubation with indomethacin ( 100  m ) did not significantly change cys - lt release when compared with spontaneous release in either il-13-treated or untreated cells ( p > 0.05 ) .  
as expected , the calcium ionophore a23187 ( calcimycin , 1um ) boosted the mean release of total cys - lts by about 10-fold compared with spontaneous release .
mean release of total cys - lts after 30  min of a23187 stimulation was significantly greater in il-13-treated cells ( 7770  630  pg / million cells ) than in cells not treated with il-13 ( 5480  670  pg / million cells ) ( p = 0.005 ) , a mean increase of 42  10% ( figure 2 ) .
coincubation of indomethacin ( 100  m ) and a23187 ( 1  m ) however did not show different values for cys - lt release compared with a23187 alone in either il-13 pretreated or untreated cells .
interleukin ( il)-13 is a th2 cytokine with well - established roles in promoting airway responsiveness , mucus secretion , and chemokine production in the allergic lung , acting principally via a receptor shared with il-4 and leading to phosphorylation of the transcription factor stat6 .
il-4 can powerfully upregulate ltc4 synthase expression and activity in human cord - blood mast cells , while il-13 can upregulate the principal receptor for cysteinyl - leukotrienes , cyslt1r , on human airway smooth muscle cells   and on macrophages .
this suggested that increased il-4/il-13 activity may be responsible for upregulating tissue expression of ltc4 synthase , as observed in the upper and lower airways in aerd patients [ 7 , 8 ] , and for enhancing responsiveness to cys - lts by increasing the expression of cyslt1 receptors , as described in aerd nasal biopsies .  
this study therefore explored whether il-13 can upregulate ltc4 synthase transcriptional expression in macrophages isolated from resected human lung and whether it leads to a higher release of cys - lts , either spontaneously or in response to a calcium ionophore , compared with macrophages not cultured with il-13 .
taqman gene expression assays failed to show significant changes in ltc4s mrna , standardised to the beta - actin housekeeping gene ( actb ) , in il-13 cultures of macrophages from twelve lung tissue donors ( figure 1 ) .
increases in ltc4s mrna are detectable after six hours of il-4 treatment in human mast cells derived from cord blood mononuclear cells , and maximal at 24 to 120 hours , suggesting that the 16-hour culture with il-13 employed in our experiments was reasonable
. il-13 may be less potent on myeloid cells than il-4 , and our cells were mature lung macrophages from mainly elderly subjects undergoing lobectomy or bullectomy ; these cells may not be as responsive to il-13 or other stimuli as cord - blood - derived mononuclear cells from healthy neonates .   despite the lack of effect on ltc4s transcription , culture of lung macrophages with il-13
did cause significant increases in cys - lt release , an effect that was apparent both on the low levels of spontaneous cys - lt production in unstimulated cells and on the tenfold higher levels of cys - lt release measured in cells stimulated with calcimycin a23187 ( figure 2 ) .
calcimycin acts as a receptor - independent trigger of arachidonate release from membrane phospholipids ; a high turnover of substrate through the 5-lo / ltc4 synthase pathway was intended to simulate rate - limiting conditions , such that an increase in ltc4 synthase enzyme expression induced by il-13 could be revealed by a raised ceiling of cys - lt synthesis . in the event ,
the lack of upregulation of ltc4s mrna suggests either a nontranscriptional effect of il-13 or an action on other components of the pathway , possibly on 5-lo activating protein ( flap ) , which is inducible by cytokines and increases markedly during human alveolar macrophage maturation .
the increases in cys - lt release seen in the il-13-cultured macrophages were relatively modest ( 4252% ) , but suggest that il-13 could contribute to increased cys - lt releasability in macrophages in the allergic asthmatic or aerd lung .  
nsaids such as indomethacin are proposed to cause acute aerd reactions by shunting of arachidonate from the inhibited cox pathway to the 5-lo / ltc4 synthase pathway or by suppressing synthesis of an inhibitory prostanoid such as pge2 , thus enhancing cys - lt production . although isolated anomalies in cox isozyme expression , prostanoid synthesis , and ep receptor signalling have been described in aerd cells and tissues [ 19 , 20 ] , a coherent picture of a systemic prostanoid defect in aerd has yet to emerge .
we postulated that endogenous cox pathways in human lung macrophages may therefore provide an adequate target for nsaid action , resulting in enhanced cys - lt release if the 5-lo / ltc4 synthase pathway has previously been induced by il-13 .
no effect of indomethacin was observed however on either spontaneous or a23187-stimulated cys - lt release in either il-13-treated or untreated cells ( figure 2 ) . in the absence of an effect of il-13 on ltc4s mrna levels ,
it is not possible to reject our hypothesis that ltc4 synthase overexpression in a single cell type could provide a simple cellular model of the key functional changes within the aerd lung . in vivo
, intercellular interactions may be required , possibly with pge2 derived from airway epithelial cells   or it may depend on the recruitment of new populations of ltc4-synthase - expressing cells , such as eosinophils . it is nevertheless intriguing that human mast cells derived in culture from the blood mononuclear cells of aerd subjects show an intrinsically raised capacity for cys - lt synthesis and that this is suppressed by pge2 , suggesting that the aerd paradigm can be detected at the level of a single cell type and that it persists in prolonged cell culture .
further comparative studies are required in primary lung cell populations from normal and aerd subjects .
the key features of aerd have recently been replicated in ovalbumin - sensitised ltc4 synthase transgenic mice ( ltc4s - tg ) , including its overexpression in airway macrophages and other leukocytes , leading to increased cys - lt synthesis both before and after nsaid challenge , accompanied by dramatically increased th2 cytokines , including il-13 .
this model suggests that primary dysregulation of ltc4 synthase in resident lung cells including macrophages may initiate overproduction of cys - lts in patients with aerd .
the cys - lts may then promote the secondary synthesis of il-4 , il-5 , and il-13 from lymphocytes   and eosinophils , leading to myocyte cyslt1r expression , suppression of epithelial pge2 synthesis and ep2 expression , and further induction of ltc4 synthase . while the ltc4s 444a / c promoter polymorphism   has been discounted as an aetiological factor in most aerd populations , other genetic , immunological , and microbial factors that could directly dysregulate ltc4 synthase in human lung cells merit further investigation .
 aspirin - exacerbated respiratory disease ( aerd ) is associated with constitutively elevated synthesis of bronchoconstrictor cysteinyl - leukotrienes , associated with increased expression of leukotriene ( lt)c4 synthase and th2 cytokines and airway eosinophilia . 
 we examined whether interleukin-13 can increase ltc4 synthase gene transcription and cysteinyl - leukotriene synthesis in macrophages isolated from resected human lung tissue and whether an nsaid ( indomethacin ) can trigger further cysteinyl - leukotriene synthesis in these cells . 
 overnight culture of human lung macrophages with il-13 ( 10  ng / ml ) increased spontaneous and ionophore - stimulated production of cysteinyl - leukotrienes by 42% ( p = 0.02 ) and 52% ( p = 0.005 ) , respectively , as quantified by enzyme immunoassays , but pcr gene transcription assays did not demonstrate an effect on ltc4s mrna . 
 the addition of indomethacin ( 100  m ) did not modulate cysteinyl - leukotriene production in either il-13-treated or untreated macrophages . 
 we conclude that while il-13 enhances cysteinyl - leukotriene synthesis in human lung macrophages , it does not replicate the enhanced ltc4 synthase expression observed in the aerd lung nor confer sensitivity to nsaids . 
1. Introduction
2. Methods
3. Results
4. Discussion