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Alveolar macrophage-epithelial cell interaction following exposure to atmospheric particles induces the… Ishii, Hiroshi; Hayashi, Shizu; Hogg, James C; Fujii, Takeshi; Goto, Yukinobu; Sakamoto, Noriho; Mukae, Hiroshi; Vincent, Renaud; van Eeden, Stephan F Aug 1, 2005

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ralssBioMed CentRespiratory ResearchOpen AcceResearchAlveolar macrophage-epithelial cell interaction following exposure to atmospheric particles induces the release of mediators involved in monocyte mobilization and recruitmentHiroshi Ishii1,2, Shizu Hayashi1, James C Hogg1, Takeshi Fujii2, Yukinobu Goto1, Noriho Sakamoto1, Hiroshi Mukae2, Renaud Vincent†3 and Stephan F van Eeden*1Address: 1James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, St. Paul's Hospital, University of British Columbia, 1081 Burrard Street, Vancouver, BC, V6Z 1Y6, Canada, 2Second Department of Internal medicine, Nagasaki University School of Medicine, Nagasaki, Japan and 3Environmental Health Directorate, Health Canada, Ottawa, Ontario, CanadaEmail: Hiroshi Ishii - hishii2@mac.com; Shizu Hayashi - SHayashi@mrl.ubc.ca; James C Hogg - JHogg@mrl.ubc.ca; Takeshi Fujii - tmks@ims.u-tokyo.ac.jp; Yukinobu Goto - ygoto@mail2.accsnet.ne.jp; Noriho Sakamoto - NSakamoto@mrl.ubc.ca; Hiroshi Mukae - hmukae@net.nagasaki-u.ac.jp; Renaud Vincent - Renaud_vincent@hc-sc.gc.ca; Stephan F van Eeden* - SVaneeden@mrl.ubc.ca* Corresponding author    †Equal contributorsAbstractBackground: Studies from our laboratory have shown that human alveolar macrophages (AM) andbronchial epithelial cells (HBEC) exposed to ambient particles (PM10) in vitro increase their production ofinflammatory mediators and that supernatants from PM10-exposed cells shorten the transit time ofmonocytes through the bone marrow and promote their release into the circulation.Methods: The present study concerns co-culture of AM and HBEC exposed to PM10 (EHC-93) and theproduction of mediators involved in monocyte kinetics measured at both the mRNA and protein levels.The experiments were also designed to determine the role of the adhesive interaction between these cellsvia the intercellular adhesion molecule (ICAM)-1 in the production of these mediators.Results: AM/HBEC co-cultures exposed to 100 µg/ml of PM10 for 2 or 24 h increased their levels ofgranulocyte-macrophage colony-stimulating factor (GM-CSF), M-CSF, macrophage inflammatory protein(MIP)-1β, monocyte chemotactic protein (MCP)-1, interleukin (IL)-6 and ICAM-1 mRNA, compared toexposed AM or HBEC mono-cultures, or control non-exposed co-cultures. The levels of GM-CSF, M-CSF,MIP-1β and IL-6 increased in co-cultured supernatants collected after 24 h exposure compared to controlcells (p < 0.05). There was synergy between AM and HBEC in the production of GM-CSF, MIP-1β and IL-6. But neither pretreatment of HBEC with blocking antibodies against ICAM-1 nor cross-linking of ICAM-1 on HBEC blocked the PM10-induced increase in co-culture mRNA expression.Conclusion: We conclude that an ICAM-1 independent interaction between AM and HBEC, lung cellsthat process inhaled particles, increases the production and release of mediators that enhance bonemarrow turnover of monocytes and their recruitment into tissues. We speculate that this interactionamplifies PM10-induced lung inflammation and contributes to both the pulmonary and systemic morbidityassociated with exposure to air pollution.Published: 01 August 2005Respiratory Research 2005, 6:87 doi:10.1186/1465-9921-6-87Received: 04 January 2005Accepted: 01 August 2005This article is available from: http://respiratory-research.com/content/6/1/87© 2005 Ishii et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 12(page number not for citation purposes)Respiratory Research 2005, 6:87 http://respiratory-research.com/content/6/1/87BackgroundExposure to ambient particulate matter with a diameter ofless than 10 µm (PM10) is strongly associated withincreased morbidity and mortality, particularly in subjectswith pre-existing pulmonary and cardiovascular diseases[1,2]. This increase in mortality induced by PM10 exposurewas present even when adjusted for the other major riskfactors such as cigarette smoking [1]. A recent report [3]has shown that environmentally relevant concentrationsof PM2.5 induced airway inflammation even in healthysubjects with a selective influx of monocytes.Although the biological mechanisms are still unclear,PM10 are known to stimulate the production of reactiveoxygen species and inflammatory mediators by alveolarmacrophages (AM) [4-7] and epithelial [7-10] and otherlung cells [11]. When AM and airway epithelial cells aredirectly exposed to inhaled atmospheric particles thesesmall particles are phagocytized by both cells [10,12].Both cell types can synthesize a variety of pro-inflamma-tory cytokines that induce airway inflammation and con-tribute to the airway lesions in asthma and chronicobstructive pulmonary diseases [9]. In vitro, AM and lungepithelial cells interact in response to PM10 and this inter-action has been implicated in amplifying their mediatorproduction [7,13]. Studies from our laboratory haveshown that the PM10(EHC-93)-induced interaction ofhuman AM and bronchial epithelial cells (HBEC)enhances the synthesis and release of a variety of pro-inflammatory cytokines and that supernatants from theseco-cultures instilled into rabbit lungs induces a systemicinflammatory response [13].We recently showed that deposition of PM10 (EHC-93 andinert carbon particles) in the lung shortened the transittime of monocytes through the bone marrow andenhanced their release into the circulation [14,15]. Fur-thermore, we also showed that monocytes are the pre-dominant inflammatory cells that accumulate in thealveoli following repeated PM10 exposure [16]. Thepresent study was designed to determine whether, and ifso, which interactions between AM and HBEC (AM/HBECco-cultures) amplify the response to PM10 exposure, espe-cially the synthesis of inflammatory mediators thatenhance bone marrow turnover of monocytes and theirrecruitment into the lung. We used primary cultures ofHBEC and human AM freshly isolated from lobectomy orpneumonectomy specimens and measured the expressionof inflammatory mediators relevant to monocyte kinetics.We further evaluated the potential role of the intercellularadhesion molecule (ICAM)-1 in the production of medi-ators by AM/HBEC co-cultures exposed to PM10.MethodsUrban air particles (PM10)PM10 particles were collected in an urban environment(EHC-93) and obtained from the Environmental HealthDirectorate, Health Canada, Ottawa, Ontario. A detailedanalysis of the EHC-93 has been presented elsewhere [17].Particles were suspended at a concentration of 1 mg/ml inhydrocortisone-free supplemented bronchial epithelialcell growth medium (BEGM; Clonetics, San Diego, CA)and sonicated 3 times for 1 min each at maximal poweron a Vibra Cell VC-50 sonicator (Sonics and MaterialsInc., Danbury, CT) prior to adding to the cells. The endo-toxin content of the PM10 suspension of 100 µg/ml was6.4 ± 1.8 EU/ml or less than 3.0 ng/ml [10,13]. This doseof LPS has been shown not to activate either AM or lungepithelial cells to produce cytokines [10].Isolation of HBEC and human AMBronchial tissue and broncho-alveolar lavage (BAL) fluidwas obtained from a total of ten patients who underwentlobectomy or pneumonectomy for small peripheral nod-ules at St. Paul's Hospital, Vancouver. Informed consentwas obtained from all subjects and these studies wereapproved by the Human Ethics Committee of the Univer-sity of British Columbia. All subjects were current smokersand were asked to abstain from smoking for 6 weeks priorto the operation. Their mean age was 67.2 yr (range 56–74 yr) (6 women and 4 men). Primary HBEC were iso-lated from bronchial tissues according to a previouslydescribed procedure [10]. In brief, pieces of excisedhuman bronchial tissue approximately 1 cm long wereincubated at 4°C for 24 h with 0.1% protease (Type14;Sigma) solution prepared in BEGM containing Fungizone(1 µg/ml; GIBCO BRL, Gaithersburg, MD). The epithelialcells were harvested, washed with BEGM with added anti-biotics(100 U/ml of penicillin and 100 µ/gml of strepto-mycin; Sigma)and Fungizone, and cultured in a 25-cm2cell culture flask until 80 to 90% confluent. Then the cellswere trypsinized and placed in 100-mm cell culture dishesand cultured in BEGM. Light microscopy showed that95% of the isolated cells had features of bronchial epithe-lial cells, that is they formed a monolayer of ciliated cells.Also, by trypan blue exclusion, >95% of these cells wereviable. Human AM were harvested from BAL fluidobtained from lung segments or lobes that were free of thetumor using a method previously described in detail[7,13]. The BAL fluid cells were >90% viable (trypan blueexclusion method) and consisted of 90–95% AM (asassessed by Wrights-Giemsa stain) and less than 2% neu-trophils. AM mono-cultures and AM/HBEC co-cultureswere suspended in BEGM. BEGM used throughout thisstudy was without hydrocortisone.Page 2 of 12(page number not for citation purposes)Respiratory Research 2005, 6:87 http://respiratory-research.com/content/6/1/87Exposure of cells to PM10Primary HBEC from the third or fourth passage of cellsfrom each patient were cultured to 90–100 % confluencein 100-mm cell culture dishes (approximately 2.5–3.0 ×106 cells/dish) then exposed for 2 and 24 h to fresh stocksuspensions of 100 µg/ml PM10 (EHC-93) prepared inBEGM.AM (1.0 × 107) from each patient were placed in 100-mmcell culture dishes and allowed to adhere to the plasticdish for 30 min in humidified incubator (5% CO2 at37°C). The non-adherent cells less than 1.0 × 106) werethen removed by rinsing twice with BEGM and adherentAM (>98% AM) were incubated in 10 ml of BEGM with orwithout 100 µg/ml of PM10 for 2 and 24 h.In co-culture experiments, freshly prepared AM (5.0 ×106) were directly placed on the confluent HBEC monol-ayers which were grown in 100-mm cell culture dishes.The AM were allowed to adhere to HBEC and the non-adherent cells were removed by washing twice withBEGM. The AM/HBEC co-cultured cells were incubated in10 ml of BEGM with or without 100 µg/ml of PM10 for 2and 24 h. Cell viability was determined following the 24h PM10 exposure in all experiments using the trypan blueexclusion method.RNase protection assay (RPA)After 2 or 24 h treatment, total RNA was isolated from thecells using a single-step phenol/chloroform extractionprocedure (Trizol, Life Technologies, Inc., Grand Island,NY). The levels of inflammatory mediator mRNA weredetermined using the RiboQuant™ multi-probe system(PharMingen, San Diego, CA) following the instructionsof the supplier. Two customized template sets were usedthat included mRNAs of the following inflammatorymediators: human regulated on activation, normal T-cellsexpressed and secreted (RANTES), macrophage inflamma-tory protein (MIP)-1β, granulocyte-macrophage colony-stimulating factor (GM-CSF), M-CSF, monocyte chemo-tactic protein (MCP)-1, interleukin (IL)-6 and leukemiainhibitory factor (LIF). Human ICAM-1 mRNA was deter-mined using a separate template set. Internal controlsincluded mRNAs of the ribosomal protein L32 and glycer-aldehyde-3-phosphate dehydrogenase (GAPDH). In brief,10 µg of total cellular RNA was hybridized overnight tothe [α-32P] UTP-labeled riboprobes which had been syn-thesized from the supplied template sets. Single-strandedRNA and free probe remaining after hybridization weredigested by a mixture of RNase A and T1. The protectedRNA was then phenolized, precipitated, and analyzed ona 5% denaturing polyacrylamide gel. Following electro-phoresis, the gel was dried under vacuum and subjected to1.63 software (National Institutes of Health, Bethesda,MD). Results were normalized to the expression of theinternal control, GAPDH. For the densitometric analysiseach RPA was repeated four to six times.ELISA measurementsCell culture supernatants were collected 24 h after addi-tion of 100 µg/ml of PM10 suspension, centrifuged, fil-tered through a syringe filter with pore size of 0.22 µm(Corning, Cambridge, MA) to eliminate as much as possi-ble any remaining particles and stored at -80°C until use.MIP-1β, GM-CSF, M-CSF, MCP-1 and IL-6 levels weremeasured by the Cytokine Core Laboratory (Baltimore,MD) using an ELISA based on a biotin-strepavidin-perox-idase detection system as previously described [10]. Allmeasurements were done in triplicate and values cor-rected for the number of AM used in each experiment arereported as the means of five experiments.ImmunocytochemistryTo demonstrate cell surface ICAM-1 (CD54) expressionon HBEC and CD11b on AM, cells were placed or grownon coverslips in 6-well plates and incubated for 2 or 24 hwith 100 µg/ml of PM10. Cells were fixed with 1% parafor-maldehyde for 10 min and immunocytochemistry wasperformed by the alkaline phosphatase anti-alkalinephosphatase method using mouse anti-human CD54monoclonal antibody (Immunotech, Marseille, France)and mouse anti-human CD11b monoclonal antibody(DAKO, Copenhagen, Denmark) to identify cell surfaceexpression of ICAM-1 and CD11b.Cell adhesion blockers and ICAM-1 cross-linkingIn experiments testing whether anti-CD54 and anti-CD11b block mediator production by co-cultured AM/HBEC, HBEC and AM were preincubated for 1 h beforePM10 exposure with control IgG F(ab')2 fragments (2 µg/ml; Jackson ImmunoResearch Laboratories, PA), mouseanti-human monoclonal CD54 F(ab')2 fragments, and/ormonoclonal CD11b F(ab')2 fragments (1 µg/ml, respec-tively; Caltag Laboratories, CA). Cells were then co-cul-tured and exposed to PM10 for 24 h in the presence of theblocking antibodies before analysis by RPA. To determinewhether ligand binding to CD54 on HBEC in of itself con-tributes to the enhanced mediator response of these cellsto PM10 stimulation cross-linking antibodies to CD54were used to simulate this possibility. We used previouslyreported methods of cross-linking CD54 which resultedin intracellular signaling [18,19]. After 2 h of exposure toPM10, HBEC were incubated for 1 h with 1 µg/ml mouseanti-human CD54 or 1 µg/ml control mouse non-specificIgG (DAKO). Cells were washed and then incubated for 4h with 10 µg/ml rabbit anti-mouse IgG (DAKO) to cross-Page 3 of 12(page number not for citation purposes)autoradiography. The quantity of protected labeled RNAwas determined using densitometry and the NIH imagelink the bound anti-CD54 and mRNA mediator expres-sion was assessed as above.Respiratory Research 2005, 6:87 http://respiratory-research.com/content/6/1/87Statistical AnalysisData are expressed as mean values ± SE. The minimumnumber of replicates for all measurement was at leastthree. For RPA and ELISA, differences between matchedpairs (control versus PM10 treated) were compared by Wil-coxon signed ranked test. To compare mediator produc-tion by co-cultures to that by AM plus HBEC mono-cultures, we used the Mann-Whitney U test. Differencesbetween multiple groups were compared by one-wayanalysis of variance (ANOVA). The post hoc test for mul-tiple comparisons was the Dunnett's test. Significance wasassumed at p < 0.05.ResultsAM/HBEC co-cultures and PM10We previously showed that the majority of AM and HBECwere in contact with each other in our co-culture system[13]. Both cells internalized PM10 particles with manycells containing more than one particle. The 100 µg/mlconcentration of PM10 used throughout this experimentwas not toxic to either AM or HBEC and >90% of cellswere viable after 24 h exposure as assessed by the trypanblue exclusion method.Expression of mRNA induced by PM10Representative autoradiographs of mRNA expression byAM or HBEC mono-cultures and AM/HBEC co-culturesafter 2 and 24 h incubation in medium alone (control) ora 100 µg/ml of PM10 suspension (PM10) are shown in Fig-ure 1. Because the RPA kit is not provided with an internalcontrol to account for variation between autoradiographsof different pairs of control versus PM10 treated cells suchas those shown in Figure 1 and due to that fact that cellsfrom a single but different patient are represented in eachdifferent pair, as documented by the differences in inten-sity of the control L32 band(s) compared to that of thecorresponding GAPDH bands as well as differences in theL32 banding pattern (Fig. 1), expected large variations indensitometric data between corresponding pairs of auto-radiographs were found. Despite these variations the com-piled densitometric analyses of these autoradiographsyielded statistically significant results. However, becauseof the unavoidable variations, the compiled densitometricresults for a few mediators differed from that depicted inthe representative autoradiographs. In Figure 1 mRNAexpression of the inflammatory mediators of interest,RANTES, MIP-1β, GM-CSF, M-CSF, MCP-1, IL-6 and LIF,was not altered after 2 h of PM10 exposure of neither AMnor HBEC mono-cultures and this result was confirmedafter densitometric analysis (n = 4, data not shown). Onlythe expression of ICAM-1 mRNA by HBEC at this time-point appears to be marginally increased (Fig. 1) but afterdensitometric analysis this change was not found to beco-cultured AM/HBEC increased MIP-1β, GM-CSF, M-CSF, IL-6, LIF and ICAM-1 mRNA expression (Fig. 1) and,of these, densitometric analysis of six such RPA experi-ments confirmed that increases in MIP-1β, GM-CSF, IL-6,and ICAM-1 were significant (Fig. 2A), as well as that ofMCP-1 (Fig. 2A) which was not detected in the represent-ative autoradiograph (Fig. 1).After 24 h exposure to PM10 the representative autoradio-graphs showed that increases in mRNA expression by AMmono-cultures were restricted to that of LIF and ICAM-1(Fig. 1) but this was not confirmed after statistical analysisof the densitometric results (n = 4, data not shown). Incontrast, the increases in GM-CSF, LIF and ICAM-1 byHBEC mono-cultures (Fig. 1) were found to be statisti-cally significant (p < 0.05 and n = 4, respectively)(Fig. 2B).Co-cultures exposed to PM10 at this time-point showedstrong increases in MIP-1β, GM-CSF, M-CSF, IL-6 andICAM-1 mRNA and minor increases in those of MCP-1and LIF (Fig. 1). Except for IL-6, the strong increases wereconfirmed by the densitometric analysis of six RPA exper-iments (Fig. 2A).Mediator production induced by PM10Figure 3 shows the GM-CSF, IL-6, MIP-1β, MCP-1 and M-CSF protein levels in supernatants of AM/HBEC co-cul-tures, AM mono-cultures and HBEC mono-cultures incu-bated for 24 h with medium alone (control) or with 100µg/ml of PM10. GM-CSF, IL-6, MIP-1β and M-CSF produc-tion by AM/HBEC co-cultures stimulated by PM10 weresignificantly increased compared to control levels. GM-CSF and IL-6 production by AM mono-cultures stimu-lated with PM10 suspension increased significantly com-pared to controls. MIP-1β production by HBEC mono-culture stimulated by PM10 were significantly increasedover control levels.The GM-CSF, IL-6 and MIP-1β produced by AM/HBEC co-cultures in response to PM10 stimulation were more thanthe sum of the respective mediator produced by PM10exposed AM and HBEC mono-cultures alone suggesting asynergistic effect in production of these cytokines (p <0.05). This synergistic effect was not seen in the produc-tion of M-CSF. MCP-1 production was not significantlyincreased by PM10 in either mono-cultures or AM/HBECco-cultures but its expression by AM tended to bedecreased by co-culturing (Fig. 3).Expression of ICAM-1 induced by PM10Figure 4 shows immunocytochemically stained CD54 onHBEC (to identify ICAM-1) and CD11b on AM. In theabsence of PM10, HBEC express low levels of ICAM-1 ontheir cell surface (Fig. 4A). After stimulation with 100 µg/Page 4 of 12(page number not for citation purposes)significant (n = 4, data not shown). In contrast to theresults from the mono-cultures, PM10 exposure for 2 h ofml of PM10 for 24 h many more cells stained positively forRespiratory Research 2005, 6:87 http://respiratory-research.com/content/6/1/87RNase protection assay of mRNA expression by AM and HBECFigure 1RNase protection assay of mRNA expression by AM and HBEC. Representative autoradiographs of RNase protection assays (RPAs) showing mediator expression by AM/HBEC co-cultures, AM mono-cultures and HBEC mono-cultures after 2 and 24 h incubation in medium alone (control) or a 100 µg/ml of PM10 suspension (PM10). After 24 h exposure AM showed increased expression of LIF and ICAM-1 mRNA. Expression of GM-CSF, LIF and ICAM-1 mRNA by HBEC was increased by 24 h PM10 stimulation compared to their respective controls. MIP-1β, GM-CSF, M-CSF, MCP-1, IL-6, LIF and ICAM-1 mRNA expression by AM/HBEC co-cultures was increased 2 and/or 24 h after incubation with PM10 compared to control. L32 and Page 5 of 12(page number not for citation purposes)GAPDH were used as controls for lane loading.Respiratory Research 2005, 6:87 http://respiratory-research.com/content/6/1/87Densitometric analysis of bands on RPAsFigure 2Densitometric analysis of bands on RPAs. (A): the density of the bands representing the mediator mRNAs in AM/HBEC co-cultures on autoradiographs such as that shown in Figure 1A was compared to that of the GAPDH mRNA band in the same lane and the resulting ratio (PM10; black bars) is shown as the percentage change from control values (white bars). The mean densitometric value confirmed that the mRNA levels of MIP-1β, GM-CSF, MCP-1, IL-6 and ICAM-1 at 2 h and those of MIP-1β, GM-CSF, M-CSF and ICAM-1 after 24 h exposure were significantly higher than control values. Values are means ± SE of six experiments representing the AM/HBEC co-culture group. (B): the mean densitometric value confirmed that the mRNA levels of GM-CSF, LIF and ICAM-1 at 24 h exposure were significantly higher than control values. Values are means ± SE of four Page 6 of 12(page number not for citation purposes)experiments representing the HBEC mono-culture group. *p < 0.05 compared with control.Respiratory Research 2005, 6:87 http://respiratory-research.com/content/6/1/87Mediator protein levels in supernatants of AM and HBECFigure 3Mediator protein levels in supernatants of AM and HBEC. GM-CSF, IL-6, MIP-1β, MCP-1, and M-CSF protein levels in supernatants of AM mono-cultures, HBEC mono-cultures and AM/HBEC co-cultures incubated for 24 h with medium alone (control; white bars) or 100 µg/ml of PM10 (black bars). GM-CSF and IL-6 production by AM mono-cultures and AM/HBEC co-cultures stimulated by PM10 increased significantly compared to controls. Exposure to PM10 also increased MIP-1β production by HBEC mono-cultures and AM/HBEC co-cultures and M-CSF production by AM/HBEC co-cultures. The GM-CSF, IL-6 and MIP-1β produced by exposed AM/HBEC co-cultures significantly exceeded the sum of those produced by AM and HBEC mono-cultures exposed separately. Values are means ± SE of five experiments. * p < 0.05 compared with control. † p < 0.05 Page 7 of 12(page number not for citation purposes)for exposed AM/HBEC co-cultures compared to the sum of the exposed HBEC and AM mono-cultures.Respiratory Research 2005, 6:87 http://respiratory-research.com/content/6/1/87ICAM-1 and their intensity of staining was increased (Fig.4B). Most AM expressed surface CD11b and this expres-sion was unaffected by 2 and 24 h stimulation with PM10(Fig. 4C, D and data not shown).ICAM-1 and PM10-induced mediator production by AM/HBEC co-culturesTo determine the role of β2-integrin/ICAM-1 interactionmolecules before PM10 exposure. Representative autoradi-ographs of mRNA expression by such AM/HBEC co-cul-tures after 24 h incubation in a 100 µg/ml of PM10suspension are shown in Figure 5A. They include pretreat-ment of neither cell before co-culture, of only AM withcontrol IgG or anti-CD11b antibody, of only HBEC withcontrol IgG or anti-CD54 antibody, and of both cell typeswith both antibodies. The increased mRNA expression inSurface expression of ICAM-1 on HBEC and CD11b on AMFigur 4Surface expression of ICAM-1 on HBEC and CD11b on AM. Photomicrographs of primary cultured HBEC and human AM on coverslips. Immunocytochemistry was performed using mouse anti-human CD54 monoclonal antibody on HBEC and mouse anti-human CD11b monoclonal antibody on AM. In the absence of PM10 stimulation HBEC rarely expressed CD54 (A). After stimulation with 100 µg/ml of PM10 for 24 h the majority of cells stained positively (arrows, pink cells) for CD54 (B). Expression of surface CD11b on AM (C) was unaffected by 2 h stimulation with PM10 (D). The scale bars represent 20 µm.Page 8 of 12(page number not for citation purposes)in mediator production by AM/HBEC co-cultures, AM andHBEC were incubated with inhibitors of these adhesionPM10-stimulated AM/HBEC co-cultures was not affectedby any of the pretreatments with these antibodies. InRespiratory Research 2005, 6:87 http://respiratory-research.com/content/6/1/87AM/HBEC co-culture responses after pretreatment with cell adhesion blockersFigure 5AM/HBEC co-culture responses after pretreatment with cell adhesion blockers. (A): autoradiographs from RNase protection assay of mediator mRNA expression by AM/HBEC co-cultures pretreated before 24 h incubation in a 100 µg/ml of PM10 suspension including no pretreatment (no treatment) before co-culture, AM pretreated with control IgG (AM-IgG), HBEC with control IgG (HBEC-IgG), AM with anti-CD11b antibody (AM-CD11b), HBEC with anti-CD54 antibody (HBEC-CD54) and both cell types with respective antibodies (both antibodies). The mRNA expression in PM10-exposed AM/HBEC co-cultures was not affected by any pretreatments with these antibodies. (B): in the absence of AM, pretreatment of HBEC to cross-link CD54 with antibody followed by 2 h exposure to PM10 (PM10-CD54) did not alter mediator expression compared Page 9 of 12(page number not for citation purposes)with HBEC pretreated with control IgG (PM10-IgG) and non-pretreated HBEC (control).Respiratory Research 2005, 6:87 http://respiratory-research.com/content/6/1/87addition, as shown in Figure 5B, CD54 cross-linking itselfin the absence of AM did not induce mediator expressionin HBEC exposed to PM10.DiscussionAM and lung epithelial cells play a key role in processinginhaled particulate matter. In the present study we con-firmed that exposing co-cultures of human AM and HBECatmospheric particles to for 2 hr increased mRNA expres-sion of GM-CSF, MCP-1 and IL-6 [13]. The current addi-tion of mRNAs, that of M-CSF and MIP-1β, to this list ofthese mediators involved in the marrow production,mobilization and recruitment of monocytes that areincreased in response to PM10 exposure reinforces thehypothesis that exposure of the lung to environmentalpollutants can stimulate a systemic inflammatoryresponse [4]. Besides these bone marrow oriented media-tors, mRNA expression of ICAM-1, an adhesion moleculepotentially involved in an interaction between AM andHBEC to amplify marrow-related mediator expression,was increased. Another important hitherto unreportedfinding, that of sustained increased expression of many ofthese mediator mRNAs, MIP-1β, GM-CSF, M-CSF, andICAM-1, over 24 h of exposure, supports the robustincrease in the expression of the corresponding mediatorproteins that we observed. These included MIP-1β, M-CSF, ICAM-1 as well as the previously reported GM-CSFand IL-6 [13]. Furthermore, the synergistic increases inGM-CSF, IL-6 and MIP-1β secretion by the co-culturescompared to the sum of the mono-cultures in response toPM10 exposure indicate an interaction between these cellswith ICAM-1 possibly contributing to this interaction.IL-6, the hematopoietic growth factors GM-CSF and M-CSF, and the C-C chemokine MIP-1 are important media-tors in the production and mobilization of monocytesfrom the bone marrow [20-22]. IL-6 is considered animportant multifunctional cytokine involved in the regu-lation of a variety of cellular responses, including being apermissive factor for monocytic colony formation byhuman hematopoietic progenitor cells in combinationwith GM-CSF [23]. Monocytes recruited into the lung playa critical important role in clearing foreign material suchas particles from the lung which underscores the impor-tance of mediators such as GM-CSF as both a pro-inflam-matory but also an anti-inflammatory mediator. This anti-inflammatory role is supported by studies that showedthat GM-CSF has a protective role against pulmonaryfibrosis [24] or hyperoxic lung injury [25] in animal mod-els. Both IL-6 and GM-CSF stimulate the marrow to pro-duce and release monocytes while the acute responsecytokines, IL-1 and TNF-α, secreted in response to PM10stimulation by AM [7,13] induce the production ofsimilar to MIP-1α [22]. Because PM10 did not induce MIP-1β production in human AM [4] or its mRNA in HBEC inthe current study, increased MIP-1β expression in the co-cultures most likely relies on an interaction between thesetwo cells. The significance of such an interaction is rein-forced by our finding that the production of this chemok-ine in response to PM10, along with that of GM-CSF andIL-6, is synergistically increased, as noted above, when AMand HBEC are co-cultured. Such a synergistic increase inmediator production could augment the release of bothmonocytes and polymorphonuclear leukocytes from thebone marrow observed after stimulation by mediatorsproduced by AM incubated alone with EHC-93 ex vivo [6]and thus contribute to a similar response to in vivo expo-sure to the ambient particles [6,15].MCP-1 was the other C-C chemokine that we studied.Along with additional support from results from our lab-oratory [15], Rosseau and colleagues [30] have shownthat the induction of MCP-1 in AM is a major contributorto the recruitment of peripheral blood monocytes into thealveolar compartment. In the present study we showedthat production of MCP-1 by AM was just marginallyincreased by PM10 exposure (p = 0.07). Interestingly, theproduction and release of MCP-1 by AM/HBEC co-cul-tures tended (not significant) to be lower than by AMalone (Fig. 3). In AM/HBEC co-cultures, expression ofMCP-1 mRNA was significantly increased by PM10 after 2h but not 24 h exposure suggesting suppression of MCP-1expression following prolonged exposure of lung cells toparticles. This suggests a translational or post-transla-tional control of MCP-1 production and could be animportant immunomodulatory pathway by which thelocal inflammatory reaction in the lung is controlled afterPM10 exposure. Together, our findings suggest that bothcolony stimulating factors and chemokines are releasedfrom lung cells following the inhalation of atmosphericparticles and that these mediators are critically importantin the production and the release of monocytes from themarrow as well as their recruitment into the lung.The close proximity of AM and epithelial cells in the lungsuggests that interaction between these cells is criticallyimportant in generating inflammatory mediators inresponse to noxious stimuli. Previous studies from ourlaboratory [13] support this concept showing that AM andepithelial cells in co-culture interact to amplify their pro-inflammatory mediator mRNA generation in response toPM10 exposure compared to exposure of mono-cultures ofthese cells. That soluble factors contribute to this interac-tion was shown when conditioned media from PM10-stimulated AM induced increases in mRNA expression ofmany of these mediators in HBEC [13]. On the otherPage 10 of 12(page number not for citation purposes)monocytic chemoattractants such as MCP-1 [20,21,26-29]. MIP-1β is a chemotactic factor for human monocyteshand, that cellular contact between different lung cells(e.g., epithelial, endothelial cells, and fibroblast) isRespiratory Research 2005, 6:87 http://respiratory-research.com/content/6/1/87necessary for cell activation and cytokine production[11,31] has also been demonstrated. Along these lines, werecently showed increased expression of ICAM-1 mRNAafter incubation of HBEC with conditioned media fromPM10-stimulated AM and this response was blocked byneutralizing antibodies to TNF-α and IL-1β [7]. WhileTNF-α and IL-1β appear to be major players in the inter-action between AM and HBEC in response to PM10, ourresults suggest that ICAM-1 may play an important role infacilitating the AM-HBEC interaction via these solublefactors.We postulated that adhesive interactions between CD11/CD18 on AM with ICAM-1 on HBEC contribute to theamplified production of cytokines from AM/HBEC co-cul-tures observed in the current study. Previous studies havedemonstrated that cross-linking CD11b/CD18 on the sur-face of phagocytes using a combination of either its ligandICAM-1 or anti-ICAM-1 antibodies primes phagocytes forincreased respiratory burst and release of reactive oxygenintermediates [32-34]. In the present study we showedincreased epithelial cell surface expression of ICAM-1induced by PM10 exposure, while CD11b was constitu-tively expressed on the surface of AM. Cross-linkingICAM-1 on HBEC did not change their PM10-inducedmRNA expression. Furthermore, blocking CD11b/CD18and one of its ligands, ICAM-1 (CD54), did not block ordecrease the PM10-induced mRNA expression in AM/HBEC co-cultures. These results are consistent with thoseof Tao and co-workers [35] who demonstrated that TNF-α and MIP-2 responses to urban air particles in rat AM andRLE (rat alveolar type II epithelial cell line) co-cultureswere not blocked with anti-CD18 (β2-integrins)/CD54,arginine-glycine-aspartate peptide (against β1/β3-integrins) and heparin (non-specific anti-inflammatoryagent). Paine and colleagues [36] demonstrated thatblocking ICAM-1 (anti-CD54 F(ab')2 fragments)decreased rat AM phagocytosis of beads and their planarchemotaxis over the surface of rat alveolar type I epithelialcells. This suggests that ICAM-1 is important for the effi-cient phagocytosis of particles by AM and promotesmobility of AM on airway epithelial cell surface in thealveolus. Together these studies showed that the β2-integrin/ICAM-1 interaction between AM and lung epi-thelial cells are important in the chemotaxis of AM in thelung and their phagocytosis of inhaled particles but thatthis adhesive interaction may not contribute to the medi-ator production and release by AM and lung epithelialcells. These findings do not exclude the possibility thatother adhesive interactions or simultaneous adhesiveinteractions of more that one adhesion molecule areinvolved in the particle-induced AM-bronchial epithelialcell mediator response.ConclusionExposure of AM/HBEC co-cultures to ambient particlesincreased the expression and release of a variety of inflam-matory mediators including GM-CSF, M-CSF, IL-6 andMIP-1β that enhance bone marrow production of mono-cytes and their recruitment into the lung. In addition thistype of exposure resulted in synergistic production of GM-CSF and IL-6 in the co cultured cells. The adhesive interac-tion between ICAM-1 on epithelial cells with the β2-integrin CD11b on AM did not contribute to this synergis-tic mediator production. We speculate that the interactionbetween AM and lung epithelial cells amplifies PM10-induced lung inflammation and contributes to thepulmonary morbidity associated with exposure to partic-ulate matter air pollution. This enhanced lung inflamma-tion may also contribute to the systemic inflammatoryresponse as well as the cardiovascular morbidity and mor-tality induced by air pollution [1,2,37].Authors' contributionsHI carried out all through the experiments and drafted themanuscript. TF, YG, NS and HM participated in the designof the study. SH and JCH participated in its design andhelped to draft the manuscript. SFVE conceived of thestudy, participated in its design and coordination andhelped to draft the manuscript. RV provided EHC-93. Allauthors read and approved the final manuscript.AcknowledgementsThe authors thank Dr. W. Mark Elliott for technical support and Health Canada for providing the EHC-93. The work was supported by grants from the National Institutes of Health (HL407201), BC Lung Association and the Wolfe & Gita Churg Foundation. SF van Eeden is the recipient of a Career Investigators Award from the American Lung Association and the William Thurlbeck Distinguished Researcher Award.References1. Dockery DW, Pope CA, Xu X, Spengler JD, Ware JH, Fay ME, FerrisBG, Speizer FE: An association between air pollution and mor-tality in six U.S. cities.  N Engl J Med 1993, 329(1):753-759.2. Pope CA 3rd: Epidemiology of fine particulate air pollutionand human health: biologic mechanisms and who's at risk?Environ Health Perspect 2000, 108:713-723.3. 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