RSV dose-dependent IL-8 and IL-6 release from A549 cells. Furthermore, the .... (0 3 M sodium chloride plus 0 03 M sodium citrate) and 1% SDS at 650; (2) ... mean ng/ml ± SEM; n = 5; *P < 0 05 versus supernatants from non- infected cells.
Immunology 1994 82 126-133
Interleukin-8, interleukin-6, and soluble tumour necrosis factor receptor type I release from a human pulmonary epithelial cell line (A549) exposed to respiratory syncytial virus R. ARNOLD, B. HUMBERT,* H. WERCHAU,* H. GALLATIt & W. KONIG Department of Medical Microbiology and Immunology, AG Infektabwehr and *Medical Microbiology and Virology, Ruhr-Universitdt Bochum, Bochum, Germany, and f Hoffmann-La Roche, Pharmaceutical Research, New Technologies, Basel, Switzerland
SUMMARY The release of interleukin-8 (IL-8), interleukin-6 (IL-6) and the soluble forms of the tumour necrosis factor receptor (sTNF-R) from human pulmonary type II-like epithelial cells (A549) after respiratory syncytial virus (RSV) infection was analysed. RSV infection alone induced a time- and RSV dose-dependent IL-8 and IL-6 release from A549 cells. Furthermore, the soluble form of the TNF-RI was also secreted in a time- and RSV dose-dependent fashion. The soluble TNF-RII was not detected in the cell supernatant of infected epithelial cells. The effect of various cytokines [ILla/fl, TNF-a/#, IL-3, IL-6, interferon-y (IFN-y), transforming growth factor-#2 (TGF-,B2)] and colony-stimulating factors [granulocyte (G)-CSF; granulocyte-macrophage (GM)-CSF] on the IL-8 release from A549 cells was also studied. Our data show that the proinflammatory cytokines IL-la/, and TNF-a/fl induced an IL-8 release in non-infected A549 cells, and increased the IL-8 release of RSV-infected A549 cells synergistically. In addition, IL-3, G-CSF, IFN-y and TGF-fl2, albeit at high concentrations, induced a low IL-8 release from non-infected A549 cells. The enhanced IL-8 secretion rates were accompanied with elevated cytoplasmic IL-8 mRNA steady state levels, as was shown by Northern blot analysis. Cellular co-culture experiments performed with A549 cells and polymorphonuclear granulocytes or peripheral blood mononuclear cells revealed that increased IL-8 amounts were secreted in the co-culture of non-infected as well as RSV-infected cells. The present study suggests a central role for the airway epithelium during RSV infection with regard to cytokine and cytokine receptor release, resulting in a recruitment and activation of inflammatory and immune effector cells. Our data also suggest that paracrine cytokine networks and cell-cell contact are involved in the regulation of IL-8 secretion within the microenvironment of the bronchial epithelium.
INTRODUCTION Respiratory syncytial virus (RSV) is one of the most important respiratory tract pathogens of infants and young children.' RSV infection is associated with bronchitis, bronchiolitis and pneumonia in infants 2-6 months of age.2 Also, re-infection of
adults and children with RSV despite serological host immunity is common.3 It has been shown that RSV is not a potent inducer of interferon-y (IFN-y) production by human mononuclear leucocytes, suggesting that IFN-y plays only a minor role in human immunological defence against RSV infection.4 Quite recently, it was observed that cell supernatants from macrophages exposed to RSV contained interleukin-l (IL-l) and IL-l inhibitor activity.5 Furthermore, leukotrienes in the nasopharyngeal secretions from RSV-infected patients, and secretion of histamine-releasing factors from human peripheral blood leucocytes after RSV infection, were observed.6'7 These factors may be involved in the proinflammatory process of virus-induced bronchospasm. The airway epithelial cells are the primary target cells for RSV infection. A large growing body of evidence suggests that the epithelium is not only a physical barrier, but has the potential to synthesize a variety of cytokines, e.g. IL-8, IL-6, granulocyte-macrophage colony-stimulating factor (GMCSF) and transforming growth factor (TGF).8'9 In addition,
Received 3 August 1993; revised 14 December 1993; accepted 22 December 1993. Abbreviations: G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN, interferon; IL, interleukin; IP, infectious particles; mRNA, messenger ribonucleic acid; PBMC, peripheral blood mononuclear cells; PMN, polymorphonuclear neutrophil granulocytes; RSV, respiratory syncytial virus; SDS, sodium dodecyl sulphate; TGF, transforming growth factor; TNF, tumour necrosis factor. Correspondence: Professor Dr W. K6nig, Lehrstuhl fur Medizinische Mikrobiologie & Immunologie, Arbeitsgruppe Infektabwehrmechanismen, Ruhr-Universitait Bochum, Universitutsstrape 150, 4630 Bochum, Germany.
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Respiratory syncytial virus chemotactic active lipid mediators for proinflammatory effector cells are also released by human airway epithelial cells. 10-12 The purpose of the study was to analyse the release of the proinflammatory cytokines IL-8, IL-6 and the soluble forms of the TNF receptors (sTNF-RI, sTNF-RII) from human epithelial cells after RSV infection. We used the pulmonary type II epithelial cell line A549 for our study. In addition, the priming pattern of different cytokines, e.g. IL-la/fl, TNF-a/fl, IL-3, IL-6, IFN-y, TGF-fl2, granulocyte colony-stimulating factor (G-CSF) and GM-CSF, for IL-8 release from RSVinfected and non-infected A549 cells was analysed. Furthermore, cell-cell interaction studies were performed with RSV-infected A549 cells in co-incubation with polymorphonuclear granulocytes (PMN) as well as peripheral blood mononuclear cells (PBMC). These studies might help to evaluate pathophysiological events occuring under in vivo situations in the microenvironment of the infected airway epithelium. MATERIALS AND METHODS Materials The reagents used were from the following sources. Ficoll 400 was from Pharmacia (Uppsala, Sweden); Macrodex (6%) was from Schiwa (Glansdorf, Germany); metrizoate (75%, w/v) was from Nycomed (Oslo, Norway). dCTP (a-32P; 6000 Ci/ mmol) was purchased from New England Nuclear (NEN; Dreieich, Germany). IL-la, IL-1,B, IL-6, IFN-y, TNF-a, and TNF-P were purchased from Boehringer Mannheim (Mannheim, Germany). TGF-P2 was purchased from Genzyme (Cambridge, MA). IL-3 and GM-CSF were a generous gift from Sandoz (Niirnberg, Germany). G-SCF was kindly provided by Hoffman-La Roche (Basel, Switzerland). Recombinant human IL-8 was purchased from Calbiochem (Bad Soden, Germany). Murine anti-human monoclonal IL-8 antibodies and goat alkaline phosphatase-labelled anti-human IL-8 antibodies were a generous gift from Dr M. Ceska (Sandoz-Forschungsinstitut, Wien, Austria). The IL-8 cDNA'3 and the IL-8R type II cDNA14 inserted into a bluescript vector (pBluescript II SK) were a generous gift from Dr D. P. Cerretti (Immunex, Seattle, WA). All media and supplements were purchased from Gibco Europe Ltd (Karlsruhe, Germany). All fine chemicals were purchased from Sigma (Deisenhofen, Germany). Preparation of cells PBMC. These were prepared from peripheral venous blood of healthy donors. Platelets were separated by isolation of platelet-rich plasma. Cells were purified on a Ficoll-metrizoate density gradient centrifugation according to B6yum.'5 The cell fraction consisting of lymphocytes (88-93%), monocytes (712%) and basophilic granulocytes (1-2%) (PBMC) was washed three times with RPMI-1640 medium containing 2 mM L-glutamine, 100 ug/ml streptomycin, 100 IU/ml penicillin and 20 mm sodium hydrogen carbonate. The contaminating erythrocytes were removed by hypotonic lysis. PMN. The human PMN were separated from the erythrocytes by dextran sedimentation. The remaining erythrocytes were lysed by exposing the cells to hypotonic conditions. This method led to >98% pure PMN (1-2% eosinophils). The cell preparations were routinely analysed by
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morphological examination of Wright-stained smears and nonspecific esterase staining. Less than 1% mononuclear cells were present. A549 pulmonary epithelial cells. The cells of the human A549 pulmonary epithelial cell line (ATCC, Rockville, MD) show features of type II alveolar epithelial cells and produce surfactant.16 The cells were grown in FK12 medium, containing 10% (v/v) fetal calf serum (FCS), 100 Mg/ml streptomycin, 100 IU/ml penicillin and 20 mm sodium hydrogen carbonate. Culture conditions The PBMC, PMN and A549 cells were cultured in a water saturated atmosphere containing 5% CO2 at 37°. Only cell preparations with a viability greater than 95%, as analysed by trypan blue exclusion test, were used. The buffers and cell media were prepared using pyrogen-free water. Virus preparation For crude virus preparation, RSV, long strain (ATCC), was grown and titrated in HEp-2 cells.'7 The RSV titre was analysed in a plaque-forming unit (PFU) assay.18 The stock titre of the virus pool used in the performed study was 5 x 106 PFU/ml. The stock solution was stored at -70° until use. IL-8, IL-6, IL-lI# and TNF-a were not present in the stock solution, as analysed by commercial ELISAs. Culture medium of uninfected HEp-2 cells was used as control. The HEp-2 cell culture fluid did not induce cytokine release from A549 cells, PBMC or PMN. In order to exclude any influence from the cell supernatants of RSV-infected HEp-2 cells on the cytokine release from A549 cells, PMN or PBMC control experiments were performed with purified RSV particles. The RSV particles were purified according to Fernie & Gerin.19 Effect of RSV on A549 cells. Prior to the infection, A549 monolayers were washed with fresh medium and exposed to RSV using a multiplicity of infection (MOI) of 0 01-1. As controls served cells incubated alone with medium. After 1 hr incubation time, the cells were washed extensively with medium, resuspended in RPMI-1640 with antibiotics and incubated for up to 72 hr. During the experiments RPMI1640 medium with antibiotics and without FCS was used to prevent any further growth of the cells or alteration of the functional status of the cells. Interaction of cytokines with A549 cells. Different concentrations of recombinant human IL-la/, (1-100 U), TNF-a/f, (10-1000 U), IL-3 (0 5-50 ng), IL-6 (10-1000 U), IFN-y (10-1000 U), TGF-f2 (0-2-20 ng), G-CSF (0-1-10 ng) and GM-CSF (0- 1-10 ng), diluted in medium, or the medium alone as control, were added to the A549 monolayers in a volume of 600 Mil. After 15 min, RSV [2-5 x 105 infectious particles (IP)/ 100 pl; MOI = 0 5] or medium was added. After 6 hr, cellconditioned media were harvested and analysed for IL-8 release. Cellular co-incubation studies (A549 + PMN; A549 + PBMC). Co-incubation experiments with A549 cells and PBMC or PMN were performed in six-well tissue culture plates (Costar, Cambridge, MA) for 3 hr. Confluent monolayers of A549 cells (5 x 105 cells/well) were co-cultured with 1 x 106 PBMC or 1 x 106 PMN in a volume of 600 M1 with 1 x 104 or 1 x 105 IP of RSV. Controls of the co-culture experiments were carried out by incubation of each cell type/ cell fraction with only medium or RSV.
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Cytokine immunoassays IL-8 ELISA. Antigenic IL-8 was analysed using a double ligand method as previously described.20 This ELISA method detected IL-8 concentrations above 30 pg/ml. IL-6 and sTNF-R ELISA. IL-6, sTNF-RI and sTNF-RII were analysed by ELISA as previously described.21 The detection limit of the ELISAs was < 100 pg/ml. RNA extraction and Northern blot analysis RNA extraction. Total cellular RNA was extracted essentially by the protocol of Chomczynski & Sacchi.22 Northern blot analysis. Between 10 and 20 ,ug of total RNA was electrophoresed under denaturing conditions in the presence of formaldehyde, according to Maniatis et al.23 The RNA was blotted onto Gene-Screen Plus membranes (NEN, Dupont, Hamburg Germany) by capillary transfer. The cDNA fragments were 32P radiolabelled by random priming.24 Northern blot analysis was performed under the following stringent washing conditions: (1) twice for 20 min with 2 x SSC (0 3 M sodium chloride plus 0 03 M sodium citrate) and 1% SDS at 650; (2) twice for 20 min with 0-1 x SSC at room temperature. Standardization was performed with respect to 28 S and 18 S rRNA. 13"425 DNA loading and transfer were checked by ultraviolet examination of the gels and blots.
Analysis of data All experiments were made at least three times. The data were calculated as mean ± standard error of the mean (SEM). The significance was evaluated by the two-tailed Student's t-test for independent means; P > 0-05 was considered not significant. RESULTS IL-8 release from bronchial epithelial cells (A549) Confluent monolayers of bronchial epithelial cells were exposed to different amounts of RSV. 5 x 105 epithelial cells were infected with 5 x 103-5 x 105 (MOI = 0-01-1) IP for up to 72 hr. Figure 1 shows the IL-8 release of the A549 at several time-points after infection. As can be seen, the epithelial cells secreted IL-8 in a time- and RSV dose-dependent manner. After 24 hr following infection up to 15-7 ± 1-2 ng IL-8/ml was released from A549 cells after exposure to 5 x 105 RSV particles. The IL-8 secretion peaked at 48 hr following
116Z _-
18 . 1
c
0i
T
1O
_
5x 103
5x10'
5x
IL-8-i
28 S 18S
Figure 2. Cytoplasmic IL-8 mRNA steady state levels in human A549 epithelial cells cultured for 1 5 hr (lanes 1-3) and 3 hr (lanes 4-6). The epithelial cells were incubated with (1) medium, (2) 104 RSV, (3) i05 RSV, (4) medium, (5) 104 RSV, and (6) 105 RSV; bottom, ethidium bromide-stained RNA after blotting onto the filter.
infection and declined after 72 hr post-infection. Since the viability of the cells decreased after 48 h incubation time, the IL-8 release after 24 hr is presented. Non-infected epithelial cells showed a constitutive IL-8 secretion after 4 hr incubation time (0 55 ± 0-15 ng/ml); and after 24 hr an IL-8 amount of 4-1 ±0 6 ng/ml) was measured in the cell supernatant from non-infected cells. HEp-2 cells did not secrete IL-8 after RSV infection; therefore, titrated RSV pools grown up in HEp-2 monolayers were devoid of IL-8 and did not interfere with the cytokine measurement. The amount of IL-8 released from A549 cells was similar using either purified RSV infectious particles or RSV-titrated HEp-2 supernatant. RSV dose-dependent expression of IL-8 mRNA by A549 cells To elucidate the molecular background of IL-8 secretion from A549 cells after RSV infection, Northern blot analysis was performed. Figure 2 shows a representative IL-8 Northern blot of A549 cells. It was evident that non-infected epithelial cells possessed cytoplasmic IL-8 gene transcripts, demonstrating a constitutive IL-8 gene expression resulting in a constitutive IL-8 secretion pattern (lanes 1 and 4). The epithelial cells accumulated IL-8 mRNA in the cytoplasm after RSV infection dependent on the infection dose of virus added to the cells (lanes 2 and 3). Furthermore, the IL-8 mRNA steady state level increased with a prolonged infection time (lanes 2 and 5, 3 and 6). Therefore, the observed increased secretion rates for IL-8, which were dependent on the infection in a RSV dose- and time-dependent manner, were accompanied by cytoplasmic IL-8 mRNA accumulation.
iO5
IP
Figure 1. IL-8 release from human A549 epithelial cells (1 x 106 cells) cultured for 2 (m), 4 (o), 6 (o) and 24 (o) hr in the presence of medium or 5 x 103, 5 x 104 or 5 x 105 infectious RSV particles, respectively; mean ng/ml ± SEM; n = 5; *P < 0 05 versus supernatants from noninfected cells.
IL-6 release from bronchial epithelial cells (A549) In order to further investigate the active role of epithelial cells with regard to effector cell recruitment and activation, we analysed the release of the proinflammatory cytokines TNF-ac and IL-6. Our studies revealed that the cells of the pulmonary
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Respiratory syncytial virus
A549 monolayers. The sTNF-R amount in the titrated RSV pools grown up in HEp-2 monolayers was under the detection limit.
3
2-s5
I2 1.5
c
IL-8 release from RSV-infected and non-infected epithelial cells (A549) after priming with cytokines and colony-stimulating
05
ci5
x 103
3 5x 104
-
.-C
5x105
IP
Figure 3. IL-6 release from human A549 epithelial cells (1 x 106 cells) cultured for 2 (u), 4 (9), 6 (o) and 24 (o) hr in the presence of 5 x 103, 5 x 104 and 5 x 105 infectious RSV particles or medium, respectively; mean ng/ml + SEM; n = 3; *P < 0-05 versus supernatants from noninfected cells.
cell line A549 did not secrete TNF-a into the cell supernatant after 24 hr post-infection. In contrast, as was shown for IL-8, the epithelial cells secreted IL-6 in a time- and RSV dosedependent manner. Figure 3 shows the IL-6 release of the cells at different time-points after infection. Up to 3-3 ng IL-6/ ml x 106 cells was secreted into the cell supernatant after 24 hr post-infection. A constitutive IL-6 secretion was not observed. The amounts of IL-6 in the titrated RSV pools which were derived from HEp-2 cells ranged below the detection limit; therefore, a correction of the obtained IL-6 secretion rates from A549 cells was not necessary. Control experiments revealed that purified RSV particles induced the same cytokine release pattern.
sTNF-RI release from bronchial epithelial cells (A549) Since epithelial cells express TNF-a/p binding sites on their cell surfaces, we analysed the release of the soluble forms of TNF receptors, e.g. the 55,000 MW TNF receptor (sTNF-RI) and the 75,000 MW TNF receptor (sTNF-RII). We observed a low constitutive secretion of the 55,000 MW TNF receptor. The release increased after infection of the cells in a time- and RSV dose-dependent manner similar to the IL-6 and IL-8 release. Figure 4 shows the secretion pattern of the human sTNF-RI after infection with purified RSV particles. The human sTNFRII was not detected in the cell supernatants of non-infected or RSV-infected cells. The same sTNF-RI release was induced after addition of RSV-titrated HEp-2 cell supernatants to the
factors To assess the influence of cytokines and colony-stimulating factors which play a role in inflammatory and immune responses on IL-8 release from epithelial cells, A549 cells were primed for 15 min prior to the infection. The following cytokines and factors were used: IL-la/fl, TNF-a/fi, TGF-f32, IFN-y, IL-6, IL-3, G-CSF and GM-CSF. Table 1 shows the IL-8 release from A549 cells which were primed with the indicated amounts of cytokines and then incubated for 6 hr with 2 5 x 105 IP RSV (MOI = 0-5) or medium. As can be seen the proinflammatory cytokines, IL-la/f and TNF-x/fl induced the highest IL-8 release by themselves. In addition, the priming with these cytokines induced a synergistic IL-8 release ofvirus-infected cells. IL-Ia/P and TNF-a/p were also active at lower concentrations, e.g. 10 U for IL-la/f and TNF-a, and 15 ng for TNF-fl. But the synergistic effect on IL-8 release was only observed at higher cytokine concentrations. IL-6 had no influence on the IL-8 release from RSVinfected and non-infected cells. In contrast, the cytokines IFN-y and TGF-f2 induced an IL-8 secretion from non-infected cells when they were applied at high amounts, e.g. 1000 U IFN-y and 20 ng TGF-f2. RSV-infected A549 cells, primed with IFN-y or TGF-,B2, showed an additive IL-8 release. Also, the colonystimulating factor G-CSF and IL-3 stimulated the epithelial cells for an enhanced IL-8 release. Furthermore, G-CSF and IL-3 were only active at concentrations higher than 1 ng/ml. RSV-infected cells primed with IL-3 showed an additive IL-8 release. In contrast, G-CSF and GM-CSF did not prime the RSV-infected epithelial cells for an increased IL-8 secretion, and GM-CSF by itself had no stimulatory effect on A549 cells with regard to IL-8 release. Table 1. IL-8 release from A549 cellst
Control 5
c3 H
2
*
WIL z
nl
100U
IL-l1
100 U 1000 U 15 ng 20 ng 1000 U 1000 U 5 ng 10 ng 10 ng
TNF-a TNF-,B TGF-,B2 IFN-y
-4
Iii
IL-la
~ ~ ~ 1~~~~~* ----
5x103
5x 10'
50x05
IL-6 IL-3 G-CSF GM-CSF
Medium
RSV
1 1 ± 0-3 20-8 ± 4-1* 22-5 ± 8-1* 13-5 ± 4.4* 6-4 ± 0.5* 1-8 ± 0.3* 21 ± 0.3* 0 9 ± 0-1 1-9 ± 0-2* 3-1 ± 0-6* 1-1 ± 0 4
51 ± 1-1 499 ± 7.3* 63-1 ± 50* 35.4 ± 4.5* 20-3 ± 1-4* 8-6 ± 0.9* 7-6 + 03* 4 5 ± 0-4 8-1 + 0.4* 6-2 ± 09 4-9 ± 0 7
IP
Figure 4. Release of sTNF-RI from human A549 epithelial cells (1 x 106 cells) cultured for 2 (o), 4 (o), 6 (o) and 24 (o) hr in the presence of 5 x 103, 5 x 104 and 5 x 105 infectious RSV particles or medium, respectively; mean ng/ml ± SEM; n = 3; *P < 0-05 versus supernatants from non-infected cells.
tThe incubation time was 6 hr. Cytokine release was analysed by ELISA. Values are mean ± SEM for three experiments and are expressed as ng/ml and 106 epithelial cells. 5 x 105 epithelial cells were infected with 2 5 x 105 IP RSV (MOI = 0-5). *Significant difference from the controls (P < 0-05).
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IL-8 mRNA accumulation of RSV-infected and non-infected A549 cells primed with IFN-y, TGF-P2, IL-3, IL-6, G-CSF and GM-CSF In order to analyse whether the increased IL-8 secretion rates were accompanied by elevated cytoplasmic IL-8 mRNA levels, we performed an IL-8 Northern blot analysis. Figure 5 shows representative Northern blots. As can be seen, non-infected A549 cells accumulated IL-8 mRNA after priming with IFN-y, TGF-#2, IL-3 and IL-6 (lanes 7-10). Furthermore, cytokineprimed RSV-infected A549 cells expressed elevated IL-8 mRNA steady state levels (lanes 2-5) in comparison to the medium control (lane 1). Although after IL-6 priming no elevated IL-8 secretion was observed, an increased IL-8 mRNA level was measured (lane 1O). As shown in Fig. 5b, the colonystimulating factor G-CSF induced an increased cytoplasmic IL-8 mRNA level in A549 cells in comparison to the medium
IL-8
2
1
(a)
3
4
i~t
6
5
7
8
9 10
t
a
'.
28 S
IL-
receptor gene expression in epithelial cells (A549)
Since A549 cells showed a potent IL-8 secretion pattern, we analysed the gene coding for the IL-8 receptor II (IL-8R) by Northern blot analysis. No cytoplasmic IL-8R mRNA was detected with the radiolabelled cDNA probe, either in noninfected cells or in RSV-infected cells. Using the cDNA for IL8R mRNA detection in PBMC, we obtained weak but detectable hybridization signals (data not shown).
IL-8 secretion of A549 cells co-cultured with PMN or PBMC during RSV infection In order to analyse whether cell-cell interaction influenced the IL-8 release from epithelial cells (A549), PMN and PBMC, we
18 S
I
2
(b)
control (lanes and 2); and RSV-infected epithelial cells primed with this colony-stimulating factor also expressed an increased steady state level of IL-8 gene transcripts (lane 5). GM-CSF priming induced no IL-8 mRNA accumulation in A549 cells by itself (lane 3), but led to an enhanced IL-8 mRNA steady state level in RSV-infected cells in comparison to unprimed RSV-infected cells (lanes 4 and 6). The cytokines ILla/fB and TNF-a/fl, which were the most active stimuli for IL-8 secretion (Table 1), also induced a high IL-8 mRNA accumulation in A549 cells (data not shown). Thus, all investigated cytokines which triggered an IL-8 release from A549 epithelial cells led to elevated cytoplasmic IL-8 mRNA levels in these cells. The only exceptions were IL-6 and GMCSF which triggered an IL-8 mRNA accumulation in noninfected cells, e.g. IL-6, or RSV-infected cells, e.g. GM-CSF, but did not influence the IL-8 secretion profile.
3
4
5
6
Al
IL- 8
28 18
c
i
J
Figure 5. (a) Expression of IL-8 mRNA in A549 cells cultured for 4 hr in the absence of RSV (lanes 1, 7-10) or in the presence of 105 RSV particles (lanes 2-6). The non-infected A549 cells were cultured for 15 min with medium (lane 1) or primed for 15 min with the following cytokines: IFN-y (100 U/ml; lane 7), TGF-/B2 (20 ng/ml; lane 8), IL-3 (50 ng/ml; lane 9) or IL-6 (103 U/ml; lane 10) and then incubated for 4 hr. Also, prior to RSV infection, for 4 hr the A549 cells were cultured for 15 min with medium (lane 6) or primed with IFN-y (lane 2), TGF-#2 (lane 3), IL-3 (lane 4) or IL-6 (lane 5); bottom, ethidium bromidestained RNA after blotting onto the filter. (b) Expression of IL-8 mRNA in A549 cells cultured for 4 hr in the absence of RSV particles (lanes 1-3) or in the presence of 105 RSV particles (lanes 4-6). The noninfected A549 cells were cultured for 15 min with medium (lane 1) or primed with G-CSF (10 ng/ml; lane 2) or GM-CSF (10 ng/ml; lane 3) and then incubated for 4 hr. Prior to RSV infection for 4 hr the A549 cells were cultured for 15 min with medium (lane 4), or primed with GCSF (lane 5) or GM-CSF (lane 6). Bottom, ethidium bromide-stained RNA after blotting onto the filter.
RPMI 5x104 5x105 IP IP Control
RPMI 5x104 5x105 IP IP Co-culture
Figure 6. (a) IL-8 release from A549 (5 x 105) cells and PBMC (1 x 106) exposed to 5 x 104 and 5 x 105 infectious RSV particles or medium. (b) Sum of the IL-8 released from A549 cells and PBMC cultured with medium or RSV, e.g. controls, and the IL-8 amount secreted into the cell supernatant of the co-cultured cells in the presence of RSV or medium. The bars represent the IL-8 concentrations/ml after 3 hr of culture; mean values ± SEM. (a) *P < 0 05 versus non-infected cells; (b) *P < 0 05 versus controls.
Respiratory syncytial virus 7
become re-infected, the precise role of T-cell-mediated immunity for protection is not well understood. Quite recently, it has been shown that the human nasal epithelium and alveolar macrophages accumulate IL-8 mRNA
(a)
6 5
4
and secrete this proinflammatory cytokine after RSV infec-
3
tion.30'3' We analysed the role of human epithelial cells derived from the respiratory tract during the onset of RSV infection. Therefore, we examined the release of cytokines, e.g. IL-6, IL-8, TNF-a, and the soluble TNF receptors from the epithelial cells
2
M.l
I
0 7E [ilk RPMI 5x 10 C
F5
131
IP
5x105 IP
2
RPMI 5x104 >x M 5x105 5x IP IP A549 PMN 1be
20 15 10
(Fig. 2).
5
0
of the human cell lines A549 infected with RSV. It is evident that A549 cells secrete IL-8 in a RSV dose- and time-dependent fashion (Fig. 1). UV-inactivated RSV particles did not induce an IL-8 secretion (data not shown). Therefore, the infection process is a prerequisite for the IL-8 release from A549 cells. The increased IL-8 secretion rate was accompanied by elevated cytoplasmic IL-8 mRNA steady state levels in RSVinfected A549 cells, as verified by Northern blot analysis
RPMl 5xl104 5x105 IP IP Control
RPMI 5x 104 5x105 IP IP Co-culture
Figure 7. (a) IL-8 release from A549 (5 x 10-5) cells and PMN (1 X 106) exposed to 5 x 104 and 5 x 105 infectious RSV particles or medium. (b) Sum of the IL-8 released from A549 cells and PMN cultured with medium or RSV, e.g. controls, and the IL-8 amount secreted into the cell supernatant of the co-cultured cells in the presence of RSV or medium. The bars represent the IL-8 concentration/ml after 3 hr of culture; mean values ± SEM. (a) *P < 005 versus non-infected cells; (b) *P < 0-05 versus controls.
performed co-incubation studies in 6-well tissue culture plates. Figures 6 and 7 show the IL-8 secretion of A549 cells, PBMC and PMN after 3 hr of co-culture. All cell types and cell fractions secreted IL-8 into the cell supernatant post-RSV infection (Figs 6a and 7a). It is shown that the co-culture of A549 cells with PBMC or PMN in the absence of a viral stimulus increased the IL-8 release from these cells significantly (Figs 6b and 7b). Furthermore, RSV-infected co-cultured cells showed an increased IL-8 release compared to the controls, e.g. the sum of IL-8 released from the infected but not co-cultured cell types. In addition, co-culture experiments performed in Transwell tissue culture plates revealed that the increased IL-8 secretion pattern was also dependent on soluble factors released from these cells (data not shown). But the IL-8 release induced in the Transwell system reached only 30% of the IL-8 amount released in direct cell-cell co-culture experiments. Therefore, soluble factors as well as cellular adhesive interactions are apparently responsible for the increased IL-8 release from the co-cultured cells.
DISCUSSION Since the isolation of human RSV in 1956, few data have accumulated concerning the inflammatory response after RSV infection, and a satisfactory animal model for the pathobiology of the lower respiratory tract illness in humans is not available.26'27 It is well known from the animal infection model that T-cell-mediated immunity plays an important role in the recovery from disease.28'29 Although human adults often
IL-8 is chemotactic for T lymphocytes and PMN.32 Furthermore, PMN become activated by IL-8, e.g. expression of CDl lb/CD18, release of proteases and reactive oxygen products are inducible cellular responses.33 Therefore, during the acute RSV infection epithelial cell-derived IL-8 might be responsible for the chemotactic recruitment and activation of PMN in the alveolar space of RSV-infected patients suffering from bronchiolitis. The A549 cells were also stimulated for IL-6 secretion after RSV infection (Fig. 3). As was observed for IL-8, the IL-6 was synthesized and secreted in a time- and RSV dose-dependent manner. The multifunctional proinflammatory cytokine IL-6 stimulates T-cell proliferation and T/B-cell differentiation. 34-36 Therefore, epithelial cells may be involved in the local immune response of the mucosa-associated lymphoid tissue. The cell supernatants of RSV-infected A549 cells did not contain TNF-a, as was analysed by ELISA. However, the soluble form of the TNF-RI, unlike sTNF-RII, was secreted from A549 cells. Similar to IL-6 and IL-8, the sTNF-RI was produced in a time- and RSV dose-dependent fashion (Fig. 4). The detection of sTNF-RI is in agreement with published results.37 The 55,000 MW TNF-RI induces TNF responses such as cytotoxicity, antiviral activity, and activation of transcription factors.38-40 Therefore, the secretion of sTNFRI might interfere with these TNF-a/,B responses.4' Since epithelial cells synthesize the colony-stimulating factors G-CSF and GM-CSF, and the cytokines IL-1, IL-6 and TGF-fi2, we analysed the priming pattern of these biologically active molecules with regard to IL-8 production from A549 cells. In addition, the priming patterns of IFN-y, TNF-a/0 and IL-3 were also analysed. The data depicted in Table 1 show that IL-Ia/# and TNF-a/p are the most active cytokines for the induction of IL-8 secretion from epithelial cells. Our results indicate that both IL-1,B and IL-la, which is mainly present in a cell-bound manner, are comparable with regard to their activity for IL-8 secretion from epithelial cells. In addition, TNF-fl/lymphotoxin produced by activated lymphocytes is as active as TNF-a, and RSVinfected A549 cells primed with these cytokines showed a synergistically increased IL-8 secretion rate (Table 1). Therefore, epithelial cells behave like endothelial cells, monocytes and PMN with regard to IL-8 secretion after IL- IB and TNF-a priming. 1342,43
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The cytokines IFN-y, IL-3, G-CSF and TGF-fl2 induced the synthesis and release of IL-8 from A549 cells (Table 1). Thus, cytokines and colony-stimulating factors which might be present in the lining fluid of the respiratory epithelium during the viral infectious process are able to induce IL-8 secretion from epithelial cells. We have shown that the cell supernatants of epithelial cells co-cultured with PMN or PBMC reveal more immunoreactive IL-8 compared to the sum of the individual cell populations (Figs 6 and 7). Since PMN and PBMC interact with RSV, and also secrete IL-8 during this process (Figs 6a and 7a), the cellular source of the increased IL-8 release cannot be unequivocally determined.7'44 In addition, the IL-8 release was further increased when the cells were infected with RSV (Figs 6b and 7b). The increased IL-8 release was dependent on direct cell-cell contact as well as on soluble mediators. Coculture experiments performed in Transwell tissue chambers revealed that up to 30% of the induced IL-8 release was a consequence of secreted soluble factors (data not shown). Quite recently, it was shown that platelet-bound IL-la increased the IL-8 release from endothelial cells.45 Since IL-k/fl and TNFa/,B are potent inducers for IL-8 synthesis, their role in co-culture systems has to be established in future studies. Obviously, the use of an epithelial cell line is only a simplified model in comparison to the complex microenvironment of the lower respiratory tract. But this model may be helpful to understand the cell-cell interactions resulting in the proinflammatory mediator release during viral infection. Our results therefore suggest that the net amount of IL-8 released in the lung during RSV-infection is dependent on the cytokine and cellular pattern in the micro-environment of the mucosa.
ACKNOWLEDGMENTS This work was supported by the Deutsche Forschungsgemeinschaft, and was in partial fulfilment of R. Arnold's PhD thesis.
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