THE PNEUMOTOXICANT PARAQUAT INDUCES IL-8 mRNA IN HUMAN MONONUCLEAR CELLS

AND PULMONARY EPITHELIAL CELLS Marina Bianchi, Giamila Fantuzzi, Riccardo Bertini,* Lianna Perin,

Mario Salmona, Pietro Ghezzi

Paraquat (PQ) is a herbicide which is highly pneumotoxic by generating reactive oxygen intermediates (ROI). Pro-inflammatory cytokines, particularly IL-1 and TNF, have been implicated in some ROI-mediated pathologies, including bleomycin toxicity and ischaemia/ reperfusion injury. We have studied the effect of PQ on the expression of the neutrophil chemotactic cytokine, IL-S, by human peripheral blood mononuclear cells (PBMC). While almost no IL-8 mRNA was detected in unstimulated cells, PQ (100 p.M) induced high mRNA expression with a maximum at 24 h of incubation. While PQ did stimulate the appearance of IL-8 mRNA, no significant production of IL-8 protein was detected. However, PQ poten- tiated the production of IL-8 in the presence of 1 nglml of endotoxin (lipopolysaccharide, LPS). This was paralleled by an increased production of chemotactic activity for neutrophils, indicating that the IL-8 was actually bioactive. Stimulation of IL-8 mRNA by PQ was suppressed by IL-4 and by free radical scavengers (dimethylsulfoxide, mannitol). Increased IL-8 expression by PQ was also observed in the human pulmonary epithelial cell line A549 indicating that the effect of PQ was not specific for PBMC. These findings suggest that IL-8 might be involved in the pulmonary effects of PQ and that its production might be stimulated following an oxidative insult, and might clarify the pathogenetic mechanisms of adult respir- atory distress syndrome (ARDS) or oxidant-induced pulmonary fibrosis.

Paraquat (PQ) is a bipyridyl herbicide (l,l’- dimethyl-4,4’-bipyridylium) whose pneumotoxicity is well known. L-3 The selective toxicity of PQ for the lung is due to the fact that it is actively accumulated by lung cells ,3 where it is rapidly converted to a free radical which reacts with molecular oxygen to form toxic reactive oxygen intermediates (ROI), like super- oxide radicals, by a redox cycling.4 While the direct toxicity which was observed by exposure of cells to PQ is likely to be a result of this toxic effect of PQ, the mechanisms of its in vivo pulmonary toxicity might well be more complex.

We previously reported that PQ potentiates the production of IL-l and TNF by human PBMC in

From ‘Mario Negri’ Institute for Pharmacological Research, 20157 Milan, Italy; and * Dompt spa, L’Aquila, Italy.

Correspondence to: Pietro Ghezzi, ‘Mario Negri’ Institute, via Eri- trea 62, 20157 Milano, Italy.

Received 26 January 1993; accepted for publication 4 March 1993. 0 1993 Academi Press Limited. 1043.4666/93/050525+06 $08.00/O

KEY WORDS: IL-UParaquat/oxidants/IL-4

CYTOKINE, Vol. 5, No. 5 (September), 1993: pp 525-530

vitro.’ The induction of these cytokines might be im- portant particularly in the induction of pulmonary fibrosis by PQ2 as well as in its acute toxicity. In fact TNF was suggested to play a role in lung silicosis and in bleomycin-induced pulmonary fibrosis in mice, and anti-TNF antibodies were shown to be protective in these experimental models.6,7 Furthermore, bleomy- tin was reported to increase IL-l production by alveo- lar macrophages ex vivo.8 Another free radical- mediated pathology in which cytokines, particularly TNF, have been implicated is the post-ischaemic reperfusion injury, since in a model of perfused rat liver TNF is produced during post-ischaemic reperfu- sion, and anti-TNF antibodies protect against the resulting cellular insult.’

Since polymorphonuclear leukocytes (PMN) play a crucial role in ischaemia/reperfusion damage as well as in cytokine-induced pulmonary damage, the atten- tion in this field is being focused on chemotactic cyto- kines as pathogenetic mediators. In particular, IL-8 is a major macrophage- and endothelial cells-derived PMN chemoattractant and was reported to activate functionally PMN for their secretory functions and production of ROI. ‘” In vivo, high levels of IL-8 were

525

526 I Bianchi et al. CYTOKINE, Vol. 5, No. 5 (September 1993: 525-530)

Donor 1 Donor 2 Donor 3 Donor 4

Ic PQ ‘C PQ I

‘C! PQ I

‘C PQ LPS ’

Figure 1. PQ-induced IL-8 mRNA expression in PBMC.

PBMC prepared from four different donors were cultured for 20 h in the presence and absence of PQ. C, control; PQ, 100 PM paraquat. As a positive control, a lane of PBMC stimulated with LPS (1 rig/ml) is also shown for donor 4.

I” ”

319.1 f 175.6” 600 -

500 -

g 400 - ” 186.9 2~ 110.6

2 300- CI

200 -

100 - 15.3 f 23.4 NS

0, I

Control PQ LPS LPSIPQ

Figure 2. Increased production of immunoreactive IL-8 by PQ.

PBMC from eight donors were cultured for 20 h in the presence and absence of PQ (100 PM) and LPS (1 ng/ ml). At the end of the incubation, in 96-well tissue culture plates, samples (cells + supernatants) were lysed by freezing-thawing and total IL-8 produced was measured by ELISA. Data are expressed as rig/ml. Numbers indicate the mean k SD. NS: not significantly different. ” P < 0.05 by Student’s t-test for paired data.

observed in the circulation, following treatment with LPS or in synovial fluids from patients with rheuma- toid arthritis. ‘I3 ‘* As a key mediator in the recruit- ment and activation of inflammatory cells, IL-8 has been postulated to play a role in ARDS, a pathology often associated with infection,13 and elevated levels of IL-8 were found in bronchoalveolar lavages from patients with idiopathic pulmonary fibrosis.14 In vitro, IL-8 production by monocytes is stimulated by IL-1 and TNF, and inhibited by IL-4.1°,15 IL-8 is also induced in cells exposed to hyperoxia. l6

In the present paper we studied the effect of PQ on IL-8 production by human PBMC and by the human pulmonary epithelial cell line A549, previously shown to produce IL-8. ”

RESULTS

Induction of IL-8 Gene Expression by PQ

Figure 1 shows the effect of PQ on IL-8 mRNA levels in PBMC from four donorss. PQ (100 PM) induced significant IL-8 mRNA, in the absence of any LPS. Ethidium bromide staining of the gels indicated that equal amounts of RNA were loadeddd on the gel (data not shown). It should also be noted that PQ was free from LPS contamination (negative by LAL assay; <.5 EU). The levels of immunoreactive IL-S produced by PBMC (eight donors) stimulated with PQ with or without LPS are shown in Fig. 2. It can be seen that PQ per se did not induce a significant amount of immunoreactive IL-8, but potentiated LPS-induced

Oxidant-induced IL-8 I 527

IL-8 production. It should be noted that for these experiments, total immunoreactive IL-8 produced (cell associated + secreted) was measured, in order to evaluate the actual amount of IL-8 synthesized. The fact that PQ alone does not induce significant amounts of IL-8 and potentiates the response to LPS further indicates that an LPS contamination of the chemical cannot be responsible for the observed effect.

PQ-induced Increase of Bioactive IL-8 Production

In order to establish whether the PQ-induced increase of IL-8 production (measured by ELISA) was biologically significant and led to an actual increase in the production of chemotactic activity, supernatants from PBMC cultured for 20 h with LPS and PQ were dialysed to remove PQ that might have interfered with the assay, and tested for their PMN chemotactic activity. The results of these experiments are reported in Table 1. It can be seen that PQ actually potentiated the production of PMN chemo- tactic activity. No chemotactic activity was induced by PQ alone, while it potentiated LPS-induced IL-S.

TABLE 1. Effect of PQ on the production of chemotactic activity by LPS-stimulated PBMC.

Migrated PMNt

Sample Experiment 1 Experiment 2

Control (no addition) FMLP lo-sM

Supernatants from: PBMC (unstimulated) PBMC + PQ PBMC + LPS PBMC + PQ + LPS

34 + 6 27 + 4 118 5 5 57 + 2

34 f 6 33 f 1 31+2 33 + 3

108 + 2 47 t 2 207 + 5* 97 t 6*

Supernatants (25 ~1) from control or PBMC cultured for 20 h in the presence and absence of LPS (1 rig/ml) and PQ (100 FM) were tested for their chemo- tactic activity on human PMN. : Results are expressed as number of migrated PMN in five oil immersion fields and are the mean + SE of triplicate samples. Two separate experiments with different subject are reported. * P < 0.01 vs (PBMC + LPS) by Student’s t-test.

PQ-induced IL-8 mRNA is Inhibited by Antioxidants DISCUSSION

As shown in Fig. 3, two free radical scavengers, The data reported in this paper show that the DMSO and mannitol (two hydroxyl radical sca- pneumotoxicant PQ induces IL-S mRNA in human

Donor I Donor II I I I I

- 18s

- 28s ‘.

Figure 3. Effect of antioxidants on PQ-induced IL-8 mRNA.

PBMC were cultured for 30 min with or without one of the follow- ing agents: mannitol (MANN), 200mM; DMSO, 50 FM; superox- ide dismutase + catalase (SOD + CAT, 3.0 and 0.8 ~.LM, respect- ively). PQ (100 pM) was then added and culture was continued for 20h.

vengers) inhibited PQ-induced IL-8 mRNA. It should be noted that these two compounds did not induce any toxicity (by Trypan blue exclusion) or RNA degradation (as judged by ethidium bromide staining of the gels) (data not shown). The two antioxidant enzymes, superoxide dismutase (SOD) and catalase, at the concentrations tested, did not inhibit the effect of PQ; on the contrary they seemed to potentiate IL-8 induction.

P&-induced IL-8 is Inhibited by IL-4

We studied the effect of IL-4 on the production of IL-8 in PQ-treated cells. As shown in Fig. 4A, IL-4 (1 @ml) markedly inhibited IL-S production induced in PBMC by LPS or PQ + LPS. The inhibitory effect of IL-4 was also evident on IL-8 mRNA induction by PQ (Fig. 4B).

PQ Induces IL-8 mRNA in a Pulmonary Epithelial Cell Line

As shown in Fig. 5, PQ also induced IL-8 mRNA levels in the human pulmonary epithelial cell line A549. It should be noted that in this case, IL-lp (20ng/ml) was used as a stimulus for IL-S production since LPS did not induce any IL-8 in these cells (data not shown), in agreement with previous reports.i7

528 I Bianchi et al.

300* r

0 C PQ LPS LPWPQ C PQ LPS LPS/P(

CYTOKINE, Vol. 5, No. 5 (September 1993: 525-530)

B Donor 1 Donor 2

I s

II 3

I

‘7 ‘7 V if E v z z

- 28s

- 18s

Figure 4. Inhibition of PQ/LPS-induced immunoreactive (A) and mRNA IL-8 (B) by IL-4.

A: PBMC from two different donors were cultured for 20 h with the indicated additions (PQ, 100 (LM; LPS, 1 @ml; IL-4, 1 rig/ml) and total (intracellular + secreted) IL-S production was measured by ELISA. Each bar represents the mean of duplicate determination (variability <5%). 0, no addition; W, IL-4). B: PBMC from two donors were cultured for 20 h with PQ (100 t.r,M) in the presence and absence of IL-4 (1 q/ml).

4h

z-----l PQ IL-l

24 h

‘C PQ IL-?

28 S

18 S

Figure 5. PQ induction of IL-8 mRNA in A549 pulmonary epi- thelial cells.

A549 cells cultured as described in Materials and Methods were exposed to PQ (100 PM) or to IL-18 (20ng/ml).

PBMC and in a pulmonary epithelial cell line. This action of PQ is not simply due to a contamination of PQ with LPS for the following reasons: (a) no LPS was detected in the PQ preparation; (b) unlike LPS, PQ induced only IL-S mRNA, and very little or no IL-S protein; (c) PQ potentiated the production of IL-S in PBMC cultured with LPS; (d) PQ stimulated IL-8 gene expression also in A549 pulmonary epithelial cells that do not respond to LPS, but require IL-l (data not shown, and ref. 17).

Since in previous work we had shown that PQ

increased IL-l and TNF production in LPS-stimulated PBMC, we considered the hypothesis that stimulated IL-S mRNA might be mediated by these cytokines. This was clearly not the case since PQ stimulated IL-8 mRNA even in the absence of LPS, and PQ per se does not induce IL-1 or TNF.5

The fact that two free radical scavengers (DMSO and mannitol) inhibited PQ induction of IL-S might indicate that an increased intracellular production of free radicals is important for its action. It is difficult to draw a conclusion from the lack of effect of anti- oxidant enzymes (SOD/catalase) since these enzymes seem to potentiate IL-S mRNA expression at least in one donor. However, it is likely that these large mol- ecules do not enter the cell and therefore it is not surprising that they have no effect on a drug that stimulates intracellular free radical production.

IL-4 inhibited the observed induction of IL-8 mRNA and protein by PQ, in agreement with pre- vious data indicating an inhibitory effect of IL-4 on the production of several cytokines, including IL-&l5 and suggesting a possible way to inhibit IL-S-mediated effects of PQ. The induction of IL-S mRNA by PQ reported here, further support the idea that the syn- thesis of this cytokine can be stimulated by oxidative stress as recently reported in human monocytes exposed to hyperoxia. l6

There is a growing list of reports indicating that oxidative damage can lead to cytokines production in vivo and in vitro. These include induction of TNF by ischaemia/reperfusion in the perfused liver,’ in vitro induction of IL-l/TNF by PQ5 or of TNF by H20218 and the reported induction of IL-8 by hyperoxia.16

Oxidant-induced IL-S / 529

We also reported that hypoxia, known to induce a paradoxical oxidative damage, also potentiates IL-l and TNF production by monocytes.‘” In general, it should be noted that IL-8 is a known stimulus for the production of ROI by phagocytes, and therefore its induction might amplify the oxidative damage.

It will be interesting to compare the induction of IL-8 with that of other genes induced by PQ, includ- ing metallothionein2’ or bacterial ‘oxidative stress’ genes mapping to the soxR and soxQ locuses.21,22 It will also be important to define the mechanism by which oxidative stress induced by PQ increases IL-8 gene expression. It could be that a breakdown pro- duct of oxidative stress, like lipid peroxides, or a ROI itself, might act as second messengers. It is also poss- ible that a regulatory protein sensitive to oxidative damage (like a protein bearing a sulphhydryl group) acts as an ‘oxidant receptor’. In this respect, it was recently reported that oxygen radicals activate the NF-KB transcription factor,23 a widely used mes- sengers for the activation of several genes including cytokines. 24 The study of the mechanism by which PQ or other pro-oxidants induce cytokine gene ex- pression, as well as the identification of inhibitors of this induction, like IL-4 or antioxidants, might provide useful tools to investigate the pathogenetic role of cytokines in pulmonary pathologies like ARDS or oxidant-induced fibrosis.

MATERIALS AND METHODS

Preparation of Human PBMC

PBMC were obtained from the buffy coats of blood donations from normal volunteers through the courtesy of the Centro Trasfusionale, Ospedale Sacco, Milan, Italy, using Ficoll-Hypaque gradients as previously described.25

In the experiments where IL-8 protein was measured by ELISA or chemotactic activity, cells were cultured at 37°C 5% CO2 in 96-wells tissue culture plates at a concen- tration of 2.5 x 106/ml in RPM1 1640 medium (Flow Labor- atories, Irvine, Scotland) containing 1% fetal calf serum (FCS, Flow Laboratories). When indicated, the following additions were made at the concentrations indicated in the text: LPS (from E. coli, 055:B5, Sigma, St Louis, MO); IL-l (human recombinant IL-lB, specific activity lO’U/ml, a kind gift from Sclavo, Siena, Italy); PQ hydrochloride (a kind gift from ICI Solplant spa, Milan, Italy). Human recombinant IL-4 (10s U/mg) was a kind gift from Immunex Corporation, Seattle, WA. DMSO, mannitol, and bovine liver catalase were from Sigma; Cu-Zn superoxide dismu- tase was a kind gift from Zambeletti, Milano, Italy.

After a 20 h incubation, cells were lysed by three cycles of freezing-thawing and total IL-8 produced was measured by a commercial ELISA (British Bio-technology Ltd, Oxford, UK; sensitivity: 20pg/ml). Cell viability was

assessed on separate wells by Trypan blue exclusion. In some experiments supernatants were dialysed against two changes of PBS to remove PQ and used for the determi- nation of chemotactic activity.

For mRNA studies, cells were cultured for 20 h in 50 ml polypropylene tubes (Falcon) at the concentration of 2.5 X

lo6 cells/ml (30 ml/tube). At the end of the incubation, cells were harvested by centrifugation (500 X g, 10min) and immediately lysed in 4M guanidine isothiocyanate (see below).

Preparation of AS49 Pulmonary Epithelial Cells

Human pulmonary adenocarcinoma cell line A549 was obtained from the American Type Culture Collection, Rockville, MD. Cells were grown in Eagle’s minimum essential medium (Flow) containing 10% FCS. Cells were seeded in 75cm2 plastic culture flasks (Falcon) and used when semiconfluent. After a change of medium, cells were exposed to the various stimuli, as described in the text. At the end of the incubation, the supernatant was discarded, cells were lysed in 4M guanidine isothiocyanate and RNA isolated as described below.

Northern Blot Analyses

Total RNA was isolated from PBMC or A549 cells by centrifugation through 5.7 M caesium chloride as previously described.26 Total RNA content was quantitated using A260 spectrophotometry. RNA (20 p+g) samples were electro- phoresed in a 1% agarose/2.2M formaldehyde gel and transferred onto Gene Screen Plus transfer membranes (New England Nuclear). The cDNA probe for human IL-8 (a kind gift from Dr K. Matsushima, Kanazawa University, Kanazawa, Japan) was labelled by random priming,27 using a multiprime DNA kit from Amersham (Amersham, UK). Prehybridization and hybridization were done following the manufacturer’s protocol, and exposed at -70°C to a Kodak XAR film with intensifying screen. For each gel, the amount of RNA loaded was checked by ethidium bromide staining.

Assay for Chemotactic Activity

PMN were purified after the Ficoll-Hypaque centrifu- gation by dextran sedimentation and migration of PMN was evaluated by a microchamber technique as described else- where.28 RPM1 or cell supernatants, 25 ~1, were seeded in the lower compartment of the chemotaxis chamber, and 50 ~1 of cell suspension (1.5 x 106/ml) were placed in the upper compartment. The two compartments were separated by a 5-ym pore-size, polyvinyl-pyrolidone-free polycarbon- ate filter (Nucleopore Corp., Pleasanton, CA). The chambers were incubated at 37°C in air with 5% CO2 for 90min. At the end of incubation, filters were removed, fixed and stained with Diff-Quick (Harleco, Gibbstown, NJ), and five oil immersion fields were counted after coding samples. In each assay, N-formylmethionyl-leucyl- phenylalanine (FMLP, Sigma) at a concentration of lo-* M was used as a positive control.

530 I Bianchi et al. CYTOKINE, Vol. 5, No. 5 (September 1993: 525-530)

Acknowledgements Interleukin-8 (IL-S): The major neutrophil chemotactic factor in the lung. Exp Lung Res 17:17-23.

14. Carrt PC, Mortenson RL, King TE Jr, Noble PW, Sable CL, Riches DWH (1991) Increased expression of the interleukin-8 gene by alveolar macrophages in idiopathic pulmonary fibrosis. J Clin Invest 88:1802-1810.

15. Standiford TJ, Strieter RM, Chensue SW, Westwich J, Kasahara K. Kunkel SL (1990) IL-4 inhibits the expression of IL-8 from stimulated human monocytes. J Immunol 145:1435-1439.

16. Metinko A, Kunkel S, Standiford T, Strieter R (1991) Monocyte expression of interleukin-8 in response to oxidant stress. Faseb J 5:A704.

17. Standiford TJ, Kunkel SL, Basha MA, Chensue SW, Lynch JP III, Toews GB, Westwick J, Strieter RM (1990) Inter- leukin-8 gene expression by a pulmonary epithelial ‘cell iine. A model for cytokine networks in the lung. J Clin Invest 86:1945- 1953.

G. Fantuzzi is a recipient of a fellowship from DompCIConsorzio Biolaq, L’Aquila, Italy. This work was supported by ENELKNR within the special project ‘interaction of energetic systems with human health and environment’.

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