alveolar - Hindawi Publishing Corporation · alveolar macrophageactivation K. Pollockand M.T. WithnallcA Rh(ne-Poulenc Rorer Ltd, Dagenham Research Centre, Rainham Road South, Dagenham,

Research Paper

Mediators of Inflammation 2, 373-377 (1993)

THE selective enzyme inhibitors genistein and Re 31-8220were used to assess the importance of protein tyrosinekinase (PTK) and protein kinase C (PKC), respectively,in N-formyl-methionyl-leucyl-phenylalanine (FMLP) in-duced generation of superoxide anion and thromboxaneB2 (TXB2) in guinea-pig alveolar macrophages (AM).Genistein (3-100 pM) dose dependently inhibited FMLP(3 nM) induced superoxide generation in non-primed AMand TXB release in non-primed or in lipopolysaccharide(LPS) (10 ng/ml) primed AM to a level > 80% but had litleeffect up to 100 uM on phorbol myristate acetate (PMA)(10 nM) induced superoxide release. Re 31-8220 inhibitedPMA induced superoxide generation (ICs0 0.21 + 0.10 pM)but had no effect on or potentiated (at 3 and 10/M) FMLPresponses in non-primed AM. In contrast, when presentduring LPS priming as well as during FMLP challengeRe 31-8220 (10 pM) inhibited primed TXB release by> 80%. The results indicate that PTK activation is requiredfor the generation of these inflammatory mediators byFMLP in AM. PKC activation appears to be required forLPS priming but not for transducing the FMLP signal;rather, PKC activation may modulate the signal by anegative feedback mechanism.

Key words: Alveolar macrophage activation, Cell priming,Protein kinase C inhibitors, Protein tyrosine kinase inhibitors

Protein tyrosine kinase but notprotein kinase C inhibitionblocks receptor inducedalveolar macrophage activation

K. Pollock and M. T. WithnallcA

Rh(ne-Poulenc Rorer Ltd, Dagenham ResearchCentre, Rainham Road South, Dagenham, EssexRMIO 7XS, UK

ca Corresponding Author

Introduction

The alveolar macrophage (AM) is the mostabundant cell in the airway lumen and may be animportant contributor to the inflammation asso-ciated with pulmonary diseases such as asthma, a’2Isolated AM respond to a variety of immunologicaland non-immunological stimuli by generating bothacute-phase and pro-inflammatory mediators. Theacute-phase mediators include the spasmogensthromboxane A2, leukotriene C4 and plateletactivating factor (PAF) as well as reactive oxygenspecies and lysosomal enzymes which can causelocal tissue damage.3 Among the pro-inflammatorymediators are PAF, leukotriene B4 and a number ofcytokines which are chemoattractant to mast cells,eosinophils and lymphocytes, all of which havebeen implicated in pulmonary inflammation, a’2 Inaddition to being directly activated AM can beprimed in vitro by agents including LPS4 and gammainterferon to give an exaggerated response tosubsequent stimuli. AM primed in vitro may becomparable with AM from asthmatic subjectswhich have been shown to release greater amountsof many of the above mediators on stimulation thancells from non-asthmatic subjects.6-9The biochemical mechanisms underlying priming

are poorly understood but may involve amplifica-tion of signal transduction processes. For thechemotactic peptide FMLP three receptor-coupled

993 Rapid Communications of Oxford Ltd

signalling pathways have been identified: (i)G-protein linked phospholipase C (PLC) whichgenerates inositol 1,4,5 trisphosphate and dia-cylglycerol (DAG) from phosphoinositides,a (ii)phospholipase D generation of DAG fromphosphatidyl choline,al and (iii) activation ofnon-receptor PTK.a2’13 As PTK can activate PLCy4to generate DAG, activation of PKC couldrepresent a common end point for all of thesepathways, although other mechanisms of PTKdependent cell activation clearly exist. Both PKCand PTK have been implicated in the primingprocess in macrophages; for instance in murineperitoneal macrophages LPS priming stimulatedmyristoylation and translocation of an intracellularsubstrate for PKC thus improving the efficiency ofthis signal transduction pathway,as whilst LPSpriming in P388 D1 cells was shown to requireprotein synthesis and to be blocked by a PTKinhibitor, a6

In order to investigate the importance of PTKand PKC enzyme systems in FMLP activation ofAM we have studied the effects of the selective PTKinhibitor genistein7 and the selective PKC inhibitorRe 31-822018 on superoxide and TXB2 (as a markerfor arachidonate metabolism) generation in normalcells, and on TXB2 generation in LPS primed cells.As a control measure the effects of these compoundson superoxide release induced by FMLP werecompared with those on superoxide release induced

Mediators of Inflammation. Vol 2.1993 373

K. Pollock and M.T. Withnall

by the phorbol ester PMA, which is known to actby stimulating PKC directly.

Materials and Methods

Male Dunkin-Hartley guinea-pigs were pur-chased from David Hall, Burton-on-Trent, UK.Tissue culture reagents were obtained fromGibco; FMLP, phorbol myristate acetate (PMA),type II1 cytochrome c, LPS (E. coli 055:B5) andsuperoxide dismutase from Sigma, UK; and TXB2radioimmunoassay kits from Amersham, UK.Protein was determined after disruption of cellswith 0.1% Triton X-100 using BioRad Coomassieblue reagent. Genistein was purchased fromICN-Flow whilst Ro 31-8220 was synthesized atDagenham Research Centre. Compounds wereinitially dissolved in dimethylsulphoxide (DMSO)and diluted in Hank’s balanced salt solution (HBSS)to a final DMSO concentration in cell incubates of

Inhibition of alveolar macrophage activation

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0 0.1 10 100 1000

Antagonist (/M)FIG. 1. The effects of Ro 31-8220 and genistein on PMA (Panel A) orFMLP (Panel B) induced superoxide release from non-primed guinea-pigAM. AM (10S/well) were incubated with or without inhibitors for 2.25 hat 37C prior to addition of stimulus for 30 min. Superoxide wasdetermined by superoxide dismutase inhibitable reduction of Fe3+

cytochrome c. Mean-t-S.E.M. (n= 3) from a single representativeexperiment.

0.10 _---t- 0.01 to 1.0 +_ 0.2ng/10/g AM protein(n 4 experiments). However LPS potentiatedmarkedly (primed) the response to FMLP over thefull concentration range tested (0.3-30 nM) (Fig. 2).FMLP (3 nM), as a non-maximal stimulus, was usedto study the eEects of Ro 31-8220 and genistein onTXB2 release.When present during the 2 h pre-incubation, or

2h LPS priming, stage and the subsequentchallenge stage genistein (1-30/M) dose-depen-dently inhibited FMLP stimulated TXB2 release inboth non-primed and primed AM with similar ICs0values (17 and 18#M, respectively, in theexperiment shown in Fig. 3A). Under theseconditions Ro 31-8220 (0.1-10/,M) did not inhibitFMLP induced TXB2 release in non-primed AMbut tended rather to potentiate release at higherconcentrations (by >27% at 3 and 10 #M in theexperiment shown) (Fig. 3B). The extent ofpotentiation varied between experiments but was aconsistent observation. In LPS primed AM low

0 0.3 3 10 30

FMLP (nM)FIG. 2. Demonstration of LPS priming of AM for TXB2 release induced byFMLP. AM (2 x 10S/well) were incubated in HBSS or in HBSS containingLPS (10 ng/ml) for 2 h at 37C then the medium replaced by fresh HBSS.After 15 min at 37C FMLP was added for a further 15 min. TXB2 wasmeasured by RIA. Mean __. S.E.M. (n 4) from a single representativeexperiment.., Non-primed" I--I, primed.

concentrations of Ro 31-8220 (0.1-3 #M) had noeffect on the FMLP response whereas Ro 31-8220at 10/,M inhibited TXB. release by 80% relative tothe LPS baseline level. Since this suggested that Ro31-8220 had an inhibitory effect on a component ofLPS priming, further studies were performed inwhich Ro 31-8220 (10 M) was added to the AMonly during selected stages of priming and/oractivation (Fig. 4). Ro 31-8220 had no effect on theLPS primed response when present only duringFMLP challenge, inhibited the response by approx.50% when present only during priming, but hadgreatest effect, reducing FMLP induced TXB2release by 80 and 100% in relation to the LPSbaseline value (n--2 experiments), when presentduring both priming and challenge. The essentialrequirement for inhibition was thus that Ro 31-8220was present during the LPS priming stage.

Discussion

Ro 31-8220 and genistein were used in the presentstudy as selective inhibitors of PKC and PTK,respectively. Ro 31-8220 is reported to have a100-fold selectivity for PKC over protein kinase A(PKA) and a 1 000-fold selectivity over Ca2+-calmodulin dependent kinase18 and has been used byseveral groups as a selective PKC inhibitor in intactcells.2-22 Genistein inhibits a number of PTKs but


K. Pollock and M.T. Withnall

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(B)

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con FMLP 0.1/M 1/M 3/M 10/MRo 318220

FIG. 3. The effects of genistein (Panel A) or Ro 31-8220 (Panel B) on FMLPinduced TXB2 release from non-primed or LPS-primed AM. AM wereprimed with LPS (10 ng/ml) and stimulated with FMLP (3 nM) as describedin the legend to Fig. 2. Genistein or Ro 31-8220 were added during bothLPS priming and FMLP stimulation. Mean -!- S.E.M. (n 4) from a singlerepresentative experiment. I-3, Non-primed;., primed.

FIG. 4. Influence of the stage of LPS priming, or FMLP challenge at whichRo 31-8220 was added, on its ability to inhibit TXB2 release. AM wereprimed with LPS (10 ng/ml) and stimulated with FMLP (3 nM) as describedin the legend to Fig. 2. LPS -I- (Ro -I- FMLP)" Ro 31-8220 added for 15 minprior to, and during FMLP stimulation, but not during priming"(Ro -t- LPS) -t- FMLP" Ro 31-8220 added during LPS priming but removedbefore FMLP stimulation" (Ro + LPS) + (Ro + FMLP)" Ro 31-8220 addedduring LPS priming and for 15 min prior to, and during, FMLP stimulation.Mean -I- S.E.M. (n 4) from a single representative experiment.

shows very weak activity against serine andthreonine kinases including PKC, PKA andphosphorylase kinase. 17 At the concentrationsemployed neither Ro 31-8220 nor genistein reducedAM viability, thus ruling out nonspecific cytotoxi-city as an explanation of any observed effects of thecompounds.The finding that Ro 31-8220 inhibited PMA

induced superoxide generation but not FMLPinduced superoxide generation or TXB2 release innon-primed AM indicates that PKC either does nottransduce the FMLP signal or that it has feed backas well as feed forward et:fects on signaltransduction. The fact that Ro 31-8220 consistentlyincreased superoxide or TXB2 release (albeit notmarkedly) rather than merely being without eecton release leads us to favour the latter explanation.There is increasing evidence from a number of celltypes that PKC inhibition can increase cellularactivation, apparently by blocking inhibitory eectsof PKC on phospholipase C. For instance, Ro31-8220 was shown to potentiate PAF stimulatedTXB2 release from platelets,2 IgE stimulatedhistamine release from human basophils,23 andzymosan induced phospholipase C activity in livermacrophages.1 In addition, in the latter study theactivation of phospholipase C by zymosan wasenhanced by chronic pretreatment of the macro-phages with PMA to down-regulate PKC. It isunlikely that a Ro 31-8220 insensitive isoform ofPKC was involved in FMLP stimulated superoxideand TXB2 production in the AM studied in thepresent work since Ro 31-8220 is not isozymeselective and, in addition, the compound inhibitedthe superoxide response to PMA.

In contrast to the complex activity of Ro 31-8220,the consistent inhibitory effects of genistein onFMLP responses in both non-primed and primedAM support only a proactive role for a PTK inFMLP stimulus-response coupling in AM, as hasrecently been reported for neutrophils2’24 and forHL-60 cells. 13 Whether the pathways leading tosuperoxide or TXB2 release are separately regulatedby PTK or whether PTK action affects an earlycommon event such as PLCy activation,4 or PLDactivation,is each of which has been demonstratedin other cell types, remains to be determined.Nonetheless, the ability of genistein to inhibitsuperoxide production and TXB2 release almostcompletely emphasizes the key importance of PTKactivation following FMLP receptor stimulation.The observation that Ro 31-8220 was able to

inhibit FMLP induced TXB2 production in primedAM provided that the compound was presentduring the priming event is of interest indemonstrating that LPS induces priming by aprocess which is positively regulated by PKC. Thisdoes not appear surprising in view of other reports

376 Mediators of Inflammation. Vol 2.1 993

Inhibition of alveolar macrophage activation

that LPS can activate PKC in macrophages26 andthat responses to LPS such as cytokine productionare blocked by PKC inhibitors.27 It does, however,differ from findings in P388 D1 macrophages wherethe non-selective PKC inhibitor H7 did not affectcell priming. 16 The fact that Ro 31-8220 presentduring FMLP challenge as well as during primingincreased the inhibition of TXB2 release contrastswith the potentiation of release seen in non-primedAM and probably reflects the complex interplaybetween feed forward and feed back effects of PKCon mediator release in these cells.

In summary, using selective enzyme inhibitors itwas found that activation of PTK is a key event inFMLP generation of superoxide and TXB2 inguinea-pig AM whereas PKC either is not activated,or is activated but has counter-balancing feed backand feed forward effects on mediator release innon-primed cells. On the other hand activation ofPKC appears to be required for LPS priming ofthese cells for enhanced TXB2 release. Thus bothPTK and PKC inhibitors could conceivably beeffective in reducing the release of these mediatorsfrom macrophages in the airways in vivo underconditions where priming has occurred during aninflammatory response.

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and monocytes with granulocytes in asthma. Eur Respir J 1991; 4 (Suppl13): 85s-90s.

2. Fuller RW. Pharmacological regulation of airway macrophage function. OlinExp Allergy 1991; 21: 651-654.

3. Fels AO, Cohn ZA. The alveolar macrophage. J App! Physiol 1986; 60:353-369.

4. Aderem AA, Cohen DS, Wright SD, Cohn ZA. Bacterial lipopolysaccharidesprime macrophages for enhanced release of arachidonic acid metabolites. JExp Med 1986; 164: 165-179.

5. Eden E, Turino GM. Interleukin secretion by human alveolar macrophagesstimulated with endotoxin is augmented by recombinant immune (gamma)interferon. A Rev Respir Dis 1986; 133: 455-460.

6. Cluzel M, Damon M, Le Doucen C, Michel FB, Crastes de Poulet A, GodardP. Enhanced alveolar cell luminol-dependent chemiluminescence in asthma.J Allergy Clin Immunol 1987; 80: 195-201.

7. Joseph M, Tonnel AB, Torpier G, Capron A, Amoux B, Benveniste J. Theinvolvement of IgE in the secretory processes of alveolar macrophages fromasthmatic patients. J Clin Invest 1983; 71: 221-230.

8. Godard P, Chantreuil J, Damon Met al. Functional assessment of alveolarmacrophages: comparison of cells from asthmatic and normal subjects. JAllergy Clin Immunol 1982; 70: 88-93.

9. Howell CJ, Pujol JL, Crea AEG et al. Identification of alveolarmacrophage derived activity in bronchial asthma which enhances leukotrieneC4 generation by human eosinophils stimulated by ionophore (A23187)granulocyte-macrophage-colony-stimulating factor (GM-CSF). Am RevRespir Dis 1989; 140: 1340-1347.

10. Hamilton TH, Adams DO. Molecular mechanisms of signal transduction inmacrophages. Immunol Today 1987; 8: 151-158.

11. Thompson NT, Tateson JE, Randall RW, Spacey GD, Bonser RW, GarlandLG. The temporal relationship between phospholipase activation,diradylglycerol formation and superoxide production in the humanneutrophil. Biochem J 1990; 271: 209-213.

12. Kusunoki T, Higashi H, Hosai S et al. Tyrosine phosphorylation and itspossible role in superoxide production by human neutrophils stimulated withFMLP and IgG. Biochem Biophys Res Commun 1992; 183: 789-796.

13. Offermanns S, Seifert R, Metzger JW et al. Lipopeptides effectivestimulators of tyrosine phosphorylation in human myeloid cells. Biochem J1992; 282: 551-557.

14. Klausner RD, Samelson LE. T cell antigen receptor activation pathways:the tyrosine kinase connection. Cell 1991; 64: 875-878.

15. Aderem AA, Albert KA, Keum MM, Wang JK, Greengard P, Cohn ZA.Stimulus-dependent myristoylation of major substrate for protein kinaseC. Nature 1988; 332: 362-364.

16. Glaser KB, Asmis R, Dennis EA. Bacterial lipopolysaccharide priming ofP388 D1 macrophage-like cells for enhanced arachidonic acid metabolism. JBiol Chem 1990; 268: 8658-8664.

17. Akiyama T, Ishida J, Nakagawa S. Genistein, specific inhibitor oftyrosine-specific protein kinases. J Biol Chem 1987; 262: 5592-5595.

18. Twomey B, Muid RE, Nixon JS, Sedgwick AD, Wilkinson SE, Dale MM.The effect of potent selective inhibitors of protein kinase C theneutrophil respiratory burst. Biocbem Biophys Res Commun 1990; 171:1087-1092.

19. Metcalf JA, Gallin JI, Nauseef WM, Root RK. Laboratory Manual ofNeutropbil Function. New York: Raven Press, 1986; 109-114.

20. Murphy CT, Westwick J. Selective inhibition of protein kinase C. Effectplatelet-activating-factor-induced platelet functional responses. Biochem J

1992; 283: 159-164.21. Dieter P, Fitzke E. Ro 31-8220 and Ro 31-7549 show improved selectivity

for protein kinase C staurosporine in macrophages. Biocbem Biophys ResCommun 1991; 181: 396-401.

22. Walker TR, Watson SP. Effect of Ro 31-8220, novel PKC inhibitor,human platelet phosphorylation and secretion. BrJ Pbarmacol 1991 104: 88P.

23. Amon U, Stebut E, Dietz KR, Bauer FW, Wolff HH. Pharmacologicalstudies the role of protein kinase C in signal transduction. Int Arch AllergyImmunol 1992; 98: 349-354.

24. Gomez-Cambronero J, Huang CK, Bonak VA et al. Tyrosine phosphoryla-tion in human neutrophil. Biochem Biophys Res Commun 1989; 162:1478-1485.

25. Uings j, Thompson NT, Randall RW et al. Tyrosine phosphorylation isinvolved in receptor coupling to phospholipase D but not phospholipase Cin the human neutrophil. Biochem J 1992; 281: 597-600.

26. Wightman PD, Raetz CRH. The activation of protein kinase C bybiologically active moieties of lipopolysaccharide. J Biol Chem 1984; 289:10048-10052.

27. Coffey RG, Weakland LL, Alberts VA. Paradoxical stimulation andinhibition by protein kinase C modulating agents of lipopolysaccharideevoked production of tumour necrosis factor in human monocytes.Immunology 1992; 76: 48-54.

ACKNOWLEDGEMENT. We thank Emma Burke for secretarial assistancein the preparation of this manuscript.

Received 16 June 1993;accepted in revised form 27 July 1993


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