Mark A. Lindsay

Superinduction of COX‐2 mRNA by cycloheximide and interleukin‐1β involves increased transcription and correlates with increased NF‐κB and JNK activation

Many primary response genes, including cyclooxygenase‐2 (COX‐2), exhibit mRNA superinduction following agonist stimulation in the presence of translational blockers such as cycloheximide. This is widely assumed to result from mRNA stabilisation. However, superinduction of IL‐1β‐induced COX‐2 mRNA levels by cycloheximide in pulmonary type II A549 cells occurred by increased transcription and not by mRNA stabilisation. Furthermore, equivalent effects were observed on NF‐κB binding to COX‐2 promoter κB sites and activation of the Jun N‐terminal kinases (JNK), p54 and p46. These signalling pathways play important roles in COX‐2 induction and may therefore account for the observed increases in COX‐2 transcription. These data are consistent with negative feed‐back involving down‐regulation of NF‐κB by de novo IκBα synthesis and suggest that JNK activation may also be down‐regulated by a cycloheximide sensitive process.

The MAP kinase inhibitors, PD098059, UO126 and SB203580, inhibit IL-1β-dependent PGE2 release via mechanistically distinct processes

In common with human bronchial epithelial cells, pulmonary A549 cells release prostaglandin (PG) E2 in response to pro‐inflammatory cytokines. We have therefore used these cells to examine the effect of the selective mitogen activated protein (MAP) kinase inhibitors; PD098059, a mitogen activated and extracellular regulated kinase kinase (MEK) 1 inhibitor, UO126, a dual MEK1 & MEK2 inhibitor, and SB203580, a p38 MAP kinase inhibitor in the IL‐1β‐dependent release of PGE2. Following IL‐1β treatment the extracellular regulated kinases (ERKs) and the p38 MAP kinases were rapidly phosphorylated. PD09059, UO126 and SB203580 prevented IL‐1β‐induced PGE2 release at doses that correlated closely with published IC50 values. Small or partial effects at the relevant doses were observed on induction of cyclo‐oxygenase (COX) activity or COX‐2 protein suggesting that the primary effects were at the level of arachidonate availability. Neither PD098059 nor SB203580 showed any effect on IL‐1β‐induced arachidonate release. We therefore speculate that the MEK1/ERK and p38 kinase cascades play a role in the functional coupling of arachidonate release to COX‐2. In contrast, UO126 was highly effective at inhibiting IL‐1β‐dependent arachidonate release, implicating MEK2 in the activation of the PLA2 that is involved in IL‐1β‐dependent PGE2 release. We conclude that the MEK1, MEK2 and p38 MAP kinase inhibitors, PD098059, UO126 and SB203580, are highly potent in respect of inflammatory PG release. Finally, we conclude that these inhibitors act via mechanistically distinct processes, which may have anti‐inflammatory benefits.

p38 MAP kinase and MKK-1 co-operate in the generation of GM-CSF from LPS-stimulated human monocytes by an NF-κB-independent mechanism

The extent to which the p38 mitogen‐activated protein (MAP) kinase and MAP kinase kinase (MKK)‐1‐signalling pathways regulate the expression of granulocyte/macrophage colony‐stimulating factor (GM‐CSF) from LPS‐stimulated human monocytes has been investigated and compared to the well studied cytokine tumour necrosis factor‐α (TNFα). Lipopolysaccharide (LPS) evoked a concentration‐dependent generation of GM‐CSF from human monocytes. Temporally, this effect was preceded by an increase in GM‐CSF mRNA transcripts and abolished by actinomycin D and cycloheximide. LPS‐induced GM‐CSF release and mRNA expression were associated with a rapid and time‐dependent activation of p38 MAP kinase, ERK‐1 and ERK‐2. The respective MKK‐1 and p38 MAP kinase inhibitors, PD 098059 and SB 203580, maximally suppressed LPS‐induced GM‐CSF generation by >90%, indicating that both of these signalling cascades co‐operate in the generation of this cytokine. Electrophoretic mobility shift assays demonstrated that LPS increased nuclear factor κB (NF‐κB) : DNA binding. SN50, an inhibitor of NF‐κB translocation, abolished LPS‐induced NF‐κB : DNA binding and the elaboration of TNFα, a cytokine known to be regulated by NF‐κB in monocytes. In contrast, SN50 failed to affect the release of GM‐CSF from the same monocyte cultures. Collectively, these results suggest that the generation of GM‐CSF by LPS‐stimulated human monocytes is regulated in a co‐operative fashion by p38 MAP kinase‐ and MKK‐1‐dependent signalling pathways independently of the activation of NF‐κB.

‘Outside‐in’ signalling mechanisms underlying CD11b/CD18‐mediated NADPH oxidase activation in human adherent blood eosinophils

Incubation of human eosinophils in BSA‐coated tissue culture plates resulted in time‐dependent adhesion and attendant activation of the NADPH oxidase that were both inhibited (by >85%) by blocking antibodies raised against CD11b and CD18. SB 203580, an inhibitor of p38 mitogen‐activated protein (MAP) kinase, did not influence adhesion but inhibited superoxide anion generation (pIC50=−6.57). PP1, an inhibitor of the src‐family of protein tyrosine kinases, inhibited adhesion and CD11b/CD18‐mediated superoxide anion generation with similar potencies (pEC50s=−5.53 and −5.99 respectively) suggesting that inhibition of the NADPH oxidase was a direct consequence of blocking adhesion. The protein kinase C (PKC) inhibitors Ro‐31 8220 (broad spectrum inhibitor), GF 109203X (inhibitor of conventional and novel isoforms) and Gö 6976 (inhibitor of conventional isoforms) suppressed adhesion‐dependent NADPH oxidase activation (pIC50s=−6.61, −6.05 and −4.89 respectively) without affecting adhesion. Based upon the selectivity of these drugs PKCδ and PKCε are implicated in the suppression of oxidant production. Wortmannin, an inhibitor of phosphatidylinositol 3‐kinase (PtdIns 3‐kinase), abolished superoxide anion production in adherent eosinophils (pEC50=−9.06). Similarly, CD11b/CD18‐dependent adhesion was suppressed with the same potency (pEC50=−9.29) although the maximum effect did not exceed 50% implying that wortmannin also had an affect on those processes that govern adhesion‐driven oxidase activation. PD 098059 and piceatannol, inhibitors of MAP kinase kinase‐1 and the syk tyrosine kinase respectively, had no effect on CD11b/CD18‐mediated adhesion or NADPH oxidase activation. The results of this study demonstrate that human eosinophils adhere to BSA‐coated plastic by a CD11b/CD18‐dependent mechanism, which is responsible for activation of the NADPH oxidase. Although the signalling pathway(s) utilized by CD11b/CD18 is still to be elucidated, the data presented herein implicate p38 MAP kinase, novel PKCs and PtdIns 3‐kinase.

Synergy between Tumor Necrosis Factor α and Interleukin 1β in Inducing Transcriptional Down-regulation of Muscarinic M2 Receptor Gene Expression INVOLVEMENT OF PROTEIN KINASE A AND CERAMIDE PATHWAYS

Stimulation of HEL 299 cells with tumor necrosis factor α (TNF-α) or interleukin 1β (IL-1β) had no effect on M2 muscarinic receptor expression. However, the combination of these two cytokines markedly down-regulated muscarinic M2 receptor protein and mRNA expression and uncoupled M2 receptors from adenylyl cyclase. There was no effect of TNF-α and IL-1β on the m2 muscarinic receptor mRNA stability, and nuclear run-on assays showed reduced m2 receptor gene transcription. Sequential cytokine addition suggests that the synergy involves postreceptor events. Although the cAMP-dependent protein kinase inhibitor H8 provided a significant protection against receptor down-regulation, the protein kinase C inhibitor GF109203X had no effect. The ceramide analog C2-ceramide (N-acetylsphingosine) was without effect on m2 receptor expression. However, a strong synergistic effect was demonstrated when cells were treated with the combination of C2-ceramide and TNF-α or IL-1β. TNF-α and/or IL-1β combination also activated the 46- and 55-kDa c-Jun NH2-terminal protein kinases and to a lesser extent p42 and p44 mitogen-activated protein kinase isoforms. Cycloheximide abolished the TNF-α and IL-1β effect, suggesting that de novo protein synthesis is required for receptor down-regulation. These results suggest that the TNF-α and IL-1β synergize to induce transcriptional down-regulation of the M2 muscarinic receptor, which seems to be mediated through activation of both ceramide and cAMP-dependent protein kinase pathways. Furthermore, these results suggest that M2 receptor expression is under the control of a cytokine network.

ROLE OF THE MITOGEN-ACTIVATED PROTEIN KINASES AND TYROSINE KINASES DURING LEUKOTRIENE B4-INDUCED EOSINOPHIL ACTIVATION

Exposure of guinea‐pig eosinophils to leukotriene B4 (LTB4; 1 μM) resulted in a rapid generation of H2O2 (index of NADPH oxidase activation), stimulated [3H]arachidonic acid (AA) release (index of phospholipase A2 activity), and promoted CD18‐dependent homotypic aggregation. Under similar conditions, LTB4 (1 μM) induced a rapid activation of extracellular‐regulated kinases‐1 and 2 (ERK‐1/2) but not c‐jun N‐terminal kinases 46 and 54 (JNK‐46/54) or p38 mitogen‐activated protein kinase (p38 MAP kinase). To examine the role of ERK‐1/2 in the mechanism of eosinophil activation, a selective inhibitor of MAP kinase kinase‐1/2 (MEK‐1/2), PD098059, was employed. However, PD 098059 at concentrations that attenuated ERK‐1/2 activation had no significant affect on eosinophil activation. In contrast, a role for tyrosine kinases in LTB4‐induced eosinophil activation was suggested by studies with the tyrosine kinase inhibitors, herbimycin A and lavendustin A. However, the results of those experiments implied divergent pathways for the control of eosinophil responses because the inhibitors were more effective at attenuating H2O2 generation than [3H]AA release, and had little effect on homotypic aggregation. J. Leukoc. Biol. 64: 555–562; 1998.

Identification of the protein kinase C isoenzymes in human lung and airways smooth muscle at the protein and mRNA level

The protein kinase C (PKC) isoenzymes expressed by human peripheral lung and tracheal smooth muscle resected from individuals undergoing heart-lung transplantation were identified at the protein and mRNA level. Western immunoblot analyses of human lung identified multiple PKC isoenzymes that were differentially distributed between the soluble and particulate fraction. Thus, PKC alpha, PKC betaII, PKC epsilon, and PKC zeta were recovered predominantly in the soluble fraction whereas the eta isoform was membrane-associated together with trace amounts of PKC alpha and PKC epsilon. PKC beta1-like immunoreactivity was occasionally seen although the intensity of the band was uniformly weak. Immunoreactive bands corresponding to PKCs gamma, delta, or theta were never detected. Reverse transcription-polymerase chain reaction (RT-PCR) of RNA extracted from human lung using oligonucleotide primer pairs that recognise unique sequences in each of the PKC genes amplified cDNA fragments that corresponded to the predicted sizes of PKC alpha, PKC betaI, PKC betaII, PKC epsilon, PKC zeta, and PKC eta (consistent with the expression of PKC isoenzyme protein) and, in addition, mRNA for PKC delta; PCR fragments of the expected size for the supposedly muscle-specific isoform, PKC theta, or the atypical isoenzyme, PKC lambda, were never obtained. The complement and distribution of PKC isoforms in human trachealis were similar, but not identical, to human lung. Thus, immunoreactive bands corresponding to the alpha, betaI, betaII, epsilon, and zeta isoenzymes of PKC were routinely labelled in the cytosolic fraction. In the particulate material PKC alpha, PKC epsilon, PKC alpha, PKC eta, and PKC mu were detected by immunoblotting. With the exception of PKC zeta, RT-PCR analyses confirmed the expression of the PKC isoforms detected at the protein level and, in addition, identified mRNA for PKC delta. Collectively, these data clearly demonstrate the expression of multiple PKC isoenzymes in human lung and tracheal smooth muscle, suggesting that they subserve diverse multifunctional roles in these tissues.

Expression and activation of protein kinase C-ζ in eosinophils after allergen challenge

Protein kinase (PK) C is an increasingly diverse family of enzymes that has been implicated in a range of cellular functions within the eosinophil. Using isoform-specific polyclonal antibodies, we have explored the expression of PKC isoforms in circulating eosinophils. Initial studies demonstrated the presence of the α, βI, βII, and ζ and the low-level expression of the δ, ε, ι, and μ isoforms but no detectable expression of the γ, η, and θ isoforms in both normal and asthmatic subjects. There was no difference in the total protein expression between these two groups. Subsequent studies examined the expression and activation of PKC isoforms in circulating eosinophils from asthmatic patients before and 24 h after a late asthmatic response to an inhaled allergen. Cellular fractionation showed PKC-α and PKC-βII to be mainly located in the cytosol, whereas PKC-βI was constitutively more expressed in the membrane. No changes in expression or subcellular localization of these isoforms were seen after allergen challenge. In contrast, PKC-ζ expression was increased after allergen challenge, and we demonstrated a significant PKC-ζ translocation to the membrane, in keeping with activation of the enzyme. Our results suggest that 24 h after allergen exposure of asthmatic patients, there is increased expression and activation of eosinophil PKC-ζ that correlates with late asthmatic responses recorded between 4 and 10 h postallergen challenge.Protein kinase (PK) C is an increasingly diverse family of enzymes that has been implicated in a range of cellular functions within the eosinophil. Using isoform-specific polyclonal antibodies, we have explored the expression of PKC isoforms in circulating eosinophils. Initial studies demonstrated the presence of the alpha, betaI, betaII, and zeta and the low-level expression of the delta, epsilon, iota, and micro isoforms but no detectable expression of the gamma, eta, and theta isoforms in both normal and asthmatic subjects. There was no difference in the total protein expression between these two groups. Subsequent studies examined the expression and activation of PKC isoforms in circulating eosinophils from asthmatic patients before and 24 h after a late asthmatic response to an inhaled allergen. Cellular fractionation showed PKC-alpha and PKC-betaII to be mainly located in the cytosol, whereas PKC-betaI was constitutively more expressed in the membrane. No changes in expression or subcellular localization of these isoforms were seen after allergen challenge. In contrast, PKC-zeta expression was increased after allergen challenge, and we demonstrated a significant PKC-zeta translocation to the membrane, in keeping with activation of the enzyme. Our results suggest that 24 h after allergen exposure of asthmatic patients, there is increased expression and activation of eosinophil PKC-zeta that correlates with late asthmatic responses recorded between 4 and 10 h postallergen challenge.

Pharmacological comparison of LTB4-induced NADPH oxidase activation in adherent and non-adherent guinea-pig eosinophils

Leukotriene B4 (LTB4) stimulation of guinea‐pig peritoneal eosinophils, induced a biphasic activation of the NADPH oxidase composed of a rapid (<3 min) phase mediated by non‐adherent cells and a sustained (3 – 120 min) phase mediated by CD11b/CD18 adherent eosinophils. Studies were undertaken to compare the intracellular mechanism that mediate these responses. SB 203580 and PP1, inhibitors of p38 mitogen‐activated protein (MAP) kinase and the src‐family protein tyrosine kinases, respectively caused concentration‐dependent attenuation of both the rapid (SB203580: pD2=−6.31; PP1: pD2=−5.50) and sustained (SB203580: pD2=−6.50; PP1: pD2=−5.73) phases. Similarly, the MAP kinase kinase‐1 inhibitor, PD098059 produced partial inhibition of the both phases of superoxide generation. The protein kinase C (PKC) inhibitors Ro‐31 8220, GF 109203X and Gö 6976 attenuated the rapid NADPH oxidase response (pD2s=−6.10, −6.72, −6.15 respectively) and, to a lesser extent, (pD2s=−5.54, −6.02, −6.51 respectively) the sustained phase. An inhibitor of phosphatidylinositol 3‐kinase (PtdIns 3‐kinase), wortmannin caused concentration dependent attenuation of the sustained (pD2=−8.68) but not rapid phase of superoxide generation. In contrast, the syk kinase inhibitor, piceatannol abolished the rapid (pD2=−6.43) but not sustained respiratory responses. This study demonstrates that LTB4‐induced superoxide generation from adherent and non‐adherent eosinophils is mediated via both common (p38 MAP kinase, MEK‐1, PKC and the src kinases) and divergent intracellular pathways (syk kinases and PtdIns 3‐kinase). This suggests the possibility of therapeutic intervention to selective attenuate activation of adherent tissue eosinophils.

Hydrogen Peroxide Reverses IL-5 Afforded Eosinophil Survival and Promotes Constitutive Human Eosinophil Apoptosis

Background: Eosinophils play a central role in the induction and perpetuation of allergic inflammatory responses. The present study was performed to investigate the effects of reactive oxygen intermediates on constitutive apoptosis as well as on interleukin (IL)-5 afforded human eosinophil survival. Methods: Peripheral blood eosinophils were isolated by CD16-negative selection to >99% purity and were cultured for 48 h. The number of apoptotic eosinophils in the culture was assessed by flow cytometric analysis of relative DNA content in propidium-iodide-stained cells, annexin-V binding or by morphological analysis. Apoptosis was confirmed by the appearance of a typical ladder pattern in the DNA fragmentation assay by agarose gel electrophoresis. Results: Exogenous H2O2 reversed IL-5-afforded eosinophil survival by inducing apoptosis. Constitutive eosinophil apoptosis was inhibited by a reduction of intracellular levels of H2O2 by catalase. Exogenous H2O2 increased the rate of constitutive apoptosis. Conclusions: Our results suggest that H2O2 may play a role in the downregulation of eosinophilic inflammation by inducing eosinophil apoptosis.