Cyclin-dependent kinase inhibitors enhance the resolution of inflammation by promoting inflammatory cell apoptosis.

Apoptosis is essential for clearance of potentially injurious inflammatory cells and subsequent efficient resolution of inflammation. Here we report that human neutrophils contain functionally active cyclin-dependent kinases (CDKs), and that structurally diverse CDK inhibitors induce caspase-dependent apoptosis and override powerful anti-apoptosis signals from survival factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF). We show that the CDK inhibitor R-roscovitine (Seliciclib or CYC202) markedly enhances resolution of established neutrophil-dependent inflammation in carrageenan-elicited acute pleurisy, bleomycin-induced lung injury, and passively induced arthritis in mice. In the pleurisy model, the caspase inhibitor zVAD-fmk prevents R-roscovitine-enhanced resolution of inflammation, indicating that this CDK inhibitor augments inflammatory cell apoptosis. We also provide evidence that R-roscovitine promotes apoptosis by reducing concentrations of the anti-apoptotic protein Mcl-1. Thus, CDK inhibitors enhance the resolution of established inflammation by promoting apoptosis of inflammatory cells, thereby demonstrating a hitherto unrecognized potential for the treatment of inflammatory disorders.

E-selectin permits communication between PAF receptors and TRPC channels in human neutrophils.

The selectin family of molecules (L-, P-, and E-selectin) mediates adhesive interactions between leukocytes and endothelial cells required for recruitment of leukocytes to inflammatory sites. Soluble E-selectin levels are elevated in inflammatory diseases and act to promote neutrophil beta(2)-integrin-mediated adhesion by prolonging Ca(2+) mobilization. Although soluble E-selectin alone was unable to initiate Ca(2+) signaling, it allowed a novel "permissive" store-operative calcium entry (SOCE) following the initial platelet-activating factor (PAF)-induced release of Ca(2+) from inositol 1,4,5-trisphosphate (IP(3))-sensitive stores. This induction of permissive SOCE in response to soluble E-selectin and PAF was shown to act through a G protein-coupled receptor (GPCR) coupled to pertussis toxin-insensitive G(q/11). Furthermore, we demonstrated that permissive SOCE was mediated by canonical transient receptor potential channel (TRPC) due to its sensitivity to specific inhibition by MRS1845 and Gd(3+) and that TRPC6 was the principal TRPC family member expressed by human neutrophils. In terms of mechanism, we demonstrated that soluble E-selectin activated Src family tyrosine kinases, an effect that was upstream of phosphatidylinositol 3'-kinase in a signaling pathway that regulates permissive SOCE following exposure of neutrophils to PAF. In summary, this report provides the first evidence for communication between an inflammatory mediator and adhesion receptors at a molecular level, through selectin receptor ligation allowing permissive SOCE to occur following PAF stimulation of human neutrophils.

Thrombin induces DNA synthesis and phosphoinositide hydrolysis in airway smooth muscle by activation of distinct receptors.

Chronic airway inflammation induces numerous structural changes of the airways involving hypertrophy and hyperplasia of airway smooth muscle (ASM). Thrombin has been identified in the bronchoalveolar lavage fluid of asthmatic subjects and displays potent bronchoconstrictor and mitogenic activity towards ASM. This study has addressed which proteinase-activated receptors (PARs) and signalling pathways are involved in mediating distinct effects of thrombin. Using cultured bovine tracheal smooth muscle (BTSM) cells as a model system, thrombin stimulated a marked increase in [3H]inositol phosphate ([3H]InsPs) accumulation, which was fully mimicked by a selective PAR1 activating peptide. In contrast, PAR1, PAR2, PAR3 and PAR4 activating peptides were unable to replicate the ability of thrombin to stimulate DNA synthesis as assessed by [3H]thymidine incorporation. Further investigation demonstrated that the mitogenic effect of thrombin did not involve stimulation of PDGF secretion but did involve activation of PDGF or EGF receptors and a G(i/o)-dependent activation of phosphoinositide 3-kinase. Thrombin, but not the PAR1, PAR2, PAR3 or PAR4 activating peptides was able to stimulate PtdIns(3,4,5)P3 mass accumulation. PAR3 antisense oligonucleotides substantially inhibit thrombin-stimulated [3H]thymidine incorporation and PtdIns(3,4,5)P3 generation but had no effect on thrombin-induced phosphoinositide hydrolysis. These data indicate that while PI hydrolysis and Ca2+ mobilisation induced by thrombin operates via PAR1-dependent activation of phospholipase C, phosphoinositide 3-kinase activation and DNA synthesis occurs via a distinct proteinase-activated receptor pathway, possibly involving PAR3.

A critical 'threshold' of beta 2-integrin engagement regulates augmentation of cytokine-mediated superoxide anion release.

1. Neutrophil adhesion regulates a number of processes involved in the pathogenesis of inflammatory diseases including rheumatoid arthritis. Neutrophil destructive potential can be modulated by adhesion, allowing alteration of inflammatory cell behaviour while preserving antimicrobial defences. beta(2)-Integrin-mediated neutrophil adhesion to albumin-coated latex beads (ACLB) allows modulation of integrin clustering and ligation and analysis of the effects of adhesion on neutrophil responses. Tumour necrosis factor-alpha (TNF alpha) enhanced neutrophil binding of different diameter ACLB equally, by almost four-fold, and independently of bead size. Adhesion of neutrophils to ACLB caused a size-dependent generation and release of O(2)(-) and also potentiated TNF alpha-induced O(2)(-) release. 2. Binding of ACLB was not affected by disruption of cytoskeletal integrity with nocodazole or cytochalasin D or following blockade of tyrosine kinase activity. In contrast, tyrosine phosphorylation and an intact cytoskeleton were essential for adhesion- and cytokine-induced O(2)(-) release from neutrophils. Inhibition of adhesion- and cytokine-induced O(2)(-) release by 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazol[3,4-d]pyrimidine (PP2) indicated that a Src-family tyrosine kinase was the principal regulatory pathway mediating this response in neutrophils, a distal role for p38 MAPK was revealed by use of SB203580. 3. Tyrosine phosphorylation of c-Fgr, a Src-family tyrosine kinase, occurred following ACLB adhesion and exposure to TNF alpha, and was susceptible to inhibition by PP2. We suggest that activation of the key regulatory enzyme c-Fgr is achieved following ligation of a critical threshold of integrins following binding of large (>3 microM) ACLB.

Regulation of phosphatidylinositol 3-kinase activity and phosphatidylinositol 3,4,5-trisphosphate accumulation by neutrophil priming agents.

Neutrophil priming by agents such as TNF-alpha and GM-CSF causes a dramatic increase in the response of these cells to secretagogue agonists and affects the capacity of neutrophils to induce tissue injury. In view of the central role of phosphatidylinositol 3-kinase (PI3-kinase) in regulating NADPH oxidase activity we examined the influence of priming agents on agonist-stimulated phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) accumulation in human neutrophils. Pretreatment of neutrophils with TNF-alpha or GM-CSF, while not influencing fMLP-stimulated PtdIns(3,4,5)P3 accumulation at 5 s, caused a major increase in PtdIns(3,4,5)P3 at later times (10-60 s), which paralleled the augmented superoxide anion (O2-) response. The intimate relationship between PtdIns(3,4,5)P3 accumulation and O2- release was confirmed using platelet-activating factor, which caused full but transient priming of both responses. Likewise, LY294002, a PI3-kinase inhibitor, and genistein, a tyrosine kinase inhibitor, caused parallel inhibition of O2- generation and PtdIns(3,4,5)P3 accumulation; in contrast, radicicol, which inhibits receptor-mediated activation of p85 PI3-kinase, had no effect on either response. Despite major increases in PI3-kinase activity observed in p85 and anti-phosphotyrosine immunoprecipitates in growth factor-stimulated smooth muscle cells, no such increase was observed in primed/stimulated neutrophils. In contrast, both fMLP and TNF-alpha alone caused a 3-fold increase in PI3-kinase activity in p110gamma PI3-kinase immunoprecipitates. p21(ras) activation (an upstream regulator of PI3-kinase) was unaffected by priming. These data demonstrate that timing and magnitude of PtdIns(3,4,5)P3 accumulation in neutrophils correlate closely with O2- generation, that PI3-kinase-gamma is responsible for the enhanced PtdIns(3,4,5)P3 production seen in primed cells, and that factors other than activation of p21(ras) underlie this response.

Proteolytic action of duodenase is required to induce DNA synthesis in pulmonary artery fibroblasts.

Duodenase is a 29-kDa serine endopeptidase that displays selective trypsin- and chymotrypsin-like substrate specificity. This enzyme has been localized to epitheliocytes of Brunner's glands, and as described here, to mast cells within the intestinal mucosa and lungworm-infected lung, implying an important additional role in inflammation and tissue remodelling. In primary cultures of pulmonary artery fibroblasts, duodenase induced a concentration-dependent increase in [3H]thymidine incorporation with a maximal effect observed at 30 nm. Pretreating duodenase with soybean trypsin inhibitor abolished DNA synthesis, confirming that proteolytic activity was an essential requirement for this response. PAR1, PAR2 and PAR4 activating peptides were unable to induce [3H]thymidine incorporation in pulmonary artery fibroblasts. Likewise, pretreatment of fibroblasts with TNFalpha, known to up-regulate PAR2 expression in other systems, and IL-1beta, did not enhance the potential of duodenase to induce DNA synthesis. Furthermore, duodenase increased GTPgammaS binding to fibroblast membranes indicating that a G-protein-coupled receptor may mediate the effects of duodenase. Duodenase-induced DNA synthesis and GTPgammaS binding were both found to be inhibited by pertussis toxin, implying a role for Gi/o. Selective inhibitors of MEK1 (PD98059) and protein kinase C (GF109203X) only partially inhibited duodenase-induced DNA synthesis, but both wortmannin (100 nm) and LY294002 (10 microm) inhibited this response completely, indicating a key role for PtdIns 3-kinase. Furthermore, duodenase induced a 2.3 plus minus 0.1-fold increase in PtdIns 3-kinase activity in p85 immunoprecipitates, which was sensitive to inhibition by wortmannin. These results suggest that duodenase can induce pulmonary artery fibroblast DNA synthesis in a PtdIns 3-kinase-dependent manner via a G-protein-coupled receptor which is activated by a proteolytic mechanism.