Although reversible airway narrowing leading to wheeze, cough and shortness of breath is a hallmark of asthma, patients with longstanding and severe disease can develop fixed airways obstruction that is associated with structural changes within the airway wall. The most prominent feature of the remodelled airway is an increase in airways smooth muscle (ASM). Heard and Hossain [2] demonstrated a threefold increase in both the cross-sectional area and number of smooth-muscle cells found within the bronchial wall of patients with fatal asthma in comparison with those dying from non-respiratory conditions. Subsequent mathematical modelling suggests that this characteristic increase in smooth-muscle bulk is the major cause of narrowing of airways in such patients. Furthermore, excessive ASM DNA synthesis has been demonstrated in at least two animal models of airways disease, including that associated with antigen challenge [3,4].

Although it has long been recognised that the signalling pathways based on extracellular signal-related kinase (ERK) and protein kinase C are involved in the mitogenic response of ASM, the importance of PI-3K has only recently been demonstrated. Scott et al [5] first showed that PI-3K activity was proportional to the mitogenic response in bovine ASM cells in culture and that the inhibition of PI-3K by wortmannin substantially (more than 90%) decreased DNA synthesis. They also provided data implicating p70^S6Kas the probable downstream mediator of these effects. p70^S6K is known to be activated by PI-3K (via PDK1 and possibly PKB/AKT and/or RAC) and is now known to be essential for the progression of cells from G₁ to S phase in the cell cycle. Further studies [6] demonstrated a rapid activation of PI-3K and accumulation of PtdIns(3,4,5) P₃ after stimulation of bovine ASM by thrombin and platelet-derived growth factor (PDGF), and showed that the magnitude of these effects was closely correlated with the mitogenic potential of these two growth factors. Krymskaya et al [7*] confirmed the requirement for PI-3K activity in human ASM cell mitogenesis, and again implicated p70^S6K as an important mediator in this response. Rac 1 has also now been shown to be important as a downstream mediator of the PI-3K mitogenic effect [8], acting via the induction of cyclin D, which is required for cell cycle progression.

PI-3K activity has been shown to be critical for the integrin-mediated invasive behaviour of breast and colon carcinoma cell lines [9], and the proto-oncogene PKB/AKT [recruited by PtdIns(3,4,5) P₃ to the plasma membrane and phosphorylated by the PI-3K-dependent PDK1] has been demonstrated to be overexpressed in ovarian, breast and pancreatic cancer. Small cell lung cancer (SCLC) is the most aggressive and invasive form of lung cancer, with a highly metastatic phenotype and a 5-year survival of only 3-8%. Interest in the role of PI-3K in the malignant potential of this disease was stimulated by the observation [10] that p70^S6K is constitutively phosphorylated and activated in SCLC cells, and that rapamycin, which inhibits the activation of p70^S6K, blocks SCLC proliferation. These observations were extended by Moore et al [11**], who demonstrated a high constitutive activity of PI-3K, PKB/AKT and p70^S6K in SCLC cell lines and showed that the proliferation of SCLC cells in liquid culture was inhibited by the PI-3K inhibitor LY294002. This inhibition resulted from a combination of decreased mitogenesis and enhanced apoptosis (see below). PI-3K inhibition also decreased SCLC cell colony formation in semi-solid media. Thus the high constitutive activity of PI-3K in these cells seems to promote growth and also anchorage-independence, contributing to the highly aggressive nature of this tumour. These observations have not yet been extended to other human lung cancer cell types; further developments are awaited.

Although not resident pulmonary cells, substantial numbers of neutrophils are recruited to the lungs in many respiratory disease states and have a critical role in the pathogenesis of the acute respiratory distress syndrome (ARDS), pulmonary fibrosis, bronchiectasis and fatal asthma. Neutrophils cause tissue damage by their capacity to release toxic oxygen radicals (generated by the NADPH oxidase complex), the exocytosis of granules containing highly histotoxic compounds such as elastase and collagenases, and the elaboration and release of additional pro-inflammatory cytokines. PI-3Ks have been shown to be key regulators of both neutrophil recruitment and activation. In mice lacking the catalytic subunit of the myeloid restricted PI-3K-γ, neutrophil migration to the inflamed peritoneum was severely compromised [12**, 13**] and, although not examined directly, a similar defect in granulocyte recruitment to the lungs is likely. The accumulation of PtdIns(3,4,5) P₃ also seems to be correlated precisely with respiratory burst activity, in that neutrophil priming by agents such as tumour necrosis factor-α markedly enhanced both the size and the duration of the release of superoxide anions and the accumulation of PtdIns(3,4,5) P₃ [14]. In these cells PI-3K inhibitors abolish the production of oxygen radicals induced by physiological agonists; neutrophils from PI-3K-γ knockout mice exhibit a diminished respiratory burst, with residual activity most probably attributable to the action of remaining Class IA PI-3K. The signalling intermediates linking PtdIns(3,4,5) P₃ to activation of the oxidase are uncertain but most probably include the small GTPase Rac 2, which is both highly expressed in neutrophils and an essential component of the NADPH oxidase complex. The role of PI-3K in granule exocytosis is less clearly delineated as high concentrations of wortmannin only partialy inhibit this process, indicating that inputs from other signalling pathways might impinge on this response.

Like neutrophils, eosinophils are non-resident pulmonary cells that accumulate in the bronchial tree and lung parenchyma in a number of disease states, including asthma and eosinophilic pneumonia. Toxic eosinophil-derived mediators such as eosinophil cationic protein, major basic protein and oxygen radicals (again products of the NADPH oxidase) can damage the airway epithelium and are thought to contribute significantly to airway hyper-responsiveness. In non-allergic subjects, eosinophils are scarce in peripheral blood and have therefore proved more difficult than neutrophils to study. Despite this, IL-5-stimulated eosinophil release from bone marrow has been shown to be inhibited by both wortmannin and LY294002 [15]; the migration of eosinophils to a number of chemoattractants also seems to be sensitive to wortmannin [16]. The role of PI-3Ks in eosinophil degranulation is not known, but these enzymes are required for activation of the eosinophil NADPH oxidase complex [17].

Alveolar macrophages undertake a number of key host defence functions within the lung. These include the phagocytosis of inhaled particles and respiratory pathogens, antigen presentation, and the generation of inflammatory cytokines. Additionally, they might be important in the resolution of acute inflammation by the ingestion of apoptotic neutrophils. So far, although few studies have addressed the role of PI-3Ks in the alveolar macrophage, such data are available for monocyte-derived macrophages, macrophage cell lines and murine peritoneal macrophages. If we extrapolate these results to alveolar macrophages, it seems highly likely that PI-3Ks will again be shown to have a critical role in the response profile of these cells. Hence, murine PI-3K-γ -null macrophages show decreased migration towards a variety of chemotactic agents, and greatly diminished recruitment to the inflamed peritoneum [13**]. The induction of cytokine gene expression in monocytes stimulated by formylated peptide has also been shown to be sensitive to PI-3K inhibition [18]. Most importantly, the consequences of excessive PI-3K activation have also been explored; mice deficient in SHIP (SH2-containing inositol-5-phosphatase), an enzyme that hydrolyses PtdIns(3,4,5) P₃, suffer from lethal infiltration of the lungs by myeloid cells, principally macrophages [19]. A remarkably similar phenotype is seen in mice with a deletion of the tyrosine phosphatase SHP-1 (Src homology 2 domain phosphatase-1); macrophages from these mice display a 10-15-fold increase in the 3-phosphorylated products of PI-3K, with enhanced integrin-dependent adhesive properties [20*]. Thus, as with neutrophils and eosinophils, it seems that one or more of the PI-3Ks is required for macrophage recruitment to the lung and for at least a subset of activation responses.

Although comparatively little is known about the function of the PI-3Ks in other pulmonary cells, several reports have emerged and indicate the global importance of this signalling pathway in other settings in the lung. For example, Liu et al [21] have demonstrated that PI-3K is a downstream mediator of PDGF-stimulated glycosaminoglycan synthesis in rat foetal lung fibroblasts, suggesting a role in the maintenance of the lung extracellular matrix. Similarly, PI-3K has been reported to mediate lung epithelial cell differentiation and surfactant protein expression fibroblast induced by growth factor-2 [22], although subsequent reports have suggested that PI-3K inhibits surfactant secretion from type II alveolar cells [23]. Future work will doubtless help to clarify the role of PI-3Ks in the differentiation and function of pulmonary epithelial cells.