Anti-inflammatory therapy in asthma is reliant on corticosteroids, particularly in their inhaled form. However, steroids are rather non-specific in their actions and they also raise concerns regarding compliance and side-effect Issues. Furthermore, a small proportion of patients with asthma fail to respond to oral glucocorticoids even at high doses. This Article will review the role that steroids and membrane receptor ligation play in the induction of eosinophil apoptosis together with the mechanisms by which corticosteroids enhance the disposal of apoptotic eosinophils by both professional and non-professional phagocytes. Eosinophils are thought to be the major pro-inflammatory effector cell in asthma and their persistence in the airways is probably enhanced by the presence of several asthma-relevant cytokines that prolong eosinophil survival by inhibition of apoptosis (interleukin (IL)-3, IL-5, granulocyte-macrophage colony-stimulating factor, IL-9, IL-13, IL-15). In contrast, a number of signals have been described that accelerate apoptosis in human eosinophils including corticosteroids or ligation of membrane receptors (CD95, CD45, CD69). Thus, the load of lung eosinophils in asthmatic disease is likely to be related to a balance in the tIssue microenvironment between pro- and anti-apoptotic signals. Furthermore, removal of apoptotic eosinophils by phagocytosis by alveolar macrophages or bronchial epithelial cells in a specific receptor-mediated way is as important as the process of apoptosis induction. Corticosteroids enhance the recognition and engulfment of apoptotic eosinophils by macrophages or bronchial epithelial cells. Caspases are key intracellular molecules in the control of apoptosis and defects in caspase-induced apoptosis in eosinophils from steroid-resistant individuals may contribute to the molecular mechanisms underlying glucocorticoid insensitivity in these cells. These findings point the way to new and more targeted anti-inflammatory therapy for asthma and may provide important clues for the development of alternative therapies for glucocorticoid resistance.

We have studied changes in the IGF axis in an ovine model of myocardial infarction (MI), in order to determine the relationship between time-based changes in post-infarct myocardium and IGF levels. IGF localization was studied by immunocytochemistry, production by in situ hybridization, and specific binding by radioligand studies. In surviving tissue, IGF-I peptide localized to cardiomyocytes, with strongest immunostaining at 1 and 2 days post-infarct in the immediate border area adjoining the infarct, where IGF-I mRNA also increased, reaching a maximum at 2 days. Binding of radiolabelled IGF-I in surviving tissue was initially lower than that seen in cardiomyocytes in control myocardium, subsequently increasing to become significantly greater by 6 days post-infarct. In necrotic tissue, IGF-I peptide was still detectable in cardiomyocytes at 0.5 days post-infarct, but had cleared from this area by 1 day, becoming detectable again at 6 days post-infarct in macrophages and fibroblasts infiltrating the repair zone. IGF-I mRNA was not detected in necrotic tissue until 6 days, when probe hybridized to macrophages and fibroblasts. Within the necrotic zone, high levels of radiolabelled IGF-I binding to a combination of receptors and binding proteins were observed in cardiomyocytes in islands of viable tissue located close to the border. Weak immunostaining for IGF-II was observed in cardiomyocytes of the surviving tissue. IGF-II mRNA was not detected in either surviving or necrotic areas. Binding of radiolabelled IGF-II was predominantly to macrophages in both surviving and infarct areas, although as with IGF-I, high levels of binding of radiolabelled IGF-II to a combination of receptors and binding proteins were observed in islands of viable tissue close to the border within the necrotic area. We conclude that, following MI, surviving cardiomyocytes at the infarct border show marked changes in IGF-I localization, production, and specific binding, indicating that the IGF axis is directly involved in post-infarct events, possibly in the maintenance of cardiac function by the induction of hypertrophy and in cell survival by decreasing apoptotic cell death, which has been demonstrated in other cell types.

Preovulatory surges of both prolactin (PRL) and progesterone have been suggested to be necessary for the induction of apoptosis in the regressing corpus luteum of the cyclic rat. The aim of these experiments was to study whether the administration of PRL and/or progesterone on the morning of pro-oestrus reproduces the regressive changes that happen in the cyclic corpus luteum (CL) during the transition from pro-oestrus to oestrus, and to analyse the temporal relationships between two characteristic features of structural luteolysis (luteal cell apoptosis and accumulation of macrophages). Cyclic rats (treated at 0900 h with an LHRH antagonist to block LH secretion) were injected at 1000 h with PRL and progesterone and killed at 0, 30, 60, 90 and 180 min after treatment. The number of apoptotic cells increased progressively from 60 min after treatment onward in hormone-treated rats, whereas the number of macrophages did not change throughout the period of time considered. Rats injected with PRL plus progesterone showed significantly greater numbers of apoptotic cells than those injected with PRL alone. The luteolytic effects of progesterone were in keeping with the presence of luteal endothelial cells showing progesterone receptor (PR) immunoreactivity in pro-oestrus. Treatment of rats during dioestrus and pro-oestrus with the specific antioestrogens LY117018 and RU58668 decreased the luteolytic effects of PRL and progesterone and the number of luteal endothelial cells immunostained for PR. These results strongly suggest that the preovulatory PRL surge and the preovulatory increase in progesterone together trigger structural regression of the corpus luteum. This seems to be dependent on oestrogen-driven cyclic changes in PRs in luteal endothelial cells.

Macrophage inflammatory protein-1alpha (MIP-1alpha) is a member of the CC chemokines. We have previously reported the use of a whole bone marrow culture system to show that MIP-1alpha stimulates the formation of osteoclast-like multinucleated cells. Here we use rat bone marrow cells deprived of stromal cells, and clones obtained from murine macrophage-like cell line RAW264 to show that MIP-1alpha acts directly on cells in osteoclast lineage. We obtained several types of RAW264 cell clones, one of these clones, designated as RAW264 cell D clone (D clone), showed an extremely high response to receptor activator of NFkappaB ligand (RANKL) and tumor necrosis factor-alpha (TNF-alpha), while the other clone, RAW264 cell N clone (N clone), demonstrated no response to RANKL or TNF-alpha. Although both clones expressed receptor activator NFkappaB (RANK) before being stimulated for differentiation, only the D clone expressed cathepsin K when cells were stimulated to differentiate to osteoclasts. MIP-1alpha stimulated the formation of mononuclear preosteoclast-like cells from rat bone marrow cells deprived of stromal cells. MIP-1alpha also stimulated formation of osteoclast-like multinucleated cells from the D clone, when these cells were stimulated with RANKL and TNF-alpha. These findings provide strong evidence to show that MIP-1alpha acts directly on cells in the osteoclast lineage to stimulate osteoclastogenesis. Furthermore, pretreatment of RAW264 cell D clone with MIP-1alpha significantly induced adhesion properties of these cells to primary osteoblasts, suggesting a crucial role for MIP-1alpha in the regulation of the interaction between osteoclast precursors and osteoblasts in osteoclastogenesis.

An inflammatory process may be involved in nitric oxide production in skeletal muscle of type 2 diabetic patients. Nitric oxide generation in skeletal muscle was assessed in 14 non-complicated type 2 diabetic patients and in 12 healthy subjects. In samples of quadriceps femoris muscle, endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), nitrite, nitrate and nitrotyrosine were determined. The macrophage-specific antigen CD163, the T-cell membrane factor CD154 and tumour necrosis factor-alpha (TNF-alpha) were also assayed. In six patients, ultrastructural analysis of muscle was performed. Nitrites and nitrates were increased in patients as compared to controls (22.7+/-4.5 and 32.7+/-7.0 vs 16.0+/-2.9 and 22.8+/-4.0 micromol/mg protein; P<0.001, Mann-Whitney U test). Endothelial NOS was similar in diabetic and control subjects (36.4+/-13.8 vs 36.3+/-6.8 ng/mg protein), contrasting with the significant increase of iNOS recorded in patients (34.3+/-13.0 vs 8.5+/-2.8 ng/mg protein, P<0.00002). Nitrotyrosine levels were higher in the patient than in the control group (42.1+/-24.4 vs 10.3+/-2.5 ng/mg protein, P<0.00002), as were CD163 (10-fold) and TNF-alpha (fourfold) levels. Furthermore, CD154 levels were detectable only in the patient samples (10.2+/-5.3 ng/mg protein). By multiple-regression analysis, changes in glycated haemoglobin values could predict 96% variation in nitrotyrosine. Macrophages were present in all muscle samples analysed by electromicroscopy. The increased levels of CD163, CD154 and TNF-alpha indicate that an inflammatory process occurs in skeletal muscle of type 2 diabetic patients. This may contribute to iNOS induction, muscle damage and insulin resistance.

Total activity of nitric oxide (NO) synthase (NOS) and expression of both endothelial (eNOS) and inducible (iNOS) isoforms were examined in corpora lutea (CL) of rabbits across pseudopregnancy by quantitative RT-PCR analysis, Western blot and immunohistochemistry. CL were collected at early- (day 4), mid- (day 9) and late- (day 13) luteal phases of pseudopregnancy. The PCR product of rabbit luteal eNOS was cloned and its direct sequence exhibited 90% homology with those of other species. The steady-state mRNA levels encoding eNOS remained fairly constant throughout both early- and mid-luteal stages of pseudopregnancy but dropped almost to half (P</=0.05) by day 13. By contrast, luteal eNOS proteins increased 2-fold (P</=0.05) from the early- to late-luteal phase. Independently of CL age, iNOS mRNA was very poorly expressed while protein levels gradually declined from the early- to late-luteal stage. Intense eNOS-like immunoreactivity was detected in large luteal cells, while iNOS staining was targeted to a few, isolated cells, probably macrophages. Basal NOS activity was greater in day 4 CL than in both day 9 and day 13 CL. These data are the first to characterize in rabbit CL the temporal expression patterns of NOS isoforms across different luteal stages of pseudopregnancy and, collectively, suggest the existence of an expressional control for this constitutive isoform, which might have a physiological role in regulating CL function during development.

Chronic corticosteroid treatment is known to induce bone loss and osteoporosis. Osteoclasts are specialised bone-resorbing cells that are formed from mononuclear phagocyte precursors that circulate in the monocyte fraction. In this study we have examined the effect of the synthetic glucocorticoid, dexamethasone, on human osteoclast formation and bone-resorbing activity. Human monocytes were cultured for up to 21 days on glass coverslips and dentine slices, with soluble receptor activator for nuclear factor kappaB ligand (RANKL; 30 ng/ml) and human macrophage-colony stimulating factor (M-CSF; 25 ng/ml) in the presence and absence of dexamethasone (10(-8) M). The addition of dexamethasone over a period of 7 and 14 days of culture of monocytes (during which cell proliferation and differentiation predominantly occurred) resulted in a marked increase in the formation of tartrate-resistant acid phosphatase-positive multinucleated cells and an increase in lacunar resorption. The addition of dexamethasone to monocyte cultures after 14 days (when resorptive activity of osteoclasts had commenced) reduced the extent of lacunar resorption compared with cultures to which no dexamethasone had been added. The addition of dexamethasone to osteoclasts isolated from giant cell tumours of bone significantly inhibited resorption pit formation. Our findings indicate that dexamethasone has a direct effect on osteoclast formation and activity, stimulating the proliferation and differentiation of human osteoclast precursors and inhibiting the bone-resorbing activity of mature osteoclasts.

Angiogenesis and vascular transformation are important processes in the normal development of the placenta. Vascular endothelial growth factor (VEGF) is a potent angiogenic growth factor and is thought to be important for placental development. Recently several new members of this family have been described. In this study we used in situ hybridisation to localise which cells in the placenta expressed mRNA for VEGF, placenta growth factor (PlGF), VEGF-B and VEGF-C. We were unable to find any message for either VEGF-B or VEGF-C in the placenta, suggesting that only low levels are produced which this method was unable to detect. The mRNA encoding VEGF was found to be produced by cells within the villous mesenchyme, decidual macrophages and decidual glands but, in contrast to our previous findings, not by trophoblast. The mRNA encoding PlGF was produced in large amounts by villous cytotrophoblast, syncytiotrophoblast and extravillous trophoblast. The mRNAs encoding VEGF and PlGF were thus not co-localised and it appears that there is unlikely to be any significant production of VEGF/PlGF heterodimer in the placenta.

Peroxisome proliferator-activated receptor gamma (PPARgamma) colocalizes with oxidized low-density lipoprotein (LDL) in foam cells in atherosclerotic lesions. We have explored a potential role of oxidized fatty acids in LDL as PPARgamma activators. LDL from patients suffering from intermittent claudication due to atherosclerosis was analyzed using HPLC and gas chromatography/mass spectrophotometry and found to contain 9-hydroxy and 13-hydroxyoctadecadienoic acid (9- and 13-HODE), as well as 5-hydroxy-, 12-hydroxy- and 15-hydroxyeicosatetraenoic acid (5-, 12- and 15-HETE respectively). PPARgamma was potently activated by 13(S)-HODE and 15(S)-HETE, as judged by transient transfection assays in macrophages or CV-1 cells. 5(S)- and 12(S)-HETE as well as 15-deoxy-Delta(12,14)-prostaglandin J(2) also activated PPARgamma but were less potent. Interestingly, the effect of the lipoxygenase products 13(S)-HODE and 15(S)-HETE as well as of the drug rosiglitazone were preferentially enhanced by the coactivator CREB-binding protein, whereas the effect of the cyclooxygenase product 15-deoxy-Delta(12,14)-prostaglandin J(2) was preferentially enhanced by steroid receptor coactivator-1. We interpret these results, which may have relevance to the pathogenesis of atherosclerosis, to indicate that the lipoxygenase products on the one hand and the cyclooxygenase product on the other exert specific effects on the transcription of target genes through differential coactivator recruitment by PPARgamma/9-cis retinoic acid receptor heterodimer complexes.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear receptor superfamily. PPARalpha is highly expressed in liver, skeletal muscle, kidney, heart and the vascular wall. PPARgamma is predominantly detected in adipose tissue, intestine and macrophages. PPARs are activated by fatty-acid derivatives and pharmacological agents such as fibrates and glitazones which are specific for PPARalpha and PPARgamma respectively. PPARs regulate lipid and lipoprotein metabolism, glucose homeostasis, cell proliferation and differentiation, and apoptosis. PPARalpha controls intra- and extracellular lipid metabolisms whereas PPARgamma triggers adipocyte differentiation and promotes lipid storage. In addition, PPARs also modulate the inflammatory response. PPAR activators have been shown to exert anti-inflammatory activities in various cell types by inhibiting the expression of proinflammatory genes such as cytokines, metalloproteases and acute-phase proteins. PPARs negatively regulate the transcription of inflammatory response genes by antagonizing the AP-1, nuclear factor-kappaB (NF-kappaB), signal transducer and activator of transcription and nuclear factor of activated T-cells signalling pathways and by stimulating the catabolism of proinflammatory eicosanoids. These recent findings indicate a modulatory role for PPARs in inflammation with potential therapeutical applications in chronic inflammatory diseases.