Sandra Brunelleschi

Tachykinins activate guinea-pig alveolar macrophages: involvement of NK2 and NK1 receptors.

1 The effects of substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) were evaluated on superoxide anion () production by guinea‐pig alveolar macrophages (AM). 2 SP dose‐dependently (ED50 = 0.7 nm) evoked production from guinea‐pig AM; the N‐terminal heptapeptide, SP(1–7), was ineffective. In the presence of thiorphan (10−5 m), an enkephalinase inhibitor, the stimulating effects of SP were not significantly modified. NKA and NKB were both able to induce production from guinea‐pig AM, ED50 values being 0.1 and 1.3 nm, respectively. Therefore, the rank order of activity of natural tachykinins was NKA > SP > NKB. Tachykinin‐evoked effects were quantitatively similar to those elicited by the autacoid mediator PAF‐acether and less than those induced by the synthetic peptide N‐formylmethionyl‐leucyl‐phenylalanine (FMLP). 3 The NK2 receptor agonist [β‐Ala8]‐NKA (4–10) dose‐dependently evoked production from guinea‐pig AM; the NK1 receptor agonist [Pro9]‐SP sulphone acted only at high concentrations, while the NK3 receptor agonist [Me, Phe7]‐NKB was ineffective. 4 These findings indicate that guinea‐pig AM possess NK2 and possibly some NK1 tachykinin receptors and further suggest tachykinin involvement in lung pathophysiology.

Expression of functional NK1 receptors in human alveolar macrophages: superoxide anion production, cytokine release and involvement of NF‐κB pathway

1 Substance P (SP) is deeply involved in lung pathophysiology and plays a key role in the modulation of inflammatory‐immune processes. We previously demonstrated that SP activates guinea‐pig alveolar macrophages (AMs) and human monocytes, but a careful examination of its effects on human AMs is still scarce. 2 This study was undertaken to establish the role of SP in human AM isolated from healthy smokers and non‐smokers, by evaluating the presence of tachykinin NK1 receptors (NK‐1R) and SPs ability to induce superoxide anion (O2−) production and cytokine release, as well as activation of the nuclear factor‐κB (NF‐κB) pathway. 3 By Western blot analysis and immunofluorescence, we demonstrate that authentic NK‐1R are present on human AMs, a three‐fold enhanced expression being observed in healthy smokers. These NK‐1R are functional, as SP and NK1 agonists dose‐dependently induce O2− production and cytokine release. In AMs from healthy smokers, SP evokes an enhanced respiratory burst and a significantly increased release of tumor necrosis factor‐α as compared to healthy non‐smokers, but has inconsistent effects on IL‐10 release. The NK1 selective antagonist CP 96,345 ((2S,3S)‐cis‐2‐diphenylmethyl‐N[(2‐methoxyphenyl)‐methyl]‐1‐azabicyclo‐octan‐3‐amine)) competitively antagonized SP‐induced effects. 4 SP activates the transcription factor NF‐κB, a three‐fold increased nuclear translocation being observed in AMs from healthy smokers. This effect is receptor‐mediated, as it is reproduced by the NK1 selective agonist [Sar9Met(O2)11]SP and reverted by CP 96,345. 5 These results clearly indicate that human AMs possess functional NK‐1R on their surface, which are upregulated in healthy smokers, providing new insights on the mechanisms involved in tobacco smoke toxicity.

Macrophage Stimulating Protein (MSP) evokes superoxide anion production by human macrophages of different origin

Macrophage Stimulating Protein (MSP), a serum factor related to Hepatocyte Growth Factor, was originally discovered to stimulate chemotaxis of murine resident peritoneal macrophages. MSP is the ligand for Ron, a member of the Met subfamily of tyrosine kinase receptors. The effects of MSP on human macrophages and the role played in human pathophysiology have long been elusive. We show here that human recombinant MSP (hrMSP) evokes a dose‐dependent superoxide anion production in human alveolar and peritoneal macrophages as well as in monocyte‐derived macrophages, but not in circulating human monocytes. Consistently, the mature Ron protein is expressed by the MSP responsive cells but not by the unresponsive monocytes. The respiratory burst evoked by hrMSP is quantitatively higher than the one induced by N‐formylmethionyl‐leucyl‐phenylalanine and similar to phorbol myristate acetate‐evoked one. To investigate the mechanisms involved in NADPH oxidase activation, leading to superoxide anion production, different signal transduction inhibitors were used. By using the non selective tyrosine kinase inhibitor genistein, the selective c‐Src inhibitor PP1, the tyrosine phosphatase inhibitor sodium orthovanadate, the phosphatidylinositol 3‐kinase inhibitor wortmannin, the p38 inhibitor SB203580, the MEK inhibitor PD098059, we demonstrate that hrMSP‐evoked superoxide production is mediated by tyrosine kinase activity, requires the activation of Src but not of PI 3‐kinase. We also show that MAP kinase and p38 signalling pathways are involved. These results clearly indicate that hrMSP induces the respiratory burst in human macrophages but not in monocytes, suggesting for the MSP/Ron complex a role of activator as well as of possible marker for human mature macrophages.

Neutrophils as mediators of human skeletal muscle ischemia-reperfusion syndrome

Nine patients with aortic aneurysm undergoing arterial reconstruction with temporary aortic occlusion were studied. Since a typical condition of ischemia-reperfusion of the muscles of the lower limbs was created during this surgery, muscle biopsies from the right femoral quadriceps as well as blood samples from the homolateral saphenous vein were taken: (1) before clamping of the aorta, (2) just before declamping, and (3) 30 minutes after reperfusion. Light microscopy revealed a consistent granulocyte infiltration in the ischemic and reperfused skeletal muscle. Ultrastructural damage to the muscle fibers was seen during ischemia and became more severe upon reperfusion. The recruitment of granulocytes into the muscle tissue paralleled the activation of the blood complement system and an increase in circulating neutrophils. Although a spontaneous superoxide anion (O2-) generation from such granulocytes cannot be proved, upon stimulation with formyl-methionyl-leucyl-phenylalanine neutrophils showed a reduced ability in O2 free radical production at the end of ischemia and enhanced O2- generation at reperfusion as compared with the controls. All these findings indicate an active role of granulocytes in the genesis of reperfusion-induced tissue injuries.

On the role of autophagy in human diseases: a gender perspective

•  Introduction •  Autophagy and disease ‐  Cancer ‐  Autophagy and cancer ‐  Gender differences in cancer ‐  Autophagy, gender disparity and cancer ‐  Neurodegenerative diseases ‐  Gender differences in neurodegenerative diseases ‐  Role of autophagy in neurodegenerative diseases ‐  Gender differences and hormones ‐  Autoimmune and inflammatory diseases ‐  Gender differences in autoimmune diseases ‐  Autophagy and Autoimmunity ‐  Implication of autophagy in AID: paradigmatic examples ‐  The involvement of hormones ‐  Cardiovascular diseases ‐  Gender disparity in cardiovascular diseases ‐  Autophagy and heart diseases ‐  Hormones, gender and cardiovascular diseases ‐  Autophagy and cell sex differences •  Concluding remarks

Rhein: an anthraquinone that modulates superoxide anion production from human neutrophils.

Rhein (4,5‐dihydroxyanthraquinone‐2‐carboxylic acid), the active metabolite of diacetylrhein, which has been reported as an effective antirheumatic drug in man, inhibited superoxide anion production from human neutrophils challenged with N‐formylmethionyl‐leucyl‐phenylalanine (FMLP: IC50, 2 times 10−5 M) and A23186 (IC50, 10−5M), but not with phorbol myristate acetate. In the same concentration range (10−6‐10−3M), the drug did not affect oxy‐radical production by a cell‐free hypoxanthine‐xanthine oxidase system and exerted weak inhibitory effects on FMLP‐evoked lysosomal enzyme release. Rhein inhibitory effects on neutrophil functioning may contribute to the overall therapeutic activity of the parent drug, diacetylrhein.

Receptor tyrosine kinases as target for anti-cancer therapy.

Receptor tyrosine kinases (RTKs) are cell surface transmembrane proteins responsible for intracellular signal transduction. They are expressed in several cell types and, after activation by growth factor binding, trigger a series of intracellular pathways, leading to a wide variety of cell responses (e.g., differentiation, proliferation, migration and invasion, angiogenesis, survival). Over-expression and/or structural alteration of RTKs family members are often associated to human cancers and tumor cells are known to use RTK transduction pathways to achieve tumor growth, angiogenesis and metastasis. Therefore, RTKs represent pivotal target in approaches of cancer therapy. A number of small molecules acting as RTK inhibitors have been synthesized by pharmaceutical companies and are under clinical trials, are being analyzed in animal models or have been successfully marketed. Ligand-dependent downregulation of RTKs is a critical step for modulating their activity and the adaptor Cbl has been indicated as the key protein involved in negative regulation of RTKs, such as EGF and HGF receptors. These data suggest novel potential pharmacological targets for the treatment of human malignancies associated to oncogenic activation of RTKs.

Role of NF-κB and PPAR-γ in lung inflammation induced by monocyte-derived microparticles

Microparticles (MP) are phospholipid vesicles shed by cells upon activation or apoptosis. Monocyte-derived MP upregulate the synthesis of proinflammatory mediators by lung epithelial cells; the molecular bases of such activity are unknown. Peroxisome proliferator-activated receptors (PPAR) have been demonstrated to be involved in the modulation of nuclear factor (NF)-&kgr;B transcriptional activity and inflammation. We investigated whether the upregulation of the synthesis of proinflammatory cytokines by human lung epithelial cells induced by monocyte/macrophage-derived MP involves NF-&kgr;B activation and is modulated by PPAR-&ggr;. MP were generated by stimulation of human monocytes/macrophages with the calcium ionophore, A23187. MP were incubated with human lung epithelial cells. NF-&kgr;B translocation was assessed by electrophoretic mobility shift assay. Interleukin (IL)-8 and monocyte chemotactic protein (MCP)-1 synthesis was assessed by ELISA and RT-PCR. Stimulation of A549 alveolar cells with monocyte/macrophage-derived MP caused an increase in NF-&kgr;B activation and IL-8 and MCP-1 synthesis that was inhibited by pre-incubation with the PPAR-&ggr; agonists, rosiglitazone and 15-deoxy-&Dgr;12,14-prostaglandin-J2. Parallel experiments with normal human bronchial epithelial cells largely confirmed the results. The effects of PPAR-&ggr; agonists were reversed by the specific antagonist, GW9662. Upregulation of the synthesis of proinflammatory mediators by human lung epithelial cells induced by monocyte/macrophage-derived MP is mediated by NF-&kgr;B activation through a PPAR-&ggr; dependent pathway.

FMLP-activated neutrophils evoke histamine release from mast cells

Human neutrophils, having been activated by the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (FMLP), evoke histamine release from rat serosal mast cells. The release is dependent on FMLP concentration and it can be inhibited by disodium cromoglycate and by a flavonoid, silymarin, which displays free radical scavenging properties. Silymarin inhibition of neutrophil-mediated histamine release is dose-dependent. These results further stress the concept of a neutrophil-mast cell interaction, which may be involved in inflammatory processes.

Priming effects of mammalian tachykinins on human neutrophils

The undecapeptide substance P (SP) is known to activate different cell types involved in inflammatory and immune processes. By evaluating primed stimulation of human neutrophils, we now demonstrate that SP (10 nM-0.1 mM) dose-dependently enhances superoxide anion production from cells stimulated by the phospholipid mediator Platelet Activating Factor (PAF). We also provide evidence that neurokinin A (NKA), which is released, as well as SP, from C fibers of sensory nerves, potentiates PAF-evoked superoxide anion generation, while neurokinin B (NKB) is ineffective.