Angela Djanani

Induction of apoptosis and inhibition of migration of inflammatory and vascular wall cells by cerivastatin.

Statins are thought to play a role in directly affecting immune and mesenchymal cells. Since cerivastatins pleiotropic effects are poorly investigated, we were interested to find out whether this drug can modulate leukocyte and vessel wall cell functions. Leukocyte migration was tested in modified Boyden microchemotaxis chambers and oxygen radical production was measured fluorometrically. Transendothelial migration experiments were performed with human umbilical vein endothelial cells and neutrophils. Neutrophil, monocyte, and vascular smooth muscle cell caspase-3 activity and annexin-V binding were quantified by FIENA and FACS, respectively. Cerivastatin [10 pM to 100 microM] decreased leukocyte chemotaxis towards interleukin-8 or RANTES. Migration of cells was completely restored by addition of mevalonic acid. In neutrophils, cerivastatin [100 microM] reduced transendothelial migration, whereas treatment of endothelial cells failed to affect transmigration. Neutrophil respiratory burst activity was unaffected by cerivastatin. At concentrations of 10 nM or higher, cerivastatin increased the rate of apoptosis in phagocytes and smooth muscle cells. Results show that cerivastatin is able to inhibit leukocyte chemotaxis, and that cerivastatin induces neutrophil, monocyte, and smooth muscle cell apoptosis. The drugs impact on transendothelial migration is due to its effects on neutrophils. In addition to its lipid-lowering effects, pharmacological properties of cerivastatin may include modulatory actions in leukocytes and mesenchymal cells.

Natural killer cell functions mediated by the neuropeptide substance P

The neuropeptide substance P (SP) can modulate a number of immunological functions in vitro and in vivo. Here, we investigated if SP boosts migration and cytotoxicity of natural killer cells, thus providing a further link between innate immunity and neurogenic inflammatory processes like asthma bronchiale. We demonstrate a dose-dependent effect of SP on natural killer cell migration with a maximal response at 10(-8) M SP. SP was shown to stimulate unstimulated as well as interleukin-2 (IL-2)-activated natural killer cells. Stimulation of natural killer cell migration was neurokinin-1 receptor dependent. Furthermore, mRNA encoding the neurokinin-1 receptor was demonstrated as being present in natural killer cells using RT-PCR while mRNA of the neurokinin-2 receptor was not detectable. Additionally, SP seems to influence specific cytotoxicity against Raji and K567 effector cells by a receptor-independent mechanism. In conclusion, our data indicate that functionally active neurokinin-1 receptors can be expressed by human natural killer cells. Substance P might therefore be a novel link between neural structures and innate immunity.

The immune modulator FTY720 targets sphingosine–kinase-dependent migration of human monocytes in response to amyloid beta-protein and its precursor

Accumulation of inflammatory mononuclear phagocytes in Alzheimers senile plaques, a hallmark of the innate immune response to β‐amyloid fibrils, can initiate and propagate neurodegeneration characteristic of Alzheimers disease. Phagocytes migrate toward amyloid β‐ protein involving formyl peptide receptor like‐1‐dependent signaling. Using human peripheral blood monocytes in Boyden chamber micropore filter assays, we show that the amyloid β‐ protein‐ and amyloid β‐precursor protein‐induced migration was abrogated by dimethylsphingosine, a sphingosine kinase inhibitor. Amyloid β‐protein stimulated in monocytes the gene expression for sphingosine‐1‐phosphate receptors 2 and 5, but not 1, 3, and 4. FTY720 that acts as a sphingosine‐1‐phosphate receptor agonist after endogenous phosphorylation by sphingosine kinase, as well as various neuropeptides that are known to be monocyte chemoattractants, dose‐dependently inhibited amyloid β‐protein‐induced migration. These data demonstrate that the migratory effects of β‐amyloid in human monocytes involve spingosine‐1‐ phosphate signaling. Whereas endogenous neuropeptides may arrest and activate monocytes at sites of high β‐amyloid concentrations, interference with the amyloid β‐protein‐dependent sphingosine‐1‐phosphate pathway in monocytes by FTY720, a novel immunomodulatory drug, suggests that FTY720 may be efficacious in β‐amyloid‐related inflammatory diseases.

Cd40-ligand-dependent induction of Cox-2 gene expression in endothelial cells by activated platelets: inhibitory effects of atorvastatin

Increasing evidence shows the importance of platelet–endothelial cell interactions in the progression of atherosclerosis. Platelets contribute to coronary events both as major components of thrombi and as a triggering factor in inflammation that leads to plaque vulnerability. Recent data suggest that statins, besides their lipid-lowering properties, exert pleiotropic effects that may be beneficial in atherosclerosis. Whether activated platelets influence cyclooxygenase-2 (COX-2) expression in human umbilical vein endothelial cells (HUVEC), the effect of atorvastatin, and possible mechanisms were investigated. COX-2 gene expression in HUVEC was studied using real-time polymerase chain reaction. CD40 ligand surface expression of platelets was tested by fluorescence-activated cell sorting analyses. Activated platelets significantly up-regulated COX-2 gene expression in HUVEC. Co-incubation of platelets with atorvastatin was shown to reverse this up-regulation via reduction of CD40 ligand surface expression on platelets. Data suggest that atorvastatin influences CD40–CD40-ligand-dependent platelet–endothelial interaction and that this influence affects platelet-induced COX-2 expression in HUVEC.

Sphingosine kinase-dependent directional migration of leukocytes in response to phorbol ester

Syndecan-4 participates in focal adhesion by non-G protein-dependent activation of protein kinase C. Ligation of syndecan-4 with antithrombin elicits pertussis toxin-sensitive chemotaxis of leukocytes. As activation of protein kinase C stimulates release of sphingosine-1-phosphate, a chemoattracting G protein-coupled receptor agonist, we studied directional migration of leukocytes in response to phorbol myristate acetate (PMA), a direct activator of protein kinase C. Human peripheral blood neutrophils, monocytes, and lymphocytes were purified and tested for chemotactic migration in micropore filter assays in response to PMA. Dose-dependent stimulation of migration was seen only when leukocytes were exposed to concentration gradients of PMA; in the absence of such a gradient, inhibition of random migration was induced. Dimethylsphingosine inhibited PMA-induced leukocyte chemotaxis, indicating that activation of sphingosine kinase for enhanced production of sphingosine-1-phosphate mediates the chemotactic response to PMA. Pertussis toxin abrogated the chemotactic response to PMA, suggesting involvement of G protein-coupled sphingosine-1-phosphate receptor. Dimethylsphingosine also inhibited leukocyte chemotaxis toward antithrombin, indicating that similar mechanisms may be involved upon syndecan-4 ligation. Data show that protein kinase C-dependent activation of sphingosine kinase may play a central role in leukocyte chemotaxis toward non-G protein-coupled receptor agonists.

Inhibition of Neutrophil Migration and Oxygen Free Radical Release by Metipranolol and Timolol

Propanolol and metoprolol exert adrenoceptor-independent effects including scavenging of free radicals and inhibition of protein kinase C leading to inhibition of leukocyte migration and radical release as a consequence. Whether topically used metipranolol and timolol exert such effects is unknown. Neutrophil chemotaxis was tested using modified Boyden microchemotaxis chambers. Respiratory burst activity of neutrophils was detected fluorometrically. Radical scavenging properties were tested using 2′,7′-dichlorofluorescein diacetate. Metipranolol and timolol inhibited neutrophil chemotaxis at doses in the micromolar range, oxygen free radical production triggered with formyl-Met-Leu-Phe was inhibited at higher concentration. Protein kinase C involvement, suggested to trigger free radical production with phorbol myristate acetate, was antagonized. A direct radical scavenging effect of the β-blockers was also seen. Inhibition of neutrophil chemotaxis and free radical production is a novel mode of action of metipranolol and timolol that may be relevant for beneficial effects in the topical treatment of eye disease.

Leukocyte motility in response to neuropeptides is heparan sulfate proteoglycan dependent

Activation of neuropeptide receptors on leukocytes induces chemotaxis. We determined in Boyden chambers with micropore filters, whether in human monocytes and lymphocytes this migratory response is heparan sulfate proteoglycan (HSPG) dependent. Chemotaxis toward calcitonin gene-related peptide, secretoneurin, vasoactive intestinal peptide (VIP), and substance P (SP) was abolished by removal of heparan sulfate side chains from cell surface proteoglycans or by addition of anti-syndecan-4 antibodies. Inhibition of neuropeptide-induced chemotaxis by dimethyl sphingosine (DMS), an inhibitor of sphingosine kinase, indicates transactivation of the sphingosine-1-phosphate chemotaxis pathway which was previously identified as being syndecan-4-related. Data suggest that HSPGs are involved in neuropeptide-induced chemotaxis of leukocytes.

Signal transduction pathways in directed migration of human monocytes induced by human growth hormone in vitro

The human growth hormone (GH) was shown to modulate leukocyte functions such as stimulating directed migration of human monocytes in vitro. Dimerisation of GH-receptors leads to the activation of various signalling mechanisms. As transduction of GH signals to monocytes is unknown, we investigated GH signalling mechanisms in monocyte migration using a modified Boyden chamber chemotaxis assay. Inhibition of tyrosyl phosphorylation of GH receptor-associated tyrosine kinase by tyrphostin-23 or staurosporine blocked GH-stimulated monocyte migration down to random levels. Furthermore, pre-incubation with effective concentrations of 4B-phorbol-12-myristate-13-acetate (PMA), staurosporine and bisindolylmaleimide I, inhibitors of protein kinase C, significantly decreased GH-induced migration, suggesting that PKC is involved in the signalling cascade. Additionally, phosphatidylinositol 3-kinase and mitogen-activated protein kinase (MAPK) activation seems to be required. This study revealed signalling pathways in monocyte movement toward GH in vitro.

Heparan Sulfate Proteoglycan–Involving Immunomodulation by Cathelicidin Antimicrobial Peptides LL-37 and PR-39

Antimicrobial peptides, which are categorized by homologous structural motifs, are effector molecules of innate immunity with direct antimicrobial activity and multiple other functions. The most prominent families are defensins and cathelicidins. These peptides are expressed in the granules of several types of leukocytes and in a wide variety of tissue types[1]. Peptide antibiotics of the cathelicidin family are characterized by a highly conserved signal sequence and proregions (“cathelin” = cathepsin L inhibitor), but show substantial heterogeneity in the carboxy-terminal domain that encodes the mature peptide[2,3,4]. Based on amino acid sequences, cathelicidin peptides are categorized in (a) linear, a-helical peptides without cysteines, e.g., LL-37/human cathelicidin antimicrobial peptide (hCAP)-18 from human; (b) peptides with an even number of cysteines linked by disulfide bridges, e.g., protegrins (porcine cathelicidin peptides); and (c) peptides with an unusually high proportion of one or two amino acids, e.g., PR-39 from porcine leukocytes (Table 1).

Agonist function of the neurokinin receptor antagonist, [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P, in monocytes.

G-protein-coupled bombesin receptors are capable of signaling through the G(i) protein even when receptor-coupling to G(q) is blocked by [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P (SpD), a neurokinin-1 receptor antagonist and biased agonist to bombesin receptors. As bombesin is a monocyte and tumor cell attractant, we were interested in the effects of SpD on cell migration. Chemotaxis of monocytes was tested in micropore filter assays. SpD was a dose-dependent agonist in monocyte migration and was not inhibited by antagonists to neurokinin-1 or -2 receptors. SpD failed to inhibit chemotaxis toward bombesin, suggesting that inhibition of bombesin receptor coupling to G(q) with SpD does not impair migratory responses elicited by bombesin. As pertussis toxin inhibited migration, coupling of receptors to G(i) may signal migration. Chemotaxis toward SpD was inhibited by bombesin receptor antagonists as well as by blocking signaling enzymes downstream of G(q) (phospholipase-3 and protein kinase C with wortmannin and bisindolylmaleimide, respectively), suggesting transactivation of G(q)-mediated chemotaxis signaling by SpD via bombesin receptors. Protein kinase C that induces sphingosine kinase activation and production of sphingosine-1-phosphate, which may lead to G(q)-dependent chemoattraction, was involved in SpD-dependent migration. Inhibition of sphingosine-1-phosphate production with dimethylsphingosine inhibited monocyte migration toward SpD. Data suggest that SpD induces migration in monocytes and signaling events involving activation of sphingosine kinase in a G(i) protein- and protein kinase C-dependent fashion. Biased agonism of SpD at bombesin receptors may affect normal and tumor cell migration.