Monocyte migration: a novel effect and signaling pathways of catestatin.

Several members of the neuropeptide family exert chemotactic actions on blood monocytes consistent with neurogenic inflammation. Furthermore, chromogranin A (CgA) containing Alzheimer plaques are characterized by extensive microglia activation and such activation induces neuronal damage. We therefore hypothesized that the catecholamine release inhibitory peptide catestatin (hCgA(352-372)) would induce directed monocyte migration. We demonstrate that catestatin dose-dependently stimulates chemotaxis of human peripheral blood monocytes, exhibiting its maximal effect at a concentration of 1 nM comparable to the established chemoattractant formylated peptide Met-Leu-Phe (fMLP). The naturally occurring catestatin variants differed in their chemotactic property insofar as that the Pro370Leu variant was even more potent than wild type, whereas the Gly364Ser variant was less effective. Specificity of this effect was shown by inhibition of catestatin-induced chemotaxis by a specific neutralizing antibody. In addition, catestatin mediated effect was blocked by dimethylsphingosine and treatment with endothelial differentiation gene (Edg)-1 and Edg-3 antisense RNA as well as by incubation with pertussis toxin and genistein indicating involvement of tyrosine kinase receptor-, G-protein- and sphingosine-1-phosphate signaling. Catestatin also stimulated Akt- and extracellular signal related kinase (ERK)-phosphorylation and catestatin-induced chemotaxis was blocked by blockers of phosphoinositide-3 (PI-3) kinase and nitric oxide as well as by inhibition of the mitogen-activated protein kinases (MAPK) system indicating involvement of these signal transduction pathways. In summary, our data indicate that catestatin induces monocyte chemotaxis by activation of a variety of signal transduction pathways suggesting a role of this peptide as an inflammatory cytokine.

Syndecan-1 is involved in osteoprotegerin-induced chemotaxis in human peripheral blood monocytes.

Chronic inflammation is characterized by tissue infiltration with monocytes/macrophages, which possess broad proinflammatory, destructive, and remodeling capacities. Elevated levels of osteoprotegerin, an important regulator of differentiation and activation of osteoclasts that also affects different cells of the immune system, were found in the serum of patients with chronic inflammatory diseases. The study of whether osteoprotegerin affects monocyte locomotion in vitro and the possible mechanisms and pathways involved was investigated using Boyden microchemotaxis chambers and Western blot analyses. Osteoprotegerin significantly stimulated monocyte chemotaxis, whereas preincubation of monocytes with osteoprotegerin inhibited monocyte migration toward optimal concentrations of regulated upon activation normal T cell expressed and secreted, monocyte chemotactic protein -1, and procalcitonin. The effects of osteoprotegerin were abolished by pretreating cells with heparinase I and chondroitinase or antibodies against the ectodomain of syndecan-1. Osteoprotegerin signaling was shown to involve protein kinase C, phosphatidylinositol 3-kinase/Akt, and tyrosine kinase. Data suggest that osteoprotegerin affects monocyte mi-gration and protein kinase C and phosphatidylinositol 3-kinase/Akt activation via syndecan-1. Osteoprotegerin-induced deactivation of monocyte chemotaxis toward different chemokines is due to interaction of osteoprotegerin with heparan sulfate and chondroitin sulfate.

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 Alzheimer's senile plaques, a hallmark of the innate immune response to beta-amyloid fibrils, can initiate and propagate neurodegeneration characteristic of Alzheimer's disease. Phagocytes migrate toward amyloid beta-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 beta-protein- and amyloid beta-precursor protein-induced migration was abrogated by dimethylsphingosine, a sphingosine kinase inhibitor. Amyloid beta-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 beta-protein-induced migration. These data demonstrate that the migratory effects of beta-amyloid in human monocytes involve spingosine-1-phosphate signaling. Whereas endogenous neuropeptides may arrest and activate monocytes at sites of high beta-amyloid concentrations, interference with the amyloid beta-protein-dependent sphingosine-1-phosphate pathway in monocytes by FTY720, a novel immunomodulatory drug, suggests that FTY720 may be efficacious in beta-amyloid-related inflammatory diseases.

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.

Modulation of inflammation by vasoactive intestinal peptide and bombesin: lack of effects on neutrophil apoptosis.

Inhibition of neutrophil apoptosis has been identified as a prominent feature in chronic inflammation, parenchymal damage, and unresolved organ dysfunction. Lung injury animal models suggest that the neuropeptides vasoactive intestinal peptide and bombesin are protective. Therefore, in vitro effects of VIP and bombesin on apoptosis of normal human neutrophils were tested. For measuring effects on cell survival and apoptosis, trypan dye exclusion, colorimetric MTT assay to assess cell survival, and caspase-3 assay and annexin-V binding for analysing apoptosis rates were used. Foetal calf serum, Fas ligand, and tumour necrosis factor-alpha served as modulatory control agents; survival-promoting and apoptosis-inducing activities of the respective agents were confirmed. Vasoactive intestinal peptide and bombesin, however, failed to significantly affect cell death in neutrophils. Data suggest that direct regulation of neutrophil apoptosis is unlikely to be among the mechanisms of lung-protective actions of VIP and bombesin.