Myristoylated alanine-rich C-kinase substrate (MARCKS) protein regulation of human neutrophil migration.

Neutrophil migration into infected tissues is essential for host defense, but products of activated neutrophils can be quite damaging to host cells. Neutrophil influx into the lung and airways and resultant inflammation characterizes diseases such as chronic obstructive pulmonary disease, bronchiectasis, and cystic fibrosis. To migrate, neutrophils must reorganize the actin cytoskeleton to establish a leading edge pseudopod and a trailing edge uropod. The actin-binding protein myristoylated alanine-rich C-kinase substrate (MARCKS) has been shown to bind and cross-link actin in a variety of cell types and to co-localize with F-actin in the leading edge lamellipodium of migrating fibroblasts. The hypothesis that MARCKS has a role in the regulation of neutrophil migration was tested using a cell-permeant peptide derived from the MARCKS myristoylated aminoterminus (MANS peptide). Treatment of isolated human neutrophils with MANS significantly inhibited both their migration and beta2 integrin-dependent adhesion in response to N-formyl-methionyl-leucyl-phenylalanine (fMLF), IL-8, or leukotriene (LT)B(4). The IC(50) for fMLF-induced migration and adhesion was 17.1 microM and 12.5 microM, respectively. MANS significantly reduced the F-actin content in neutrophils 30 seconds after fMLF stimulation, although the peptide did not alter the ability of cells to polarize or spread. MANS did not alter fMLF-induced increases in surface beta2 integrin expression. These results suggest that MARCKS, via its myristoylated aminoterminus, is a key regulator of neutrophil migration and adhesion.

p38 mitogen-activated kinase (MAPK) is essential for equine neutrophil migration.

Equine laminar tissues do not contain resident neutrophils and have less superoxide dismutase (SOD) activity than other equine tissues, which makes them inherently more vulnerable to damage induced by reactive oxygen species (ROS) produced by neutrophils that enter the tissues. In the advanced clinical stages of acute laminitis, pathologic events in affected feet include a breakdown in the basement membrane, neutrophil infiltration, and platelet-neutrophil aggregates in laminar dermal veins, highlighting the contribution of neutrophils to the pathophysiology of the disease. The aim of this study was to determine the role of p38 MAPK in the mechanism underlying equine neutrophil migration to potentially reveal therapeutic targets that may limit lamellar damage from the neutrophil influx that occurs in acute laminitis. We determined that the endogenous chemoattractant LTB(4) transiently activated p38 MAPK and induced chemotaxis of equine primary neutrophils. Inhibition with the p38 MAPK specific inhibitor SB203580 reduced LTB(4)-induced migration in a dose-dependent manner with an IC(50) of 2.8 microM. We then examined the potential mechanisms underlying the ability of SB203580 to abolish migration. We determined that inhibition of p38 MAPK with 10 microM SB203580 disrupted the ability of neutrophils to polarize in response to LTB(4) and PAF. In contrast, p38 MAPK did not appear to be required for chemoattractant- or PKC-induced beta2 integrin-dependent adhesion or chemoattractant-induced upregulation of surface beta2 integrins, but was required for TNFalpha-induced adhesion. These findings support a function for p38 MAPK in equine neutrophil migration and suggest the potential for the ability of p38 MAPK inhibition to limit neutrophilic inflammation in the laminae during acute laminitis.

Role of p38 MAPK in LPS induced pro-inflammatory cytokine and chemokine gene expression in equine leukocytes.

Endotoxemia occurs when bacterial lipopolysaccharide (LPS) in the blood induces a dysregulated inflammatory response, resulting in circulatory shock and multi-organ failure. Laminitis is a common complication in endotoxemic horses and is frequently the reason for humane euthanasia of these cases. Blood leukocytes are a principal target of LPS in endotoxemia leading to activation of multiple signal transduction pathways involved in the induction of a number of pro-inflammatory genes. In other animal models, the p38 mitogen activated protein kinase (MAPK) pathway has been associated with induced expression of tumor necrosis factor-alpha (TNFalpha), interleukin (IL)-1beta, IL-6 and IL-8. The goal of this study was to determine the role of the p38 MAPK pathway in the induction of these pro-inflammatory cytokine and chemokine genes in LPS-stimulated equine leukocytes. Stimulation of equine peripheral blood leukocytes resulted in an increase in TNFalpha, IL-1beta, IL-6 and IL-8 mRNA levels. Pharmacological inhibition of p38 MAPK activity with SB203580 or SB202190 reduced the ability of LPS stimulation to increase mRNA concentrations for all four genes. However, only SB203580 pretreatment significantly reduced LPS-stimulated IL-1beta and IL-8 mRNA expression and only pretreatment with SB202190 significantly reduced LPS-stimulated TNFalpha and IL-6 mRNA expression. From this study we conclude TNFalpha, IL-1beta, IL-6 and IL-8 are induced upon LPS stimulation of equine leukocytes and that this induction of gene expression is dependent on the p38 MAPK pathway. However, there are differences in the efficacy of the p38 inhibitors tested here that may be explained by differences in specificity or potency. This study provides evidence for the use of selective p38 MAPK inhibitors as potential therapeutics for the treatment of equine endotoxemia.

Regulation of VASP serine 157 phosphorylation in human neutrophils after stimulation by a chemoattractant.

Vasodilator-stimulated phosphoprotein (VASP) is a cAMP-dependent protein kinase A (PKA) substrate, which links cellular signaling to cytoskeletal organization and cellular movement. VASP is phosphorylated by PKA on serine 157 (Ser 157), which is required for VASP function in platelet adhesion and fibroblast motility. Our hypothesis is that PKA regulates neutrophil migration through VASP Ser 157 phosphorylation. The objective of this study was to characterize VASP Ser 157 phosphorylation in chemoattractant-stimulated neutrophils. fMLF, IL-8, leukotriene B(4), or platelet-activating factor stimulation resulted in an initial increase in VASP Ser 157 phosphorylation, which was maximal by 30 s and was followed by a return to baseline Ser 157 phosphorylation by 10 min. In contrast, stimulation with the nonchemoattractant, proinflammatory cytokine TNF-alpha did not affect Ser 157 phosphorylation. The kinetics of fMLF-induced VASP Ser 157 phosphorylation levels closely matched the kinetics of the fold-change in F-actin levels in fMLF-stimulated neutrophils. fMLF-induced Ser 157 phosphorylation was abolished by pretreatment with the PKA inhibitor H89 and the adenylyl cyclase inhibitor SQ22536. In contrast, fMLF-induced Ser 157 phosphorylation was unaffected by the PKC inhibitors calphostin and staurosporine, the PKG inhibitors Rp-8-pCPT-cGMP and KT5823, and the calmodulin-dependent protein kinase II inhibitor KN-62. Inhibition of adhesion with EDTA or the anti-beta2-integrin antibody IB4 did not alter fMLF-induced VASP phosphorylation or dephosphorylation. These data show that chemoattractant stimulation of human neutrophils induces a rapid and transient PKA-dependent VASP Ser 157 phosphorylation. Adhesion does not appear to be an important regulator of the state of VASP Ser 157 phosphorylation in chemoattractant-stimulated neutrophils.

The role of p38 mitogen-activated kinase (MAPK) in the mechanism regulating cyclooxygenase gene expression in equine leukocytes.

The goal of this study was to define the role for p38 mitogen-activated kinase (MAPK) in the signaling mechanism regulating pro-inflammatory cyclooxygenase (COX) gene expression in lipopolysaccharide (LPS)-activated equine leukocytes for the purposes of identifying novel targets for anti-inflammatory therapy in endotoxemic horses. The p38 MAPK has been shown to positively regulate inflammatory gene expression in human leukocytes and can be activated by a variety of stimuli including LPS, TNF-alpha, and IL-1. Activation-associated phosphorylated p38 MAPK has been implicated in the up-regulation of several inflammatory genes, including COX-2 which ultimately results in the production of prostanoids that are responsible for the pathophysiology associated with endotoxemia. Our hypothesis is that activation of p38 MAPK is essential for LPS-induced COX-2 expression in equine peripheral blood leukocytes. We tested our hypothesis by investigating the effects of the specific p38 MAPK inhibitors SB203580 and SB202190 on LPS-induced COX-2 protein expression and PGE(2) production in equine leukocytes. LPS stimulation activated p38 MAPK and increased COX-2 expression in a dose-dependent manner with maximal activation observed after 30min and 4h, respectively, at a concentration of 10 ng/ml LPS. In contrast, LPS stimulation did not affect COX-1 protein expression. Pretreatment with SB203580 or SB202190 significantly inhibited LPS-induced activation-associated p38 MAPK phosphorylation, COX-2 mRNA and protein levels, and PGE(2) production in equine leukocytes. Maximal inhibition of LPS-induced COX-2 protein expression was achieved at a concentration of 10 microM SB203580. We concluded that p38 MAPK is essential for LPS-induced COX-2 expression suggesting that p38 MAPK is a potential target for anti-inflammatory therapy during equine endotoxemia.