Samuel L. Jones

Two Signaling Mechanisms for Activation of αMβ2 Avidity in Polymorphonuclear Neutrophils

Circulating polymorphonuclear neutrophils (PMN) are quiescent, nonadherent cells that rapidly activate at sites of inflammation, where they develop the capacity to perform a repertoire of functions that are essential for host defense. Induction of integrin-mediated adhesion, which requires an increase in integrin avidity, is critical for the development of these effector functions. Although a variety of stimuli can activate integrins in PMN, the signaling cascades involved are unclear. Phosphatidylinositol (PI) 3-kinase has been implicated in integrin activation in a variety of cells, including PMN. In this work, we have examined activation of the PMN integrin αMβ2, assessing both adhesion and generation of the epitope recognized by the activation-specific antibody CBRM1/5. We have found that PI 3-kinase has a role in activation of αMβ2 by immune complexes, but we have found no role for it in αMβ2 activation by ligands for trimeric G protein-coupled receptors, including formylmethionylleucylphenylalanine (fMLP), interleukin-8, and C5a. Cytochalasin D inhibition suggests a role for the actin cytoskeleton in immune complex activation of αMβ2, but cytochalasin has no effect on fMLP-induced activation. Similarly, immune complex activation of the Rac/Cdc42-dependent serine/threonine kinase Pak1 is blocked by PI 3-kinase inhibitors, but fMLP-induced activation is not. These results demonstrate that two signaling pathways exist in PMN for activation of αMβ2. One, induced by FcγR ligation, is PI 3-kinase-dependent and requires the actin cytoskeleton. The second, initiated by G protein-linked receptors, is PI 3-kinase-independent and cytochalasin-insensitive. Pak1 may be in a final common pathway leading to activation of αMβ2.

A functional granulocyte colony-stimulating factor receptor is required for normal chemoattractant-induced neutrophil activation

Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor that is widely used to treat neutropenia. In addition to stimulating polymorphonuclear neutrophil (PMN) production, G-CSF may have significant effects on PMN function. Because G-CSF receptor (G-CSFR)-deficient mice do not have the expected neutrophilia after administration of human interleukin-8 (IL-8), we examined the effect of the loss of G-CSFR on IL-8-stimulated PMN function. Compared with wild-type PMNs, PMNs isolated from G-CSFR-deficient mice demonstrated markedly decreased chemotaxis to IL-8. PMN emigration into the skin of G-CSFR-deficient mice in response to IL-8 was also impaired. Significant chemotaxis defects were also seen in response to N-formyl-methionyl-leucyl-phenylalanine, zymosan-activated serum, or macrophage inflammatory protein-2. The defective chemotactic response to IL-8 does not appear to be due to impaired chemoattractant receptor function, as the number of IL-8 receptors and chemoattractant-induced calcium influx, actin polymerization, and release of gelatinase B were comparable to those of wild-type PMNs. Chemoattractant-induced adhesion of G-CSFR-deficient PMNs was significantly impaired, suggesting a defect in beta2-integrin activation. Collectively, these data demonstrate that selective defects in PMN activation are present in G-CSFR-deficient mice and indicate that G-CSF plays an important role in regulating PMN chemokine responsiveness.

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.

The effect of lidocaine on in vitro adhesion and migration of equine neutrophils.

The effect of lidocaine on in vitro migration and adhesion of equine neutrophils was evaluated. Neutrophils were isolated from equine whole blood using a Percoll-gradient centrifugation protocol. Purified neutrophils were incubated with lidocaine at concentrations from 0.1 to 1000 microg/ml for 30 min at 37 degrees C, after calcein loading. Neutrophil integrin-mediated adhesion in response to stimulation with 100 nM LTB(4), 100 nM PAF, or 100 ng/ml IL-8, or integrin-mediated migration in response to stimulation with 100 nM LTB(4), 150 nM PAF, or 100 ng/ml IL-8 was assessed. Statistical significance was set at P<0.05. Neutrophil adhesion was significantly increased in response to all three stimulants. IL-8-stimulated adhesion was significantly increased when neutrophils were incubated with 1mg/ml lidocaine, compared to lower lidocaine concentrations. LTB(4)-stimulated adhesion was significantly increased when neutrophils were incubated with 1mg/ml lidocaine compared to that at 5 microg/ml lidocaine. Migration was significantly increased in response to IL-8. IL-8 and LTB(4) stimulated migration was significantly increased when neutrophils were incubated with 1mg/ml lidocaine, compared to lower lidocaine concentrations. In conclusion, lidocaine did not inhibit neutrophil migration or adhesion in vitro at therapeutic concentrations, and increased migration and adhesion at higher concentrations.

Protective Role of Neutrophils in Mice Experimentally Infected with Rhodococcus equi

ABSTRACT Neutrophils are important in controlling early infections with the intracellular bacterium Rhodococcus equi. Antineutrophil monoclonal antibody (RB6-8C5)-induced neutrophil deficiency during the first week after experimental infection of mice with R. equi resulted in more severe disease and significantly increased tissue concentrations of R. equi.

Protein kinase A regulates β2 integrin avidity in neutrophils

The adhesive phenotype of neutrophils (PMN) depends largely on activating and deactivating intracellular signals regulating β2 integrin avidity for ligand. Our hypothesis is that PKA is a negative regulator of β2 integrin avidity. In this work, we examined the role of PKA in PMN αMβ2 integrin activation. Elevation of cAMP inhibited αMβ2 integrin‐dependent adhesion of PMN to immune complexes (IC), but not PMA‐induced adhesion. The PKA inhibitor KT5720 reversed the ability of cAMP to suppress adhesion to IC. Moreover, inhibition of PKA activity was sufficient to activate αMβ2 integrin‐dependent adhesion and increase β2 integrin expression and binding of the monoclonal antibody CBRM1/5, which recognizes activated αMβ2 specifically. However, PKA activity was necessary for sustained adhesion. Disruption of A kinase‐anchoring, protein‐PKA binding with a cell‐permeant peptide derived from the AKAP Ht31 also activated adhesion. Unlike pharmacologic inhibition of PKA, AKAP peptide‐induced adhesion was PKC dependent and did not affect β2 integrin expression or CBRM1/5 binding. These data demonstrate that PKA appears to have a dual role in the mechanism regulating αMβ2 integrin avidity and adhesion.

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.

Expression and Activity of COX-1 and 2 and 5-LOX in Joint Tissues from Dogs with Naturally Occurring Coxofemoral Joint Osteoarthritis

Understanding the neurobiology of pain in naturally occurring models of osteoarthritis (OA) may improve the understanding of human OA pain. Both COX and LOX have been associated with joint pain. This study evaluated COX‐1, COX‐2, and 5‐LOX expression and activity in a naturally occurring canine model of secondary OA. Hip joint capsule with synovial tissue (HJC) and femoral head subchondral bone (FH) was collected from normal dogs and dogs undergoing total hip replacement for coxofemoral joint OA. Tissues were analyzed for COX‐1, COX‐2, and LOX protein, and PGE2 and LTB4. Significantly more COX‐2 protein was present in OA HJC than normal joints (p = 0.0009). There was no significant difference in COX‐1 or LOX protein, although LOX protein was increased (p = 0.069). PGE2 concentration in normal and OA HJC was similar (p = 1.0). LTB4 concentration in OA HJC was significantly greater than normal HJC (p = 0.028). Significantly more COX‐1 (p = 0.0098), COX‐2 (p = 0.0028), and LOX (p = 0.0095) protein was present in OA FH tissue compared to normal FH tissue. There were no differences in PGE2 or LTB4 concentration in normal and OA FH tissue (p = 0.77 and p = 0.11). Together, these data suggest both COX‐2 and 5‐LOX are appropriate targets for the management of pain associated with naturally occurring OA.

The effects of cyclo‐oxygenase inhibitors on bile‐injured and normal equine colon

A potential adverse effect of cyclo-oxygenase (COX) inhibitors (nonsteroidal anti-inflammatory drugs [NSAIDs]) in horses is colitis. In addition, we have previously shown an important role for COX-produced prostanoids in recovery of ischaemic-injured equine jejunum. It was hypothesised that the nonselective COX inhibitor flunixin would retard repair of bile-injured colon by preventing production of reparative prostaglandins, whereas the selective COX-2 inhibitor, etodolac would not inhibit repair as a result of continued COX-1 activity. Segments of the pelvic flexure were exposed to 1.5 mmol/l deoxycholate for 30 min, after which they were recovered for 4 h in Ussing chambers. Contrary to the proposed hypothesis, recovery of bile-injured colonic mucosa was not affected by flunixin or etodolac, despite significantly depressed prostanoid production. However, treatment of control tissue with flunixin led to increases in mucosal permeability, whereas treatment with etodolac had no significant effect. Therefore, although recovery from bile-induced colonic injury maybe independent of COX-elaborated prostanoids, treatment of control tissues with nonselective COX inhibitors may lead to marked increases in permeability. Alternatively, selective inhibition of COX-2 may reduce the incidence of adverse effects in horses requiring NSAID therapy.

Detection of Bartonella henselae in the Blood of 2 Adult Horses

BACKGROUND Bartonella spp. are emerging zoonotic agents that have been found in a wide variety of domestic animals and wildlife and cause a number of clinical syndromes. Bartonella sp. infection has been identified in a growing number of animal species, including cats, rodents, porpoises, and canids, but has not been reported in horses. OBJECTIVE To document the presence of Bartonella sp. in the blood of horses. ANIMALS One horse with chronic arthropathy and 1 horse with presumptive vasculitis. METHODS Blood samples were tested for the presence of Bartonella sp. by a combination of multiplex real-time polymerase chain reaction and enrichment culture technique. RESULTS Bartonella henselae was isolated or detected in the blood of both horses. CONCLUSION AND CLINICAL IMPORTANCE Bartonella henselae infection should be investigated as the cause of disease in horses.