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1.
Front Vet Sci ; 4: 159, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29034248

RESUMO

In many equine inflammatory disease states, neutrophil activities, such as adhesion, migration, and reactive oxygen species (ROS) production become dysregulated. Dysregulated neutrophil activation causes tissue damage in horses with asthma, colitis, laminitis, and gastric glandular disease. Non-steroidal anti-inflammatory drugs do not adequately inhibit neutrophil inflammatory functions and can lead to dangerous adverse effects. Therefore, novel therapies that target mechanisms of neutrophil-mediated tissue damage are needed. One potential neutrophil-targeting therapeutic is the PGE1 analog, misoprostol. Misoprostol is a gastroprotectant that induces intracellular formation of the secondary messenger molecule cyclic AMP (cAMP), which has been shown to have anti-inflammatory effects on neutrophils. Misoprostol is currently used in horses to treat NSAID-induced gastrointestinal injury; however, its effects on equine neutrophils have not been determined. We hypothesized that treatment of equine neutrophils with misoprostol would inhibit equine neutrophil adhesion, migration, and ROS production, in vitro. We tested this hypothesis using isolated equine peripheral blood neutrophils collected from 12 healthy adult teaching/research horses of mixed breed and gender. The effect of misoprostol treatment on adhesion, migration, and respiratory burst of equine neutrophils was evaluated via fluorescence-based adhesion and chemotaxis assays, and luminol-enhanced chemiluminescence, respectively. Neutrophils were pretreated with varying concentrations of misoprostol, vehicle, or appropriate functional inhibitory controls prior to stimulation with LTB4, CXCL8, PAF, lipopolysaccharide (LPS) or immune complex (IC). This study revealed that misoprostol pretreatment significantly inhibited LTB4-induced adhesion, LTB4-, CXCL8-, and PAF-induced chemotaxis, and LPS-, IC-, and PMA-induced ROS production in a concentration-dependent manner. This data indicate that misoprostol-targeting of E-prostanoid (EP) receptors potently inhibits equine neutrophil effector functions in vitro. Additional studies are indicated to further elucidate the role of EP receptors in regulating neutrophil function. Overall, our results suggest misoprostol may hold promise as a novel anti-inflammatory therapeutic in the horse.

2.
Front Vet Sci ; 4: 160, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29034249

RESUMO

Pro-inflammatory cytokines including tumor necrosis factor α (TNFα), IL-1ß, IL-6, and IL-8 are potent immune mediators that exacerbate multiple equine diseases such as sepsis and laminitis. Unfortunately, safe and effective cytokine-targeting therapies are lacking in horses; therefore, novel mechanisms of inhibiting cytokine production are critically needed. One potential mechanism for inhibiting cytokine synthesis is elevation of intracellular cyclic AMP (cAMP). In human leukocytes, intracellular cAMP production is induced by activation of E-prostanoid (EP) receptors 2 and 4. These receptors can be targeted by the EP2/4 agonist and prostaglandin E1 analog, misoprostol. Misoprostol is currently used as a gastroprotectant in horses but has not been evaluated as a cytokine-targeting therapeutic. Thus, we hypothesized that misoprostol treatment would inhibit pro-inflammatory cytokine production by lipopolysaccharide (LPS)-stimulated equine leukocytes in an in vitro inflammation model. To test this hypothesis, equine leukocyte-rich plasma (LRP) was collected from 12 healthy adult horses and used to model LPS-mediated inflammatory signaling. LRP was treated with varying concentrations of misoprostol either before (pretreated) or following (posttreated) LPS stimulation. LRP supernatants were assayed for 23 cytokines using an equine-specific multiplex bead immunoassay. Leukocytes were isolated from LRP, and leukocyte mRNA levels of four important cytokines were evaluated via RT-PCR. Statistical differences between treatments were determined using one-way RM ANOVA (Holm-Sidak post hoc testing) or Friedman's RM ANOVA on Ranks (SNK post hoc testing), where appropriate (p < 0.05, n = 3-6 horses). These studies revealed that misoprostol pre- and posttreatment inhibited LPS-induced TNFα and IL-6 protein production in equine leukocytes but had no effect on IL-8 protein. Interestingly, misoprostol pretreatment enhanced IL-1ß protein synthesis following 6 h of LPS stimulation, while misoprostol posttreatment inhibited IL-1ß protein production after 24 h of LPS stimulation. At the mRNA level, misoprostol pre- and posttreatment inhibited LPS-induced TNFα, IL-1ß, and IL-6 mRNA production but did not affect IL-8 mRNA. These results indicate that misoprostol exerts anti-inflammatory effects on equine leukocytes when applied before or after a pro-inflammatory stimulus. However, the effects we observed were cytokine-specific and sometimes differed at the mRNA and protein levels. Further studies are warranted to establish the inhibitory effects of misoprostol on equine cytokine production in vivo.

3.
Vet Immunol Immunopathol ; 192: 33-40, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29042013

RESUMO

Inhibition of prostaglandin E2 (PGE2) production effectively limits inflammation in horses, however nonspecific prostaglandin blockade via cyclooxygenase (COX) inhibition elicits deleterious gastrointestinal side effects in equine patients. Thus, more selective PGE2 targeting therapeutics are needed to treat inflammatory disease in horses. One potential target is microsomal prostaglandin E-synthase-1 (mPGES-1), which is the terminal enzyme downstream of COX-2 in the inducible PGE2 synthesis cascade. This enzyme has yet to be studied in equine leukocytes, which play a pivotal role in equine inflammatory disease. The objective of this study was to determine if mPGES-1 is a PGE2-selective anti-inflammatory target in equine leukocytes. To evaluate this objective, leukocyte-rich plasma (LRP) was isolated from equine whole blood collected via jugular venipuncture of six healthy adult horses of mixed breeds and genders. LRP was primed with granulocyte-monocyte colony-stimulating factor (GM-CSF) and stimulated with lipopolysaccharide (LPS) in the presence or absence of an mPGES-1 inhibitor (MF63), a COX-2 inhibitor (NS-398), or a nonselective COX inhibitor (indomethacin). Following treatment, mPGES-1 and COX-2 mRNA and protein levels were measured via qPCR and western blot, respectively, and PGE2, thromboxane (TXA2) and prostacyclin (PGI2) levels were measured in cellular supernatants via ELISA. This study revealed that LPS significantly increased mPGES-1 mRNA, but not protein levels in equine LRP as measured by qPCR and western blot, respectively. In contrast, COX-2 mRNA and protein were coordinately induced by LPS. Importantly, treatment of LPS-stimulated leukocytes with indomethacin and NS-398 significantly reduced extracellular concentrations of multiple prostanoids (PGE2, TXA2 and PGI2), while the mPGES-1 inhibitor MF63 selectively inhibited PGE2 production only. mPGES-1 inhibition also preserved higher basal levels of PGE2 production when compared to either COX inhibitor, which might be beneficial in a clinical setting. In conclusion, this work identifies mPGES-1 as a key regulator of PGE2 production and a PGE2-selective target in equine leukocytes. This study demonstrates that mPGES-1 is a potentially safer and effective therapeutic target for treatment of equine inflammatory disease when compared to traditional non-steroidal anti-inflammatory drugs.


Assuntos
Dinoprostona/antagonistas & inibidores , Inflamação/veterinária , Prostaglandina-E Sintases/antagonistas & inibidores , Animais , Western Blotting/veterinária , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática/veterinária , Cavalos , Inflamação/metabolismo , Inflamação/fisiopatologia , Leucócitos/metabolismo , Microssomos/enzimologia , Prostaglandina-E Sintases/metabolismo , Prostaglandina-E Sintases/fisiologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária
4.
Inflammation ; 38(3): 1126-41, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25515270

RESUMO

Dysregulated release of neutrophil reactive oxygen species and proteolytic enzymes contributes to both acute and chronic inflammatory diseases. Therefore, molecular regulators of these processes are potential targets for new anti-inflammatory therapies. We have shown previously that myristoylated alanine-rich C-kinase substrate (MARCKS), a well-known actin binding protein and protein kinase C (PKC) substrate, is a key regulator of neutrophil functions. In the current study, we investigate the role of PKC-mediated MARCKS phosphorylation in neutrophil migration and adhesion in vitro. We report that treatment of human neutrophils with the δ-PKC inhibitor rottlerin significantly attenuates f-Met-Leu-Phe (fMLF)-induced MARCKS phosphorylation (IC50=5.709 µM), adhesion (IC50=8.4 µM), and migration (IC50=6.7 µM), while α-, ß-, and ζ-PKC inhibitors had no significant effect. We conclude that δ-PKC-mediated MARCKS phosphorylation is essential for human neutrophil migration and adhesion in vitro. These results implicate δ-PKC-mediated MARCKS phosphorylation as a key step in the inflammatory response of neutrophils.


Assuntos
Adesão Celular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Proteína Quinase C-delta/metabolismo , Acetofenonas/farmacologia , Benzopiranos/farmacologia , Movimento Celular , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Humanos , Inflamação/imunologia , Substrato Quinase C Rico em Alanina Miristoilada , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Ativação de Neutrófilo/imunologia , Fosforilação , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Proteína Quinase C-delta/antagonistas & inibidores
5.
Vet Immunol Immunopathol ; 160(3-4): 167-76, 2014 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-24857637

RESUMO

Neutrophil infiltration is a prominent feature in a number of pathologic conditions affecting horses including recurrent airway obstruction, ischemia-reperfusion injury, and laminitis. Cell signaling components involved in neutrophil migration represent targets for novel anti-inflammatory therapies. In order to migrate into tissue, neutrophils must respond to chemoattractant signals in their external environment through activation of adhesion receptors (i.e. integrins) and reorganization of the actin cytoskeleton. Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS), a highly conserved actin-binding protein, has a well demonstrated role in cytoskeletal dependent cellular functions (i.e. adhesion, spreading, and migration), but the details of MARCKS involvement in these processes remain vague. We hypothesized that MARCKS serves as a link between the actin cytoskeleton and integrin function in neutrophils. Using a MARCKS-specific inhibitor peptide known as MANS on equine neutrophils in vitro, we demonstrate that inhibition of MARCKS function significantly attenuates ß2-integrin-dependent neutrophil functions including migration, adhesion, and immune complex-mediated respiratory burst. The MANS peptide did not, however, inhibit the ß2-integrin-independent PMA mediated respiratory burst. These results attest to the essential role of MARCKS function in regulating neutrophil responses, and strongly implicate MARCKS as a potential regulator of ß2-integrins in neutrophils.


Assuntos
Antígenos CD18/fisiologia , Cavalos/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas de Membrana/fisiologia , Infiltração de Neutrófilos/fisiologia , Sequência de Aminoácidos , Animais , Complexo Antígeno-Anticorpo/fisiologia , Antígenos CD18/imunologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Sequência Conservada , Cavalos/imunologia , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Dados de Sequência Molecular , Substrato Quinase C Rico em Alanina Miristoilada , Infiltração de Neutrófilos/efeitos dos fármacos , Infiltração de Neutrófilos/imunologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacologia , Explosão Respiratória/efeitos dos fármacos , Homologia de Sequência de Aminoácidos , Acetato de Tetradecanoilforbol/farmacologia
6.
PLoS One ; 8(6): e66512, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23840497

RESUMO

Myristoylated alanine-rich C-kinase substrate (MARCKS) is a ubiquitously expressed substrate of protein kinase C (PKC) that is involved in reorganization of the actin cytoskeleton. We hypothesized that MARCKS is involved in regulation of fibroblast migration and addressed this hypothesis by utilizing a unique reagent developed in this laboratory, the MANS peptide. The MANS peptide is a myristoylated cell permeable peptide corresponding to the first 24-amino acids of MARCKS that inhibits MARCKS function. Treatment of NIH-3T3 fibroblasts with the MANS peptide attenuated cell migration in scratch wounding assays, while a myristoylated, missense control peptide (RNS) had no effect. Neither MANS nor RNS peptide treatment altered NIH-3T3 cell proliferation within the parameters of the scratch assay. MANS peptide treatment also resulted in inhibited NIH-3T3 chemotaxis towards the chemoattractant platelet-derived growth factor-BB (PDGF-BB), with no effect observed with RNS treatment. Live cell imaging of PDGF-BB induced chemotaxis demonstrated that MANS peptide treatment resulted in weak chemotactic fidelity compared to RNS treated cells. MANS and RNS peptides did not affect PDGF-BB induced phosphorylation of MARCKS or phosphoinositide 3-kinase (PI3K) signaling, as measured by Akt phosphorylation. Further, no difference in cell migration was observed in NIH-3T3 fibroblasts that were transfected with MARCKS siRNAs with or without MANS peptide treatment. Genetic structure-function analysis revealed that MANS peptide-mediated attenuation of NIH-3T3 cell migration does not require the presence of the myristic acid moiety on the amino-terminus. Expression of either MANS or unmyristoylated MANS (UMANS) C-terminal EGFP fusion proteins resulted in similar levels of attenuated cell migration as observed with MANS peptide treatment. These data demonstrate that MARCKS regulates cell migration and suggests that MARCKS-mediated regulation of fibroblast migration involves the MARCKS amino-terminus. Further, this data demonstrates that MANS peptide treatment inhibits MARCKS function during fibroblast migration and that MANS mediated inhibition occurs independent of myristoylation.


Assuntos
Quimiotaxia/fisiologia , Fibroblastos/citologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas de Membrana/fisiologia , Células 3T3 , Animais , Becaplermina , Quimiotaxia/efeitos dos fármacos , Colágeno/metabolismo , Fibronectinas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Substrato Quinase C Rico em Alanina Miristoilada , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-sis/farmacologia
7.
Am J Respir Cell Mol Biol ; 48(3): 314-21, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23221047

RESUMO

Myristoylated alanine-rich C kinase substrate (MARCKS) is a ubiquitously expressed protein kinase C substrate that has emerged as a potential therapeutic target for the amelioration of mucin secretion and inflammation in patients with chronic obstructive pulmonary disease. MARCKS also plays a key role in regulating the adhesion, migration, and degranulation of neutrophils. Moreover, given its biological role in epithelial and immune cells, we hypothesized that MARCKS may play an integral role in cytokine secretion by neutrophils. Because the amino terminus of MARCKS is highly conserved across vertebrate species, we successfully applied the well-characterized human MARCKS inhibitory peptide, myristoylated N-terminal sequence (MANS), to attenuate the function of MARCKS in isolated canine neutrophils. Pretreatment of canine neutrophils with MANS peptide significantly reduced both mRNA and protein expression in a broad range of LPS-induced cytokines, including IL-8, a chemokine (C-X-C motif) ligand-1 orthologue, and TNF-α, in comparison with untreated cells or those treated with a control peptide. This reduction in cytokine expression was observed even when neutrophils were treated with MANS 2 hours after LPS exposure. The observed reduction in cytokine secretion was not attributable to protein retention or cell death, but was associated with reduced cytokine transcript synthesis. These observations identify MARCKS protein as a promising therapeutic target in the treatment of inflammatory diseases or syndromes attributed to neutrophil influx and inflammatory cytokine production, such as sepsis, acute lung injury, and acute respiratory distress syndrome.


Assuntos
Interleucina-8/biossíntese , Interleucina-8/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Neutrófilos/metabolismo , Peptídeos/farmacologia , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genética , Animais , Cães , Humanos , Interleucina-8/metabolismo , Lipopolissacarídeos/farmacologia , Substrato Quinase C Rico em Alanina Miristoilada , Neutrófilos/efeitos dos fármacos , Peptídeos/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Fator de Necrose Tumoral alfa/metabolismo
8.
J Zoo Wildl Med ; 43(2): 289-95, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22779232

RESUMO

This pilot study was designed to determine whether cyclooxygenase (COX)-1, COX-2, or both are expressed in normal turtle tissues and whether level of expression changes when tissue becomes inflamed. Five eastern box turtles, Terrapene carolina carolina, that either died or were euthanatized due to disease or injuries were used for this work. Tissues were obtained from the five turtles. Western blot analysis was used to evaluate tissues for COX-1 and COX-2 proteins. Densiometric analysis was used to compare Western blot bands within each turtle. COX-1 and COX-2 were found in the liver, kidney, grossly normal muscle, and grossly traumatized (inflamed) muscle of all study turtles. In all cases, COX-1 and COX-2 proteins were increased in traumatized muscle over grossly normal nontraumatized muscle. The highest levels of COX-1 and COX-2 proteins were found in kidney and liver. There was no statistical difference between the amount of COX-1 protein in liver and kidney, but traumatized muscle compared with grossly normal muscle had significantly greater COX-1 but not COX 2 protein concentrations. There was no statistical difference between the amount of COX-2 protein in liver and kidney. Traumatized muscle expressed nonstatistically significant greater amounts of COX-2 compared with grossly normal muscle. COX-1 and COX-2 proteins are expressed in turtle tissues, and both isoforms are upregulated during inflammation of muscle tissue. Traditional nonsteroidal anti-inflammatory drugs (NSAIDs) that block both COX isoforms might be more efficacious than COX-2-selective drugs. This work suggests that NSAIDs should be evaluated for potential liver and kidney toxicity in turtles.


Assuntos
Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Tartarugas , Ferimentos e Lesões/veterinária , Animais , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 2/genética , Músculo Esquelético/enzimologia , Ferimentos e Lesões/metabolismo
9.
J Leukoc Biol ; 92(3): 633-9, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22623357

RESUMO

A role for MARCKS protein in directed migration of macrophages toward a chemoattractant was investigated. A peptide identical to the N-terminus of MARCKS (the MANS peptide), shown previously to inhibit the function of MARCKS in various cell types, was used. We investigated whether this MARCKS-related peptide could affect migration of macrophages, using the mouse macrophage-like J774A.1 cell line and primary murine macrophages. Both of these cell types migrated in response to the chemoattractants macrophage/MCPs, MCP-1 (25-100 ng/ml) or C5a (5-20 ng/ml). Cells were preincubated (15 min) with MANS or a mis-sense control peptide (RNS), both at 50 µM, and effects on migration determined 3 h after addition of chemoattractants. The movement and interactions of MARCKS and actin also were followed visually via confocal microscopy using a fluorescently labeled antibody to MARCKS and fluorescently tagged phalloidin to identify actin. MANS, but not RNS, attenuated migration of J774A.1 cells and primary macrophages in response to MCP-1 or C5a, implicating MARCKS in the cellular mechanism of directed migration. Exposure of cells to MCP-1 resulted in rapid phosphorylation and translocation of MARCKS from plasma membrane to cytosol, whereas actin appeared to spread through the cell and into cell protrusions; there was visual and biochemical evidence of a transient interaction between MARCKS and actin during the process of migration. These results suggest that MARCKS is involved in directed migration of macrophages via a process involving its phosphorylation, cytoplasmic translocation, and interaction with actin.


Assuntos
Quimiotaxia de Leucócito/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Citoesqueleto de Actina/imunologia , Citoesqueleto de Actina/metabolismo , Animais , Western Blotting , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Macrófagos/imunologia , Proteínas de Membrana/imunologia , Camundongos , Microscopia Confocal , Substrato Quinase C Rico em Alanina Miristoilada , Transporte Proteico/fisiologia
10.
Anat Rec (Hoboken) ; 294(9): 1511-24, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21809467

RESUMO

Myristoylated alanine-rich C-kinase substrate (MARCKS) is an actin binding protein substrate of protein kinase C (PKC) and critical for mouse and Xenopus development. Herein two MARCKS paralogs, marcksa and marcksb, are identified in zebrafish and the role of these genes in zebrafish development is evaluated. Morpholino-based targeting of either MARCKS protein resulted in increased mortality and a range of gross phenotypic abnormalities. Phenotypic abnormalities were classified as mild, moderate or severe, which is characterized by a slight curve of a full-length tail, a severe curve or twist of a full-length tail and a truncated tail, respectively. All three phenotypes displayed abnormal neural architecture. Histopathology of Marcks targeted embryos revealed abnormalities in retinal layering, gill formation and skeletal muscle morphology. These results demonstrate that Marcksa and Marcksb are required for normal zebrafish development and suggest that zebrafish are a suitable model to further study MARCKS function.


Assuntos
Embrião não Mamífero/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Morfolinos/farmacologia , Peixe-Zebra/crescimento & desenvolvimento , Sequência de Aminoácidos , Animais , Embrião não Mamífero/citologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Dados de Sequência Molecular , Família Multigênica , Substrato Quinase C Rico em Alanina Miristoilada , Fenótipo , Filogenia , Homologia de Sequência de Aminoácidos , Peixe-Zebra/genética
11.
Am J Respir Cell Mol Biol ; 42(5): 586-94, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-19574534

RESUMO

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.


Assuntos
Quimiotaxia de Leucócito , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Neutrófilos/citologia , Neutrófilos/metabolismo , Actinas/metabolismo , Antígenos CD18/metabolismo , Adesão Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Polaridade Celular/efeitos dos fármacos , Fatores Quimiotáticos/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Humanos , Substrato Quinase C Rico em Alanina Miristoilada , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Peptídeos/farmacologia , Acetato de Tetradecanoilforbol/farmacologia
12.
Vet Immunol Immunopathol ; 129(1-2): 137-42, 2009 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-19136156

RESUMO

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.


Assuntos
Anestésicos Locais/farmacologia , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Cavalos/imunologia , Lidocaína/farmacologia , Neutrófilos/efeitos dos fármacos , Animais , Adesão Celular/imunologia , Movimento Celular/imunologia , Cavalos/sangue , Interleucina-8/imunologia , Leucotrieno B4/imunologia , Ativação de Neutrófilo/efeitos dos fármacos , Ativação de Neutrófilo/imunologia , Neutrófilos/imunologia , Fator de Ativação de Plaquetas/imunologia
13.
Vet Immunol Immunopathol ; 129(3-4): 181-91, 2009 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-19095309

RESUMO

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.


Assuntos
Quimiotaxia de Leucócito/fisiologia , Cavalos/fisiologia , Neutrófilos/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Antígenos CD18/metabolismo , Adesão Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/fisiologia , Imidazóis/farmacologia , Inflamação/metabolismo , Leucotrieno B4/metabolismo , Piridinas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
14.
Vet Immunol Immunopathol ; 129(3-4): 192-9, 2009 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-19070370

RESUMO

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.


Assuntos
Citocinas/metabolismo , Regulação da Expressão Gênica/fisiologia , Cavalos/metabolismo , Leucócitos/metabolismo , Lipopolissacarídeos/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Células Cultivadas , Quimiocinas/genética , Quimiocinas/metabolismo , Citocinas/genética , Cavalos/sangue , Cavalos/imunologia , Leucócitos/efeitos dos fármacos , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
15.
J Leukoc Biol ; 83(4): 964-71, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18218860

RESUMO

Activation of beta2 integrins is necessary for neutrophil adhesion and full activation of neutrophil effector functions. We demonstrated previously that inhibition of protein kinase A (PKA) activity in quiescent neutrophils is sufficient to increase beta2-integrin cell surface expression, affinity, and adhesion. Thus, a tonic level of PKA activity prevents inappropriate activation of beta2 integrins in unstimulated neutrophils. Myosin light-chain (MLC) phosphorylation is an important regulator of leukocyte integrin function and adhesion. Moreover, PKA regulates MLC phosphorylation via inhibiting MLC kinase (MLCK) and MLC dephosphorylation via effects on the Rho kinase (ROCK)/MLC phosphatase pathway. We hypothesize that the tonic inhibitory effect of PKA on beta2-integrin activation neutrophils operates via its inhibition of MLC phosphorylation. We demonstrate here that inhibition of PKA activity with KT5720 activated beta2 integrins and adhesion coincident with an increase in MLC serine 19 (Ser 19) phosphorylation. KT5720-induced activation of beta2 integrins, adhesion, and MLC Ser 19 phosphorylation was abolished by pretreatment with the MLCK inhibitor ML-7 and specific MLCK inhibitory peptides but not the ROCK inhibitor Y-27632. These findings demonstrate that tonic PKA activity prevents activation of beta2 integrins and adhesion by inhibiting MLC phosphorylation via a MLCK-dependent but ROCK-independent pathway.


Assuntos
Antígenos CD18/sangue , Proteínas Quinases Dependentes de AMP Cíclico/sangue , Quinase de Cadeia Leve de Miosina/sangue , Neutrófilos/fisiologia , Quinases Associadas a rho/sangue , Adulto , Antígenos CD18/efeitos dos fármacos , Carbazóis/farmacologia , Adesão Celular , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Homeostase , Humanos , Indóis/farmacologia , Neutrófilos/enzimologia , Oligopeptídeos/farmacologia , Fosforilação , Pirróis/farmacologia
16.
J Leukoc Biol ; 82(5): 1311-21, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17684042

RESUMO

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.


Assuntos
Moléculas de Adesão Celular/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , GMP Cíclico/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas dos Microfilamentos/metabolismo , Neutrófilos/efeitos dos fármacos , Fosfoproteínas/metabolismo , Serina/metabolismo , Actinas/metabolismo , Western Blotting , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Adesão Celular , Moléculas de Adesão Celular/genética , Células Cultivadas , Quimiotaxia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , GMP Cíclico/análogos & derivados , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de GMP Cíclico/fisiologia , Humanos , Interleucina-8/farmacologia , Leucotrieno B4/farmacologia , Proteínas dos Microfilamentos/genética , N-Formilmetionina Leucil-Fenilalanina/análogos & derivados , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/metabolismo , Fosfoproteínas/genética , Fosforilação/efeitos dos fármacos , Fator de Ativação de Plaquetas/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/fisiologia , Serina/química , Serina/genética , Transdução de Sinais , Fator de Necrose Tumoral alfa/farmacologia
17.
Vet Immunol Immunopathol ; 118(3-4): 294-303, 2007 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-17614138

RESUMO

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.


Assuntos
Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Regulação Enzimológica da Expressão Gênica , Cavalos , Leucócitos/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Células Cultivadas , Ativação Enzimática , Epoprostenol/metabolismo , Cavalos/sangue , Cavalos/genética , Imidazóis/farmacologia , Prostaglandinas/genética , Prostaglandinas/metabolismo , Piridinas/farmacologia , RNA Mensageiro/metabolismo , Fatores de Tempo
18.
J Vet Intern Med ; 21(3): 367-77, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17552439

RESUMO

Nonsteroidal anti-inflammatory drugs (NSAIDs) have long been used for the treatment of pain and inflammation because of their inhibitory effects on cyclooxygenase (COX). For almost as long as NSAIDs have been in use, multiple adverse effects have been noted. Assessment of many of these adverse effects have been complicated because of the discovery of multiple splice variants of the cox gene, and a greater array of COX inhibitors, especially the COX-2 selective inhibitors have become available. Some of these adverse effects cannot be readily explained by the effect of these drugs on COX. This has sparked a new field of investigation into the COX-independent effects of the COX inhibitors. The major noncyclooxygenase targets of the COX inhibitors of particular relevance to inflammation and the gastrointestinal tract are phosphatidylinositol 3'-kinase Akt signaling, uncoupling of oxidative phosphorylation, PPARgamma, nuclear factor KB, mitogen activated protein kinases, and heat shock proteins.


Assuntos
Inibidores de Ciclo-Oxigenase/metabolismo , Regulação Enzimológica da Expressão Gênica , Mucosa Intestinal/metabolismo , Transdução de Sinais , Animais , Anti-Inflamatórios não Esteroides/efeitos adversos , Anti-Inflamatórios não Esteroides/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Inibidores de Ciclo-Oxigenase/efeitos adversos , Inibidores de Ciclo-Oxigenase/farmacologia , Ativação Enzimática , Proteínas de Choque Térmico/metabolismo , NF-kappa B/metabolismo , PPAR gama/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
19.
Physiol Rev ; 87(2): 545-64, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17429041

RESUMO

Mucosal repair is a complex event that immediately follows acute injury induced by ischemia and noxious luminal contents such as bile. In the small intestine, villous contraction is the initial phase of repair and is initiated by myofibroblasts that reside immediately beneath the epithelial basement membrane. Subsequent events include crawling of healthy epithelium adjacent to the wound, referred to as restitution. This is a highly regulated event involving signaling via basement membrane integrins by molecules such as focal adhesion kinase and growth factors. Interestingly, however, ex vivo studies of mammalian small intestine have revealed the importance of closure of the interepithelial tight junctions and the paracellular space. The critical role of tight junction closure is underscored by the prominent contribution of the paracellular space to measures of barrier function such as transepithelial electrical resistance. Additional roles are played by subepithelial cell populations, including neutrophils, related to their role in innate immunity. The net result of reparative mechanisms is remarkably rapid closure of mucosal wounds in mammalian tissues to prevent the onset of sepsis.


Assuntos
Enteropatias/patologia , Mucosa Intestinal/patologia , Animais , Células Epiteliais/fisiologia , Humanos , Inflamação/patologia , Enteropatias/fisiopatologia , Mucosa Intestinal/citologia , Mucosa Intestinal/fisiopatologia , Membrana Mucosa/citologia , Membrana Mucosa/fisiologia
20.
Vet Microbiol ; 121(1-2): 177-81, 2007 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-17204376

RESUMO

Effusive, fibrinous pericarditis is an uncommon disease entity in horses. In 2001, pericarditis occurred in conjunction with an epizootic in central Kentucky that was associated with exposure to eastern tent caterpillars (ETCs). Bacterial isolation from equine pericardial fluid samples was attempted using an insect cell culture growth medium (ICCGM). Using previously cultured, stored frozen samples from four horses with fibrinous pericarditis, inoculation of 10% blood agar plates yielded no growth, whereas simultaneous inoculation of ICCGM resulted in the isolation of Proprionibacterium acnes, Staphylococcus equorum, a Streptococcus sp. and Pseudomonas rhodesiae from pericardial fluid samples. A similar or novel caterpillar-associated bacteria was not identified; however, use of an ICCGM might enhance isolation of bacteria from equine pericardial fluid.


Assuntos
Bactérias/isolamento & purificação , Infecções Bacterianas/veterinária , Surtos de Doenças/veterinária , Doenças dos Cavalos/microbiologia , Pericardite/veterinária , Animais , Bactérias/genética , Infecções Bacterianas/microbiologia , Infecções Bacterianas/patologia , Meios de Cultura , Técnicas de Cultura/veterinária , Doenças dos Cavalos/patologia , Cavalos , Insetos/citologia , Insetos/microbiologia , Kentucky/epidemiologia , Derrame Pericárdico/microbiologia , Derrame Pericárdico/patologia , Pericardite/microbiologia , Pericardite/patologia , Projetos Piloto , Estudos Retrospectivos
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