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1.
Atherosclerosis ; 258: 40-50, 2017 03.
Article in English | MEDLINE | ID: mdl-28189040

ABSTRACT

BACKGROUND AND AIMS: Diabetes is a major risk factor for the development of atherosclerosis. Hyperglycemia stimulates vascular smooth muscle cells (VSMC) to secrete ligands that bind to the αVß3 integrin, a receptor that regulates VSMC proliferation and migration. This study determined whether an antibody that had previously been shown to block αVß3 activation and to inhibit VSMC proliferation and migration in vitro, inhibited the development of atherosclerosis in diabetic pigs. METHODS: Twenty diabetic pigs were maintained on a high fat diet for 22 weeks. Ten received injections of anti-ß3 F(ab)2 and ten received control F(ab)2 for 18 weeks. RESULTS: The active antibody group showed reduction of atherosclerosis of 91 ± 9% in the left main, 71 ± 11%, in left anterior descending, 80 ± 10.2% in circumflex, and 76 ± 25% in right coronary artery, (p < 0.01 compared to lesions areas from corresponding control treated arteries). There were significant reductions in both cell number and extracellular matrix. Histologic analysis showed neointimal hyperplasia with macrophage infiltration, calcifications and cholesterol clefts. Antibody treatment significantly reduced number of macrophages contained within lesions, suggesting that this change contributed to the decrease in lesion cellularity. Analysis of the biochemical changes within the femoral arteries that received the active antibody showed a 46 ± 12% (p < 0.05) reduction in the tyrosine phosphorylation of the ß3 subunit of αVß3 and a 40 ± 14% (p < 0.05) reduction in MAP kinase activation. CONCLUSIONS: Blocking ligand binding to the αVß3 integrin inhibits its activation and attenuates increased VSMC proliferation that is induced by chronic hyperglycemia. These changes result in significant decreases in atherosclerotic lesion size in the coronary arteries. The results suggest that this approach may have efficacy in treating the proliferative phase of atherosclerosis in patients with diabetes.


Subject(s)
Coronary Artery Disease/prevention & control , Diabetes Mellitus, Experimental/drug therapy , Diabetic Angiopathies/prevention & control , Immunoglobulin Fab Fragments/pharmacology , Integrin alphaVbeta3/antagonists & inhibitors , Muscle, Smooth, Vascular/drug effects , Animals , Cell Proliferation/drug effects , Coronary Artery Disease/etiology , Coronary Artery Disease/metabolism , Coronary Vessels/drug effects , Coronary Vessels/metabolism , Coronary Vessels/pathology , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/metabolism , Diabetic Angiopathies/etiology , Diabetic Angiopathies/metabolism , Femoral Artery/drug effects , Femoral Artery/metabolism , Femoral Artery/pathology , Immunoglobulin Fab Fragments/administration & dosage , Injections, Subcutaneous , Integrin alphaVbeta3/immunology , Integrin alphaVbeta3/metabolism , Ligands , Macrophages/drug effects , Macrophages/metabolism , Male , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/pathology , Neointima , Phosphorylation , Plaque, Atherosclerotic , Protein Binding , Sus scrofa
2.
J Vet Intern Med ; 29(3): 908-16, 2015.
Article in English | MEDLINE | ID: mdl-25871966

ABSTRACT

BACKGROUND: Studies of some human prothrombotic diseases suggest that phosphatidylserine-positive (PS+) and tissue factor-positive (TF+) microparticles (MPs) might play a role in the pathogenesis of thrombosis or serve as biomarkers of thrombotic risk. HYPOTHESIS/OBJECTIVES: To determine if circulating levels of PS+MP and procoagulant activity (PCA) associated with PS+MPs and TF+ MPs are increased in dogs with IMHA. ANIMALS: Fifteen dogs with primary or secondary IMHA and 17 clinically healthy dogs. METHODS: Prospective case-controlled observational study. Circulating PS+MPs were measured by flow cytometry. PCA associated with PS+MPs and TF+MPs was measured by thrombin and Factor Xa generating assays, respectively. RESULTS: Circulating numbers of PS+MPs were not significantly higher in dogs with IMHA [control median 251,000/µL (36,992-1,141,250/µL); IMHA median 361,990/µL (21,766-47,650,600/µL) P = .30]. However, PS+MP PCA [control median 2.2 (0.0-16.8) nM PS eq; IMHA median 8.596, (0-49.33 nM PS eq) P = .01] and TF+MP PCA [control median 0.0, (0.0-0.0 pg/mL); IMHA median 0.0; (0-22.34 pg/mL], P = .04) were increased. Intravascular hemolysis, which we showed might increase PS+ and TF+MP PCA, was evident in 3 of 5 dogs with PS+MP PCA and 2 of 4 dogs with TF+MP PCA higher than controls. Underlying disease in addition to IMHA was detected in 1 of 5 dogs with PS+PCA and 3 of 4 dogs with TF+MP PCA higher than controls. CONCLUSIONS AND CLINICAL IMPORTANCE: TF+ and PS+MP PCA is increased in some dogs with IMHA. Further studies that determine if measuring TF+ and PS+ MP PCA can help identify dogs at risk for thrombosis are warranted.


Subject(s)
Anemia, Hemolytic, Autoimmune/veterinary , Coagulants/therapeutic use , Dog Diseases/drug therapy , Anemia, Hemolytic, Autoimmune/drug therapy , Animals , Blood Coagulation Tests/veterinary , Case-Control Studies , Coagulants/administration & dosage , Dogs , Female , Flow Cytometry , Hematocrit/veterinary , Male , Memory, Episodic , Microspheres
3.
Haemophilia ; 18(6): 941-7, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22812621

ABSTRACT

The objective of the present study was to evaluate the pharmacokinetic (PK) and pharmacodynamic (PD) profiles of the new recombinant FVIII compound turoctocog alfa and a Glyco-PEGylated FVIII derivative thereof (N8-GP) in Haemophilia A dogs. Six haemophilic dogs divided into two groups were included in the study. Each dog was administered a dose of 125 U kg(-1) , blood samples were collected at predetermined time points for both pharmacokinetic (FVIII measured by one-stage aPTT assay) and pharmacodynamic [whole blood clotting time (WBCT)] evaluations. After intravenous administration to haemophilic dogs, the plasma concentration at the first sampling point was comparable for turoctocog alfa and N8-GP, and the clearance was estimated to be 6.5 and 3.9 mL h(-1) kg(-1) for turoctocog alfa and N8-GP respectively. Both turoctocog alfa and N8-GP were able to reduce the WBCT time to normal levels (<20 min), however, the reduced clearance was reflected in the WBCT, which returned to baseline at a later time point for N8-GP as compared with dogs dosed with turoctocog alfa. The clearance was 40% reduced for N8-GP as compared with turoctocog alfa. Simulations of a multiple dosing regimen in dogs, suggest that to maintain WBCT <20 min N8-GP can be dosed at reduced intervals, e.g. with 4 days between doses, whereas turoctocog alfa will have to be dosed with 2½ day between doses. Data thereby supports N8-GP as an alternative to standard rFVIII replacement therapy, with a more convenient dosing regimen.


Subject(s)
Factor VIII/pharmacokinetics , Hemophilia A/drug therapy , Polyethylene Glycols/pharmacokinetics , Animals , Dogs , Factor VIII/analysis , Factor VIII/therapeutic use , Female , Half-Life , Male , Partial Thromboplastin Time , Polyethylene Glycols/analysis , Polyethylene Glycols/chemistry , Polyethylene Glycols/therapeutic use , Thrombelastography , Whole Blood Coagulation Time
4.
J Thromb Haemost ; 10(8): 1591-9, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22726310

ABSTRACT

BACKGROUND: Prophylaxis of hemophilia B, at present, requires multiple infusions of human factor (F)IX concentrates per week. A FIX molecule with a prolonged half-life has the potential to greatly improve the convenience of, and adherence to, prophylaxis. OBJECTIVES: The aim of our studies was to investigate the pharmacokinetic (PK) and pharmacodynamic (PD) profile of a recombinant fusion protein linking coagulation FIX with albumin (rIX-FP). METHODS: Cynomolgus monkeys and hemophilia B dogs received single intravenous doses of rIX-FP (50-500 IU kg(-1)). rIX-FP plasma levels were determined by an activity-based assay (dogs only) and anti-FIX ELISA methods. Additionally, activated partial thromboplastin time (APTT) was determined in hemophilia B dogs. Data were compared with a direct study comparator (recombinant FIX [rFIX]) or previously published data. RESULTS: The terminal half-life of rIX-FP was prolonged in both species compared with FIX reference data. In hemophilia B dogs, human FIX antigen levels remained above 0.05 IU mL(-1) more than three times longer after rIX-FP (7.3 days) compared with rFIX (2.3 days), whereas respective calculations based on activity levels confirmed the observed superior profile. Prolonged PDs of rIX-FP were demonstrated with APTT<60 s sustained around four times longer with rIX-FP (5.9 days) than rFIX (1.5 days). CONCLUSIONS: These studies indicate that the recombinant albumin fusion technology successfully improves the PK profile of FIX. Clinical studies will test whether the improved kinetics result in a significant half-life extension in patients with hemophilia B.


Subject(s)
Factor IX/pharmacokinetics , Hemophilia B/drug therapy , Hemostatics/pharmacokinetics , Serum Albumin/pharmacokinetics , Animals , Area Under Curve , Blood Coagulation/drug effects , Disease Models, Animal , Dogs , Enzyme-Linked Immunosorbent Assay , Factor IX/administration & dosage , Female , Half-Life , Hemophilia B/blood , Hemostatics/administration & dosage , Hemostatics/blood , Humans , Injections, Intravenous , Macaca fascicularis , Male , Metabolic Clearance Rate , Models, Biological , Partial Thromboplastin Time , Recombinant Fusion Proteins/pharmacokinetics , Serum Albumin/administration & dosage , Serum Albumin, Human
6.
Haemophilia ; 17(5): e963-8, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21682818

ABSTRACT

N8, a new recombinant factor VIII (rFVIII) compound developed for the treatment of haemophilia A, is produced in Chinese hamster ovary (CHO) cells and formulated without human- or animal-derived materials. The aim of the present study was to compare the pharmacokinetics (PK) and the procoagulant effect, measured by ex vivo whole blood clot formation, of N8 and a commercial rFVIII in a cross-over study in haemophilia A dogs. N8 and Advate® (100 IU kg⁻¹) were administered intravenously to three haemophilia A dogs. Blood was sampled between 0 and 120 h postdose and FVIII:C analysed. PK parameters maximum plasma concentration, area under the curve, half-life (t(½)), clearance, mean residence time (MRT) and volume of distribution and incremental recovery were calculated. Whole blood clotting time (WBCT) and thromboelastography (TEG®) were used to determine the haemostatic potential. No adverse reactions were observed with N8 or Advate ®. N8 and Advate® exhibited similar PK parameters, with t(½) 7.7-11 h and MRT 11-14 h. Both rFVIII compounds corrected the prolonged WBCT (> 48 min) to the range of normal dogs (8-12 min), i.e. N8 to 7.5-10.5 min and Advate® to 7.5-11.5 min. N8 and Advate® also normalized the whole blood clot formation according to TEG®. The native whole blood clotting assays (WBCT, TEG®) appeared to be more sensitive to low concentrations of FVIII than assays in citrated plasma samples. In conclusion, comparison of N8 and Advate ® in haemophilia A dogs revealed similar safety, similar PK and similar effects in whole blood clot formation assays.


Subject(s)
Blood Coagulation/drug effects , Factor VIII/pharmacokinetics , Hemophilia A/blood , Recombinant Proteins/pharmacokinetics , Animals , Cross-Over Studies , Dog Diseases/blood , Dog Diseases/drug therapy , Dogs , Drug Evaluation, Preclinical/methods , Female , Half-Life , Hemophilia A/drug therapy , Hemophilia A/immunology , Hemophilia A/veterinary , Hemostasis/drug effects , Injections, Intravenous , Male , Metabolic Clearance Rate
7.
Haemophilia ; 16 Suppl 3: 19-23, 2010 May.
Article in English | MEDLINE | ID: mdl-20586797

ABSTRACT

Dogs with haemophilia A or haemophilia B exhibit spontaneous bleeding comparable with the spontaneous bleeding phenotype that occurs in humans with severe haemophilia. The phenotypic and genotypic characteristics of haemophilic dogs have been well-described, and such dogs are suitable for testing prophylactic protein replacement therapy and gene transfer strategies. In dogs with haemophilia, long-term effects on spontaneous bleeding frequency (measured over years) can be used as an efficacy endpoint in such studies. Although complete correction of coagulopathy has not been achieved, published data show that prophylactic factor replacement therapy and gene transfer can markedly reduce the frequency of spontaneous bleeding in haemophilic dogs. Further studies are currently ongoing.


Subject(s)
Factor IX/therapeutic use , Genetic Therapy , Hemophilia A/therapy , Hemophilia B/therapy , Hemorrhage/prevention & control , Animals , Dogs , Genetic Therapy/methods
8.
Haemophilia ; 14(5): 968-77, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18680527

ABSTRACT

von Willebrand factor (VWF) is a multimeric glycoprotein that mediates platelet adhesion and is decreased in von Willebrand disease (VWD). 1-8 deamino-d-arginine vasopressin (DDAVP), the most common treatment for VWD, is limited by tachyphylaxis and inconvenience, and in 20% of the patients, unresponsiveness. Recombinant human interleukin-11 (rhIL-11), a gp-130 signalling cytokine with haematopoietic and anti-inflammatory activity, increases VWF antigen and its activity in heterozygous VWF(+/-) mice and dogs. To determine the biological efficacy and safety of rhIL-11 in non-bleeding human subjects with mild VWD, we conducted a phase II prospective open-label trial of rhIL-11 at 10, 25 and 50 mug kg(-1) subcutaneously (s.c.), given daily for 7 days in nine subjects with mild VWD. VWF and factor VIII (FVIII) levels increased gradually and progressively after s.c. rhIL-11, which was sustained through 7 days of dosing to 1.5- to 3-fold over baseline. Following intravenous DDAVP, 0.3 mug kg(-1), on day 7 there was a further boost in VWF and FVIII levels, suggesting that the mechanism of rhIL-11 differs from that of DDAVP. Platelet VWF mRNA expression measured by quantitative PCR increased from two- to eightfold over baseline, suggesting that the mechanism of rhIL-11 effect may be upregulation of VWF mRNA. VWF and FVIII levels returned to baseline by day 14. rhIL-11 was well tolerated with less than grade-1 hypertension, hypokalaemia and fluid retention. Recombinant IL-11 increases VWF levels in humans with mild VWD, justifying future clinical trials to determine its potential in preventing or reducing bleeding in this patient population.


Subject(s)
Interleukin-11/administration & dosage , von Willebrand Diseases/drug therapy , Adult , Deamino Arginine Vasopressin/adverse effects , Deamino Arginine Vasopressin/therapeutic use , Dose-Response Relationship, Drug , Drug Administration Schedule , Factor VIII/metabolism , Female , Hemostatics/adverse effects , Hemostatics/therapeutic use , Humans , Injections, Subcutaneous , Interleukin-11/adverse effects , Interleukin-11/therapeutic use , Male , Platelet Count , Polymerase Chain Reaction/methods , Prospective Studies , RNA, Messenger/genetics , Recombinant Proteins/therapeutic use , Young Adult , von Willebrand Diseases/blood , von Willebrand Factor/biosynthesis , von Willebrand Factor/genetics , von Willebrand Factor/metabolism
9.
Endocrinology ; 148(10): 5002-10, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17640990

ABSTRACT

IGF-I has been shown to play a role in the progression of atherosclerosis in experimental animal models. IGF-binding protein-4 (IGFBP-4) binds to IGF-I and prevents its association with receptors. Overexpression of a protease-resistant form of IGFBP-4 has been shown to inhibit the ability of IGF-I to stimulate normal smooth muscle cell growth in mice. Based on these observations, we prepared a protease-resistant form of IGFBP-4 and infused it into hypercholesterolemic pigs. Infusion of the protease-resistant mutant inhibited lesion development by 53.3 +/- 6.1% (n = 6; P < 0.01). Control vessels that received an equimolar concentration of IGF-I and the protease-resistant IGFBP-4 showed no reduction in lesion size compared with control lesions that were infused with vehicle. Infusion of a nonmutated form of IGFBP-4 did not significantly inhibit lesion development. Proliferating cell nuclear antigen analysis showed that the mutant IGFBP-4 appeared to inhibit cell proliferation. The area occupied by extracellular matrix was also reduced proportionally compared with total lesion area. Immunoblotting revealed that the mutant IGFBP-4 remained intact, whereas the wild-type IGFBP-4 that was infused was proteolytically cleaved. Further analysis of the lesions revealed that a marker protein, IGFBP-5, whose synthesis is stimulated by IGF-I, was decreased in the lesions that received the protease-resistant, IGFBP-4 mutant, whereas there was no change in lesions that received wild-type IGFBP-4 or the mutant protein plus IGF-I. These findings clearly illustrate that infusion of protease-resistant IGFBP-4 into the perilesion environment results in inhibition of cell proliferation and attenuation of the development of neointima. The findings support the hypothesis that inhibiting IGFBP-4 proteolysis in the lesion microenvironment could be an effective means for regulating neointimal expansion.


Subject(s)
Hypercholesterolemia/pathology , Insulin-Like Growth Factor Binding Protein 4/chemistry , Insulin-Like Growth Factor Binding Protein 4/pharmacology , Insulin-Like Growth Factor I/antagonists & inhibitors , Peptide Hydrolases/metabolism , Tunica Intima/pathology , Animals , Carotid Arteries/drug effects , Carotid Arteries/metabolism , Carotid Arteries/pathology , Cell Proliferation/drug effects , Drug Resistance , Female , Femoral Artery/drug effects , Femoral Artery/metabolism , Femoral Artery/pathology , Hypercholesterolemia/metabolism , Hyperplasia , Insulin-Like Growth Factor Binding Protein 4/genetics , Insulin-Like Growth Factor Binding Protein 5/antagonists & inhibitors , Insulin-Like Growth Factor I/metabolism , Insulin-Like Growth Factor I/pharmacology , Male , Mutation , Swine , Transfection , Tunica Intima/drug effects , Tunica Intima/metabolism
10.
Article in English | MEDLINE | ID: mdl-11795630

ABSTRACT

A variety of platelet substitutes (e.g., rehydrated, lyophilized (RL) platelets, thromboerythrocytes, plateletsomes, infusible platelet membranes, synthocytes, fibrinogen-coated microcapules) are potentially useful as hemostatic agents in transfusion medicine. However, as "foreign" particles, platelet substitutes interact to varying extents with elements of the reticulo-endothelial system for clearance, reducing hemostatic efficacy. Experiments were performed to better understand the interaction of RL platelets with elements of the innate and acquired immune systems. The infusion of heterologous RL platelets into rats resulted in rapid clearance from the free circulation with half-life values of minutes. The clearance of RL platelets was inhibited when macrophages were rendered apoptotic with gadolinium. Transmission EM analysis of splenic tissue after infusion of lyophilized cells, as well as in vitro mixing studies with splenic macrophages and RL platelets, indicated that macrophage-mediated phagocytosis mechanisms were operant in RL platelet clearance by the reticulo-endothelial system. Studies with IV IgG, as a competitive inhibitor of the macrophage Fc receptor, provides evidence that RL platelet destruction is in part mediated by platelet surface bound IgG. This hypothesis was further supported by the finding that RL platelets react with IgG class antibodies that are pre-existing in naïve animals. These studies provide a rational basis for prolonging the circulation time of RL platelets and other platelet substitutes.


Subject(s)
Blood Platelets/cytology , Blood Preservation/methods , Platelet Transfusion , Spleen/cytology , Animals , Blood Platelets/immunology , Freeze Drying , Immunoglobulin G/analysis , Immunoglobulins, Intravenous , Macrophages/immunology , Phagocytosis , Rats , Rats, Sprague-Dawley , Spleen/immunology
11.
Br J Haematol ; 111(1): 167-74, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11091197

ABSTRACT

This study aimed to evaluate the effect of cross-linking and lyophilization on intracellular signalling processes in rehydrated, lyophilized (RL) platelets, which are under development as a platelet substitute for transfusion. Exposure of RL platelets to thrombin resulted in enhanced phosphorylation of several proteins, including 18 kDa and 42 kDa kinase substrates that were shown to be the substrates of myosin light chain and protein kinase C respectively. Cross-linking and lyophilization depleted the platelets of free cytoplasmic ADP and ATP, but had less effect on protein-bound nucleotides. The surface membrane of RL platelets was found to be permeable to poly dT probes less than approximately 3 kDa in size; larger nucleotide probes and proteins did not penetrate the surface membrane. Taken together, our results indicate that RL platelets retain some of the haemostatic stimulus-response functions of fresh platelets and are capable of feedback amplification in coagulation.


Subject(s)
Blood Platelets/physiology , Freeze Drying , Signal Transduction , Thrombin/pharmacology , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Autoradiography , Blood Platelets/drug effects , Blotting, Western , Cell Membrane/physiology , Cytoplasm/enzymology , Humans , Myosin Light Chains , Oligonucleotide Probes/metabolism , Permeability , Phosphorylation , Platelet Transfusion , Protein Kinase C
12.
Mol Biol Cell ; 9(10): 2933-47, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9763453

ABSTRACT

In neutrophils activated to secrete with formyl-methionyl-leucyl-phenylalanine, intermediate filaments are phosphorylated transiently by cyclic guanosine monophosphate (cGMP)-dependent protein kinase (G-kinase). cGMP regulation of vimentin organization was investigated. During granule secretion, cGMP levels were elevated and intermediate filaments were transiently assembled at the pericortex to areas devoid of granules and microfilaments. Microtubule and microfilament inhibitors affected intermediate filament organization, granule secretion, and cGMP levels. Cytochalasin D and nocodazole caused intermediate filaments to assemble at the nucleus, rather than at the pericortex. cGMP levels were elevated in neutrophils by both inhibitors; however, with cytochalasin D, cGMP was elevated earlier and granule secretion was excessive. Nocodazole did not affect normal cGMP elevations, but specific granule secretion was delayed. LY83583, a guanylyl cyclase antagonist, inhibited granule secretion and intermediate filament organization, but not microtubule or microfilament organization. Intermediate filament assembly at the pericortex and secretion were partially restored by 8-bromo-cGMP in LY83583-treated neutrophils, suggesting that cGMP regulates these functions. G-kinase directly induced intermediate filament assembly in situ, and protein phosphatase 1 disassembled filaments. However, in intact cells stimulated with formyl-methionyl-leucyl-phenylalanine, intermediate filament assembly is focal and transient, suggesting that vimentin phosphorylation is compartmentalized. We propose that, in addition to changes in microfilament and microtubule organization, granule secretion is also accompanied by changes in intermediate filament organization, and that cGMP regulates vimentin filament organization via activation of G-kinase.


Subject(s)
Cyclic GMP/blood , Cytoplasmic Granules/physiology , Intermediate Filaments/ultrastructure , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/physiology , Neutrophils/ultrastructure , Actin Cytoskeleton/drug effects , Actin Cytoskeleton/physiology , Actin Cytoskeleton/ultrastructure , Aminoquinolines/pharmacology , Cyclic GMP-Dependent Protein Kinase Type I , Cyclic GMP-Dependent Protein Kinases/blood , Cytochalasin D/pharmacology , Cytoplasmic Granules/drug effects , Cytoplasmic Granules/ultrastructure , Enzyme Inhibitors/pharmacology , Humans , In Vitro Techniques , Intermediate Filaments/drug effects , Intermediate Filaments/physiology , Kinetics , Microtubules/drug effects , Microtubules/physiology , Microtubules/ultrastructure , Models, Biological , Neutrophils/drug effects , Nocodazole/pharmacology , Phosphorylation , Signal Transduction , Vimentin/blood
13.
Cell Biochem Biophys ; 28(2-3): 251-75, 1998.
Article in English | MEDLINE | ID: mdl-9515168

ABSTRACT

The compartmentalization of cAMP in human neutrophils during phagocytosis of serum-opsonized zymosan suggests that cAMP is an important second messenger for regulating phagocytosis. Type 4 cAMP-specific phosphodiesterase (PDE-4), cAMP-dependent protein kinase (PKA), and adenylate cyclase are the principal effector molecules for cAMP regulation in phagocytes. Immunofluorescence microscopy demonstrated that PDE-4 isoforms (HSPDE-4A, HSPDE-4B, HSPDE-4D) were targeted to the forming phagosome in neutrophils, and were colocalized with the catalytic subunit of PKA and degranulated myeloperoxidase. Phagocytosis and accumulation of PDE-4 and PKA near adherent zymosan were inhibited by elevating cAMP levels with forskolin or rolipram. cAMP, PDE-4, and PKA were localized at sites of zymosan adherence in cells treated with cytochalasin D to inhibit phagosome formation, suggesting that zymosan engagement to Fc/CR3 receptors triggers cAMP elevations at sites of phagocytosis. HSPDE-4A, HSPDE-4B, HSPDE-4D, and PKA also were localized at the forming phagosome in monocyte-derived macrophages, and the lysosomal marker CD63 demonstrated the absence of PDE-4 around internalized phagolysosomes. These results suggest that cAMP levels are focally regulated by PDE-4 at the nascent phagosome, and that PKA may phosphorylate proteins associated with pseudopodia formation and phagosome internalization.


Subject(s)
3',5'-Cyclic-AMP Phosphodiesterases/analysis , Cell Compartmentation/immunology , Cyclic AMP-Dependent Protein Kinases/analysis , Macrophages/enzymology , Neutrophils/enzymology , Cell Adhesion , Colforsin/pharmacology , Cyclic AMP/analysis , Cyclic GMP/analysis , Cyclic Nucleotide Phosphodiesterases, Type 4 , Cytochalasin D/pharmacology , Humans , Isoenzymes/analysis , Macrophages/immunology , Neutrophils/immunology , Nucleic Acid Synthesis Inhibitors/pharmacology , Peroxidase/analysis , Phagocytosis , Phagosomes/enzymology , Phosphodiesterase Inhibitors/pharmacology , Pyrrolidinones/pharmacology , Rolipram , Signal Transduction/immunology , Zymosan
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