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1.
Invest Ophthalmol Vis Sci ; 39(5): 777-85, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9538885

ABSTRACT

PURPOSE: The objectives of this study were to examine whether preconditioning can decrease ischemic damage to the retina, by electroretinographic assessment of visual function and by histologic examination of retinal structure; to investigate the time course of the effectiveness of preconditioning; and to determine whether protein synthesis is involved. METHODS: Retinal ischemia was produced for 60 minutes in anesthetized Sprague-Dawley rats. Recovery after ischemia was measured by electroretinography for a maximum period of 7 days. Retinal sections that were sliced 1 micron thick were examined 7 days after ischemia. Retinal ischemia for 5 minutes constituted the preconditioning stimulus. To assess the time course of preconditioning, animals first underwent preconditioning and then 60 minutes of ischemia 1, 24, 72, or 168 hours later; or they underwent a 5-minute sham experiment and 60 minutes of ischemia 24 hours later. An additional group of rats received 0.4 mg/kg cycloheximide, the protein synthesis inhibitor, intraperitoneally before preconditioning and underwent 60 minutes of ischemia 24 hours later. RESULTS: In contrast to the nonpreconditioned rats, preconditioned rats had complete recovery of the a- and b-waves compared with preischemic baseline amplitudes, and ischemia-induced histologic damage was completely prevented when preconditioning was performed 24 or 72 hours (but not 168 hours) before ischemia. Separation of preconditioning and 60 minutes of ischemia by 1 hour caused an even greater impairment of functional retinal recovery compared with that seen in sham-preconditioned rats. Severe histologic damage was also noted. Block of protein synthesis by cycloheximide completely attenuated the protective effect of preconditioning. CONCLUSIONS: Preconditioning induces profound retinal tolerance to ischemia in vivo. The absence of a protective effect of preconditioning when there was a 1-hour or a 168-hour separation between the preconditioning stimulus and ischemia and the inhibition of preconditioning by cycloheximide support the hypothesis that a transient change in protein expression is necessary to provide this protection.


Subject(s)
Ischemic Preconditioning , Reperfusion Injury/prevention & control , Retinal Vessels/physiopathology , Animals , Cycloheximide/pharmacology , Electroretinography , Eye Proteins/physiology , Protein Synthesis Inhibitors/pharmacology , Rats , Rats, Sprague-Dawley , Reperfusion Injury/pathology , Reperfusion Injury/physiopathology , Retina/physiology , Retinal Diseases/pathology , Retinal Diseases/physiopathology , Retinal Diseases/prevention & control
2.
Vision Res ; 36(18): 2979-94, 1996 Sep.
Article in English | MEDLINE | ID: mdl-8917798

ABSTRACT

Nitric oxide synthase (NOS), the enzyme that catalyzes the formation of nitric oxide from L-arginine, exists in three major isoforms, neuronal, endothelial, and immunologic. Neuronal and endothelial isoforms are constitutively expressed, and require calcium for activation. Both of these isoforms can be induced (i.e., new protein synthesis occurs) under appropriate conditions. The immunologic isoform is not constitutively expressed, and requires induction usually by immunologic activation; calcium is not necessary for its activation. Neuronal and immunologic NOS have been detected in the retina. Neuronal NOS may be responsible for producing nitric oxide in photoreceptors and bipolar cells. Nitric oxide stimulates guanylate cyclase of photoreceptor rod cells and increases calcium channel currents. In the retina of cats, NOS inhibition impairs phototransduction as assessed by the electroretinogram. Inducible nitric oxide synthase, found in Müller cells and in retinal pigment epithelium, may be involved in normal phagocytosis of the retinal outer segment, in infectious and ischemic processes, and in the pathogenesis of diabetic retinopathy. Nitric oxide contributes to basal tone in the retinal circulation. To date, findings are conflicting with respect to its role in retinal autoregulation. During glucose and oxygen deprivation, nitric oxide may increase blood flow and prevent platelet aggregation, but it may also mediate the toxic effects of excitatory amino acid release. This reactive, short-lived gas is involved in diverse processes within the retina, and its significance continues to be actively studied.


Subject(s)
Nitric Oxide/physiology , Retina/physiology , Animals , Choroid/blood supply , Cyclic GMP/physiology , Cytokines/pharmacology , Humans , Hyperemia/physiopathology , Ischemia/metabolism , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/metabolism , Photoreceptor Cells/physiology , Pigment Epithelium of Eye/enzymology , Rabbits , Rats , Retinal Diseases/metabolism , Retinal Vessels , Vision, Ocular/physiology
3.
Invest Ophthalmol Vis Sci ; 36(12): 2396-403, 1995 Nov.
Article in English | MEDLINE | ID: mdl-7591629

ABSTRACT

PURPOSE: To determine the effect of nitric oxide synthase inhibition on blood flow in the cat retina and on changes in the electroretinogram after 1 hour of ocular ischemia. METHODS: After the induction of general anesthesia, one eye in each of 18 cats was subjected to 60 minutes of ischemia by raising intraocular pressure above systolic arterial pressure. One group (n = 9) was administered 30 mg/kg L-NG-nitro-arginine-methylester (L-NAME) intravenously 60 minutes before ischemia. The other group (n = 9) was administered an equivalent volume of saline intravenously. Five minutes after the end of ischemia, blood flow in the retina, choroid, and iris-ciliary body was measured using injections of radioactively labeled microspheres. Electroretinographic studies were carried out before, during, and 1, 2, 3, and 4 hours after ischemia ended. Arterial blood gas tension, systemic arterial pressure, hematocrit, and anesthetic levels were controlled in each experiment. RESULTS: Basal blood flow in iris-ciliary body was decreased by L-NAME, whereas retinal and choroidal blood flow was unchanged. Postischemic hyperemia was unaltered in the choroid and reduced in the retina. There was enhancement of postischemic hyperemia in the iris-ciliary body, but this effect may have resulted from a decrease in basal flow in the L-NAME group. Electroretinographic a- and b-wave recoveries were not altered by L-NAME; L-NAME infusion significantly decreased b-wave amplitude. CONCLUSIONS: Nitric oxide appears to be a significant factor in the regulation of uveal circulation and may contribute to the regulation of retinal, but not choroidal, blood flow after ischemia. The authors could not detect a difference in outcome by electroretinography with or without nitric oxide synthase inhibition.


Subject(s)
Arginine/analogs & derivatives , Enzyme Inhibitors/pharmacology , Ischemia/physiopathology , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide/physiology , Retinal Vessels , Uvea/blood supply , Animals , Arginine/pharmacology , Blood Flow Velocity/drug effects , Cats , Electroretinography , Injections, Intravenous , Microspheres , NG-Nitroarginine Methyl Ester , Ocular Hypertension , Regional Blood Flow/drug effects , Regional Blood Flow/physiology , Retina/physiology , Retinal Vessels/drug effects , Retinal Vessels/physiology
4.
Mol Carcinog ; 10(2): 82-7, 1994 Jun.
Article in English | MEDLINE | ID: mdl-8031468

ABSTRACT

The expression and DNA binding activity of members of the activating protein-1 (AP-1) and activating transcription factor (ATF) families of transcription factors were analyzed in sham and ultraviolet (UV)-irradiated subclones of the B16 mouse melanoma cell system. The four subclones we used represent sequential stages in the development and progression of malignant melanoma and exhibit differences in growth and metastatic potential. Western blot analysis revealed differential expression of some AP-1 (c-jun, jun-B, and jun-D) and ATF (43- and 47-kDa cyclic AMP-responsive element binding protein (CREB) family members) in the different subclones; while c-jun expression was noted in the subclones with the greater malignant potential, jun-D was expressed in those with the lesser malignant potential. Furthermore, a delicate balance between the two forms of CREB was noted; the 47-kDa CREB appeared, when expressed exclusively, in subclones that exhibit a greater malignant potential. Electrophoretic mobility shift assays using AP-1, CRE, and UV-responsive element (URE) consensus sequences indicated that distinct complexes were formed with extracts from each of the four subclones. The complexes were competitively inhibited by each of the target sequences used, suggesting that "cross-talk" occurs between some AP-1 and ATF family members in this cell system. Moreover, a multimer of the URE sequence, cloned upstream of a chloramphenicol acetyltransferase reporter gene, was transcriptionally active and responsive to UV irradiation in two of the four subclones. UV-related transcriptional activation was directly correlated with the expression of a 43-kDa CREB. Together, these observations identify members of AP-1 and CREB families whose expression and activities correlate with the malignant potential of subclones that represent different stages in melanoma development and progression.


Subject(s)
DNA-Binding Proteins/physiology , Melanoma, Experimental/genetics , Transcription Factors/physiology , Transcription, Genetic , Animals , Antibodies , Base Sequence , Cyclic AMP Response Element-Binding Protein/immunology , Cyclic AMP Response Element-Binding Protein/physiology , DNA, Neoplasm/metabolism , Electrophoresis , Gene Expression , Melanoma, Experimental/pathology , Mice , Molecular Sequence Data , Proto-Oncogene Proteins c-jun/physiology , Tumor Cells, Cultured , Ultraviolet Rays
5.
J Clin Invest ; 89(4): 1257-67, 1992 Apr.
Article in English | MEDLINE | ID: mdl-1556187

ABSTRACT

Although acute asbestos-induced pleurisy is characterized by an influx of neutrophils, the identity of the factors that attract these cells to the pleural space and the source of the factors are unknown. We found that instillation of crocidolite asbestos into the pleural space of rabbits led to the appearance in pleural liquid of chemotactic activity for neutrophils, and that this chemotactic activity was inhibited significantly by a neutralizing antibody to human interleukin 8 (IL-8). Cultured rabbit pleural mesothelial cells incubated with crocidolite asbestos also released chemotactic activity for neutrophils, which was inhibited significantly by the anti-IL-8 antibody. To determine whether rabbit pleural mesothelial cells synthesize IL-8, we generated a probe for rabbit IL-8 mRNA by amplifying cDNA prepared from stimulated pleural mesothelial cells using the polymerase chain reaction (PCR) and primers based on homologous sequences in human and sheep IL-8 cDNAs. Homology-based PCR yielded a single cDNA fragment with a nucleotide sequence 88% identical to that of a corresponding region of human IL-8 cDNA. With the radiolabeled PCR product as a probe, we demonstrated rapid induction of IL-8 mRNA expression in pleural mesothelial cells exposed to asbestos. As expected, tumor necrosis factor-alpha also led to the appearance of IL-8 in the rabbit pleural space and stimulated cultured pleural mesothelial cells to synthesize and release IL-8. We conclude that asbestos directly stimulates pleural mesothelial cells to synthesize IL-8 and that mesothelial cell-derived IL-8 may play an important role in mediating asbestos-induced pleural inflammation.


Subject(s)
Asbestos/adverse effects , Interleukin-8/physiology , Pleurisy/etiology , Animals , Base Sequence , DNA/isolation & purification , Epithelium/physiology , Interleukin-8/genetics , Male , Molecular Sequence Data , RNA, Messenger/analysis , Rabbits , Tumor Necrosis Factor-alpha/pharmacology
6.
Eur J Immunol ; 21(8): 1911-6, 1991 Aug.
Article in English | MEDLINE | ID: mdl-1831131

ABSTRACT

The low-affinity Fc receptor on human peripheral blood monocytes (Fc gamma RIIA) is polymorphic with respect to its ability to bind murine IgG1. The two allelic forms of the receptor, high responder (HR) and low responder (LR), yield characteristic patterns after isoelectric focusing and react differently with the anti-Fc gamma RII monoclonal antibody (mAb), 41H16. We recently cloned cDNA encoding the extracellular domains of Fc gamma RIIA on monocytes from one HR and two LR donors, and found that they differed at only a single base. The cDNA isolated from the HR donor had a G at position 519 and would be expected to encode an aginine at residue 133 in the mature protein, while the cDNA isolated from both LR donors had an A at position 519 and would be expected to encode a histidine at the same residue. To determine whether this single amino acid substitution actually accounts for the functional polymorphism involving Fc gamma RIIA, we transfected COS cells with full-length HR and LR Fc gamma RIIA cDNA, and examined them for their ability to react with anti-Fc gamma RIIA mAb and to bind red blood cells (RBC) coated with either murine IgG2b or murine IgG1. Whereas COS cells transfected with either the HR cDNA or the LR cDNA reacted with the anti-Fc gamma RII mAb, IV.3, and bound murine IgG2b-coated RBC, only COS cells transfected with the HR cDNA formed rosettes with murine IgG1-coated RBC and reacted strongly with mAb 41H16. A total of nine LR donors were identified, and all were homozygous for the A substitution at position 519. We conclude that at an A at position 519 in the cDNA encoding Fc gamma RIIA is the primary molecular basis for the LR form of the receptor, and that the amino acid at residue 133 determines whether Fc gamma RIIA efficiently binds murine IgG1.


Subject(s)
Antigens, Differentiation/analysis , Immunoglobulin G/metabolism , Monocytes/chemistry , Receptors, Fc/analysis , Adult , Antigens, Differentiation/genetics , Base Sequence , Humans , Molecular Sequence Data , Polymerase Chain Reaction , Polymorphism, Genetic , Receptors, Fc/genetics , Receptors, IgG , Structure-Activity Relationship , Transfection
7.
Blood ; 77(12): 2682-7, 1991 Jun 15.
Article in English | MEDLINE | ID: mdl-1710518

ABSTRACT

Human neutrophils express two polymorphic forms (NA1 and NA2) of Fc receptor III (FcRIII), which differ structurally and antigenically. We recently isolated FcRIII cDNAs from NA1NA1 and NA2NA2 homozygotes and determined that they differ only at five nucleotides, predicting four amino acid substitutions. To determine whether the cDNAs that we isolated actually encode proteins that differ structurally and that react appropriately with anti-NA1 and anti-NA2 antibodies, we transfected Chinese hamster ovary (CHO) cells with constructs containing either the NA1 FcRIII cDNA or the NA2 FcRIII cDNA. The receptors on transfected CHO cells were then compared with the receptors on normal human neutrophils from an NA1NA2 heterozygote. After immunoprecipitation and treatment with N-glycanase, receptors isolated from surface-labeled CHO cells transfected with the NA1 FcRIII cDNA had an apparent molecular mass of 29 Kd after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), while the receptors isolated from CHO cells transfected with the NA2 FcRIII cDNA had an apparent molecular mass of 33 Kd. Identical 29-Kd and 33-Kd bands were observed when receptors isolated from surface-labeled neutrophils of an NA1NA2 heterozygote were treated similarly. Using a cell-free rabbit reticulocyte lysate system, we translated NA1 FcRIII and NA2 FcRIII RNAs in vitro and also found differences in the apparent molecular masses of the two forms of the receptor. Finally, reactivity of transfected CHO cells with anti-NA monoclonal and alloantibodies confirmed that the cDNAs we isolated actually encode the NA1 and NA2 forms of neutrophil FcRIII.


Subject(s)
Neutrophils/chemistry , Receptors, Fc/genetics , Transfection , Amidohydrolases/metabolism , Animals , Cell Line , Cricetinae , DNA/genetics , Electrophoresis, Polyacrylamide Gel , Epitopes/immunology , Flow Cytometry , Fluorescent Antibody Technique , Heterozygote , Humans , Immunosorbent Techniques , Molecular Weight , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase , Protein Biosynthesis , Receptors, Fc/chemistry , Receptors, Fc/immunology , Rosette Formation
8.
J Clin Invest ; 86(1): 341-6, 1990 Jul.
Article in English | MEDLINE | ID: mdl-1694867

ABSTRACT

In the course of examining the structure and function of Fc receptors on peripheral blood cells of patients with systemic lupus erythematosus, we identified a patient whose neutrophils did not react with either monoclonal or polyclonal antibodies to Fc receptor III. However, neutrophils from the patient were comparable to neutrophils from healthy controls with respect to their expression of Fc receptor II, complement receptor 1, complement receptor 3, and the phosphatidylinositol-linked, complement regulatory protein, decay-accelerating factor. The abnormality of expression of Fc receptor III was limited to the patient's neutrophils (her natural killer cells reacted normally with anti-Fc receptor III antibodies), and was associated with abnormal recognition and binding of IgG-coated erythrocytes. Analysis of genomic DNA revealed evidence that failure of the patient's neutrophils to express Fc receptor III was most likely due to an abnormality of the gene that encodes this receptor.


Subject(s)
Antigens, Differentiation/genetics , Lupus Erythematosus, Systemic/genetics , Neutrophils/physiology , Receptors, Fc/genetics , Adult , Antigens, Differentiation/classification , Blotting, Southern , CD55 Antigens , Female , Genes , Humans , Leukocytes, Mononuclear/physiology , Membrane Proteins/metabolism , Polymerase Chain Reaction , Receptors, Fc/classification , Receptors, IgG , Restriction Mapping
9.
J Clin Invest ; 84(5): 1688-91, 1989 Nov.
Article in English | MEDLINE | ID: mdl-2478590

ABSTRACT

Two polymorphic forms of Fc receptor III (FcR III) are expressed on human neutrophils. These differ with respect to their apparent molecular masses after digestion with N-glycanase, and with respect to their reactivity with MAb Gran 11 and alloantisera which recognize determinants (NA1 and NA2) of the biallelic neutrophil antigen (NA) system. To determine the molecular basis for this polymorphism we isolated RNA from neutrophils of NA1NA1 and NA2NA2 homozygotes and synthesized corresponding cDNAs. cDNAs encoding FcR III were then amplified using the polymerase chain reaction, cloned, and sequenced. The cDNA that encodes FcR III on NA1NA1 neutrophils differed from the cDNA that encodes FcR III on NA2NA2 neutrophils at five nucleotides, predicting four amino acid substitutions. As a result, NA1 FcR III has only four potential N-linked glycosylation sites as compared with six in NA2 FcR III. The amino acid substitutions and differences in the number of potential N-linked glycosylation sites probably account for the different forms of neutrophil FcR III observed after digestion with N-glycanase and for the antigenic heterogeneity of this receptor.


Subject(s)
Antigens/genetics , DNA/genetics , Neutrophils/immunology , Polymorphism, Genetic , Receptors, Fc/genetics , Amidohydrolases/metabolism , Animals , Antibodies, Monoclonal , Base Sequence , Cloning, Molecular , Epitopes/genetics , Glycosylation , Humans , Mice , Molecular Weight , Nucleic Acid Hybridization , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase , Polymerase Chain Reaction , RNA/genetics , RNA/isolation & purification
10.
Arthritis Rheum ; 32(10): 1193-6, 1989 Oct.
Article in English | MEDLINE | ID: mdl-2803322
11.
J Immunol ; 143(5): 1731-4, 1989 Sep 01.
Article in English | MEDLINE | ID: mdl-2527271

ABSTRACT

IgG Fc receptor II (Fc gamma RII) on human monocytes is polymorphic with respect to its appearance on gels after isoelectric focusing and with respect to its ability to mediate T lymphocyte proliferation induced by murine anti-CD3 mAb of the IgG1 isotype (i.e., its ability to bind murine IgG1). To determine the molecular basis for this polymorphism, we isolated total cellular RNA from PBMC of responders and nonresponders (defined by Leu-4-induced [3H] thymidine incorporation) and synthesized corresponding cDNA. Sequences encoding the extracellular domain of Fc gamma RII were then amplified using the Taq polymerase chain reaction. Amplified DNA fragments were cloned into pUC vectors, and sequenced. Analysis of clones from two nonresponders revealed a single base change (G for A) at position 519, which would result in the substitution of a histidine for an arginine at residue 133 in the mature Fc gamma RII protein. These findings suggest that the polymorphism involving human monocyte Fc gamma RII results from allelic variation of a single gene.


Subject(s)
Antigens, Differentiation/genetics , Immunoglobulin G/metabolism , Monocytes/metabolism , Polymorphism, Genetic , Receptors, Fc/genetics , Adult , Antigens, Differentiation, T-Lymphocyte/immunology , Base Sequence , Cloning, Molecular , Erythrocytes/metabolism , Gene Amplification , Humans , Lymphocyte Activation , Molecular Sequence Data , Receptors, IgG , Rosette Formation , T-Lymphocytes/immunology
12.
J Clin Invest ; 83(5): 1676-81, 1989 May.
Article in English | MEDLINE | ID: mdl-2523415

ABSTRACT

We characterized Fc receptor III (FcR III) on human neutrophils and found it to be heavily glycosylated and polymorphic. In some individuals, FcR III that had been digested with N-glycanase appeared after SDS-PAGE under reducing conditions as two bands with apparent molecular masses of 33 and 29 kD. In other individuals, N-glycanase-treated FcR III appeared as a single band with an Mr of either 33 or 29 kD. After SDS-PAGE of N-glycanase-treated FcR III under nonreducing conditions, the apparent Mr of each structural type was decreased, suggesting the presence of intramolecular disulfide bonds. Digestion of the 33-kD band and the 29-kD band with Staphylococcus aureus V8 protease yielded similar, but not identical, peptide maps. Thus, at least two polymorphic forms of FcR III are expressed on human neutrophils. The structural polymorphism of neutrophil FcR III correlated with previously described antigenic polymorphisms detected by monoclonal antibody Gran 11 and by alloantisera which recognize epitopes of the biallelic, neutrophil antigen (NA) system. Individuals whose neutrophils expressed the two-band structural type of FcR III were NA1NA2 heterozygotes. Individuals whose neutrophils expressed the single 33-kD band structural type were NA2NA2 homozygotes, and individuals whose neutrophils expressed the single 29-kD band structural type were NA1NA1 homozygotes. These findings indicate that antigenic and structural polymorphisms of human neutrophil FcR III are related and can be accounted for by differences at the level of primary protein structure.


Subject(s)
Antigens, Differentiation/isolation & purification , Immunoglobulin G/metabolism , Polymorphism, Genetic , Receptors, Fc/isolation & purification , Antibodies, Monoclonal , Antigen-Antibody Reactions , Antigens, Differentiation/genetics , Antigens, Differentiation/immunology , Antigens, Heterophile/immunology , Glycosylation , Humans , Isoantibodies , Molecular Weight , Neutrophils/immunology , Neutrophils/metabolism , Receptors, Fc/genetics , Receptors, Fc/immunology , Receptors, IgG , Structure-Activity Relationship
14.
J Appl Physiol (1985) ; 64(3): 929-35, 1988 Mar.
Article in English | MEDLINE | ID: mdl-3366749

ABSTRACT

We studied the effects of bovine catalase on increased lung vascular permeability to fluid and protein during air emboli in unanesthetized sheep. Pulmonary arterial and left atrial pressures, cardiac output, lung lymph flow, lymph and plasma protein concentrations, arterial PO2, and numbers of arterial leukocytes were measured in paired experiments in which each sheep served as its own control. We found an increase in protein-rich lung lymph flow during embolization in untreated sheep, indicating an increase in microvascular permeability. When sheep were pretreated with intraperitoneal injections of catalase (50 mg/kg divided over the 24 h before air infusion), vascular pressures, arterial PO2, and leukocyte counts were not different from when the sheep were untreated, but the expected increases in transvascular fluid and protein flow during emboli were significantly attenuated (by approximately 50%). This effect required catalase enzyme activity, as demonstrated by the failure of enzymatically inactivated catalase (by reaction in vitro with aminotriazole in the presence of H2O2) or catalase vehicle (0.1% thymol in water) to affect the lung lymph response to air emboli. We conclude that H2O2 plays a role in the pathogenesis of the acute lung injury caused by intravenous air infusions into unanesthetized sheep. Because both catalase and superoxide dismutase have protected sheep lungs from air emboli-induced increased vascular permeability, a possible specific cause of microvascular barrier injury could be hydroxyl radicals formed from reactions between H2O2 and superoxide anion.


Subject(s)
Capillary Permeability/drug effects , Catalase/pharmacology , Embolism, Air/physiopathology , Lung/blood supply , Pulmonary Edema/etiology , Animals , Blood Gas Analysis , Blood Pressure , Cardiac Output , Female , Hydrogen Peroxide/metabolism , Leukocyte Count , Lung/physiopathology , Lymphatic System/physiopathology , Sheep , Vascular Resistance
15.
Am Rev Respir Dis ; 137(2): 420-8, 1988 Feb.
Article in English | MEDLINE | ID: mdl-3277502

ABSTRACT

Administration of endotoxin intravenously to unanesthetized sheep causes an acute lung injury characterized by increased microvascular barrier permeability and subsequent pulmonary edema. Endotoxin-induced sheep lung injury can be attenuated by leukocyte depletion, and may be mediated by toxic metabolites of oxygen. We studied effects of administering catalase, which catalyzes conversion of hydrogen peroxide to oxygen and water, to sheep subsequently infused with endotoxin to test the hypothesis that hydrogen peroxide plays a role in the pathogenesis of lung injury. We found that infusions of endotoxin (1 microgram/kg) into untreated sheep caused the expected biphasic response, a transient, early, marked pulmonary arterial hypertension followed by a prolonged increase in protein-rich lung lymph flow characteristic of increased microvascular permeability filtration in the lungs. Intraperitoneal injections of catalase (50 mg/kg) prior to infusing endotoxin in these same sheep resulted in substantial catalase activity in plasma and in lung lymph, and attenuated the expected changes in pulmonary arterial pressure, lung lymph flow, and arterial leukocyte counts and oxygen tension after endotoxin infusions. Furthermore, mechanical elevation of hydrostatic pressure in the lungs of a catalase-treated sheep infused with endotoxin resulted in increased lung lymph flow with a decreased protein concentration, indicating that the microvascular barrier to fluid and protein was functionally intact. Administration of catalase that was inactivated by reaction with hydrogen peroxide in the presence of aminotriazole or administration of the catalase vehicle, thymol, had no effects on the sheep responses to endotoxin. We conclude that hydrogen peroxide plays a role in the pathogenesis of endotoxin-induced acute lung injury in sheep.


Subject(s)
Catalase/therapeutic use , Endotoxins/toxicity , Lung Diseases/etiology , Sheep Diseases/etiology , Acute Disease , Anesthesia , Animals , Catalase/antagonists & inhibitors , Catalase/metabolism , Cell Adhesion/drug effects , Cells, Cultured , Drug Evaluation, Preclinical , Escherichia coli , Female , Hydrogen Peroxide/metabolism , Lung/drug effects , Lung/enzymology , Lung Diseases/physiopathology , Lung Diseases/prevention & control , Lymph/drug effects , Lymph/enzymology , Neutrophils/drug effects , Neutrophils/enzymology , Sheep , Sheep Diseases/physiopathology , Sheep Diseases/prevention & control , Thymol/pharmacology
16.
Infect Immun ; 55(12): 2956-61, 1987 Dec.
Article in English | MEDLINE | ID: mdl-3679539

ABSTRACT

We have examined the role played by human peripheral blood monocytes in mediating responses of human polymorphonuclear leukocytes (PMN) to bacterial lipopolysaccharide (LPS) in vitro. When incubated with Salmonella typhimurium LPS at 37 degrees C, human PMN suspended in serum-free buffer released the specific granule constituent lactoferrin into the surrounding medium. Release of lactoferrin from PMN varied with the concentration of LPS (1 to 1,000 ng/ml) as well as with the duration of incubation (2 to 60 min) and was not accompanied by significant release of the cytoplasmic enzyme lactate dehydrogenase. LPS-induced release of lactoferrin from PMN was augmented significantly when cell suspensions were supplemented with additional monocytes and lymphocytes. Only monocytes, however, secreted significant amounts of lactoferrin-releasing activity (in a time- and concentration-dependent manner) when incubated separately with LPS. Lactoferrin-releasing activity was heat (80 degrees C for 15 min) labile, eluted after chromatography on Sephadex G-100 with an apparent molecular weight of approximately 60,000, and was inhibited by antibodies to tumor necrosis factor alpha. Thus, LPS-induced noncytotoxic release of lactoferrin from human PMN suspended in serum-free buffer is mediated, at least in part, by tumor necrosis factor alpha derived from contaminating monocytes.


Subject(s)
Lactoferrin/metabolism , Lactoglobulins/metabolism , Lipopolysaccharides/pharmacology , Monocytes/physiology , Neutrophils/metabolism , Tumor Necrosis Factor-alpha/physiology , Cells, Cultured , Humans , In Vitro Techniques , Leukocytes, Mononuclear/physiology , Monokines , Proteins/physiology
17.
J Rheumatol Suppl ; 14 Suppl 13: 53-8, 1987 Jun.
Article in English | MEDLINE | ID: mdl-3612652

ABSTRACT

Studies completed thus far indicate that normal human serum contains a specific anionic polypeptide (cochemotaxin) that permits low concentrations of C5a desarg to exhibit chemotactic activity for human polymorphonuclear leukocytes. Under conditions of limited complement activation in vivo, the cochemotaxin may play an important physiologic role by amplifying the chemotactic activity of C5a desarg. The "complex" of C5a desarg plus cochemotaxin probably accounts for most of the chemotactic activity that is generated in human serum after complement activation and also appears to be the target of the heat stable cationic protein inhibitor (Bb fragment of Factor B) that is present in serum from some patients with active systemic lupus erythematosus.


Subject(s)
Chemotaxis, Leukocyte , Lupus Erythematosus, Systemic/immunology , Neutrophils/immunology , Complement C5/analogs & derivatives , Complement C5/physiology , Complement C5a, des-Arginine , Humans
18.
J Biol Chem ; 262(16): 7850-8, 1987 Jun 05.
Article in English | MEDLINE | ID: mdl-2953727

ABSTRACT

We have used Ca2+-dependent binding to a phospholipid vesicle affinity column to isolate a mixture of three synexin-like proteins from the cytosol of human polymorphonuclear leukocytes (PMN), with relative molecular weights of approximately 67,000, 47,000, and 28,000. Rabbit antibodies raised against bovine liver synexin recognized the 47,000 molecular weight PMN protein. These PMN proteins, like bovine liver synexin, promoted aggregation of isolated PMN specific granules in the presence of Ca2+ and increased the overall rate of Ca2+-induced fusion of liposomes composed of phosphatidate (PA)/phosphatidylethanolamine (PE) (1:3) and phosphatidylserine/PE (1:3), but decreased the rate of spermine-induced fusion of PA/PE (1:3) liposomes. Using fluorescent lipid probes, rapid fusion of PA/PE liposomes with PMN specific granules (50% maximum signal within a few minutes) was observed when 1 mM Ca2+ was added in the presence of both synexin and free arachidonic acid. Dilution of the aqueous contents of liposomes was also observed under the same conditions. The rate of fusion increased monotonically with Ca2+ and arachidonic acid concentrations, but synexin exhibited an optimum concentration. Lack of any one of the components precluded rapid fusion. These results suggest that PMN contain a protein similar to, or identical with, synexin that may be involved in calcium-dependent fusion of intracellular membranes.


Subject(s)
Blood Proteins , Calcium-Binding Proteins/blood , Cytoplasmic Granules/metabolism , Neutrophils/metabolism , Animals , Annexin A7 , Cattle , Cell Membrane/metabolism , Chromatography, Affinity , Cytoplasmic Granules/ultrastructure , Humans , Kinetics , Liposomes , Liver/metabolism , Proteins/isolation & purification , Spermine
19.
J Clin Invest ; 78(6): 1589-95, 1986 Dec.
Article in English | MEDLINE | ID: mdl-3782473

ABSTRACT

The chemotactic activity of human C5a des Arg is enhanced significantly by an anionic polypeptide (cochemotaxin) in normal human serum and plasma. We have found that the cochemotaxin attaches to the oligosaccharide chain of native C5a des Arg to form a complex with potent chemotactic activity for human polymorphonuclear leukocytes. Although capable of enhancing the chemotactic activity of native C5a des Arg, the cochemotaxin had no effect on the chemotactic activity of either deglycosylated C5a des Arg, native C5a, or N-formyl-methionyl-leucyl-phenylalanine. Of the known components of the oligosaccharide chain, only sialic acid prevented enhancement by the cochemotaxin of the chemotactic activity exhibited by native C5a des Arg. Sialic acid also prevented the formation of C5a des Arg-cochemotaxin complexes, detected by acid polyacrylamide gel electrophoresis, molecular sieve chromatography on polyacrylamide gels, and sucrose density gradient ultracentrifugation.


Subject(s)
Anaphylatoxins , Blood Proteins/pharmacology , Chemotactic Factors/pharmacology , Chemotaxis/drug effects , Complement C5/analogs & derivatives , Peptides , Blood Physiological Phenomena , Blood Proteins/metabolism , Chromatography, Gel , Complement C5/metabolism , Complement C5/pharmacology , Complement C5a , Complement C5a, des-Arginine , Electrophoresis, Polyacrylamide Gel , Humans , Iodine Radioisotopes , Molecular Weight , N-Acetylneuraminic Acid , Sialic Acids/pharmacology
20.
J Immunol ; 136(9): 3412-9, 1986 May 01.
Article in English | MEDLINE | ID: mdl-3514759

ABSTRACT

We have used a new centrifugation assay to examine the effects of highly purified human C5a and C5a des Arg, as well as effects of N-formyl-methionyl-leucyl-phenylalanine (FMLP), on both the extent and strength of human polymorphonuclear leukocyte (PMN) adherence to monolayers of cultured human umbilical vein endothelial cells. At concentrations that were chemotactic for PMN, C5a (0.1 nM), C5a des Arg (5.0 nM), and FMLP (1.0 nM) significantly reduced the percentage of PMN that adhered to endothelial monolayers. Adherence also was reduced by C5a des Arg that was generated by incubating (37 degrees C, 30 min) fresh human serum with either zymosan or purified C5a. High concentrations of C5a (greater than 1.0 nM) and FMLP (greater than 50 nM) that diminished PMN chemotaxis significantly enhanced the percentage of PMN that adhered tightly to endothelial cells (adherent cells resisted a dislodgment force of 1200 X G). Tight adherence of PMN to endothelial cells also was increased by high concentrations of C5a that were added to human serum in which carboxypeptidase N activity was destroyed by heating (56 degrees C, 30 min), and by C5a that was generated by incubating (37 degrees C, 30 min) fresh human serum with zymosan in the presence of the carboxypeptidase N inhibitor, epsilon-aminocaproic acid. High concentrations of C5a des Arg (up to 80 nM) neither enhanced adherence of PMN to endothelial cells nor decreased PMN migration. Thus, a reciprocal relation exists between PMN migration and PMN adherence to endothelial cells in response to chemotactic factors. At concentrations that are chemotactic for human PMN, C5-derived peptides and FMLP reduce the adherence of PMN to endothelial monolayers. Only at concentrations that decrease PMN migration do C5a and FMLP augment PMN adherence.


Subject(s)
Chemotactic Factors/physiology , Endothelium/physiology , Neutrophils/physiology , Adult , Cell Adhesion/drug effects , Cells, Cultured , Chemotaxis, Leukocyte/drug effects , Complement C5/analogs & derivatives , Complement C5/physiology , Complement C5a , Complement C5a, des-Arginine , Endothelium/cytology , Humans , Interleukin-8 , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Time Factors , Umbilical Veins
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