36 results on '"Cochrane C"'
Search Results
2. Domains of the human neutrophil N-formyl peptide receptor involved in G protein coupling. Mapping with receptor-derived peptides.
- Author
-
Schreiber RE, Prossnitz ER, Ye RD, Cochrane CG, and Bokoch GM
- Subjects
- Amino Acid Sequence, Binding Sites, Humans, In Vitro Techniques, Molecular Sequence Data, Peptides metabolism, Receptors, Formyl Peptide, GTP-Binding Proteins metabolism, N-Formylmethionine Leucyl-Phenylalanine metabolism, Neutrophils metabolism, Receptors, Immunologic metabolism, Receptors, Peptide metabolism
- Abstract
Chemotactic signaling by the human neutrophil N-formyl peptide receptor requires its association with heterotrimeric G protein. Synthetic peptides and a fusion protein derived from the intracellular regions of the receptor were used to identify sites which interact with G protein. A peptide derived from the second intracellular loop (C12R), and peptides (F15R and S22L) and a fusion protein derived from the receptor's carboxyl terminus inhibited binding of anti-Gi alpha antibody (R16,17) to Gi alpha in a competitive enzyme-linked immunoassay, and antagonized pertussis-toxin catalyzed ADP-ribosylation of Gi alpha. C12R also inhibited G protein-dependent, high affinity ligand binding to the receptor and physical coupling of receptor to G protein. In contrast, a peptide consisting of the entire third loop of the N-formyl peptide receptor was totally inactive in these assays. Collectively, these data suggest that the second intracellular loop and the carboxyl-terminal tail are important for effective N-formyl peptide receptor/G protein coupling, but that the third intracellular loop is less important in coupling, unlike previous findings with other G protein-coupled receptor systems. The chemoattractant receptor family may rely on different structural determinants to interact with GTP-binding proteins.
- Published
- 1994
3. Reconstitution of recombinant N-formyl chemotactic peptide receptor with G protein.
- Author
-
Schreiber RE, Prossnitz ER, Ye RD, Cochrane CG, Jesaitis AJ, and Bokoch GM
- Subjects
- Animals, Brain metabolism, Cattle, Cell Line, Centrifugation, Density Gradient, Fibroblasts metabolism, GTP-Binding Proteins isolation & purification, Humans, Immunoassay, Kinetics, Mice, Molecular Weight, Receptors, Formyl Peptide, Receptors, Immunologic genetics, Receptors, Immunologic isolation & purification, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Transfection, GTP-Binding Proteins metabolism, N-Formylmethionine Leucyl-Phenylalanine metabolism, Neutrophils metabolism, Receptors, Immunologic metabolism
- Abstract
A recombinant human neutrophil N-formyl peptide receptor (rFPR) expressed in transfected mouse fibroblasts (TX2 cells) was analyzed for its ability to couple physically with the heterotrimeric G protein, Gi. Immunoprecipitation of photoaffinity-labeled rFPR and endogenous neutrophil formyl peptide receptor (nFPR) with an anti-FPR peptide antibody demonstrated that the receptors were identical in both size and extent of glycosylation. Coupling of rFPR with endogenous TX2 Gi was demonstrated by coimmunoprecipitation of the two proteins with an anti-Gi antibody. Moreover, rFPR was able to form a physical complex with purified Gi in a soluble reconstitution system. We observed similar affinities of rFPR and nFPR for Gi. This report provides the first direct evidence that rFPR associates physically with Gi and provides a foundation for analysis of the G protein coupling capacity of mutant rFPRs.
- Published
- 1993
- Full Text
- View/download PDF
4. The rabbit neutrophil N-formyl peptide receptor. cDNA cloning, expression, and structure/function implications.
- Author
-
Ye RD, Quehenberger O, Thomas KM, Navarro J, Cavanagh SL, Prossnitz ER, and Cochrane CG
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Calcium metabolism, Cloning, Molecular, DNA genetics, GTP-Binding Proteins metabolism, Gene Expression, Humans, Membrane Glycoproteins genetics, Molecular Sequence Data, RNA, Messenger genetics, Rabbits, Receptors, Formyl Peptide, Receptors, Immunologic metabolism, Second Messenger Systems, Sequence Alignment, Signal Transduction, Structure-Activity Relationship, Transfection, N-Formylmethionine Leucyl-Phenylalanine metabolism, Neutrophils physiology, Receptors, Immunologic genetics
- Abstract
The rabbit neutrophil N-formyl peptide receptor (FPR) has been well studied for its ligand binding properties. Recent gene cloning experiments have established the existence of a subfamily of G protein-coupled receptors that share extensive sequence homology with the FPR, yet lack the capability of high affinity binding to FMLP. These findings prompted us to identify the structural requirement for formyl peptide ligand binding by delineation of the primary structure of the rabbit FPR. A rabbit neutrophil cDNA library was screened with a cloned human FPR cDNA probe and the insert of one positive isolate (B6) was sequenced. The 1268-bp cDNA insert encodes a peptide of 352 amino acids. Stably transfected L cell fibroblasts expressing the rabbit cDNA displayed specific binding of the ligand fMet-Leu-[3H]Phe with two affinities (Kd = 0.31 and 7.5 nM). Addition of the nonhydrolyzable guanosine triphosphate analogue, GTP gamma S, converted > or = 85% of the high affinity sites to the low affinity sites. FMLP induced mobilization of intracellular calcium in the transfected cells (EC50 = 0.5 nM), a response sensitive to pertussis toxin. FMLP stimulation desensitized the receptor such that subsequent stimulation with the same ligand produced a significantly reduced signal. These results indicate that the cloned rabbit receptor represents a high affinity FPR, and that FPR-mediated early signal transduction events can be fully reconstituted in transfected mammalian cells. The rabbit FPR sequence is 78% identical to that of the human FPR, and 68% identical to FPR2, a homologue of FPR with a low binding affinity (Kd > or = 400 nM) for FMLP. Analysis of the aligned sequences of these three proteins revealed that: 1) the amino termini and the second extracellular loops have the lowest sequence homology; 2) sequence in the intracellular domains that couple to G protein are highly conserved; and 3) the first and the third extracellular loops and their adjacent transmembrane domains of the FPR may contain residues essential for the high affinity binding of FMLP.
- Published
- 1993
5. Ligand/receptor internalization: a spectroscopic analysis and a comparison of ligand binding, cellular response, and internalization by human neutrophils.
- Author
-
Sklar LA, Jesaitis AJ, Painter RG, and Cochrane CG
- Subjects
- Cell Membrane physiology, Flow Cytometry, Fluorescein, Fluoresceins, Humans, Hydrogen-Ion Concentration, Kinetics, Pancreatic Elastase metabolism, Receptors, Formyl Peptide, Spectrometry, Fluorescence, Superoxides metabolism, Neutrophils metabolism, Oligopeptides metabolism, Receptors, Cell Surface metabolism
- Abstract
We have compared the kinetics of the responses of neutrophils to the kinetics of ligand-receptor interaction and internalization, using as a model ligand the fluoresceinated hexapeptide N-CHO-Nle-Leu-Phe-Nle-Tyr-Lys-Fluorescein (Nle, norleucine). Cellular responses, ie, membrane depolarization, enzyme (elastase) secretion, and superoxide anion (O-2) generation, are all initiated within 10 sec of the exposure of cells to stimulus. In the cases of membrane depolarization and secretion (in cytochalasin B-treated cells), full responses are elicited by binding which occurs within 15 sec of peptide addition. Ligand binding and internalization have been analyzed over the same time frame with new spectroscopic techniques. The association of ligand and receptor is monitored using an antibody to fluorescein. The antibody to fluorescein specifically quenches the ligand which is in solution, but receptor-bound ligand is inaccessible to the antibody. The internalization of the receptor-bound ligand is monitored by the accessibility of the fluoresceinated peptide to quenching by an external pH change (7.4 leads to 4.0). Ligand which is either outside or on the cell surface is instantaneously quenched while intracellular peptide (or intracellular fluorescein derived from fluorescein diacetate) is only slowly quenched. No internalization is observed until 1 min after binding begins and internalization proceeds at a rate of up to 5,000 receptors/min/cell following a near optimal stimulatory ligand concentration (approximately 1 nM) while the occupied receptors are being cleared from the surface. A comparison of the kinetics of internalization and the cellular responses suggests that internalization of the ligand is too slow to be involved in the triggering of the cellular responses.
- Published
- 1982
- Full Text
- View/download PDF
6. Fluoresceinated chemotactic peptide and high-affinity antifluorescein antibody as a probe of the temporal characteristics of neutrophil stimulation.
- Author
-
Sklar LA, Oades ZG, Jesaitis AJ, Painter RG, and Cochrane CG
- Subjects
- Antigen-Antibody Complex, Fluoresceins, Humans, Immunologic Techniques, Ligands, Time Factors, Chemotactic Factors metabolism, Chemotaxis, Leukocyte, Neutrophils physiology, Oligopeptides metabolism
- Abstract
Antifluorescein antibody molecules were used to interrupt the stimulation of neutrophils by a fluoresceinated chemotactic peptide. From the results we construct a semiquantitative relationship among ligand-receptor interaction, the time course of cell triggering and response, and aspects of cellular adaptation. The interaction of the antibody with the free fluoresceinated peptide is complete within a few seconds and the peptide-antibody complex neither stimulates the cells nor inhibits subsequent stimulation by unlabeled peptide. When antibody is added to a cell suspension that has been stimulated with the fluoresceinated peptide, we observe that: (i) the apparent membrane depolarization response monitored by a fluorescent dye can be inhibited only if antibody is added within 30 sec of stimulation; (ii) the superoxide response can be inhibited even if antibody is added more than 1 min after stimulation and decays with an intrinsic half-life of about 12 sec; (iii) responses to a second dose of nonfluoresceinated peptide are enhanced if the antibody is added within 2 min of stimulation by the fluoresceinated peptide. These results suggest that different neutrophil responses depend in individual ways on the time course and extent of ligand binding to its receptor. A comparison of these data with the time course of binding permits an estimate of the number of receptors involved in these responses.
- Published
- 1981
- Full Text
- View/download PDF
7. Photoaffinity labeling of the N-formyl peptide receptor binding site of intact human polymorphonuclear leukocytes. A label suitable for following the fate of the receptor-ligand complex.
- Author
-
Schmitt M, Painter RG, Jesaitis AJ, Preissner K, Sklar LA, and Cochrane CG
- Subjects
- Azides chemical synthesis, Cell Membrane metabolism, Humans, Molecular Weight, Oligopeptides chemical synthesis, Receptors, Cell Surface drug effects, Receptors, Cell Surface isolation & purification, Receptors, Formyl Peptide, Affinity Labels pharmacology, Azides pharmacology, Neutrophils metabolism, Oligopeptides metabolism, Oligopeptides pharmacology, Receptors, Cell Surface metabolism
- Published
- 1983
8. The kinetics of neutrophil activation. The response to chemotactic peptides depends upon whether ligand-receptor interaction is rate-limiting.
- Author
-
Sklar LA, Jesaitis AJ, Painter RG, and Cochrane CG
- Subjects
- Chemotaxis, Humans, Kinetics, Membrane Potentials drug effects, Neutrophils drug effects, Peptides pharmacology, Spectrometry, Fluorescence, Superoxides blood, Valinomycin pharmacology, Neutrophils physiology
- Published
- 1981
9. Survey of plant inhibitors of polymorphonuclear leukocyte elastase, pancreatic elastase, cathepsin G, cathepsin B, Hageman factor fragments, and other serine proteinases.
- Author
-
Hojima Y, Pisano JJ, and Cochrane CG
- Subjects
- Cathepsin B, Cathepsin G, Cathepsins antagonists & inhibitors, Factor XII antagonists & inhibitors, Factor XIIa, Humans, In Vitro Techniques, Pancreatic Elastase antagonists & inhibitors, Peptide Fragments antagonists & inhibitors, Serine Endopeptidases, Species Specificity, Neutrophils enzymology, Pancreas enzymology, Plant Extracts pharmacology, Protease Inhibitors
- Abstract
Various flower bulbs and vegetable and legume seeds were tested for inhibitors of polymorphonuclear leukocyte elastase, pancreatic elastase, cathepsin G, cathepsin B, trypsin, alpha-chymotrypsin, Hageman factor fragments, plasma kallikrein, and plasmin. Calla bulbs contained a 33,000 dalton polymorphonuclear leukocyte elastase inhibitor and a 4,000 dalton cathepsin G inhibitor. Seeds of some members in the Cruciferae family, such as radish and broccoli, were found to contain one or more 2,500-4,000 dalton inhibitors which inhibited cathepsin G, trypsin, Hageman factor fragments, and plasmin, but not plasma kallikrein. These seeds also contained a 1,000 dalton cathepsin B inhibitor. The above inhibitors were probably polypeptides which inhibited proteinases by making an enzyme-inhibitor complex, with the exception of the cathepsin B inhibitor. These newly found inhibitors with their characteristic profiles of inhibition should be useful in biochemical and pathophysiological studies on granulocyte proteinases and enzymes of the coagulation and fibrinolytic pathways.
- Published
- 1983
- Full Text
- View/download PDF
10. Evidence for N-formyl chemotactic peptide-stimulated GTPase activity in human neutrophil homogenates.
- Author
-
Hyslop PA, Oades ZG, Jesaitis AJ, Painter RG, Cochrane CG, and Sklar LA
- Subjects
- Adenylyl Cyclases metabolism, Cell-Free System, GTP-Binding Proteins, Humans, Kinetics, Receptors, Formyl Peptide, GTP Phosphohydrolases metabolism, Neutrophils enzymology, Phosphoric Monoester Hydrolases metabolism, Receptors, Cell Surface physiology
- Abstract
Neutrophil homogenates contained a high affinity guanosine triphosphatase (GTPase) that was stimulatable (+27%) by the addition of 100 nM N-formyl chemotactic peptide (CHO-pep), but not by 1 microgram X ml-1 phorbolmyristate acetate (PMA). Kinetic analysis of the stimulation demonstrated an apparent lagtime of 14.3 +/- 6.9 s between the addition of CHO-pep and the optimal GTPase stimulation. The GTPase activity (but not CHO-pep-stimulated GTPase activity) was preserved in a highly purified plasma membrane fraction of the homogenate. From these observations we suggest that both a high affinity guanine nucleotide binding protein and GTPase are closely associated with the plasma membrane CHO-pep receptor. The possibility that GTPase activity may influence guanine nucleotide regulation of adenylate cyclase during CHO-pep stimulation of neutrophils is discussed.
- Published
- 1984
- Full Text
- View/download PDF
11. Photoaffinity labeling of the N-formyl peptide receptor of human polymorphonuclear leukocytes.
- Author
-
Painter RG, Schmitt M, Jesaitis AJ, Sklar LA, Preissner K, and Cochrane CG
- Subjects
- Cell Fractionation, Cell Membrane analysis, Glycoproteins analysis, Humans, Lectins, Neutrophils metabolism, Neutrophils ultrastructure, Oligopeptides metabolism, Oligopeptides pharmacology, Receptors, Cell Surface metabolism, Receptors, Formyl Peptide, Superoxides metabolism, Wheat Germ Agglutinins, Affinity Labels, Neutrophils analysis, Receptors, Cell Surface analysis
- Abstract
Quantitative analysis of ligand-occupied receptor interactions with elements of the cytoskeleton and with intracellular compartments requires a sensitive and simple method of identifying the receptor-ligand complex in living cells. Toward this goal, we have prepared a photoactivatable arylazide derivative of the chemotactic peptide N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys, which can be radiolabeled to high specific activity with 125I. This derivative was biologically active as judged by its ability to elicit superoxide anion production by human PMNL at nanomolar concentrations (ED50 approximately 0.7 nM). When incubated at 0 degree C with whole PMNL, radioactive ligand became specifically and saturably associated with a 60-70,000-dalton species (as assessed by SDS-PAGE) after exposure to UV light. Addition of 10-100-fold excess of unlabeled parent or unlabeled azidopeptide derivative completely blocked uptake into this species. Approximately 20-40% of the available surface receptor-binding sites were covalently labeled under these conditions. Subcellular fractionation of the labeled cells on sucrose gradients after homogenization showed that the labeled species was primarily associated with plasma membrane-rich fractions. The labeled receptor could be completely solubilized with Triton X-100 in a form which eluted as a single species with a Stoke's radius of less than 50 A on Sepharose 4B columns. In addition, the solubilized receptor-ligand complex bound specifically to wheat germ agglutinin, indicating that it is probably a glycoprotein. The ability to label the receptor in living PMNL with a high efficiency should facilitate the study of receptor dynamics and receptor physiochemical properties in this system.
- Published
- 1982
- Full Text
- View/download PDF
12. A continuous, spectroscopic analysis of the kinetics of elastase secretion by neutrophils. The dependence of secretion upon receptor occupancy.
- Author
-
Sklar LA, McNeil VM, Jesaitis AJ, Painter RG, and Cochrane CG
- Subjects
- Cytochalasin B pharmacology, Detergents pharmacology, Humans, Kinetics, Octoxynol, Pancreatic Elastase blood, Polyethylene Glycols pharmacology, Spectrometry, Fluorescence, Neutrophils enzymology, Pancreatic Elastase metabolism
- Abstract
We have developed a continuous spectroscopic method to analyze the kinetics of elastase secretion by human neutrophils. We have used the elastase-specific substrate methylsuccinylalanylalanylprolylvalylmethylcoumarin amide (Castillo, M. J., Nakajima, K., Zimmerman, J., and Powers, J. C. (1979) Anal. Biochem. 99, 53-64), which liberates the fluorophore, aminomethylcoumarin, when cleaved by elastase. We find that secretion of elastase in cytochalasin B-treated cells is initiated within approximately 5 s of exposure of the cells to the fluoresceinated chemotactic peptide stimulus, N-formyl-norleucylleucylphenylalanylnorleucyltyrosyllysine-fluorescein, and that secretion is completed within 30 s. The kinetics of this response is only slightly dependent on the concentration of the stimulus. Up to 100% (approximately 0.5 pg/neutrophil) of the elastase can be released in a dose-dependent manner by stimulated cells. We have also used this fluoresceinated stimulus and an antibody to fluorescein (Sklar, L. A., Oades, Z. G., Jesaitis, A. J., Painter, R. G., and Cochrane, C. G. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 7540-7544) to probe the temporal relationship between the binding of stimulus to the receptors and the cellular response. We find that the entire response is elicited by the binding which occurs within the first 15 s after the addition of stimulus, regardless of the dose. We estimate that an occupancy of no more than 20% of the cellular receptors for the stimulus is required to evoke the optimal response.
- Published
- 1982
13. Cell-dependent chemiluminescence. Modulation of the N-formyl chemotactic peptide (FNLPNTL) mediated oxidative burst in human polymorphonuclear leukocytes (PMNL) by murine monoclonal antibody NMS-1.
- Author
-
Schmitt M and Cochrane CG
- Subjects
- Antibodies, Monoclonal immunology, Humans, In Vitro Techniques, Luminescent Measurements, Luminol, Neutrophils drug effects, Neutrophils immunology, Chemotactic Factors pharmacology, Neutrophils metabolism, Oligopeptides pharmacology, Oxygen blood
- Abstract
Binding of purified monoclonal antibody (moAB) IgM NMS-1 to suspended initially spherical living human PMNLs is not associated with the generation of chemiluminescence but was found to enhance the chemiluminescence response to the N-formyl chemotactic peptide FNLPNTL. We investigated quantitatively the kinetics of oxygen metabolite generation by PMNLs stimulated with FNLPNTL +/- moAB NMS-1 using luminol-dependent chemiluminescence as a very sensitive detection system. Chemiluminescence detection allowed the analysis of the time sequence of onset and development of reactive oxygen metabolites following stimulation of PMNLs by FNLPNTL in the presence of moAB NMS-1. The increase of response of PMNLs stimulated with FNLPNTL in the presence of moAB NMS-1 depended on the concentration of the antibody and the sequence of stimulus addition. Stimulation of human PMNLs by 10 nM FNLPNTL induced a rapid burst of chemiluminescence which peaked approximately 5 min after stimulus addition. The subsequent addition of moAB NMS-1 (greater than or equal to 2 micrograms/ml DPBS(+) - 0.1% HSA, 37 degrees C) to FNLPNTL-stimulated PMNLs - after the FNLPNTL-mediated response had already decayed (16-18 min) - without delay induced a second burst of oxygen metabolite generation. The magnitude of this second peak of activation was dose-dependent. Treatment of PMNLs with moAB NMS-1 (greater than or equal to 1 microgram/ml DPBS(+) - 0.1% HSA, 3 min, 37 degrees C) - prior to FNLPNTL (10 nM) stimulation - increased rate and magnitude of the FNLPNTL-mediated response. This response is biphasic with the first peak at the FNLPNTL position and a second, higher peak approximately 16 min after FNLPNTL addition. The magnitude of response was dose-dependent. The latency (lag time) of the response was not changed compared to controls which received no moAB NMS-1 treatment. The observed moAB NMS-1 dependent increase in FNLPNTL-mediated chemiluminescence is transient (50-60 min), persistent activation was not detected.
- Published
- 1987
- Full Text
- View/download PDF
14. The dynamics of ligand-receptor interactions. Real-time analyses of association, dissociation, and internalization of an N-formyl peptide and its receptors on the human neutrophil.
- Author
-
Sklar LA, Finney DA, Oades ZG, Jesaitis AJ, Painter RG, and Cochrane CG
- Subjects
- Cell Membrane metabolism, Flow Cytometry, Humans, Kinetics, Ligands, Protein Binding, Receptors, Formyl Peptide, Temperature, Fluoresceins metabolism, Neutrophils metabolism, Oligopeptides metabolism, Receptors, Immunologic metabolism
- Abstract
Parallel cytometric and fluorimetric analyses of the interaction of a fluoresceinated N-formyl hexapeptide (N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein, Nle = norleucine) with its receptors on human neutrophils are presented. The cytometric analyses take advantage of the ability of the fluorescence flow cytometer to discriminate free and receptor-bound ligand in a homogeneous real-time assay. The spectrofluorometric analysis relies on a high affinity antibody to fluorescein to discriminate free and bound ligand. We find that the number of receptors for formyl peptides on the surface of a resting cell is 53,000 +/- 13,000 (Kd approximately 0.6 +/- 0.2 nM). We use commercially available cytometric standards to calibrate the cytometer and we obtain similar values for the number of receptors. The temperature dependence of the kinetics of ligand-receptor interactions have been examined. The association rate constant varies from approximately 3 X 10(8) M-1 min-1 at 4 degrees C to approximately 10(9) M-1 min-1 at 37 degrees C (delta H approximately 8 kcal/mol). While ligand internalization is blocked at 4 degrees C, at 37 degrees C internalization proceeds at an initial rate of approximately 24% of the occupied receptors/min following a latency period of approximately 20-30 s. Intermediate rates and longer latency periods are found at 15 and 25 degrees C. Dissociation of the ligand is heterogeneous and depends both on the length of time of association and the temperature. After short periods of association, the ligand dissociates with t1/2 approximately 1-5 min. After longer periods (30 min at 15 degrees C or 100 min at 4 degrees C), but while the ligand-receptor complex remains on the cell surface, t1/2 increases to greater than 30 min. It appears that the ligand-receptor complex undergoes an alteration in affinity, with a time course at elevated temperatures, which parallels or lags behind the time course of the participation of the occupied receptors in cell activation.
- Published
- 1984
15. Damage to the bases in DNA induced by stimulated human neutrophils.
- Author
-
Jackson JH, Gajewski E, Schraufstatter IU, Hyslop PA, Fuciarelli AF, Cochrane CG, and Dizdaroglu M
- Subjects
- Catalase pharmacology, Chemical Phenomena, Chemistry, DNA drug effects, DNA metabolism, Deferoxamine pharmacology, Dimethyl Sulfoxide pharmacology, Free Radicals, Gas Chromatography-Mass Spectrometry, Humans, Hydroxides pharmacology, Hydroxyl Radical, Iron pharmacology, Molecular Structure, Superoxide Dismutase pharmacology, Tetradecanoylphorbol Acetate pharmacology, DNA Damage, Neutrophils physiology
- Abstract
Leukocyte-induced DNA damage may partially account for the known association between chronic inflammation and malignancy. Since elucidation of the chemical nature of leukocyte-induced DNA damage may enhance our understanding of the mechanisms underlying leukocyte-induced DNA damage and the carcinogenesis associated with inflammation, the present study was undertaken to characterize the chemical modifications that occur in DNA exposed to stimulated human neutrophils. Calf thymus DNA was exposed to phorbol myristate acetate (PMA)-stimulated neutrophils in the presence or absence of exogenously added iron ions. DNA samples were subsequently hydrolyzed, derivatized and analyzed by gas chromatography-mass spectrometry with selected-ion monitoring. A variety of base modifications including cytosine glycol, thymine glycol, 4,6-diamino-5-formamidopyrimidine, 8-hydroxyadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 8-hydroxyguanine were identified. The yield of these various base products was increased by the addition of iron ions. Specifically, in the presence of physiologic quantities of iron ions, approximately 7 of every 1,000 DNA bases were modified. Addition of the superoxide anion scavenger, superoxide dismutase, the hydrogen peroxide scavenger, catalase, the hydroxyl scavenger, dimethylsulfoxide, or the iron chelator, deferoxamine, to DNA mixtures containing PMA, neutrophils, and iron ions, greatly decreased the yield of the damaged DNA base products. Our results indicate that stimulated human neutrophils can damage each of the four bases in DNA. It is likely that hydroxyl radical, generated via an iron catalyzed Haber-Weiss reaction, mediates neutrophil-induced DNA base damage, since: (a) the chemical structure of neutrophil-induced DNA base damage is consistent with a hydroxyl radical-mediated mechanism, (b) hydroxyl radical generated via ionizing radiation in aqueous solution produces DNA base modifications that are identical to neutrophil-induced DNA base modifications, (c) iron ions increase neutrophil-induced DNA base damage, and (d) iron chelators or scavengers of superoxide anion, hydrogen peroxide or hydroxyl radical decrease neutrophil-induced DNA base damage.
- Published
- 1989
- Full Text
- View/download PDF
16. Inflammatory cells in solid murine neoplasms. II. Cell types found throughout the course of Moloney sarcoma regression or progression.
- Author
-
Russell SW, Gillespie GY, Hansen CB, and Cochrane CG
- Subjects
- Animals, Cell Count, Cell Separation, Immunoglobulins, Leukocyte Count, Male, Mice, Mice, Inbred BALB C, Moloney murine leukemia virus, Remission, Spontaneous, Eosinophils immunology, Macrophages immunology, Neutrophils immunology, Sarcoma, Experimental pathology, T-Lymphocytes immunology
- Abstract
Regressing and progressing Moloney sarcomas, induced in BALB/c mice by the injection of cultured sarcoma cells (MSC)1, were sampled for histologic analysis and then disaggregated using mixtures of trypsin, collagenase and DNAse or collagenase and DNAse alone. The types of inflammatory cells (IC) found in resultant cell suspensions were determined 6, 11, 14 and 18 days post inoculation. Inflammatory infiltrates were composed almost exclusively of three cell types; neutrophils, T lymphocytes and macrophages. The extent to which each was found in tumors was related to the time post inoculation. Neutrophils were part of an early acute inflammatory response seen in both developing regressing and progressing sarcomas. The onset of regression was associated histologically with the appearance within tumors of a mononuclear inflammatory infiltrate. T lymphocytes and macrophages were the principal constituents. A higher percentage of T lymphocytes was recovered at all sampling times from regressing, compared to progressing, sarcomas. During development of the mononuclear inflammatory infiltrate there were relatively more large T cells in regressing, than in progressing tumors, and the percentage of macrophages was higher. Thereafter, the proportion of macrophages in the recovered cell population was approximately the same for both types of tumor. Such equality was more apparent than real, however, since IC were restricted to the peripheries of progressing sarcomas after the acute inflammatory phase, but continued to be found throughout regressing neoplasms. The effective ratio of macrophages and T lymphocytes to tumor cells therefore was much lower in progressing sarcomas than was suggested by percentage figures. The data presented support the concept that T lymphocytes are instrumental in causing the regression of Moloney sarcomas, possibly through interactions with macrophages.
- Published
- 1976
- Full Text
- View/download PDF
17. Activation of neutrophils by N-formyl chemotactic peptides.
- Author
-
Painter RG, Sklar LA, Jesaitis AJ, Schmitt M, and Cochrane CG
- Subjects
- Antibodies, Monoclonal, Cyclic AMP blood, Flow Cytometry, Humans, Kinetics, Neutrophils drug effects, Receptors, Cell Surface drug effects, Receptors, Cell Surface isolation & purification, Receptors, Formyl Peptide, Spectrometry, Fluorescence, Chemotactic Factors pharmacology, Chemotaxis, Leukocyte drug effects, Neutrophils immunology, Receptors, Cell Surface physiology
- Abstract
The response of neutrophils to N-formyl peptides is mediated via a specific 50,000- to 60,000-dalton (Mr) receptor. Real-time kinetic analysis indicated that most of the cellular responses elicited by this ligand began within 5-10 s of addition to the cells at 37 C. Of three possible biochemical changes measured that could serve as transducers or second messengers, two, i.e., increases in cyclic AMP (cAMP) and intracellular free Ca2+, occurred within 5 s of stimulus addition. In contrast, internalization of the ligand by cells showed a latency time of 20-30 s, which indicates that it probably plays no role in triggering later responses. Using pulse binding techniques that allow the level of a given response to be measured as a function of the measured level of surface receptor occupancy, we found that O2- production required up to 30% receptor occupancy to elicit 50% of maximal response. In contrast, secretion, cAMP changes, Ca2+ changes, and membrane potential changes required less than 5% occupancy. Within 5 s, occupied receptors were converted at the cell surface to a slowly dissociating form. The receptors, exhibiting apparent higher affinity, were transiently associated with the cell cytoskeleton as defined by their conversion to a Triton X-100-insoluble form. Internalized receptor-ligand complexes were transported, in large part, to the Golgi apparatus. Further analyses of the mechanism of stimulation of leukocytes have been performed with monoclonal antibodies directed against the neutrophil surface. Data with these antibodies, which are not directed to the N-formyl peptide receptor, reveal that some modulated the N-formyl peptide-mediated responses and other antibodies initiated responses of the cells.
- Published
- 1984
18. The presence of neutrophil elastase and evidence of oxidation activity in bronchoalveolar lavage fluid of patients with adult respiratory distress syndrome.
- Author
-
Cochrane CG, Spragg RG, Revak SD, Cohen AB, and McGuire WW
- Subjects
- Adult, Body Fluids, Humans, Respiratory Distress Syndrome physiopathology, Therapeutic Irrigation, Bronchi enzymology, Neutrophils enzymology, Oxidation-Reduction, Pancreatic Elastase analysis, Pulmonary Alveoli enzymology, Respiratory Distress Syndrome enzymology
- Published
- 1983
- Full Text
- View/download PDF
19. Signal transduction and ligand-receptor dynamics in the human neutrophil. Transient responses and occupancy-response relations at the formyl peptide receptor.
- Author
-
Sklar LA, Hyslop PA, Oades ZG, Omann GM, Jesaitis AJ, Painter RG, and Cochrane CG
- Subjects
- Actins metabolism, Aminoquinolines, Calcium metabolism, Cell Aggregation, Cyclic AMP analysis, Free Radicals, Humans, Ligands, Light, Pancreatic Elastase metabolism, Receptors, Formyl Peptide, Receptors, Immunologic analysis, Scattering, Radiation, Neutrophils metabolism, Receptors, Immunologic metabolism
- Abstract
The responses of neutrophils to formyl peptides are initiated and in many cases achieve a maximal level prior to equilibrium receptor occupancy. In order to begin to understand the linkage between receptor occupancy and cell response we have used a pulsed binding procedure to analyze: 1) the number of receptors contributing to three potential signalling events and six functional responses and 2) the evolution of these responses once ligand binding is interrupted. We find that the half-optimal elevations of the potential signals are produced by less than 1% occupancy (Ca2+) or 1-3% occupancy (cAMP, membrane depolarization). In contrast, actin polymerization and a rapid light scatter response are elicited by less than 0.1% occupancy. Half-optimal elastase release and degranulation require approximately 3% occupancy. While half-optimal O2- production and aggregation require approximately 30% occupancy, the half-optimal rate of O2- production requires less than 10% occupancy. To resolve the apparent lack of correlation between the responses and the signals we examined their time courses following the pulse of stimulation. At least four responses and one signal are transient and decay while occupied receptors remain on the membrane surface. These include the Quin 2-Ca2+ signal, actin polymerization, the light scatter response, O2- generation, and aggregation. Ca2+ elevation is correlated with the responses in that: 1) each of these responses is transient unless new receptors are occupied; 2) occupancy of nearly all of the receptors contributes to the time course of these responses; 3) when binding is interrupted, the responses decay with a half-time of 15 s, following a latency of approximately 10 s or less (except for disaggregation where latency is 30-40 s). We discuss evidence in support of the hypothesis that transient cell responses arise from transient receptor activation.
- Published
- 1985
20. Physicochemical properties of the N-formyl peptide receptor on human neutrophils.
- Author
-
Allen RA, Jesaitis AJ, Sklar LA, Cochrane CG, and Painter RG
- Subjects
- Affinity Labels, Azides metabolism, Chromatography, Gel, Detergents, Glycoproteins isolation & purification, Humans, Oligopeptides metabolism, Receptors, Formyl Peptide, Solubility, Neutrophils analysis, Receptors, Immunologic isolation & purification
- Abstract
In order to investigate the physicochemical properties of the N-formyl peptide receptor of human neutrophils, the receptor was specifically and covalently labeled with an iodinated, photoactivatable derivative of the chemotactic hexapeptide, N-formyl-norleucylleucyl- phenylalanyl-norleucyl-[125I]iodotyrosyl-N epsilon-(6- (4'-azido-2'-nitrophenylamino) hexanoyl)-lysine. After labeling isolated neutrophil membranes, the receptor was extracted with Triton X-100, digitonin, or octyl glucoside and subjected to gel filtration on a calibrated Ultrogel AcA 34 column. The Triton X-100- and digitonin-extracted receptor eluted as single molecular species, with Stokes radii of 40 and 33 A, respectively. This material was subjected to further physicochemical analysis. When octyl glucoside-extracted material was gel-filtered, a second peak containing specifically labeled material eluted in the void volume. Subsequent sodium dodecyl sulfate-polyacryl-amide amide gel electrophoresis analysis indicated that this species was the result of disulfide bonded aggregates containing the monomeric species. Sedimentation equilibrium analysis was carried out in H2O and D2O/H2O mixtures, yielding an apparent molecular mass of 63,000 daltons for both Triton X-100- and digitonin-extracted receptor. This agrees closely with the reduced sodium dodecyl sulfate-polyacrylamide gel electrophoretic value of 50,000-60,000 daltons, indicating that the receptor extracted from unstimulated membranes is monomeric in these detergents. From the sedimentation equilibrium data, the partial specific volume (v) and frictional ratio (f/f0) were calculated. The v is high in both Triton X-100 (0.880) and digitonin (0.829), indicating that the receptor may be associated with tightly bound endogenous lipid or that it is a hydrophobic membrane protein. This latter likelihood is further supported by the quantitative extraction of receptor into Triton X-114 by a phase-separation method. The frictional ratio of 1.1-1.3 is consistent with an elongated globular protein having an axial ratio of approximately 3:1. This in conjunction with the Stokes radius of 40 A would indicate that the receptor is capable of spanning the 35-40-A nonpolar center of the lipid bilayer. The state of the receptor in situ is discussed.
- Published
- 1986
21. Monoclonal antibody NMS-1 increases N-formyl chemotactic peptide-mediated oxidative burst generation in human neutrophils.
- Author
-
Schmitt M, Painter RG, von Tscharner V, Sklar LA, Jesaitis AJ, Fayle DH, and Cochrane CG
- Subjects
- Antigens, Surface isolation & purification, Calcium analysis, Cytoplasmic Granules metabolism, Humans, Kinetics, Neutrophils metabolism, Oxygen metabolism, Antibodies, Monoclonal immunology, Antigens, Surface immunology, Hydrogen Peroxide biosynthesis, Neutrophils drug effects, Oligopeptides pharmacology
- Abstract
Murine monoclonal antibody (mAb) NMS-1 was generated which binds to the surface of living human neutrophils. The antigens on neutrophil plasma membranes recognized by mAb NMS-1 were solubilized in Nonidet P-40 and immunopurified on matrix-bound mAb NMS-1. mAb NMS-1 binds to four periodate-sensitive structures of 70,000, 95,000, 140,000, and 170,000 Da on the plasma membrane surface of human neutrophils as was shown by Western blot analysis. Binding of mAb NMS-1 to human neutrophils induced a rapid transient rise in cytosolic free calcium (Quin 2 fluorescence) but no detectable generation of reactive oxygen metabolites. The oxidative burst of N-formyl peptide-treated neutrophils, however, increased in the presence of mAb NMS-1. The kinetics of N-formyl peptide (N-formyl-norleucyl-leucyl-phenylalanyl-norleucyl-tryrosyl-lysine; FNLPNTL)-mediated hydrogen peroxide formation (p-hydroxy phenyl acetate oxidation) in the presence of mAb NMS-1 was analyzed quantitatively. 1) When neutrophils were incubated with mAb NMS-1 before FNLPNTL addition, an increase in rate, magnitude, and duration of hydrogen peroxide formation was observed compared with controls which received no mAb NMS-1 treatment. After termination of the initial linear phase of response, a second transient linear phase of hydrogen peroxide formation was induced. This second phase of activation was not observed in neutrophils which received no mAb NMS-1 treatment. The onset of the response and latency before attainment of the initial linear rate of hydrogen peroxide formation was not changed by mAb NMS-1 pretreatment. 2) When neutrophils were stimulated with FNLPNTL, the addition of mAb NMS-1--after termination of the FNLPNTL-induced response--without delay induced a second transient burst of hydrogen peroxide formation. Persistent activation of hydrogen peroxide formation by mAb NMS-1 in FNLPNTL-stimulated neutrophils was not observed.
- Published
- 1987
22. The role of polymorphonuclear leukocytes in the initiation and cessation of the Arthus vasculitis.
- Author
-
COCHRANE CG, WEIGLE WO, and DIXON FJ
- Subjects
- Animals, Guinea Pigs, Rabbits, Antibodies, Endothelium, Hypersensitivity, Immune System Diseases, Immunoglobulins, Inflammation, Leukocytes physiology, Neutrophils, Vasculitis
- Abstract
The role of polymorphs in the Arthus type hypersensitivity vasculitis has been studied. Polymorphs were found to play an essential role in not only producing the inflammatory vasculitis, but also were instrumental in ridding the damaged vessel of the antigen, probably by means of proteolytic catabolism at the inflammatory site. A temporal relationship between the disappearance of antigen from the damaged vessels and a decrease in inflammatory reaction was found. The earliest localization of antigen and its associated rabbit globulin in the Arthus vasculitis was found to be beneath the endothelium of small vessels. Since submitting this article for publication, Sorkin and Boyden (J. Immunol., 1959, 82, 332) have reported the catabolism of antigen in the presence of antibody by mononuclear cells obtained from the peritoneal cavities of guinea pigs. Evidence was presented indicating that the antigen molecule was actually broken down by the mononuclear cells. Techniques similar to those reported here were used.
- Published
- 1959
- Full Text
- View/download PDF
23. BOUND COMPLEMENT AND IMMUNOLOGIC INJURY OF BLOOD VESSELS.
- Author
-
WARD PA and COCHRANE CG
- Subjects
- Animals, Guinea Pigs, Rats, Anaphylaxis, Antibodies, Arthus Reaction, Carrageenan, Chemotaxis, Chromatography, Complement System Proteins, Immunoelectrophoresis, Neutrophils, Ovalbumin, Pathology, Phagocytosis, Polysaccharides, Research, Vascular Diseases, Zymosan, gamma-Globulins
- Abstract
Rats and guinea pigs were depleted of complement (C') by treatment with heat aggregated human gamma-globulin (agg HGG), zymosan, anti-beta1C globulin, and carrageenan. Although antigen and antibody were bound to vascular structures, Arthus reactions were inhibited. This inhibition was characterized by the lack of C' binding to walls of vessels, the lack of polymorphonuclear (PMN's) cellular infiltrates, and the lack of significant vascular damage. When the same animals were followed for several hours thereafter, levels of serum C' began to rise, C' was bound in tissues, PMN infiltrates appeared, and immunologic vasculitis developed. Blood counts, chemotaxis of PMN's induced by lysates of PMN granules, together with studies on motility and phagocytosis by PMN's obtained from C' depleted rats, failed to establish any abnormality in these cells which would account for inhibition of Arthus reactions. The specificity of C' depletion in terms of effects in the first four reacting components of guinea pig C' was studied. Treatment with agg HGG led to loss of activity in all components, whereas zymosan and anti-beta1C globulin predominately affected the third component (C'3c). Carrageenan mainly affected the first two reacting components of C'. Thus, the availability of the 3c component, or a subsequently reacting component, correlated with the attraction of PMN's to immune reactants in vivo. Various antibodies with different C' fixing capacities in vitro were tested for their ability to induce immunologic vasculitis in normal animals. In rats, only those antibodies which fixed C' in vitro possessed biological activity, whereas in guinea pigs, all antibodies tested, regardless of C' fixation in vitro, induced Arthus reactions. For a given antibody in rats the vasculitis-inducing property was reflected in its ability to bind C' in vascular structures. Rats depleted of circulating PMN's by specific antibody were tested for Arthus activity. Although concentrations of immune reactants and C' were readily detected in vascular structures, no PMN infiltration occurred and significant vascular damage was averted.
- Published
- 1965
- Full Text
- View/download PDF
24. A ROLE OF POLYMORPHONUCLEAR LEUKOCYTES AND COMPLEMENT IN NEPHROTOXIC NEPHRITIS.
- Author
-
COCHRANE CG, UNANUE ER, and DIXON FJ
- Subjects
- Animals, Rabbits, Rats, Antibodies, Basement Membrane, Complement System Proteins, Electrons, Glomerulonephritis, Kidney Glomerulus, Mechlorethamine, Microscopy, Microscopy, Electron, Nephritis, Neutrophils, Pathology, Proteinuria, Research, Toxicology
- Abstract
In acute nephrotoxic nephritis, polymorphonuclear leukocytes (polymorphs) accumulated in large numbers in the glomeruli in the first 12 hours. The endothelial cells were dislodged by the polymorphs which then came to lie immediately adjacent to the glomerular basement membranes. Ultrastructural changes in neither polymorphs nor basement membranes were observed. Depletion of polymorphs in both rats and rabbits prevented the development of proteinuria. This occurred when doses of nephrotoxic globulin were employed that produced proteinurias of as much as 1800 mg/kg/24 hours in intact rabbits, or enough to yield near maximal immediate proteinuria in intact rats. In addition, measurable glomerular damage was frequently averted until the onset of the secondary stage of NTN. Controls indicated that the polymorph depleted animals exhibited minimal non-specific changes in the blood, that the ability of their vascular beds to react to stimuli was not affected, and that deposition of nephrotoxic antibody and C' in the glomeruli was not inhibited. Elimination of polymorphs from the circulation was only partially effective in preventing glomerular damage when large doses of nephrotoxic globulin were used. This indicated that under these circumstances, a polymorph independent glomerular injury may also take place in first stage nephrotoxic nephritis. An indirect role of C', i.e., the accumulation of polymorphs, in bringing about glomerular injury in first stage nephrotoxic nephritis was apparent. When rabbit nephrotoxic globulin was injected into rats depleted of C', or when duck nephrotoxic globulin that fixed C' poorly was injected into normal rats, C' failed to bind with the antibody along glomerular basement membranes and polymorphs did not accumulate.
- Published
- 1965
- Full Text
- View/download PDF
25. Acute immunologic arthritis in rabbits.
- Author
-
DeShazo CV, Henson PM, and Cochrane CG
- Subjects
- Animals, Antigen-Antibody Complex, Arthritis immunology, Capillary Permeability, Chemotaxis, Complement System Proteins, Disease Models, Animal, Fluorescent Antibody Technique, Hindlimb, Joints, Male, Microscopy, Electron, Rabbits, Serum Albumin, Radio-Iodinated, Synovial Fluid, Synovial Membrane, Time Factors, Antibodies, Antigens, Arthus Reaction, Neutrophils immunology
- Abstract
Mediators of acute immunologic injury have been studied in vivo by producing arthritis in rabbit knee joints. A reversed passive Arthus lesion was produced by injecting antibody into the joint space and antigen intravenously. Injury was assessed by measuring leakage of serum proteins and circulating radiolabeled proteins into the joint space and by the accumulation of neutrophils in the joint fluid. Inflammatory exudate was recovered for study by a standardized irrigation technique.Maximal vascular permeability developed 2 hr after injection as neutrophils accumulated about immune complexes in venule walls to produce structural injury. After 5 hr the number of neutrophils in the joint space rose rapidly, followed by a second rise in permeability at 8 hr. Neutrophil depletion abolished both peaks of permeability. It was then possible to reconstitute the synovial lesion in neutrophil-depleted rabbits by intra-articular injection of purified suspensions of neutrophils.A requirement for complement was demonstrated in development of the lesion. Rabbits genetically deficient in C6 showed delay in vascular permeability, appearance of neutrophils, and histologic lesions. The delay was longer in normal rabbits depleted of C3. In C6-deficient rabbits depleted of C3, still further reduction in injury occurred. Evidence was obtained as well for a chemotactic attraction of neutrophils in vivo. Antigen-antibody-complement complexes in the walls of blood vessels attracted neutrophils placed in the joint space of neutrophil-depleted rabbits. Omission of either antigen or antibody from this replacement reaction prevented the migration of neutrophils.
- Published
- 1972
- Full Text
- View/download PDF
26. PATHOGENIC FACTORS IN VASULAR LESIONS OF EXPERIMENTAL SERUM SICKNESS.
- Author
-
KNIKER WT and COCHRANE CG
- Subjects
- Animals, Cattle, Rabbits, Antigens, Arteries, Cardiovascular Diseases, Complement System Proteins, Fluorescence, Fluorescent Antibody Technique, Glomerulonephritis, Immune Sera, Inflammation, Kidney, Microscopy, Microscopy, Fluorescence, Myocarditis, Neutrophils, Pathology, Proteinuria, Research, Serum Albumin, Serum Albumin, Bovine, Serum Sickness, Virulence Factors
- Abstract
The present studies suggest that polymorphonuclear leukocytes (PMN's) are essential for the development of cardiovascular lesions in serum sickness. In the absence of PMN's, necrotic vascular lesions were never seen and endothelial proliferation in arteries was inhibited. Zones of fibrinoid deposits did not occur. By contrast, at least two-thirds of the control animals exhibited endothelial proliferation, and half had necrosis of arterial walls, usually with fibrinoid deposits. In arterial lesions that involved the intima and media, the internal elastic lamina was disrupted. This was associated with accumulations of PMN's and was prevented when PMN's were depleted. The observations suggested that the elastic lamina acts as a barrier to the outward spread of inflammation in arteries and that it is an important substrate of PMN action. Although glomerulitis and proteinuria developed in PMN-depleted animals, no conclusions could be drawn concerning the pathogenic role of PMN's in renal lesions, since PMN depletion could not be effected before the onset of immune elimination without influencing the immune response itself. Host complement (beta1C-globulin) was localized along with the antigen and rabbit gamma globulin in glomeruli and arteries showing lesions. In the glomeruli these deposits formed a granular lining along the area of the basement membrane. In arteries the fluorescent amorphous particles were in the intima and media of inflamed vessels. The immune response to BSA and the incidence and severity of cardiovascular and renal lesions were enhanced by the intravenous administration of pooled rabbit antiserum to BSA given 18 hours before BSA antigen and by injecting endotoxin along with the BSA. These additions to the usual procedure of inducing serum sickness did not appear to change the quality of the disease. In normal rabbits, the peak incidence of cardiovascular lesions was early in immune elimination of antigen, at a time when levels of circulating complexes was maximal. Conversely, the severest renal injury was noted several days later, at the completion of immune elimination.
- Published
- 1965
- Full Text
- View/download PDF
27. Mediation of immunologic glomerular injury.
- Author
-
Cochrane CG
- Subjects
- Animals, Autoimmune Diseases, Basement Membrane, Chromatography, DEAE-Cellulose, Complement System Proteins, Glomerulonephritis urine, Graft Rejection, Histocompatibility, Humans, Immune Sera, Immunoglobulin M, Kidney Function Tests, Kidney Transplantation, Methods, Microbial Collagenase, Microscopy, Electron, Peptide Hydrolases metabolism, Proteinuria, Rabbits, Rats, Transplantation Immunology, Transplantation, Homologous, Venoms, Glomerulonephritis immunology, Neutrophils
- Published
- 1969
28. Isolation and characterization of permeability factors from rabbit neutrophils.
- Author
-
Ranadive NS and Cochrane CG
- Subjects
- Acrylates, Animals, Blood Platelets drug effects, Chromatography, Gel, Chromatography, Ion Exchange, Electrophoresis, Gels, Histamine H1 Antagonists pharmacology, Methods, Molecular Weight, Rabbits, Time Factors, Blood Proteins analysis, Blood Proteins pharmacology, Capillary Permeability drug effects, Histamine Release drug effects, Neutrophils analysis
- Abstract
Four basic proteins that increase vascular permeability have been isolated in purified form from rabbit neutrophilic granules. These proteins are termed band 1, 2, 3, and 4 protein according to their electrophoretic migration in acrylamide gel. Molecular weights of band 1 and 2 protein derived from amino acid composition were 4800 and 5300, respectively. These values are in good agreement with those obtained for these proteins by gel diffusion techniques. The molecular weight of band 3 protein was also in the range of 5000 by the latter technique. The molecular weight of band 4 protein determined by ultracentrifugal analysis and amino acid composition was 12,000. Although all four proteins had the capacity to induce immediate increase in vascular permeability, only band 2 protein was found to release histamine from isolated rat peritoneal mast cells. Furthermore, it has been shown that the permeability-inducing activity of band 2 protein can be inhibited by pretreating rabbits with antihistamine. Band 2 protein did not release histamine from rabbit platelets and depletion of rabbit platelets from the circulation had no influence on the permeability-inducing activity of this protein. Band 1, 3, and 4 proteins did not release histamine from isolated rat peritoneal mast cells and their capacity to increase vascular permeability remained unaffected by treatment of rabbits with antihistamine. These investigations suggest that the histamine-releasing activity of band 2 protein is a specific phenomenon and is associated with particular amino acid grouping or spacial configuration of the molecules. By the same token, the increase in vascular permeability induced by the nonhistamine-releasing band 1, 3, and 4 proteins represents a specific phenomenon (or phenomena) not particularly related to the over-all charge of these molecules.
- Published
- 1968
- Full Text
- View/download PDF
29. Immunological induction of increased vascular permeability. I. A rabbit passive cutaneous anaphylactic reaction requiring complement, platelets, and neutrophils.
- Author
-
Henson PM and Cochrane CG
- Subjects
- Animals, Antigens, Capillary Permeability, Chlorpheniramine pharmacology, Chromatography, Electrophoresis, Methysergide pharmacology, Rabbits, Serotonin Antagonists, Serum Albumin, Bovine, Venoms pharmacology, Antibodies analysis, Blood Platelets immunology, Blood Vessels immunology, Complement System Proteins, Neutrophils immunology, Passive Cutaneous Anaphylaxis drug effects
- Abstract
Passive cutaneous anaphylaxis (PCA) reactions were produced in rabbits by antibodies to bovine serum albumin. Two types of antibodies were found, each inducing increased vascular permeability, but by means of different mediation systems. One of these antibodies required the presence of complement, platelets, and neutrophils for the induction of the PCA reaction, which was inhibited by antihistamine. This antibody was heat stable, sedimented in the 7S region, and was found in both fast and slow electrophoretic fractions of rabbit gamma-globulin. Homocytotropic antibody was also detected. The PCA reactions induced by this type of antibody did not require platelets or neutrophils and were not inhibited in rabbits depleted of C3 with cobra venom factor. The lesions were, however, prevented by administration of antihistamine.
- Published
- 1969
- Full Text
- View/download PDF
30. Immunologic tissue injury mediated by neutrophilic leukocytes.
- Author
-
Cochrane CG
- Subjects
- Arteritis immunology, Arthus Reaction immunology, Chemotaxis, Connective Tissue immunology, Cytoplasmic Granules, Fluorescent Antibody Technique, Glomerulonephritis immunology, Humans, Mast Cells immunology, Microscopy, Electron, Neutrophils immunology
- Published
- 1968
- Full Text
- View/download PDF
31. The effect of complement depletion on neutrophil migration in acute immunologic arthritis.
- Author
-
DeShazo CV, McGrade MT, Henson PM, and Cochrane CG
- Subjects
- Agranulocytosis chemically induced, Animals, Arthus Reaction, Capillary Permeability, Chemotaxis, Knee immunology, Leukocyte Count, Nitrogen Mustard Compounds, Rabbits, Serum Albumin, Radio-Iodinated, Synovial Membrane immunology, Arthritis immunology, Cell Movement, Complement System Proteins, Neutrophils immunology
- Published
- 1972
32. STUDIES ON THE LOCALIZATION OF CIRCULATING ANTIGEN-ANTIBODY COMPLEXES AND OTHER MACROMOLECULES IN VESSELS. I. STRUCTURAL STUDIES.
- Author
-
COCHRANE CG
- Subjects
- Animals, Cattle, Guinea Pigs, Rabbits, Anaphylaxis, Antibodies, Antigen-Antibody Complex, Antigen-Antibody Reactions, Arthus Reaction, Blood Vessels, Complement System Proteins, Electrons, Fluorescent Antibody Technique, Glomerulonephritis, Immune Sera, Immunoelectrophoresis, Inflammation, Microscopy, Microscopy, Electron, Necrosis, Neutrophils, Ovalbumin, Pathology, Research, Serum Albumin, Serum Albumin, Bovine, Vascular Diseases, gamma-Globulins
- Abstract
A short term model in which circulating antigen-antibody complexes and host complement localized in vessel walls of guinea pigs was analyzed. Localization was accomplished by subjecting the animals to anaphylactic shock. The circulating macromolecules, such as antigen-antibody complexes, appeared to localize by being trapped in the vessel wall along the basement membrane that acted as a filter during a state of increased permeability of the vessel. It was suggested that this point of localization between the endothelial cell and the basement membrane may well represent the earliest focus of inflammation in diseases caused by the deposition of injurious macromolecules such as soluble antigen-antibody complexes from the blood stream. Complexes localized in the vessel walls did not provoke Arthus-type vasculonecrotic reactions even though in both these vessels and in cutaneous Arthus reactions antibody, antigen, and host complement (C'3c) were deposited in the vessel walls. The possibility was presented that since circulating macromolecules and probably complexes deposited in (a) relatively small amounts, and (b) in a position beneath endothelial cells, they were not strongly chemotactic toward circulating polymorphonuclear leukocytes. Vasculonecrotic reactions, therefore, were not observed. It was brought out that this may be similar to the situation in glomerulonephritis induced by localized immune complexes, in which severe necrosis is not observed. In the Arthus vascular reaction, host complement was found microscopically accumulated with the immune reactants in affected vessel walls.
- Published
- 1963
- Full Text
- View/download PDF
33. THE ROLE OF SERUM COMPLEMENT IN CHEMOTAXIS OF LEUKOCYTES IN VITRO.
- Author
-
WARD PA, COCHRANE CG, and MUELLER-EBERHARD HJ
- Subjects
- Animals, Guinea Pigs, Humans, In Vitro Techniques, Mice, Rabbits, Antigen-Antibody Complex, Antigen-Antibody Reactions, Chemotaxis, Leukocyte, Chromatography, Complement Fixation Tests, Complement System Proteins, Edetic Acid, Electrophoresis, Inflammation, Leukocytes, Neutrophils, Research, Ultracentrifugation, Zymosan, gamma-Globulins
- Abstract
By the use of chambers containing two compartments with an interposed micropore filter, chemotaxis of polymorphonuclear leukocytes (PMN's) in vitro was studied employing various agents that fixed serum complement (C'). Antigen-antibody complexes, zymosan, and aggregated human gamma globulin, in the presence of fresh rabbit, guinea pig, or mouse serum resulted in the migration of PMN's through the micropore filter. Pepsin-degraded rabbit antibody or unaltered duck serum containing antibody did not exhibit such activity after addition of antigen. Heating of the serum before treatment or the presence of EDTA prevented the generation of the chemotactic factor. The chemotactic factor could not be generated in whole serum from rabbits genetically deficient in C'. However, the defect in this rabbit serum could be corrected by addition of rabbit or human C'6. Serum of B10.D2 mice deficient in hemolytic C' also yielded poor chemotactic activity. Interaction of the first four reacting components of guinea pig C' did not result in significant chemotactic activity unless guinea pig euglobulin with heat labile components was also present. In rabbit serum, C'5 and C'6, when "activated" by interaction with the first four reacting components, behaved like a protein-protein complex and exhibited marked chemotactic activity. By employing conditions favoring dissociation of the complex, the individual components were isolated and shown to be chemotactically inactive. Upon recombination of the two components, however, activity reappeared. Using another approach, the C'5-C'6 complex was isolated intact, and shown to be chemotactically active while other fractions not containing these components were not active. It is postulated that the C'5-C'6 complex is the active chemotactic factor generated in serum after the addition of C'-fixing agents.
- Published
- 1965
- Full Text
- View/download PDF
34. Basic proteins in rat neutrophils that increase vascular permeability.
- Author
-
Ranadive NS and Cochrane CG
- Subjects
- Animals, Chromatography, DEAE-Cellulose, Chromatography, Gel, Dinitrophenols pharmacology, Electrophoresis, Histamine Release drug effects, Mast Cells drug effects, Rabbits, Rats, Stimulation, Chemical, Capillary Permeability drug effects, Neutrophils analysis, Proteins pharmacology
- Published
- 1970
35. BASIC PROTEINS IN RAT NEUTROPHILS THAT INCREASE VASCULAR PERMEABILITY.
- Author
-
N. S. Ranadive and Cochrane, C. G.
- Subjects
- *
NEUTROPHILS , *BASIC proteins , *PERMEABILITY , *HISTAMINE , *GRANULOCYTES , *BIOGENIC amines , *PHAGOCYTES - Abstract
The basic proteins of rat neutrophils possessing the capacity to induce vascular Permeability were analysed. Evidence indicating the presence of four such permeability proteins was presented. One of these induced the release of' histamine from rat mast cells. [ABSTRACT FROM AUTHOR]
- Published
- 1970
36. Further Studies on the Chemotactic Factor of Complement and its Formation <em>in vivo</em>.
- Author
-
Ward, P. A., Cochrane, C. G., and Muller-Eberhard, H. J.
- Subjects
- *
CHEMOTAXIS , *SERUM , *ELECTROPHORESIS , *NEUTROPHILS , *INTERLEUKIN-8 , *AMINO acids - Abstract
The chemotactic factor, generated in whole rabbit serum following treatment with immune precipitates, was found to be principally associated with fractions containing the fifth and sixth components of complement (C'5 and C'6) after electrophoretic separation of serum. The chemotactic factor could also be generated in the intact animal, adding credence to its importance in tissue reactions induced by immunological agents. The isolated and activated C'5 and C'6 complex was found to be at least 10-20 times more active in the chemotaxis of rabbit polymorphonuclear leucocytes (PMNs) in vitro than bradykinin, kallidin, histamine, serotonin and extracts of PMN granules. The amino acid derivatives N-CBZ-glycyl-L-phenylalanine and N-CBZ-α glutamyl-L-tyrosine inhibited formation of the chemotactic factor in serum. The latter derivative also caused loss of activity of the preformed chemotactic factor in rabbit serum and in density gradient ultracentrifugation it was found that the C'5 and C'6 complex dissociated in the presence of this inhibitor. Chemotactically active C'5 to C'6 fractions, when added to the suspension of PMNs, prevented the ability of these cells to migrate toward a chemotactic source. It was also found that the ratio of combination of C'5 and C'6 was critical for the full expression of chemotactic activity. Utilizing purified components of human C', very recently obtained data indicate the requirement of the seventh component of C' (C'7) for generation of chemotactic activity. No later reacting components of C' are required. Whether C'7 is incorporated into the chemotactically active complex which sediments rapidly in the ultracentrifuge is not yet established. Similar data with guinea-pig C' complexes were also obtained. [ABSTRACT FROM AUTHOR]
- Published
- 1966
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.