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Biomedical subjects

H R Colten

Publications and source records attributed to H R Colten.

At least 127 records · Page 7Linked to original sources
PubMed1983

Induction of granulocyte histaminase release by particle-bound complement C3 cleavage products (C3b, C3bi) and IgG.

The interaction of opsonized particles with human granulocytes promotes a number of important biologic functions, including phagocytosis, superoxide generation, and release of a variety of enzymes, including histaminase. We have previously determined that histaminase release occurs via a C3-dependent process. Although fluid-phase C3b dimers can mediate release, the relative effects of particle-bound C3b and C3bi and of IgG have not been examined. In this report we demonstrate that particle-bound C3 deposited on activators of the alternative C pathway effected histaminase release in the absence of IgG. Particle-bound C3bi and C3b were both effective as mediators of histaminase release. The extent of release varied as a function of the activating surface on which C3 was deposited (zymosan C3b was considerably more potent than C3b bound to rabbit erythrocytes, which was slightly more potent than C3b bound to neuraminidase-treated sheep erythrocytes). In contrast, C3b or C3bi deposited on nonactivating surfaces (such as sheep erythrocytes) at inputs of up to 2,000,000 molecules per granulocyte failed to induce histaminase release unless IgG was also present. The ability of C3b bound to particles that serve as activators of the alternative pathway to induce histaminase release is apparently not the result of decreased susceptibility of C3b to proteolysis or to an increased binding affinity to the C3b receptor, but may relate to the interaction of other surface structures on activating particles with the PMN membrane.

Amine Oxidase (Copper-Containing)↗
PubMed1983

Macrophage maturation: differences in complement secretion by marrow, monocyte, and tissue macrophages detected with an improved hemolytic plaque assay.

In order to examine one function of mononuclear phagocytes during maturation from bone marrow precursors to tissue macrophages, an improved hemolytic plaque assay for the detection of synthesis of the second (C2) and fourth (C4) components of C by single cells was developed. With this method, production of C2 and C4 was assessed in cell populations derived from bone marrow, blood, lung, peritoneum, and spleen. The proportion of cells producing C2 and C4 in each population varied. Approximately 10% of bone marrow cells produced C4, but not detectable C2 plaque-forming cells (PFC) were detected. Circulating monocytes yielded about 10% PFC each for C2 and C4. The proportion of C2-producing cells in tissue macrophages varied from approximately 2% in bronchoalveolar macrophages to about 45% in peritoneal and splenic macrophage populations, whereas C4 production by macrophages from lung, peritoneum, and spleen were all approximately 45%. These data suggest that differences in C biosynthesis characterize mononuclear phagocytes at different stages of maturation.

Animals↗
PubMed1983

Isolation of guinea pig macrophages bearing surface C4 by fluorescence-activated cell sorting: correlation between surface C4 antigen and C4 protein secretion.

Studies of complement (C) secretion by single cells indicate that only a subset of a guinea pig macrophage population is capable of secreting the second (C2) and fourth (C4) components of C. Moreover, cell-surface bound C4 antigen is also found on a proportion of guinea pig peritoneal macrophages. The availability of methods for the isolation of macrophages bearing surface membrane C4 antigen and for the detection of the secretion of C by single cells made it possible to ascertain the relationship between these two subsets. Approximately 25% of the freshly isolated peritoneal macrophages were surface C4 antigen positive. When incubated in conditioned medium containing C4 or incubated in small volumes to increase the concentration of fluid phase C4, the proportion of macrophages bearing surface C4 increased to approximately 80%. The surface C4 antigen was adsorbed from the medium and was predominantly native C4. The proportion of peritoneal macrophages secreting functionally active C4 or C2 was approximately 45% as measured by a hemolytic plaque assay technique. The proportion of C4-secreting cells decreased to 5% after incubation in conditioned medium containing preformed C4, whereas the proportion of C2-producing macrophages was unchanged, i.e., it remained at about 45%. The removal of secreted C4 with F(ab')2 anti-C4 or effectively decreasing C4 concentration by increasing the volume of the culture medium abrogated the decrease in the proportion of C4-secreting cells. Conditioned medium derived from cells genetically deficient in C4 had no effect on the proportion of C4- or C2-producing macrophages. The macrophage population bearing surface membrane C4, isolated by fluorescence-activated cell sorting, contained more than 90% of the C4-producing cells. Cells producing C2 were distributed equally in both subpopulations. Maintenance of the surface C4-positive, C4-producing cells in culture for 12 hr resulted in a decrease in the proportion of C4-secreting cells. Conversely, isolated macrophages initially surface C4 negative and not producing C4, developed the capacity to produce C4 in culture. C4 production in the isolated surface C4-negative population was inhibited by incubation in medium containing preformed C4. These results suggest the presence of a negative feedback effect on C4 secretion that is mediated by extracellular C4.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1983

Biosynthesis and postsynthetic processing of human C-reactive protein.

Human C-reactive protein (CRP) is one of several plasma proteins that increase after tissue injury or inflammation. The magnitude of the increase in CRP (100 to 1000-fold over the resting state) makes this an excellent model for studies of eukaryotic gene control. Accordingly, the biosynthesis and postsynthetic processing of human CRP was examined under cellfree conditions and in Xenopus oocytes injected with liver mRNA. The primary translation product is larger than native serum CRP by an 18-amino acid amino terminal extension. The sequence of this signal peptide was derived from nucleotide sequencing of a 1.8-kilobase (Kb) CRP-specific cDNA clone. Northern blot analysis revealed a CRP mRNA of approximately 2.2 Kb, a size more than three times that required (615 bases) to code for the primary translation product. These data form the basis for study of molecular control of the acute phase response.

Amino Acid Sequence↗
PubMed1983

Low ionic strength or chemical cross-linking of monomeric C3b increases its binding affinity to the human complement C3b receptor.

Quantitative studies of the interaction between a fragment of the third component of complement (C3b) and its receptor on human cells have been undertaken with a recently developed radioligand binding assay. Specific binding of fluid phase monomeric C3b was direct and saturable under low (mu = 0.0513) but not physiological (mu = 0.15) ionic strength. Dimeric C3b had a greater affinity for the C3b receptor at either ionic strength. Complexing of monomeric C3b with another protein (human IgG) did not result in increase in binding to human erythrocytes when compared with monomeric C3b alone. These data suggest that ionic forces are involved in C3b receptor-ligand interactions and that the stable C3b-receptor binding site is present in monomeric C3b. Furthermore they suggest that the greater affinity of dimeric or oligomeric C3b is a result of multimeric binding to preclustered C3b receptors. These findings provide an explanation for the relatively stable interaction of particle bound C3b and C3b receptor-bearing cells in the immune adherence reaction.

Chromatography, Gel↗
PubMed1982

Cooperative interaction of factor B and other complement components with mononuclear cells in the antibody-independent lysis of xenogeneic erythrocytes.

Synergistic cytotoxicity is a term used to describe a cytotoxic system in which xenogeneic erythrocyte target cells are lysed in the presence of nonimmune human mononuclear effector cells and antibody-depleted normal human serum. Neither the mononuclear cells nor the serum alone are cytolytic to the target erythrocytes. Previous studies have shown that the serum activity is not immunoglobulin and is heat-labile, suggesting a similarity to serum complement. In this report, sera deficient in various complement components as well as highly purified single complement components were tested with whole mononuclear cell populations and purified monocytes and lymphocytes to further characterize this cytotoxicity system. Whole mononuclear cell populations failed to mediate target cell lysis in sera deficient in C5 or factor B. However, C3-deficient serum, even in the presence of anti-C3 antibody, supported synergistic cytotoxicity normally. Purified lymphocytes were also normally cytotoxic in C3-deficient serum but failed to lyse targets in sera deficient in C5, C7, C8, or depleted of factor B. Purified monocytes failed to lyse the target cells only in factor B-depleted serum and could lyse the target cells in serum-free medium when purified factor B alone was added. Monocyte-mediated cytotoxicity induced by factor B was inhibited 73-100% by adding lymphocytes back to the purified monocytes. Thus, both lymphocytes and monocytes can serve as effector cells in this form of cytotoxicity but require cooperative interaction with different sets of complement components. In addition, lymphocytes can modulate the monocyte-mediated form of target cell lysis associated with factor B.

Animals↗
PubMed1982

Complement proteins C2, C4 and factor B. Effect of glycosylation on their secretion and catabolism.

Tunicamycin, an inhibitor of N-acetylglucosaminylpyrophosphopolyisoprenol-dependent glycosylation, was used to study the effect of glycosylation on the synthesis, post-translational modification, secretion and function of the complement proteins that are associated with the major histocompatibility complex in humans, mice and guinea pigs. Tunicamycin blocked glycosylation of pro-C4, C2 and factor B and inhibited secretion of the corresponding native complement proteins synthesized by guinea-pig peritoneal macrophages in tissue culture. In addition, underglycosylated pro-C4 was more rapidly catabolized intracellularly than the corresponding fully glycosylated pro-complement protein. C4 protein secreted by cells incubated with tunicamycin had approximately the same specific biological activity as the protein obtained from control culture media, suggesting that carbohydrate is not required for its activity in immune haemolysis. Direct studies of carbohydrate incorporation and the tunicamycin effect suggested an unequal distribution of sugar among the C4 subunits, with maximal incorporation of carbohydrate into alpha-, and less into the beta-chain of the native protein.

Animals↗
PubMed1982

Biosynthesis and processing of a human precursor complement protein, pro-C3, in a hepatoma-derived cell line.

A 180,000-dalton single-chain molecule (human pro-C3) is the precursor of the third component of human complement (C3), a disulfide-linked two-chain protein. The pro-C3 is converted by limited proteolysis to C3. The relationship between pro-C3 and C3 was established with the use of Hep G2, a cell line derived from a human hepatocellular carcinoma, which synthesizes at least 17 plasma proteins.

Animals↗
PubMed1982

Isolation of cDNA clones for the human complement protein factor B, a class III major histocompatibility complex gene product.

cDNA clones corresponding to a major histocompatibility class III antigen, the complement protein factor B, have been isolated from a human adult liver cDNA library. The clones, ranging in size from 1.0 to 2.3 kilobases, were identified by direct hybridization with two synthetic oligonucleotide mixtures. Two regions of the factor B amino acid sequence, each with minimal ambiguity in codon assignment, were chosen for synthesis of the oligonucleotides. The sequences of two clones have been partially determined. They contain coding information for the amino acid sequence of the Bb fragment of factor B and the entire 3' -untranslated region.

Adult↗
PubMed1982

Radionuclide assessment of the effects of chest physical therapy on ventilation in cystic fibrosis.

This study assesses the use of krypton 81m scintigraphy as a measurement tool in evaluating the effectiveness of bronchial drainage with percussion and vibration on peripheral ventilation in patients with cystic fibrosis. Ten patients with cystic fibrosis participated. Each patient underwent a krypton 81m ventilation study and traditional pulmonary function tests. Forty-five minutes later, these studies were repeated before and after a chest physical therapy treatment. Each patient acted as his own control. All krypton 81m scintiscans were recorded and analyzed visually and numerically using a digital computer to assess distribution of ventilation. Visual analysis of the scintiscans indicated individual variation in treatment response: in some patients ventilation improved with therapy; in others, no change was noted; still others had changes independent of treatment. Numerical data derived from the scintiscans and pulmonary function tests showed no important differences among the three studies of each patient. Airway abnormalities characteristic of cystic fibrosis, progression of the disease, sputum production, or a combination of these factors may account for the individual variation in response to treatment. Krypton 81m scintigraphy is a reliable measure of regional ventilation and should be useful for assessing the efficacy of chest physical therapy because of the consistent, high quality visual data retrieved.

Adolescent↗
PubMed1982

Complement biosynthesis by the human hepatoma-derived cell line HepG2.

The human hepatoma-derived cell line, HepG2, synthesized and secreted functional complement proteins C1r, C1s, C2, C3, C4, C5, factor B, C1 inhibitor, C3b inactivator, a small amount of C6, and trace amounts of C8; but failed to produce detectable C1q, C7, or C9. Immunochemically, C2, C3, C4, C5, and B were isolated from culture medium as proteins with molecular sizes and subunit structures identical to the corresponding components isolated from serum. C2 and factor B from cellular lysates had slightly lower molecular weights than the corresponding proteins in culture medium. C3, C4, and C5 were detected as single chain precursor molecules in cellular lysates. These results demonstrate that human C5, like C3 and C4, is synthesized as a single chain precursor that is converted by limited proteolysis to the native two-chain molecule. It also establishes the precursor-product relationship for human pro-C4 and native C4, pro-C5, and native C5.

Carcinoma, Hepatocellular↗
PubMed1982

Complement (C3b) interaction with the human granulocyte receptor: correlation of binding of fluid-phase radiolabeled ligand with histaminase release.

The interaction of C3b, the major cleavage product of C3, with its receptor on human granulocytes results in important biological functions, including phagocytosis, superoxide generation, and release of a variety of enzymes, including histaminase. We have determined the binding kinetics and isotherm of trypsin-generated fluid-phase dimeric C3b at both 0 degrees C and 37 degrees C using human polymorphonuclear leukocytes (PMN). At 37 degrees C the apparent number of receptors per cell was threefold greater than number at 0 degrees C (66,000 vs 21,000), and the affinity (Ka) was slightly less (3.5 X 10(7) M-1 vs 6 x 10(7) M-1). C3b dimer binding was specifically inhibited by a F(ab,)2 anti-C3b receptor antibody. C3b dimer induced histaminase release in a dose-dependent fashion at a concentration of 1 to 4 X 10(7) molecules/cell, whereas at lower concentrations a dose-dependent inhibition of opsonized zymosan-induced release was noted. These effects were independent of IgG. In contrast, C3b monomer failed to demonstrate measurable direct binding or to induce histaminase release. Histaminase release, however, did occur after incubation of PMN with affinity-linked monomer (anti-C3 F(ab')2-C3b complexes). Monovalent complexes did not affect release. Monomeric C3, C3c, and C3d were without effect; however, affinity-linked C3 and C3c did cause release.

Amine Oxidase (Copper-Containing)↗
PubMed1982

Complement biosynthesis in human breast-milk macrophages and blood monocytes.

The availability of techniques for establishment of primary, long term monolayers of breast milk macrophages and blood monocytes permitted a direct comparison of biosynthetic functions of human tissue macrophage and its progenitor. In addition to previously described morphological differences, four specific characteristics of the breast milk macrophage were identified: (i) a reduced rate of total protein secretion relative to the monocyte; (ii) abrogation of the 3 day lag in complement secretion regularly observed in blood monocyte cultures; (iii) an increase in the secretion of complement proteins C2 and factor B; and (iv) an increase in the ratio of C2 : factor B secretion. These differences did not result from cell-cell interactions between monocytes and macrophages, stable factors elucidated by monocyte or macrophage monolayers, or heat-stable factors in breast milk. In addition, both monocytes and macrophages synthesized and secreted C3 in an apparently native but haemolytically inactive form. The differences observed suggest that macrophages may modulate local availability of complement proteins in tissues or in the early phase of an inflammatory response.

Cells, Cultured↗
PubMed1982

Biosynthesis of a structurally abnormal C2 complement protein by macrophages from C2-deficient guinea pigs.

In order to characterize a genetic deficiency of C2 in guinea pigs, production of C2 by peritoneal macrophage cultures derived from four normal, four heterozygous deficient, and four homozygous deficient animals was measured functionally and immunochemically after metabolic labeling with 35S-methionine. Macrophage monolayers from homozygous deficient animals failed to secrete hemolytically detectable C2 up to 74 hr in culture. A single cell hemolytic plaque assay also failed to demonstrate any functional C2 production by cells from homozygous deficient animals. No C2 protein was detected in media from three of the four homozygous deficient animals, but in one, apparent C2 fragments were present. In contrast, intracellular C2 protein was identified in all four homozygous deficient cell cultures. Its mobility on SDS-PAGE was slightly faster than normal. Much less abnormal intracellular C2 protein was recovered from homozygous deficient macrophage monolayers than intracellular C2 protein from normal macrophage monolayers. Monolayers from heterozygous animals produced functional and immunochemical C2 at approximately 30% of the normal rate. Normal rates of biosynthesis and secretion of two other MHC-linked class III antigens, C4 and factor B, were detected in macrophage cultures from homozygous and heterozygous deficient animals. These data suggest that a specific defect, i.e. a structural abnormality in C2 protein, underlies C2 deficiency in guinea pigs.

Animals↗
PubMed1981

NH2-terminal structure and cleavage of guinea pig pro-C3, the precursor of the third complement component.

The NH2-terminal structures of the intracellular precursor of the third component of guinea pig complement (pro-C3), synthesized in macrophage cultures, and beta chain of C3 from guinea pig plasma were determined. Six of the first 16 residues of pro-C3 were identified by a microradiosequencing method. All six are nonpolar and are identical with the residues in beta chain of native plasma C3. This establishes beta chain as the NH2-terminal subunit in pro-C3. A comparison of primary structures of guinea pig and human C3 beta chains revealed identity of at least 8 residues within the NH2-terminal decapeptide. Incubation of plasmin with radiolabeled pro-C3 in macrophage lysate resulted in loss of pro-C3 and the generation of a two-chain molecule electrophoretically indistinguishable from native C3. These data indicate similarities between pro-C3 and the precursor of the fourth complement component, pro-C4, with respect to subunit organization, presence of phylogenetically conserved NH2-terminal regions, and the probable mechanism for generation of their respective native proteins.

Amino Acid Sequence↗