James D. Ogle

The effects of cytokines, platelet activating factor, and arachidonate metabolites on C3B receptor (CR1, CD35) expression and phagocytosis by neutrophils

The cytokines tumor necrosis factor alpha (TNF alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and interleukin 1 (IL 1) all caused an upregulation of C3b receptors (CR1) on neutrophils that ranged from around 76% (G-CSF and IL 1) to 93% (TNF alpha and GM-CSF) of the upregulation obtained by pretreatment of the neutrophils with the chemotactic peptide FMLP. However, only TNF alpha and G-CSF caused a significant increase in phagocytosis of opsonized microspheres. Platelet derived growth factor, interleukin 2, and transforming growth factor beta had no effect on either of these parameters. The mediators platelet activating factor (PAF) and leukotriene B4 (LTB4) both caused a large upregulation of CR1 (93% and 80%, respectively, of the FMLP-mediated value); however, only PAF caused a significant enhancement of phagocytosis by the neutrophils. Prostaglandin E2 and thromboxane B2 had no effect on these parameters. Considerable individual variation was observed among some of the untreated and mediator-treated neutrophil preparations regarding CR1 expression and phagocytosis. The upregulation of CR1 and associated increase in phagocytic capacity of neutrophils caused by certain cytokines and other mediators may be important in host defense. Also the lack of enhancement of phagocytosis accompanying an upregulation of CR1 is unusual and may have important implications regarding the cellular mechanisms of phagocytosis by neutrophils.

The production of C3, PGE2 and TxB2 by splenic, alveolar, and peritoneal guinea pig macrophages

The synthesis and release of C3, PGE2, and TxB2 by cultured splenic, alveolar and peritoneal guinea pig macrophages in 24 hour culture was determined. There were significant differences in C3 production among all three sources of macrophages. Splenic macrophages produced significantly less PGE2 than alveolar or peritoneal macrophages. Peritoneal macrophages produced significantly more TxB2 than splenic or alveolar macrophages. The cells from the different sources appear to be different populations of macrophages.

Studies on peptides obtained from enzymic digests of collagen with evidence for the presence of an unidentified compound in this protein.

Abstract Collagen was partially hydrolyzed by the exoenzymes of Clostridium histolyticum and by crude trypsin following heat denaturation. The hydrolyzates contained a very large number of peptides. Thirty peptides, most of which contained proline and/or hydroxyproline, were found in the acidic, neutral, and weakly basic fractions. These fractions represented 82% of the proline, 89% of the hydroxyproline, and only 15% of the arginine in collagen. With the exception of two tripeptides previously reported (1), none of the peptides accounted for over 1% by weight of collagen, indicating that these peptide residues apparently do not repeat often in the collagen molecule. The sequence of five of the peptides is given. One of the peptides contained a residue which corresponded to no known amino acid in collagen. This unidentified compound has been isolated from complete acid hydrolyzates of collagen. The results of preliminary studies on it are given.

Comparison of abilities of recombinant interleukin-1α and -β and noninflammatory IL-1β fragment 163–171 to upregulate C3b receptors (CR1) on human neutrophils and to enhance their phagocytic capacity

Both recombinant IL-1α and -β caused an upregulation of C3b receptors (CR1) on human neutrophils and caused a receptor-mediated enhancement of phagocytosis of C3b·IgG-coated microspheres by these leukocytes. Theα andβ forms of the recombinant cytokine were of comparable potency regarding CR1 upregulation, although both generally had less than 25% of the potency of FMLP in this respect. Recombinant IL-1β was slightly more potent than theα form of the cytokine regarding phagocytosis of opsonized microspheres and, again, both forms were less potent than FMLP in causing an enhancement of phagocytosis by neutrophils. The synthetic noninflammatory immunostimulatory nonapeptide corresponding to residues 163–171 of IL-1β was completely inert with respect to upregulation of CR1 on neutrophils and the enhancement of phagocytosis by these cells. Thus this domain in the intact IL-1β molecule apparently is not involved in CR1 upregulation and the ensuing enhancement in phagocytosis by neutrophils, although it is apparently important in the immunostimulatory activity regarding the proliferation of lymphocytes.

Effects of combination of tumor necrosis factor alpha and chemotactic peptide, f-Met-Leu-Phe, on phagocytosis of opsonized microspheres by human neutrophils.

Pretreatment of normal, human neutrophils with 8 units/ml of TNF-α followed by treatment with 10−8 M FMLP resulted in a synergistic effect of the combination of the two mediators on the enhancement of the phagocytic capacity of the cells. This enhancement of phagocytosis occurred without an additional increase in the upregulation of C3b receptors (CR1) beyond that caused by each mediator alone. Pretreatment of the cells with 8 units/ml of TNF-α followed by 10−6 M FMLP resulted in an additive effect of the mediators on neutrophil phagocytosis, again without an additional up-regulation of CR1. This additive effect resulted in an increase in phagocytic capacity of the neutrophils greater than that obtained by treatment of the cells with 10−6 M FMLP alone, which heretofore has resulted in the greatest enhancement of phagocytic capacity obtained by any pretreatment condition. These synergistic and additive effects of the combination of mediators could be of great importance in host defense against bacterial infections and have important implications regarding the mechanisms of receptor upregulation and phagocytosis.

Human Polymorphonuclear Leukocyte (PMN) Priming/Activation by Acute Ethanol Intoxication

The effects of chronic alcoholism on susceptibility to infections are well documented. Less is known on the effect of sporadic acute ingestion of ethanol on the immune system. One should not assume that it induces neutrophil function deficiency in vivo. One example of upregulation caused by ethanol treatment was described by Dorio (1): in vitro ethanol treatment of rat macrophages causes an acute rise in intracellular calcium, stimulates a small amount of Superoxide production, but it also inhibits concanavalin A- and phorbol ester-induced Superoxide formation. Acute ingestion of ethanol has been shown to affect some, but not all PMN functions: decreased adherence to surfaces, decreased delivery to inflammation sites, but no abnormality of chemotaxis, Superoxide generation, bacterial killing, or intracellular cAMP and cGMP levels (2,3). In other experimental studies in mice, both low dose (1 mg/Kg) and high dose (4 mg/Kg of body weight) of ethanol increased the number of sticking or plugging PMNs in sinusoids of the liver (4). A small decrease in phagocytosis has been detected by some (3), but not by others (2).

Adhesive effect of certain cytokines and other perturbants on human neutrophils

Pretreatment of normal human neutrophils with certain cytokines and other mediators caused some of the cells to become adhesive and stick to the plastic (polypropylene) incubation tubes during pretreatment and during the assay for phagocytosis of C3b·IgG-coated microspheres. Often as much as 40% of the cells were adherent to the tubes after the reaction. This sticking of the neutrophils to the plastic tubes was confirmed by increase in cytometer sipping time and by lactic dehydrogenase assay of the suspended cells and of the cells stuck on the sides of the empty incubation tubes. Only those perturbants that caused an up-regulation of C3b receptors (CR1, CD35) and in most cases caused an enhancement of phagocytosis mediated the adhesiveness of the cells. Unless these stuck cells were detached by vigorous flushing with cold buffer containing EDTA, many of the cells were not admitted into the cytometer for determination of the effect of the perturbants on binding and phagocytic capacity of the neutrophils. This observation could have implications regarding the possibility of subpopulations of neutrophils and differences in function of adherent cells versus cells in suspension. In the cases studied there was no appreciable difference between the total binding and phagocytic capacities of the adherent and suspended cells.

Preparation of properdin by affinity chromatography

Abstract A new technique is described for the preparation of properdin by immune affinity chromatography. Anti-human properdin antibody was attached via its carboxyl groups to amine-substituted Sepharose 4B with the use of a water soluble carbodiimide. The attachment of the antibody via its carboxyl groups to the solid support resulted in an attenuation of its binding of properdin, which allowed the latter to be eluted from the column under very mild conditions. This method requires considerably less time than other chromatographic procedures, and results in an almost 100% yield. Seventy % of the properdin was electrophoretically pure after one passage over a DEAE-cellulose column; the remaining 30% contained traces of impurities which were not removed by one passage over a DEAE-cellulose column.