Thermal injury increases the number of eosinophil progenitors in rat spleen and bone marrow.

We have investigated the effects of thermal injury upon myelopoiesis. IL-3, GM-CSF, and IL-5 were used to stimulate myeloid colony formation. IL-3 induces early myeloid progenitors and a more developed myeloid progenitor, the granulocyte-macrophage colony-forming unit (GM-CFU), to multiply and develop into mature myeloid cells. GM-CSF induces GM-CFU to become mature myeloid cells, while IL-5 induces eosinophil progenitors to become mature eosinophils. Stem Cell Factor (SCF) + IL-6 and FLT3 ligand, which have no effect on colony formation by themselves, were used to enhance the effects of IL-3 and GM-CSF, respectively. We found that thermal injury increased the number of early myeloid progenitors and GM-CFU in the spleen with either IL-3 or GM-CSF as a stimulant. Thermal injury increased the number of early myeloid progenitors in the bone marrow when GM-CSF, but not IL-3, was used to stimulate colony growth. Also, thermal injury increased the numbers of eosinophil progenitors in rat spleen and bone marrow and increased splenic levels of IL-5 mRNA.

Thermal injury functionally alters bone marrow-derived macrophages: a study of monocyte-hepatocyte interactions.

Thermal injury quantitatively and qualitatively alters hematopoiesis, including monocyte-macrophage lineage changes, resulting in altered mononuclear cell function. These bone marrow cells (BMCs) ultimately become fixed tissue macrophages (e.g., Kupffer cells). To study the effects of thermal injury on macrophage-hepatocyte interactions, rat BMCs were isolated 24 hours after burn injury, and myelopoiesis was induced by 7-day culture in granulocyte-macrophage colony-stimulating factor. Separate cultures included inflammatory mediators with growth factor function (IL-6 or PGE2). Cultured cells were incubated up to 96 hours with isolated normal hepatocytes (+/- lipopolysaccharide stimulation). The 96-hour exposure to postburn BMCs produced less of the acute phase proteins (APPs), C3 and transferrin, but more cytotoxicity as measured by 1-lactate dehydrogenase release. Sham BMCs cultured with added IL-6 caused higher APP release and minimal cytotoxicity, whereas burn BMCs stimulated lower APP release and retained cytotoxicity. In conclusion, myeloid cells regulate APP synthesis differently after thermal injury and may become more cytotoxic to hepatocytes.

Bone marrow and splenocyte coculture-generated cells enhance allograft survival.

BACKGROUND: Protocols that incorporate donor-specific cell infusions using bone marrow, spleen, or blood transfusion continue to enhance allograft survival and often lead to tolerance in experimental models. Clinical benefits from these modalities have not been as striking, leading to ongoing study in this field. We have explored culture techniques for the in vitro selection and development of cellular effectors capable of enhancing allograft survival. METHODS: Rat bone marrow or spleen cells cultured under a variety of conditions were screened for suppressor function. Bone marrow cells, nonadherent to plastic, cultured for 7 days with granulocyte-macrophage colony-stimulating factor, lipopolysaccharide, and with or without splenocytes were found to contain predominantly myeloid lineage cells and had the ability to suppress phytohemagglutinin or mixed lymphocyte reaction-induced splenocyte proliferation. Standard donor-specific peripheral blood transfusion was compared with cultured donor-specific bone marrow cells, splenocytes, or marrow cells cultured with splenocytes (cocultured) administered intravenously at 1 x 10(7) cells/kg the day before an ACI to Lewis heterotopic heart transplant. Cyclosporine was administered at 10 mg/kg on day -1 and 2.5 mg/kg on days 0-6 relative to transplantation. RESULTS: Mean allograft survival in cyclosporine-treated animals was 8.5 days without and 16.6 days with a donor-specific blood transfusion. Cocultured cells extended allograft survival (39.5 days), whereas bone marrow or splenocytes cultured alone did not. With Percoll gradient separation, two predominant culture subfractions, one with potent suppressor function and another with stimulator function, were identified. Flow cytometric analysis showed mixed populations enriched for macrophages but also including dendritic cells in both subfractions. The suppressive fraction extended allograft survival to 20.8 days and the stimulatory fraction was less effective, yet remixing of both fractions regained the full allograft survival advantage. CONCLUSIONS: In this model, the coculture of bone marrow cells and splenocytes with granulocyte-macrophage colony-stimulating factor and lipopolysaccharide produced functionally divergent subpopulations that synergistically enhanced allograft survival. The development of cellular effectors with enhanced ability to prolong allograft survival using in vitro culture techniques is possible, and provides a new therapeutic option in the use of cell infusion-based therapies.

Peripheral ingroup membership status and public negativity toward outgroups.

Peripheral membership status in a desirable ingroup was predicted to elevate outgroup derogation when Ss believed other ingroup members might learn of their responses. Less negativity toward outgroups was expected when peripheral members' responses were to remain private. Core ingroup members, in contrast, were not expected to show public-private differences in derogation of outgroups. The results of 2 experiments supported these predictions, with peripheral but not core ingroup members advocating the most coercion for the outgroup under public conditions in both laboratory-created ingroups (Experiment 1) and naturally occurring groups that had meaning for the participants (Experiment 2). Thus, outgroup derogation can serve a public presentation function that allows for enhancement of an insecure status within a desirable ingroup.

Effect of glutamine on phagocytosis and bacterial killing by normal and pediatric burn patient neutrophils.

Glutamine is essential for the function of lymphocytes and macrophages, where it serves, among other things, as a source of energy. Little information is available concerning the fuel that polymorphonuclear cells use for their metabolic and bactericidal functions. It was the purpose of this study to determine whether glutamine would enhance the in vitro bactericidal function of normal neutrophils and whether the amino acid would restore the observed impaired function in burn patients to or above the normal level. Twelve burn patients with total body surface area burns ranging from 32% to 87% were studied. At various postburn times, neutrophils were isolated and their ability to kill Staphylococcus aureus in the presence and absence of glutamine was determined and compared with that in normal subjects. Glutamine enhanced the bactericidal function of normal neutrophils. In every patient, at all but two postburn times, glutamine caused an improvement in the observed abnormal neutrophil bactericidal function and often restored it to or slightly above the normal level. Glutamine had no effect on the expression of C3b receptors (CR1 or CD35) or on phagocytosis by the cells. This study confirms the beneficial effects of glutamine in at least one arm of the immune system and adds evidence for the possible advantage of including this amino acid in the diets of burn and other trauma patients.

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.

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-alpha 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-alpha 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.

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.

Effects of chemotactic peptide f-Met-Leu-Phe (FMLP) on C3b receptor (CR1) expression and phagocytosis of microspheres by human neutrophils.

FMLP caused maximal upregulation of CR1 on neutrophils at a concentration of 10(-8) M but caused maximal enhancement of CR1-dependent phagocytosis of C3b.IgG-coated microspheres only at a concentration of 10(-6) M. There were positive correlations between FMLP-mediated upregulation of CR1 and FMLP-mediated enhancement of phagocytosis (correlation coefficient = 0.73, slope = 2.2) and between FMLP-mediated upregulation of CR1 and FMLP-mediated increase in total cell-associated microspheres (correlation coefficient = 0.88, slope = 1.3). The phagocytic capacity of both untreated and 10(-6) M FMLP-treated neutrophils was completely inhibited by fluid phase C3b and partially inhibited by aggregated IgG. The data suggest that CR1 upregulation is required but is not sufficient for maximal phagocytosis by the leukocytes. The data also suggest that FMLP at the higher concentrations may impart a phagocytic function to CR1, activate other phagocytic receptors, elicit phagocytosis-inducing mediators or may elicit a separate mechanism of phagocytosis. During the study, it was observed that there was considerable individual variation among different neutrophil preparations with respect to CR1 expression and binding and phagocytic capacity.

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

Both recombinant IL-1 alpha and -beta 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. The alpha and beta 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-1 beta was slightly more potent than the alpha 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-1 beta 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-1 beta 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.

Effect of antibiotics on CR1 receptor levels of human neutrophils and on the binding and phagocytosis of opsonized polystyrene microspheres by these leucocytes.

Fourteen antibiotics were studied for their effects on the following properties or functions of normal, human neutrophils: (1) the expression of CR1 receptors; (2) the total binding of C3b.IgG-coated polystyrene microspheres in the presence of cytochalasin D to inhibit phagocytosis; (3) the net phagocytosis of the opsonized microspheres; (4) the residual, external binding after phagocytosis. Fluorescence, flow cytometric methods were used to determine binding and phagocytosis of the model target particles. Only nafcillin, a penicillin, caused a decrease (33 per cent) in phagocytic capacity of the neutrophils at a physiologically significant dose (serum level); the antifungal antibiotic, amphotericin B, caused a 30 per cent increase in phagocytic capacity. These small changes in neutrophil phagocytic capacity may not be physiologically significant. There were no significant differences in the four measured parameters caused by other antibiotics tested at physiologically significant doses.

Phagocytosis of opsonized fluorescent microspheres by human neutrophils. A two-color flow cytometric method for the determination of attachment and ingestion.

A highly reproducible two-color fluorescence cytometric method is described for determining the amount of receptor-mediated and of non-specific phagocytosis by human neutrophils of polystyrene microspheres that have been covalently coated with C3b, iC3b, IgG, mixtures of these, BSA or human F(ab')2. The method includes a correction for externally bound particles and thus measures net phagocytosis. The method involves the incubation of neutrophils with coated green fluorescent microspheres in buffer alone or with cytochalasin D to inhibit phagocytosis followed by staining the externally bound microspheres with red fluorescent antibodies to the immobilized ligand, and determining the green and red fluorescence in a dual laser fluorescence activated flow cytometer. The red to green fluorescence ratio of the mixtures containing cytochalasin D allows for a correction of the green fluorescence due to externally bound microspheres in the mixtures not containing cytochalasin D. The corrected green intensities thus represent net phagocytosis. The specificity of receptor-mediated phagocytosis was confirmed by inhibition with fluid-phase C3b or iC3b or monoclonal antibodies to the receptors. The method can be applied to the determination of both adherent and suspension phagocytosis and can be used as a general model of the phagocytosis of bacteria by neutrophils.

Determination of C3b receptors on normal and patient polymorphonuclear neutrophils with C3b-coated fluorescent microspheres.

A method, devised in the authors' laboratories, for the determination of C3b receptors on normal and patient neutrophils using C3b-coated fluorescent microspheres, was applied to the quantitation of C3b receptors on the neutrophils of several patients suffering from burns and trauma and a patient with pancreatitis. From three to 11 days in the clinical course the relative number of C3b receptors was, or rose to, two to ten times the number of receptors present at later times in the clinical course and, in most of the cases studied, the increase in C3b receptor number coincided with enhanced neutrophil bactericidal function. The rise in C3b receptor number was ascribed to up-regulation by C3a and C5a des Arg from complement activation and also, in the cases where sepsis occurred, to the presence of bacterial chemotactic peptides. Preliminary experiments with zymosan-activated serum and the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine confirmed this explanation.

Studies on the binding of C3b-coated microspheres to human neutrophils.

A method is described for the quantitation of C3b receptors on human neutrophils using a mixture of C3b-coated fluorescent and C3b-coated non-fluorescent microspheres. The method measures the "sterically available' C3b receptors on the cells, for example, the receptors available to opsonized bacteria. The use of mixtures of fluorescent and non-fluorescent microspheres resulted in lowered fluorescence intensities of the microsphere-coated neutrophils that were well within the fluorescence limitations of fluorescence activated cell analyzers or sorters used in the assay procedure. These mixtures also allowed the distribution of the C3b-coated microspheres around the neutrophils to be easily visualized in the fluorescence microscope. The binding of the C3b-coated microspheres to the neutrophils was shown to be receptor mediated by typical saturable binding kinetics, by complete inhibition by fluid phase C3b, but not by other proteins and by nearly complete inhibition by anti-C3b receptor antibody. Several parameters that could affect the binding of C3b-coated microspheres to neutrophils were studied; these included time and temperature of incubation of the microspheres with the cells, the diameter of the microspheres, the C3b content of the C3b-coated microspheres, the presence of metal ions, azide, EDTA, protein (BSA, IgG), soybean trypsin inhibitor in the buffers, and the method of isolation of the neutrophils. The C3b-coated microspheres were evenly distributed around the neutrophils in almost all of the cases; however, the neutrophils used in these studies were not activated and were not phagocytosing. The method is extremely reproducible and sensitive in detecting small changes in number of C3b receptors on cells.

Modification of apolipoprotein C-II with 1,2-cyclohexanedione and 2,3-butanedione. Role of arginine in the activation of lipoprotein lipase.

Apolipoprotein C-II, the activator protein of lipoprotein lipase, contains 78 amino acids with a single residue of arginine at position 49. Chemical modification of apolipoprotein C-II with 1,2-cyclohexanedione or 2,3-butanedione results in a loss of both the arginine residue and the ability of the protein to enhance the activity of bovine milk lipoprotein lipase toward a trioleoylglycerol substrate; removal of the modifying group restores arginine and more than 70% of the activating property of the apolipoprotein. Arginine modification of apolipoprotein C-II does not effect its lipid-binding properties as assessed by its association to sonicated vesicles of dimyristoylphosphatidylcholine. Furthermore, secondary structure associated with complex formation with dimyristoylphosphatidylcholine are nearly identical for the unmodified, 1,2-cyclohexanedione-modified or modified-reversed proteins. These results suggest that arginine-49 of apolipoprotein C-II is situated at or near an amino acid sequence domain involved in the activation of lipoprotein lipase. However, a guanidinium group is not required for lipid binding.

Reduced immunoregulatory potency of low density lipoproteins with selectively modified arginine and lysine residues of apolipoprotein B.

Human plasma low density lipoproteins (LDL) suppress lymphocyte activation in vitro by inhibiting the early, membrane-associated events such as phytohemagglutinin-enhanced Ca2+ accumulation and phosphatidylinositol turnover. Chemical modification of the arginine residues of the protein constituent of LDL by 1,2-cyclohexanedione/borate or of the lysine residues by reductive methylation substantially decreases the immunosuppressive potency of LDL. The decrease in inhibitory capability of LDL correlates with a reduction in the ability of the derivatized LDL to compete with 125I-labeled LDL for lymphocyte membrane receptors. This correlation indicates that immunoregulation by LDL is the direct result of the binding of LDL to specific receptors at the cell surface. The receptor recognition site of LDL may consist of a high content of basic amino acid residues such that chemical modification of the LDL apolipoprotein reduces the LDL-lymphocyte interaction by specifically altering the change and/or steric properties of the receptor recognition site. Alternatively, chemical modification of arginine or lysine may cause a conformational change of the lipoprotein which is transmitted to the receptor recognition site. Derivatization of lysine or arginine residues does not elicit gross structural alteration of LDL, as evidenced by chemical analysis and by fluorescence quenching analysis. The intrinsic fluorescence intensity of LDL is, however, decreased by chemical modification, indicative of a minor but perhaps biologically significant structural alteration.

Binding of plasma low density lipoproteins to erythrocytes.

Low density lipoproteins (LDL) containing apolipoprotein B bind to intact, freshly isolated erythrocytes. The LDL-erythrocyte interaction is of low affinity, with a Kd of 1.1 x 10(-6) M. Binding is noncooperative. There are about 200 binding sites per cell and, within the limits of experimental uncertainty, these sites comprise a homogeneous class. Binding of LDL is a temperature-independent process. The maximum amount of LDL blood increases following proteolytic digestion of the cells with trypsin or chymotrypsin. The specificity of the binding sites for LDL is not absolute: high density lipoproteins and lipid vesicles composed of phosphatidylcholine or phosphatidylcholine/cholesterol (equimolar) complete with LDL for occupancy of 60% of the binding sites. Modification of 5--6 of the 9 apolipoprotein B arginine residues with 1,2-cyclohexanedione/borate or of 10--15 of the 20 lysine residues by reductive methylation does not alter the ability of LDL to bind to erythrocytes. Native LDL and methylated-LDL alter erythrocyte morphology. However, LDL in which the arginine residues are derivatized with 1,2-cyclohexanedione/borate do not induce the discocyte leads to echinocyte transformation. Chemically modified and native LDL exchange cholesterol with erythrocytes at equal rates and to nearly equal extents. Taken together, the data suggest that the binding sites for LDL on the erythrocyte membrane are distinct from the LDL receptors at the surface of other cells--e.g., fibroblasts and lymphocytes--which do not bind HDL and which do not recognize LDL with derivatized arginine or lysine residues. It is proposed that the biological function of the erythrocyte binding sites is to mediate the exchange of cholesterol between the cell membrane and lipoproteins.