Amplification of the activity of human leukocyte inhibitory factor (LIF) by the generation of a low molecular weight inhibitor of PMN leukocyte chemotaxis

Leukocyte inhibitory factor (LIF), which was derived from human peripheral blood lymphocytes by stimulation with concanavalin A ad partially purified by Sephadex G-100 gel filtration, inhibited the in vitro spontaneous migration and chemotaxis of human PMN leukocytes as assessed in a Boyden chamber micropore filter assay. The inhibitory activity was attributed to LIF, a principle defined in terms of its inhibition of PMN leukocyte migration from glass capillary tubes since it was preferentially directed to PMN leukocytes as compared to mononuclear leukocytes, exhibited a size comparable to LIF by gel filtration, and was inactivated by diisopropyl fluorophosphate in parallel with LIF. Incubation of PMN leukocytes with LIF released additional inhibitory activity, distinct from LIF, which resembled the neutrophil-immobilizing factor (NIF) by virtue of its approximate m.w. of 4000 by filtration on Sephadex G-25, inactivation by trypsin digestion, and preferential noncytotoxic inhibition of spontaneous migration and chemotaxis of PMN leukocytes as compared to mononuclear leukocytes. Thus LIF inhibits PMN leukocyte migration both by a direct action on the cells and by an amplification pathway that is mediated by low m.w. chemotactic inhibitors similar to NIF.     

Electrophoretic mobility distributions of normal human T and B lymphocytes and of peripheral blood lymphoblasts in acute lymphocytic leukemia: effects of neuraminidase and of solvent ionic strength.

Human T and B lymphocytes were found to be distinguishable on the basis of electrophoretic mobility, with the T cells having the higher mobility, in agreement with previous reports. The effects of the enzyme neuraminidase on the electrophoretic mobilities of T and B lymphocytes were determined. T lymphocytes showed a greater decrease in electrophoretic mobility after neuraminidase treatment; the relative mobilities of T and B cells were reversed by neuraminidase treatment, and the electrophoretic distinguishability was enhanced. The electrophoretic mobility distributions of lymphoblasts from patients with acute lymphocytic leukemia were found to differ from those of normal cells in their response to neuraminidase treatment and to changes in solution ionic strength. These results imply that the surface structure of the leukemic cells differs from that of either T or B lymphocytes from normal donors.     

Electrophoretic mobility of mouse cells and homologous isolated nuclei

The electrophoretic mobilities of Ehrlich ascites, sarcoma 37 ascites, mouse liver cells and their isolated nuclei were measured under similar environmental conditions. No differences in mobility were detected between cells and homologous nuclei from the same cell population and it was concluded that their surface charge densities were probably the same. The effect of neuraminidase on Ehrlich ascites and liver cells and nuclei was also determined; neuraminidase reduced the mobility of Ehrlich ascites cell nuclei as well as cells. The reduction in mobility of cells and nuclei prepared by a sucrose method was the same; however, the reduction in mobility of citric acid prepared nuclei was less than that of citric acid treated cells. The reduction in mobility of both liver cells and nuclei was small or insignificant. It is suggested that although cells and nuclei have similar electrophoretic mobilities, possibly different groups contribute to their surface charge.     

Red cell aging. II. Anomalous electrophoretic properties of neuraminidase treated human erythrocytes.

Desialylation of human red blood cells (RBC) by Vibrio cholerae neuraminidase (VCN) was found to produce cells with electrophoretic properties which were inconsistent with the view of simple loss of N-acetylneuraminic acid (NANA) as the sole effect of VCN treatment. Modification of human RBC with 50--350 U VCN/10(10) RBC for one hour at 37 degrees C releases 90-100% of the NANA and produces a progressive decrease towards zero in their electrophoretic mobilities when measured in 0.15 M NaCl (pH 7.2) at 25 degrees C. The appearance of positive groups on the desialylated cells was indicated by the VCN-treated cells displaying positive mobilities below approximately pH 5.5 and increased negative mobilities at approximately pH 9 as well as substantial increases in their mobility at neutral pH following treatment with formaldehyde. Adsorption of about 95% of the VCN activity at 0 degrees C to the RBC did not produce any significant change in their electrophoretic mobilities thus indicating that the observed changes in the electrophoretic properties of the RBC following VCN treatment could not be attributable to adsorption of VCN. These studies indicate that the cationic charge groups which appear at the electrophoretic surface of the RBC after VCN treatment are probably of endogenous origin. It is suggested that this alteration rather than simple NANA release may operate to shorten the in vivo survival time of desialylated red cells.     

Effect of ribonuclease and neuraminidase on the electrophoretic mobility of tissue culture cells in parasynchronous growth

Cultured mammalian cells (RPMI no. 41) in parasynchronous growth were treated, at different stages of the mitotic cycle, with neuraminidase and ribonuclease, separately and sequentially, and their electrophoretic mobilities determined. Changes in the electrophoretic mobility of these cells are probably mainly due to variations in the density of negatively charged groups susceptible to neuraminidase, although variations in groups susceptible to ribonuclease may occur. It is suggested that the observed variations in electrophoretic mobility of different cells may be due to differences in the relative lengths of different life-cycle phases. Where G₂ phase is relatively long or G₁ relatively short the cell populations will hve higher mean electrophoretic mobilities.     

Study of electrophoretic mobility of cellular membranes isolated from rat liver.

Plasma membranes as well as mitochondrial and microsomal subfractions were subjected to zone electrophoresis. Treatment with neuraminidase, phospholipase A or C does not influence the movement of plasma membranes and smooth microsomes. Trypsin increases mobility of plasma membranes and smooth by about 20%, and further treatment with phospholipase C decreases mobility of plasma membranes, total smooth and smooth I microsomes, which, however, is not the case with smooth II microsomes. Low concentrations of trypsin also solubilize enzyme proteins of smooth microsomes from phenobarbital-treated rat liver, but electrophoretic mobility is not increased, indicating structural differences in induced membranes. The mobility of the outer and inner mitochondrial membranes is significantly higher than that of submitochondrial particles. For microsomes the negative surface charge density occurs in the decreasing order of: ribosomes--rough--smooth I--smooth II. A 10 mM CsCl gradient decreases the mobility of rough microsomes by 40% and of ribosomes by 20% but has no effect on total smooth micromes. On the other hand, 5mM MgCl2 decreased the mobility of all three fractions. EDTA-treated rough and EDTA-treated smooth microsomes have the same electrophoretic mobilities. However, the mobilities of non-treated rough and smooth microsomes differ significantly from each other.     

Cytochalasin B and the sialic acids of Ehrlich ascites cells

The effect of cytochalasin B (CB) on the electrophoretic mobility and density of ionized sialic acid groups at the surface of Ehrlich ascites cells was examined together with a biochemical assay of the total sialic acid content of treated and control cells. Sialic acid assays indicated that CB-treated cells had a greater amount of total sialic acid and sialic acid sensitive to neuraminidase than control cells/cell. Equal amounts of sialic acid were removable by neuraminidase treatment from control cells and cells pretreated with neuraminidase and subsequently cultured with CB. The electrophoresis results showed a decrease in electrophoretic mobility in the presence of CB which could be reversed by growth in CB-free medium. Neuraminidase treatment did not make a significant additional reduction in the mobility of CB-treated cells. CB also prevented the recovery of electrophoretic mobility of neuraminidase treated cells. The results suggest that while CB does not inhibit sialic acid synthesis, it does alter the expression of ionized sialic acid groups at the electrokinetic surface. CB-containing culture media could be re-utilized several times suggesting that CB is not significantly bound or metabolized by Ehrlich ascites cells.     

Quantitative measurement of lipoprotein surface charge by agarose gel electrophoresis.

The electrophoretic mobilities of low density lipoprotein (LDL) and six pure proteins in a 0.5% agarose gel have been compared to literature electrophoretic mobility values determined by the Tiselius moving boundary method. There is a strong correlation (r = 0.99) between the electrophoretic mobilities determined by the two techniques. The electrophoretic behavior of charged particles smaller than very low density lipoproteins (VLDL) is not markedly perturbed by a 0.5% agarose matrix, and variations in mobility primarily reflect differences in particle valence and density of surface charge. Application of electrokinetic theory to derive protein and lipoprotein net charges from the electrophoretic mobilities in agarose yields a quantitative delineation of lipoprotein electrophoretic migration patterns wherein the beta mobility region comprises a surface potential range of -4.5 to -7.0 mV; the pre-beta region a range of -7.0 to -10.5 mV; the alpha mobility region a range of -10.5 to -12.5 mV and the serum albumin region a range of -12.5 to -14.0 mV. Because protein conformation and charge are critical in metabolic regulation, the agarose gel electrophoresis technique provides a valuable analytical tool that should help to elucidate further details of the structure-function relationships of serum lipoprotein particles.     

Production of human lymphocyte mediators after activation with periodate or neuraminidase and galactose oxidase.

Treatment of human mononuclear cells with sodium metaperiodate (NaIO4) or neuraminidase and galactose oxidase (NGO) results in lymphocyte activation and subsequent generation of supernatants rich in migration inhibitory factor (MIF) and leukocyte inhibitory factor (LIF). Preliminary characterization of these mediators by Sephadex G-100 gel filtration suggests that they are similar to antigen- and concanavalin A-induced MIF and LIF, eluting in the 25000 m.w. and 68000 m.w. regions, respectively. The possibility of galactose oxidase carryover into the supernatants has been studied and conditions are described that minimize this eventuality. A method is presented for producing control and lymphokine-rich supernatants both of which have been exposed to identical concentrations of NGO although in the control activation is blocked by the addition of 0.1 M galactose to the incubation. These findings establish NaIO4 and NGO as useful mitogens for generating human lymphokine-rich supernatants that can be used without further purification.     

Study of electrophoretic mobility of cellular membranes isolated from rat liver

Plasma membranes as well as mitochondrial and microsomal subfractions were subjected to zone electrophoresis. Treatment with neuraminidase, phospholipase A or C does not influence the movement of plasma membranes and smooth microsomes. Trypsin increases mobility of plasma membranes and smooth by about 20%, and further treatment with phospholipase C decreases mobility of plasma membranes, total smooth and smooth I microsomes, which, however, is not the case with smooth II microsomes. Low concentrations of trypsin also solubilize enzyme proteins of smooth microsomes from phenobarbital-treated rat liver, but electrophoretic mobility is not increased, indicating structural differences in induced membranes. The mobility of the outer and inner mitochondrial membranes is significantly higher than that of submitochondrial particles. For microsomes the negative surface charge density occurs in the decreasing order of: ribosomes — rough — smooth I — smooth II. A 10 mM CsCl gradient decreases the mobility of rough microsomes by 40% and of ribosomes by 20% but has no effect on total smooth microsomes. On the other hand, 5 mM MgCl₂ decreased the mobility of all three fractions. EDTA-treated rough and EDTA-treated smooth microsomes have the same electrophoretic mobilities. However, the mobilities of non-treated rough and smooth microsomes differ significantly from each other.     

Spontaneous injury in isolated sheep lungs: role of resident polymorphonuclear leukocytes.

Perfusion of isolated sheep lungs with homologous blood caused pulmonary hypertension and edema that was not altered by depletion of perfusate polymorphonuclear (PMN) leukocytes (D. B. Pearse et al., J. Appl. Physiol. 66: 1287-1296, 1989). The purpose of this study was to evaluate the role of resident PMN leukocytes in this injury. First, we quantified the content and activation of lung PMN leukocytes before and during perfusion of eight isolated sheep lungs with a constant flow (100 ml.kg-1.min-1) of homologous blood. From measurements of myeloperoxidase (MPO) activity, we estimated that the lungs contained 1.2 x 10(10) PMN leukocytes, which explained why the lung PMN leukocyte content, measured by MPO activity and histological techniques, did not increase significantly with perfusion, despite complete sequestration of 2.0 x 10(9) PMN leukocytes from the perfusate. MPO activities in perfusate and lymph supernatants did not increase during perfusion, suggesting that lung PMN leukocytes were not activated. Second, we perfused lungs from 6 mechlorethamine-treated and 6 hydroxyurea-treated sheep with homologous leukopenic blood and compared them with 11 normal lungs perfused similarly. Despite marked reductions in lung PMN leukocyte concentration, there were no differences in pulmonary arterial pressure, lymph flow, or reservoir weight between groups. Extravascular lung water was greater in both groups of leukopenic lungs. These results suggest that resident PMN leukocytes did not contribute to lung injury in this model.     

The effects of calcium2+ and magnesium2+ on the electrophoretic mobility of chromaffin granules measured by electrophoretic light scattering

Electrophoretic light scattering was used to determine the electrophoretic mobility distributions of isolated bovine adrenal chromaffin granules as a function of divalent metal ion concentrations. Changes in the electrophoretic mobility reflected changes in the surface charge density of the granules. Ca2+ and Mg2+ (0.10--2.0 mM) were equally effective in reducing the electrophoretic mobilities. These findings are consistent with recent studies of the binding of Ca2+ and Mg2+ to the surface of chromaffin granules and are further evidence that the specific role of Ca2+ in exocytosis is due to effects other than the ability of Ca2+ to decrease the electrostatic repulsion between negatively charged membranes.     

Effect of acetaldehyde and glutaraldehyde fixation on the surface properties of red blood cells as determined by partition in aqueous phases

A correlation between partition of cells in dextran/polyethylene glycol aqueous phase systems and their relative electrophoretic mobilities is often in evidence. Absence of such a correlation is indicative that partition measures surface properties other than charge at the plane of shear. Acetaldehyde- and glutaraldehyde-fixed erythrocytes were partitioned in two-polymer phases and their electrophoretic mobility examined in order to investigate the circumstances under which the above-indicated correlation holds. Aspects of the effect of fixation of cells on their surface properties was thereby obtained.

1. 1. Acetaldehyde-fixed and normal human red blood cells have similar partitions and similar electrophoretic mobilities; glutaraldehyde-fixed red cells display markedly increased partitions and mobilities.

2. 2. Lipid-extracted aldehyde-fixed cells have substantially increased partitions, but unchanged mobilities, when compared with the fixed cells from which they were prepared. This indicates that the removal of lipid exposes a higher density of charge groups deeper in the membrane than is measurable by electrophoresis under the conditions used.

3. 3. The countercurrent distribution obtained with fixed cells depends on the length of time chosen for phase settling. Short times result in a single distribution while longer settling times give rise to a two-peak distribution. The latter phenomenon probably arises from a time-dependent aggregation of the fixed cells in the phases.

4. 4. Electrophoretic examination of the glutaraldehyde-fixed cells from different cavities along the extraction train indicates that the fixation process does not eliminate differences in the relative electrophoretic mobilities of erythrocytes of different ages.

     

Cellular Electrophoretic Mobility and the Mitotic Cycle

The electrophoretic mobility of RPMI No. 41 cells grown in suspension, parasynchronized by double thymidine blocking and cold shock, is reported. No. 41 cells have a higher electrophoretic mobility during the mitotic peak phase than at other times in the mitotic cycle. Treatment of parasynchronous cells by neuraminidase reduces the mobility to the same value irrespective of the stage of the cells in the mitotic cycle. The higher electrophoretic mobility of cells in mitotic peak phase is probably due to a higher surface charge density at this time, possibly caused by a higher concentration of ionized neuraminic acid carboxyl groups at the hydrodynamic shear layer. The mobility of nonsynchronous rapidly and slowly growing cells differs; neuraminidase reduces their mobility by proportionately similar amounts. The results suggest that the differences in mobility between rapidly and slowly growing cells cannot be accounted for exclusively by differences in the amount of neuraminic acid groups at the shear layer.     

Calculation of the Electrophoretic Mobility of a Particle Bearing Bound Polyelectrolyte Using the Nonlinear Poisson-Boltzmann Equation

A numerical method for determining the electrophoretic mobility of a polyelectrolyte-coated particle is presented. The particle surface is modeled as having a permeable layer of polyelectrolyte molecules anchored to its surface. Fluid flow within the polyelectrolyte layer is subject to Stokes drag arising from the polyelectrolyte segments. The method allows arbitrary distribution of polymer segments and charge density normal to the surface to be used. The hydrodynamic plane of shear may also be varied. The potential profile is determined by a numerical solution to the nonlinearized Poisson-Boltzmann equation. The potential profile is then used in a numerical solution to the Navier-Stokes equation to give the required mobility. The use of the nonlinearized Poisson-Boltzmann equation extends the results to higher charge density/lower ionic strength conditions than previous treatments. The surface potentials and mobilities for three limiting charge distributions are compared for both the linear and nonlinear treatments to delimit the range of validity of the linear treatment. The utility of the numerical, nonlinear treatment is demonstrated by an improved fit to the electrophoretic mobility of human erythrocytes as a function of ionic strength in the range 10 to 150 mM.     

Leukocyte inhibitory factor stimulates neutrophil-endothelial cell adhesion

We investigated the ability of the human lymphokine leukocyte inhibitory factor (LIF) to modulate neutrophil-endothelial cell (EC) adherence. EC were cultured from collagenase-treated human umbilical cord veins and grown in complete medium supplemented with EC growth factor. Adherence was measured as the percent of 51Cr-labeled neutrophils remaining adherent to the EC after gentle lavage. Polymorphonuclear neutrophils (PMN) were pretreated with LIF (0.5 to 8 U/ml), extensively washed, and allowed to interact with the EC monolayers. LIF was demonstrated to induce an increase in the capacity of PMN to bind EC in a dose-dependent fashion (from 30.9 +/- 2.1% adherence with control-treated PMN to 68.6 +/- 3.0% at 4 U LIF; p less than 0.001). In subsequent experiments we demonstrated that 10 min was a sufficient preincubation time for LIF to modulate the capacity of the PMN to adhere to EC. LIF has previously been observed to up-regulate expression of C receptor type 3 on PMN, a receptor which has been shown to be involved in PMN-EC binding. Exposure of PMN to anti-C receptor type 3 antibody before their incubation with LIF abrogated its effect as did inactivation of LIF by an esterase inhibitor. We also investigated the ability of LIF to stimulate EC to bind untreated PMN. EC were pretreated with LIF (0.25 to 4 U/ml), extensively washed, and adherence measured as before. LIF was shown to induce a dose-dependent increase in the capacity of the EC to bind PMN (from 28.8 +/- 3.1% for untreated EC to 91.1 +/- 4.0% at 4 U LIF; p less than 0.001). Modulation of EC function required a minimum of 30 min and was inhibited in the presence of cycloheximide or actinomycin D. Neither anti-TNF-alpha or -beta antibodies nor polymixin B abrogated the augmentation by LIF. However, anti-IL-1 antibody partially inhibited the stimulation of EC adhesiveness by LIF, suggesting the possible involvement of this cytokine. These studies provide further evidence that LIF may mediate an important pro-inflammatory role in vivo.     

Computation of the electrophoretic mobility of proteins.

A scheme is presented for computing the electrophoretic mobility of proteins in free solution, accounting for the details of the protein shape and charge distribution. The method of Teubner is implemented using a boundary integral formulation within which the velocity distribution, the equilibrium electrical potential around the molecule, and the potential distribution due to the applied field are solved for numerically using the boundary element method. Good agreement of the numerical result is obtained for spheres with the corresponding semi-analytical specialization of Henry's analysis. For protein systems, the method is applied to lysozyme and ribonuclease A. In both cases, the predicted mobility tensors are fairly isotropic, with the resulting scalar mobilities being significantly smaller than for spheres of equal volume and net charge. Comparisons with previously published experimental results for ribonuclease show agreement to be excellent in the presence of a net charge, but poorer at the point of zero charge. The approach may be useful for evaluating approximate methods for estimating protein electrophoretic mobilities and for using electrophoretic measurements to obtain insight into charge distributions on proteins.     

Leukocyte inhibitory factor activates human neutrophils and macrophages to release leukotriene B4 and thromboxanes

Recent evidence has proved that cytokines can stimulate the production of 5-lipoxygenase products. Leukotriene B4 (LTB4) is a major mediator of leukocyte activation in acute inflammatory reactions, which produce chemotaxis, lysosomal enzyme release, and cell aggregation. Leukocyte inhibitory factor (LIF) also causes biological responses related to inflammation, i.e., LIF directly induces specific granule secretion by polymorphonuclears (PMNs) and potentiates many formyl-methionyl-leucyl-phenylalanine (FMLPs) mediated responses. Since arachidonic acid products are important mediators of inflammation, we have studied the effects of LIF on the arachidonic acid cascade products LTB4 and thromboxane A2 (TxA2). Resuspended at a final concentration of greater than 95% polymorphonuclear PMNs were isolated and tested with some cytokines on the release of LTB4 and TxA2. Peripheral blood mononuclear cells were isolated and seeded in Petri dishes and incubated for 60 min. Adherent macrophages were used for the cytokine stimulation study. Both types of leukocytes were treated with LIF, interleukin 6 (IL 6), and granulocyte-monocyte colony stimulating factor (GM-CSF) at different concentrations, and test agents A23187 and FMLP. Radioimmunoassay for LTB4 and TxB2 was determined by the resulting supernatants. Treatment of PMNs and macrophages with LIF at different concentrations proved to generate significant increases in LTB4 and TxA2 production. This was compared with IL 6 and GM-CSF, which had no effects. In these experiments, TxA2 generations could not be attributed to platelet contamination of PMN suspensions. The quantity of platelet contamination was not sufficient to influence how much TxB2 was produced. The similarities of LIF to other arachidonate stimulating cytokines suggest a similar mode of action in producing hematologic changes typical of tissue injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the mechanism of binding of human lymphocyte products to guinea pig macrophages.

When lymphocytes from a majority of patients with cancer are incubated with encephalitogenic factor, a lymphocyte product is released that reduces the anodic electrophoretic mobilities of guinea pig macrophages and fixed, tanned sheep erythrocytes. Although these reactions are not specific for cancer, it is distinctly possible that in patients with cancer, products from stimulated lymphocytes are capable of altering the surfaces of the patients' own macrophages, thereby modifying the course of their disease. In this paper, we attempt to elucidate some mechanisms for the binding of lymphocyte products to macrophages, such as occurs in the macrophage electrophoretic mobility (MEM) test, since this may be of general interest. Binding of lymphocyte product to macrophages has been monitored by measurements of their electrophoretic mobilities and by electron microscopic determination of the density of binding of electron-dense, cationic colloidal iron hydroxide particles to their surfaces. The results show that the lymphocyte products reduce the net surface negativity of the macrophages by (coulombic) binding of this net positively charged material to sialic acids at the macrophage surface. Product-binding can be prevented by prior treatment of the macrophages with neuraminidase. It appears that only a minority of sialic acids are involved in the binding process, which occurs without demonstrable blocking of adjacent sialic acids or redistribution of such sites over the macrophage surface. Parallel experiments with fixed tanned erythrocytes also suggest that binding of lymphocyte product is not solely determined by surface sialic acids, although it cannot occur without them.     

On the mechanism of binding of human lymphocyte products to guinea pig macrophages

When lymphocytes from a majority of patients with cancer are incubated with encephalitogenic factor, a lymphocyte product is released that reduces the anodic electrophoretic mobilities of guinea pig macrophages and fixed, tanned sheep erythrocytes. Although these reactions are not specific for cancer, it is distinctly possible that in patients with cancer, products from stimulated lymphocytes are capable of altering the surfaces of the patients' own macrophages, thereby modifying the course of their disease. In this paper, we attempt to elucidate some mechanisms for the binding of lymphocyte products to macrophages, such as occurs in the macrophage electrophoretic mobility (MEM) test, since this may be of general interest. Binding of lymphocyte product to macrophages has been monitored by measurements of their electrophoretic mobilities and by electron microscopic determination of the density of binding of electron-dense, cationic colloidal iron hydroxide particles to their surfaces. The results show that the lymphocyte products reduce the net surface negativity of the macrophages by (coulombic) binding of this net positively charged material to sialic acids at the macrophage surface. Product-binding can be prevented by prior treatment of the macrophages with neuraminidase. It appears that only a minority of sialic acids are involved in the binding process, which occurs without demonstrable blocking of adjacent sialic acids or redistribution of such sites over the macrophage surface. Parallel experiments with fixed tanned erythrocytes also suggest that binding of lymphocyte products is not solely determined by surface sialic acids, although it cannot occur without them.