Hansuli Keller

The phosphatidylinositol 3-kinase inhibitor wortmannin markedly reduces chemotactic peptide-induced locomotion and increases in cytoskeletal actin in human neutrophils

To define a possible role of the enzyme phosphatidylinositol 3-kinase (PI 3-kinase) in motile functions of neutrophils, we have used a potent inhibitor of this enzyme, [1S-(1alpha,6b alpha,9a beta,11alpha,11bbeta)]-1-(acetyloxy)-1,6b,7,8,9a,10,11 ,11b-octahydro-1-(methoxymethyl)-9a,11b-dimethyl-3H-furo[4,3,2-de]indeno [4,5-h]-2-benzopyran-3,6,9-trione (wortmannin). Wortmannin markedly attenuated chemotactic peptide-induced development of polarity, locomotion and increases in cytoskeletal actin and alpha-actinin in human neutrophils at low, nM, concentrations (ED50 = 4-40 nM; 0.4-3 pmol/10(6) cells). The increase in cytoskeletal actin induced by phorbol-12-myristate-13-acetate in contrast was not affected by wortmannin (18 pmol/10[6] cells). Moreover, the increase in total F-actin induced by an incubation for 1 min with chemotactic peptide was much less sensitive to wortmannin than increases in cytoskeletal actin; 80 pmol/10(6) cells were necessary for half-maximal inhibition. Wortmannin thus appears to primarily affect F-actin organization, rather than polymerization. Inhibition of development of polarity by wortmannin correlated with inhibition of production of phosphatidylinositol 3,4,5-trisphosphate. According to our findings, activation of a wortmannin-sensitive target, very likely PI 3-kinase, is required for optimal chemotactic peptide-induced neutrophil motility.

Effects of colchicine, vinblastine and nocodazole on polarity, motility, chemotaxis and cAMP levels of human polymorphonuclear leukocytes☆

We present evidence for intrinsic polymorphonuclear leukocyte (PMN) polarity manifested in presence of microtubule-disrupting drugs. Polarization in response to colchicine correlated with the known dose-dependent effects of this drug on microtubule disassembly. The response to 10(-5) M colchicine, 10(-5) M vinblastine and 10(-6) M nocodazole was associated with stimulated motility and random locomotion. Responses elicited by microtubule-disrupting drugs differed from f-Met-Leu-Phe (fMLP)-induced polarization by functional and morphological criteria. Polarization, motility and orthokinesis responses were much weaker. Furthermore, ruffling was almost absent in PMNs polarized in response to colchicine, vinblastine or nocodazole. The response was inhibited by cytochalasin B, indicating that it is microfilament-dependent. We suggest that microtubule-disrupting drugs induce motility via structural changes in the cytoskeleton which act as signals for the motor apparatus. The intrinsic polarity manifested in the presence of microtubule-disrupting drugs could be reversed by an extracellular chemotactic gradient. Stimulated locomotion and motility in response to microtubule-disrupting drugs was only observed with initially spherical PMNs but not with initially motile cells. The findings provide an explanation for the numerous conflicting statements on the chemokinetic activities of these drugs. The role of cAMP in stimulated polarization and motility has been studied. Colchicine, vinblastine and nocodazole elicited a transient elevation of cAMP levels within 1 min of stimulation. cAMP elevation and stimulated motility were not quantitatively correlated.

Re-assessment of Boyden's technique for measuring chemotaxis.

Abstract Boydens one-filter technique for measuring chemotaxis of polymorphonuclear leucocytes is based on the assumption that all cells which have migrated through the membrane remain at its lower surface where they are counted. Since a variable proportion of cells on the bottom side of the membrane becomes detached, the one-filter system is not a reliable quantitative method. A modified procedure eliminating this loss of cells by the use of two filters, the lower one being impermeable for leucocytes, is described.

Studies on the regulation of the neutrophil chemotactic response using a rapid and reliable method for measuring random migration and chemotaxis of neutrophil granulocytes

AbstractsAn economic and sensitive test system for measuring random and directional migration of human neutrophils is described. The technique, based on a modified Boyden chamber equipped with a two-filter system, permits a substantial reduction of both incubation time and sample volume. The influence of various technical factors such as the neutrophil concentration in the cell suspension, the incubation time of the chambers, the test concentration of activated plasma or serum, the presence of heparin, and the procedure for separating neutrophils from human peripheral blood, was investigated. Standardized procedures for measuring and reporting neutrophil chemotaxis are proposed.The method has been used to study the significance of factors regulating neutrophil migration such as cytotaxin inactivators and neutrophil immobilizing factors (NIF). Activity of cytotaxin inactivators as assessed in undiluted serum or plasma at pH 7.4, 6.0 or 4.0 was very low. In contrast, potent neutrophil immobilizing activity was found in human serum or diluted plasma. These factors which inhibit migration were accordingly termed neutrophil immobilizing factors of plasma (NIF-P) and neutrophil immobilizing factor of serum (NIF-S). These factors are heat-stable, non-dialysable and of high molecular weight.

The effect of endotoxin and tumour necrosis factor on erythrocyte and leucocyte deformability in vitro

Summary Microcirculatory disorders are a common finding in sepsis. We have analysed the influence of two factors released in sepsis, endotoxin and tumour necrosis factor (TNF), on rheological properties of blood cells. The deformability of mixed cell suspensions, isolated erythrocytes, mononuclear cells, or polymorphonuclear leucocytes exposed to endotoxin and TNF in vitro was assessed by filtration through pores of different sizes. Mixed blood cell suspensions showed an increase in cell rigidity when incubated with 100 ng/ml endotoxin. The filtration resistance of isolated erythrocytes, mononuclear or polymorphonuclear leucocytes was not affected by endotoxin. Incubation with TNF in physiological concentrations increased the rigidity of mixed blood cells and of isolated polymorphonuclear leucocytes in a dose‐ and time‐dependent manner, while erythrocytes and mononuclear leucocytes remained unaffected. Polymorphonuclear cells showed decreased deformability associated with shape changes (polarized and non‐polar cells with surface protrusions and a shift of F‐actin into protrusions). The decrease in deformability was reversed by cytochalasin B or xanthin derivatives such as pentoxifylline. We conclude that TNF decreases the passive deformability of polymorphonuclear leucocytes, which may affect the microcirculation in sepsis. The reversibility with xanthin derivatives may represent a new therapeutic approach for the high morbidity and mortality in sepsis.

Protrusive activity quantitatively determines the rate and direction of cell locomotion.

Locomoting blebbing cells have been used as a model to obtain novel insight into the mechanisms of cell locomotion. We tested the hypothesis that locomotion can be due to progressive one-sided protrusion of cellular volume into pseudopods. The hypothesis is supported by the finding that the rate and direction of locomotion of individual Walker carcinosarcoma cells can be predicted by sequential measurement of protrusive activity. Protrusive activity at the front is closely associated with forward movement of the rear part of the cell. During bleb formation the cell membrane of Walker carcinosarcoma cells is pushed forward faster (1.2-4.1 microns/sec) than known rates of actin elongation.

The chemokinetic effect of serum albumin.

Experiments performed by means of time lapse cinematography or the filter technique show that human serum albumin has marked chemokinetic effects on neutrophil cultured in Geys solution. The average speed of the cells, as well as the proportion of neutrophils showing locomotion, is increased. Enhanced locomotion correlates with decreased attachment to the substratum as determined by morphological and functional criteria.

Suppression of bleb formation, locomotion, and polarity of Walker carcinosarcoma cells by hypertonic media correlates with cell volume reduction but not with changes in the F-actin content.

The putative role of cellular or solvent volume in protrusive activity and locomotion has been investigated in blebbing Walker carcinosarcoma cells using hypertonic media. Blebbing, locomotion, and cell polarity are completely suppressed by 0.2 M sorbitol. The response occurs in two steps. In a first step, i.e. within 10 sec after the addition of sorbitol, blebbing and locomotion are inhibited and this is associated with an average cell volume reduction by 17% (corresponding to a reduction in solvent volume by 38%). It clearly precedes suppression of cell polarity (pre-existing protrusions, tail) occurring in a second step within 5 to 10 min after addition of sorbitol without additional reduction in the cell or solvent volume. The relative amount of F-actin does not correlate with the decrease in cell volume, suppression of blebbing, locomotion, and cell polarity. A significant decrease in the relative amount of F-actin is found only at volume reductions which are higher than those required to completely suppress blebbing, locomotion, and cell polarity. F-actin staining occurs preferentially along the cell membrane in isotonic as well as in hypertonic media. The results are best compatible with the hypothesis that hydrostatic pressure rather than actin polymerization at the front is the direct force driving the membrane forward during bleb formation. Cells with lamellipodia show a similar response to hypertonic media, suggesting that basically similar mechanisms may operate in both forms of protrusions.

Nonspecific esterase in human lymphocytes.

A substantial proportion of human peripheral lymphocytes exhibits esterase activity as demonstrated by cytochemical techniques using hexazotized pararosanilin as coupling agent and different alpha-naphthyl compounds, in particular alpha-naphthyl-acetate, as substrates. Esterase-positive lymphocytes showed one or several dots of the reaction product. Plasma cells and macrophages also exhibited marked esterase activity but with diffuse distribution of the reaction product throughout the cytoplasm. The substrate specificity, the time course and the pH optimum of the cytochemical reaction were determined. The results indicate that these lymphocyte esterases are identical with acid lipases. The number of esterase-positive cells reaches a plateau well below 100% (85% for blood lymphocytes) even with optimal staining procedures. The relation between esterase activity and other so-called lymphocyte markers such as sIg and the capacity to form spontaneous rosettes with sheep red blood cells was investigated in peripheral human blood lymphocytes. A fair correlation between rosette formation and esterase activity was found and most of the esterase-positive cells were sIg-negative. However, the fit was never complete. The results suggest that esterase activity is largely characteristic for small peripheral T cells. Almost all blast cells formed following stimulation with Con A or PHA were esterase-positive. Histochemical studies showed that the large majority of the thymocytes in the cortex lacked esterase activity. In lymph nodes, a low proportion of esterase-positive cells was found in the germinal centers, whereas most lymphocytes in the paracortical are were esterase-positive.

The filter technique for measuring leucocyte locomotion in vitro. Comparison of three modifications

Three forms of filter technique for measuring random and directional locomotion of leucocytes have been compared: (1) the conventional one filter technique of Boyden (lower surface count method); (2) the two filter system with a lower cell-impermeable filter designed to count the cells at the underside of the upper filter as well as those on the lower filter (two filter count method); and (3) two filter systems counting only cells associated with the lower filter (lower filter count method). In some instances all three methods produce qualitatively similar results. In others totally different results are reproducibly obtained with identical cell preparations, media and attractants. Compared to the two filter count method, the lower surface count method and the lower filter count method are not sufficiently reliable. The discrepancies are partly due to errors in measuring the response. They are caused by variable cell adhesion to the filters resulting in a varying distribution of cells between the upper and lower filter and/or detachment of neutrophils from the upper filter. Some of the discrepancies are not due to errors in assessing the response, but to differences in gradient formation and drift of chemokinetic and chemotactic materials from one compartment to the other.