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Which method for the assessment of spreading of polymorphonuclear leukocytes?
Journal of Immunological Methods, 135 (1990) 281-284
281
Elsevier JIM05782
Letter to the editors
Which method for the assessment of spreading of polymorphonuclear leukocytes? * Susan D. Rogers and Leon P. Bignold Department of Pathology, University of Adelaide, South Australia, Australia (Received 1 June 1990, revised received 7 September 1990, accepted 10 September 1990)
Dear Editors, Spreading of cells is an active process by which flattening of the bodies of living cells occurs on solid substrata. Spreading is associated with adhesiveness and, in those cells which move with pseudopods, with locomotion. A degree of spreading is an essential preliminary step to the movement of amoeboid cells on surfaces (Vasiliev, 1982, 1985). With respect to polymorphonuclear leukocytes (PMN), the relationships between spreading, adhesion and locomotion appear complex. While a degree of spreading is necessary for locomotion, more pronounced spreading has been reported to be associated with hyperadhesiveness and decreased motility (Keller et al., 1979). Spreading is therefore a factor to consider in relation to techniques for measuring random motility and chemotaxis of neutrophils (see Wilkinson, 1982; Bignold, 1988). In addition to its possible relationship to locomotion, spreading has been used as an indicator of function of PMN and has been found to be affected by plasma proteins (Keller et al., 1979; Valerius, 1983), N-formyl peptide (Keller et al., 1983), vitamin K (Gallin et al., 1982), taxol (Roberts et al., 1982), interleukin-1 (Sullivan et al., 1989), antibiotics (Burgaleta et al., 1985), con-
S.D. Rogers, Department of Pathology, University of Adelaide,G.P.O. Box 498, Adelaide, S.A. 5001, Australia. * This workwas supported by the AustralianResearchCouncil. Correspondence to:
genital deficiency of membrane components (Crowley et al., 1980; Buescher et al., 1985) and lipid-coating of the solid substratum (Hafeman et al., 1982). In these previous reports, various techniques for estimating spreading have been used. Visual assessments of spreading were made by Keller et al. (1979), Hafeman et al. (1982), Kruskal et al. (1986) and Wallis et al. (1986). On the other hand, quantitative techniques for assessing spreading in a population of ceils have included counting the proportion (%) of adherent cells that had a spread appearance (Crowley et al., 1980; Valerius, 1983). Other quantitative techniques have involved estimating the mean areas of cells either by measuring both the cell length and cell breadth using a microscope-eyepiece microscale (graticule) and calculating area using the formula for the area of an ellipse (Burgaleta et al., 1985) or by manually tracing the periphery of individual cells and subsequently measuring cell area with a computerised technique (Gallin et al., 1982; Roberts et al, 1982). Keller et al. (1983) assessed spreading both by counting the proportion of cells spread out on a glass substratum and by measuring their average contact area using the MOP/AMO1 morphometry system from traced outlines of the ceils seen by reflection-contrast microscopy. Studies in this laboratory of the effects of various plasma proteins on chemotaxis to formyl peptide using a novel 'sparse=pore' polycarbonate membrane in the Boyden chamber (Bignold, 1987, 1988) have shown that gamma globulins cause spreading of cells on the membrane in excess of that occurring in Hanks' solution alone (Bignold
0022-1759/90/$03.50 © 1990 ElsevierSciencePublishersB.V. (BiomedicalDivision)
282 et al., 1990). When assessed as the proportions of cells spread, there was no difference between the spreading in the highest concentration (0.5%) of g a m m a globulin and spreading in a lower concentration (0.05%). However, when assessed visually as the degree of spreading, the effect was greater in 0.5% than in other concentrations of g a m m a globulin (Fig. 1). To investigate further the possibility that detectable spreading might vary according to the method of assessment, we undertook a study of spreading of P M N on 'sparse-pore' polycarbonate m e m b r a n e (Nuclepore, California) (see Bignold, 1987) in g a m m a globulin solutions, measuring first, the proportions of cells spread and second, the area of cells measured both with a microscope eyepiece graticule and with a computerised electronic writing tablet. Neutrophil leukocytes were separated from whole blood by the one-step Hypaque-Ficoll method of Ferrante and Thong (1978; see also Bignold and Ferrante, 1987), washed in Hanks' solution and then resuspended in solutions of 0.005%, 0.05% and 0.5% g a m m a globulin (Sigma, cat. no. H-3375) in Hanks' solution buffered with 20 m M Hepes (Sigma, cat. no. G-4386). These neutrophil preparations (2000 cells in 40/xl) were pipetted onto pieces of 'sparse-pore' polycarbonate membrane (Bignold, 1987, 1988) which had been placed on the bottom of humidified wells of Linbro tissue culture plates. After 20 min
1O0
300 J
75
225
50
150
B
spread Area by gtaticule
% celts
25 I 0
t Hanks
i 0.005%
75
~
Area by tablet i 0.05%
0
t Q.5%
% gamma globulin (~/v)
Fig. 2. Comparison of spreading of polymorphonuclear leukocytes on 'sparse-pore' polycarbonate (Nuclepore) filtration membranes assessed as proportions of cells spread (mean + SE) and as cell areas (mean +_SE) by an eyepiece graticule method and by a computerised electronic tablet technique. Asterisks indicate values which differ significantly (paired t test, P < 0.05) from the value obtained in Hanks' solution by the same technique.
incubation at 37 o C, the cells were fixed by adding 4% phosphate-buffered formaldehyde to each well. The pieces of m e m b r a n e were then rinsed, stained with haematoxylin (1 h) and air dried before being mounted between a slide and coverslip and examined by bright-field light microscopy (Fig. 1). The same m e m b r a n e pieces were used for each method of assessment of spreading. In the visual assessment of PMN, the cells were classed as either spread or unspread (mainly polar with some spherical cells) and the results expressed as the proportion (%) of attached cells
e
Fig. 1. Photomicrographs of PMN attached to polycarbonate membrane in 0.05% (a) and 0.5% (b) gamma globulin in Hanks' solution. Although the proportions of cells spread are the same, the degree of spreading appears to be greater in 0.5% gamma globulin (stained with haematoxylin,bright field illumination, × 400).
283
spread out on the membrane (Fig. 2). In the method involving the graticule microscale, the dimensions of cells were measured with an Olympus Vanox microscope and a 40 × objective. Spreading was calculated according to the area of an ellipse [(~r × greatest length of cell × greatest breadth at right angles to greatest length) - 4]. In the computerised method, the same axes were measured with a Zeiss camera lucida device and microscope together with an electronic writing tablet (Houston Instruments Trugrid, Model 1011, Houston, Texas) using a Macintosh SE computer (Apple Computer, California) and the MacMeasure program (Hook and Rasband, 1987). The areas of the cells were calculated as before. The computerised tablet method was approximately ten times faster to use than the graticule eyepiece method, and also caused less fatigue to the operator. At least 100 cells per treatment were assessed and the areas averaged. Spreading in each gamma globulin concentration was compared with the spreading observed in Hanks' solution alone (paired two tailed t test). The results of the study of the effects of gamma globulin at concentrations of 0.005%, 0.05% and 0.5% (w/v) compared to Hank's solution alone are shown in Fig. 2. The proportions (means ___ standard error) of adhering PMN which had spread on polycarbonate membrane were 74.5 + 6.8%, 92.0 _ 2.9% and 90.7 + 5.2% respectively, compared with 47.6 + 5.2% in Hanks' solution alone. PMN spreading expressed as the mean area + SE of an ellipse (/~m2) using the graticule method was 125.5 + 6.7 # m 2, 137.4 + 7.0 # m 2 and 240.0 -/- 10.2 # m a respectively; in Hanks' solution alone the value was 107.8 + 7.5/~m 2. PMN spreading as measured by the electronic tablet method gave values which were similar to those obtained using the graticule method, being 139.3 _+ 2.5/zm 2, 164.2 + 2.5 # m 2 and 253.8 _ 2.8/~m 2 respectively, and 116.9 + 3.1 /~m2 in Hanks' solution alone. Statistical analysis revealed that spreading expressed as the proportion of cells spread was significantly higher in concentrations of 0.05 % w / v and 0.5% w / v gamma globulin than that in Hanks' solution alone ( P < 0.05). In contrast, spreading expressed as the mean area of cells by the graticule or writing tablet methods was increased significantly ( P < 0.05) only in the highest gamma
globulin concentration (0.5%) compared to the spreading in Hanks' solution alone. No differences were found between the results obtained with the eyepiece graticule microscale and those with the electronic writing tablet methods. These results indicate that PMN spreading measured as the proportion of adherent cells showing spread morphology differs with respect to the concentration of gamma globulin from spreading calculated as the mean area of the adherent cells. This difference appears to be explicable in terms of the aspects of cell morphology used for the assay. Measurements of the proportion of cells spread on the membrane are based on changes in the appearance of the cells from spherical or polar (both counted as unspread) to spread, irrespective of the degree of spreading. This change occurs in the range between 0.005% gamma globulin and 0.05% gamma globulin. In contrast, PMN spreading measured as the mean area of cells gives an estimate which includes the degree of spreading. At the lower concentrations, mean area does not change sufficiently to be statistically significant, i.e., the simple conversion of polar cells to spread cells involves little change in area. However, at higher concentrations of gamma globulin, the degree of spreading and hence the mean area of all cells increases accordingly. From the findings in this study, investigators should be aware that the method chosen for the measurement of P M N spreading can affect the results of the study.
References Bignold, L.P. (1987) A novel polycarbonate (Nnclepore) membrane demonstrates chemotaxis unaffected by chemokinesis in the Boyden chamber. J. Immunol. Methods 105, 275. Bignold, L.P. (1988) Measurement of chemotaxis of polymorphonuclear leukocytes in vitro. The problems of control of gradients of chemotactic factors, of the control of the cells and of the separation of chemotaxis from chemokinesis (review article) J. Immunol. Methods 108, 1. Bignold, L.P. and Ferrante, A. (1987) Mechanism of separation of polymorphonuclear leukocytes from whole blood by the one-step Hypaque-Ficoll method. J. Immunol. Methods 96, 29. Bignold, L.P., Rogers, S.D. and Harkin, D.G. (1990) Effects of plasma proteins on adhesion, spreading, polarisation in suspension, random motility and chemotaxis of neutrophil
284 leukocytes on polycarbonate (Nuclepore) filtration membrane. Eur. J. Cell Biol., in press. Buescher, E.S., Gaither, T., Nath, J. and GaUin, I. (1985) Abnormal adherence-related functions of neutrophils, monocytes, and Epstein-Barr virus-transformed B cells in a patient with C3bi receptor deficiency. Blood 65, 1382. Burgaleta, C., Sofia, H. and Martinez, F. (1985) Stimulation effects of carbenicillin and ticarcillin on the adhesion and spreading of human polymorphonuclear leukocytes. J. Antimicrob. Chemother. 15, 729. Crowley, C.A., Curnutte, J.T., Rosin, R.E., Andre-Schwartz, J., Gallin, J.I., Klempner, M., Snyderman, R., Southwick, F.S., Stossel, T.P. and Babior, B.M. (1980) An inherited abnormality of neutrophil adhesion. Its genetic transmission and its association with a missing protein. New Engl. J. Med. 302, 1163. Ferrante, A. and Thong, Y.H. (1978) A rapid one-step procedure for purification of mononuclear and polymorphonuclear leukocytes from human blood using a modification of the Hypaque-Ficoll technique. J. Immunol. Methods 24, 389. Gallin, J.l., Seligmarm, B.E., Cramer, E.B., Schiffmann, E. and Fletcher, M.P. (1982) Effects of vitamin K on human neutrophil function. J. Immunol. 128, 1399. Hafeman, D.G., Smith, L.M., Fearon, D.T. and McConnell, H.M. (1982) Lipid monolayer coated solid surfaces do not perturb the lateral motion and distribution of C3b receptors on neutrophils. J. Cell Biol. 94, 224. Hook, G.R. and Rasband, W. (1987) Macmeasure: a low-cost, easy to operate quantitative morphometrics system for the Macintosh computer. In: G.W. Bailey (Ed.), Proceedings of the 45th annual meeting of the Electron Microscopy Society of America. San Francisco Press, San Francisco, CA, p. 920. Keller, H.-U., Barandun, S., Kistler, P. and Ploem, J.S. (1979)
Locomotion and adhesion of neutrophil granulocytes. Effects of albumin, fibrinogen and gamma globulins studied by refection contrast microscopy. Exp. Cell Res. 122, 351. Keller, H.-U., Zimmermann, A. and Cottier, H. (1983) Crawling-like movements, adhesion to solid substrata and chemokinesis of neutrophil granulocytes. J. Cell Sci. 64, 89. Kruskal, B.A., Shak, S. and Maxfield, F.R. (1986) Spreading of human neutrophils is immediately preceded by a large increase in cytoplasmic free calcium. Proc. Natl. Acad. Sci. U.S.A. 83, 2919. Roberts, R.L., Nath, J., Friedman, M.M. and Gallin, J.l. (1982) Effects of taxol on human neutrophils. J. Imrnunol. 129, 2134. Sullivan, G.W., Carper, H.T., Sullivan, J.A., Murata, T. and Mandell, G.L. (1989) Both recombinant interleukin-1 (beta) and purified human monocyte interleukin-1 prime human neutrophils for increased oxidative activity and promote neutrophil spreading. J. Leukocyte Biol. 45, 389. Valerius, N.H. (1983) Chemotaxis, spreading and oxidative metabolism of neutrophils: influence of albumin in vitro. Acta Pathol. Microbiol. Scand. Sect. C 91, 43. Vasiliev, J.M. (1982) Spreading and locomotion of tissue cells: factors controlling the distribution of pseudopodia. Phil. Trans. R. Soc. Lond. B299, 159. Vasiliev, J.M. (1985) Spreading of non-transformed and transformed cells. Biochima Biophysica Acta 780: 21. Wallis, W.J., Hickstein, D.D., Schwartz, B.R., June, C.H., Ochs, H.D., Beatty, P.G., Klebanoff, S.J. and Harlan, J.M. (1986) Monoclonal antibody-defined functional epitopes on the adhesion-promoting glycoprotein complex (CDw18) of human neutrophils. Blood 67, 1007. Wilkinson, P.C. (1982) Chemotaxis in inflammation. 2nd edn, Churchill-Livingstone, Edinburgh.
281
Elsevier JIM05782
Letter to the editors
Which method for the assessment of spreading of polymorphonuclear leukocytes? * Susan D. Rogers and Leon P. Bignold Department of Pathology, University of Adelaide, South Australia, Australia (Received 1 June 1990, revised received 7 September 1990, accepted 10 September 1990)
Dear Editors, Spreading of cells is an active process by which flattening of the bodies of living cells occurs on solid substrata. Spreading is associated with adhesiveness and, in those cells which move with pseudopods, with locomotion. A degree of spreading is an essential preliminary step to the movement of amoeboid cells on surfaces (Vasiliev, 1982, 1985). With respect to polymorphonuclear leukocytes (PMN), the relationships between spreading, adhesion and locomotion appear complex. While a degree of spreading is necessary for locomotion, more pronounced spreading has been reported to be associated with hyperadhesiveness and decreased motility (Keller et al., 1979). Spreading is therefore a factor to consider in relation to techniques for measuring random motility and chemotaxis of neutrophils (see Wilkinson, 1982; Bignold, 1988). In addition to its possible relationship to locomotion, spreading has been used as an indicator of function of PMN and has been found to be affected by plasma proteins (Keller et al., 1979; Valerius, 1983), N-formyl peptide (Keller et al., 1983), vitamin K (Gallin et al., 1982), taxol (Roberts et al., 1982), interleukin-1 (Sullivan et al., 1989), antibiotics (Burgaleta et al., 1985), con-
S.D. Rogers, Department of Pathology, University of Adelaide,G.P.O. Box 498, Adelaide, S.A. 5001, Australia. * This workwas supported by the AustralianResearchCouncil. Correspondence to:
genital deficiency of membrane components (Crowley et al., 1980; Buescher et al., 1985) and lipid-coating of the solid substratum (Hafeman et al., 1982). In these previous reports, various techniques for estimating spreading have been used. Visual assessments of spreading were made by Keller et al. (1979), Hafeman et al. (1982), Kruskal et al. (1986) and Wallis et al. (1986). On the other hand, quantitative techniques for assessing spreading in a population of ceils have included counting the proportion (%) of adherent cells that had a spread appearance (Crowley et al., 1980; Valerius, 1983). Other quantitative techniques have involved estimating the mean areas of cells either by measuring both the cell length and cell breadth using a microscope-eyepiece microscale (graticule) and calculating area using the formula for the area of an ellipse (Burgaleta et al., 1985) or by manually tracing the periphery of individual cells and subsequently measuring cell area with a computerised technique (Gallin et al., 1982; Roberts et al, 1982). Keller et al. (1983) assessed spreading both by counting the proportion of cells spread out on a glass substratum and by measuring their average contact area using the MOP/AMO1 morphometry system from traced outlines of the ceils seen by reflection-contrast microscopy. Studies in this laboratory of the effects of various plasma proteins on chemotaxis to formyl peptide using a novel 'sparse=pore' polycarbonate membrane in the Boyden chamber (Bignold, 1987, 1988) have shown that gamma globulins cause spreading of cells on the membrane in excess of that occurring in Hanks' solution alone (Bignold
0022-1759/90/$03.50 © 1990 ElsevierSciencePublishersB.V. (BiomedicalDivision)
282 et al., 1990). When assessed as the proportions of cells spread, there was no difference between the spreading in the highest concentration (0.5%) of g a m m a globulin and spreading in a lower concentration (0.05%). However, when assessed visually as the degree of spreading, the effect was greater in 0.5% than in other concentrations of g a m m a globulin (Fig. 1). To investigate further the possibility that detectable spreading might vary according to the method of assessment, we undertook a study of spreading of P M N on 'sparse-pore' polycarbonate m e m b r a n e (Nuclepore, California) (see Bignold, 1987) in g a m m a globulin solutions, measuring first, the proportions of cells spread and second, the area of cells measured both with a microscope eyepiece graticule and with a computerised electronic writing tablet. Neutrophil leukocytes were separated from whole blood by the one-step Hypaque-Ficoll method of Ferrante and Thong (1978; see also Bignold and Ferrante, 1987), washed in Hanks' solution and then resuspended in solutions of 0.005%, 0.05% and 0.5% g a m m a globulin (Sigma, cat. no. H-3375) in Hanks' solution buffered with 20 m M Hepes (Sigma, cat. no. G-4386). These neutrophil preparations (2000 cells in 40/xl) were pipetted onto pieces of 'sparse-pore' polycarbonate membrane (Bignold, 1987, 1988) which had been placed on the bottom of humidified wells of Linbro tissue culture plates. After 20 min
1O0
300 J
75
225
50
150
B
spread Area by gtaticule
% celts
25 I 0
t Hanks
i 0.005%
75
~
Area by tablet i 0.05%
0
t Q.5%
% gamma globulin (~/v)
Fig. 2. Comparison of spreading of polymorphonuclear leukocytes on 'sparse-pore' polycarbonate (Nuclepore) filtration membranes assessed as proportions of cells spread (mean + SE) and as cell areas (mean +_SE) by an eyepiece graticule method and by a computerised electronic tablet technique. Asterisks indicate values which differ significantly (paired t test, P < 0.05) from the value obtained in Hanks' solution by the same technique.
incubation at 37 o C, the cells were fixed by adding 4% phosphate-buffered formaldehyde to each well. The pieces of m e m b r a n e were then rinsed, stained with haematoxylin (1 h) and air dried before being mounted between a slide and coverslip and examined by bright-field light microscopy (Fig. 1). The same m e m b r a n e pieces were used for each method of assessment of spreading. In the visual assessment of PMN, the cells were classed as either spread or unspread (mainly polar with some spherical cells) and the results expressed as the proportion (%) of attached cells
e
Fig. 1. Photomicrographs of PMN attached to polycarbonate membrane in 0.05% (a) and 0.5% (b) gamma globulin in Hanks' solution. Although the proportions of cells spread are the same, the degree of spreading appears to be greater in 0.5% gamma globulin (stained with haematoxylin,bright field illumination, × 400).
283
spread out on the membrane (Fig. 2). In the method involving the graticule microscale, the dimensions of cells were measured with an Olympus Vanox microscope and a 40 × objective. Spreading was calculated according to the area of an ellipse [(~r × greatest length of cell × greatest breadth at right angles to greatest length) - 4]. In the computerised method, the same axes were measured with a Zeiss camera lucida device and microscope together with an electronic writing tablet (Houston Instruments Trugrid, Model 1011, Houston, Texas) using a Macintosh SE computer (Apple Computer, California) and the MacMeasure program (Hook and Rasband, 1987). The areas of the cells were calculated as before. The computerised tablet method was approximately ten times faster to use than the graticule eyepiece method, and also caused less fatigue to the operator. At least 100 cells per treatment were assessed and the areas averaged. Spreading in each gamma globulin concentration was compared with the spreading observed in Hanks' solution alone (paired two tailed t test). The results of the study of the effects of gamma globulin at concentrations of 0.005%, 0.05% and 0.5% (w/v) compared to Hank's solution alone are shown in Fig. 2. The proportions (means ___ standard error) of adhering PMN which had spread on polycarbonate membrane were 74.5 + 6.8%, 92.0 _ 2.9% and 90.7 + 5.2% respectively, compared with 47.6 + 5.2% in Hanks' solution alone. PMN spreading expressed as the mean area + SE of an ellipse (/~m2) using the graticule method was 125.5 + 6.7 # m 2, 137.4 + 7.0 # m 2 and 240.0 -/- 10.2 # m a respectively; in Hanks' solution alone the value was 107.8 + 7.5/~m 2. PMN spreading as measured by the electronic tablet method gave values which were similar to those obtained using the graticule method, being 139.3 _+ 2.5/zm 2, 164.2 + 2.5 # m 2 and 253.8 _ 2.8/~m 2 respectively, and 116.9 + 3.1 /~m2 in Hanks' solution alone. Statistical analysis revealed that spreading expressed as the proportion of cells spread was significantly higher in concentrations of 0.05 % w / v and 0.5% w / v gamma globulin than that in Hanks' solution alone ( P < 0.05). In contrast, spreading expressed as the mean area of cells by the graticule or writing tablet methods was increased significantly ( P < 0.05) only in the highest gamma
globulin concentration (0.5%) compared to the spreading in Hanks' solution alone. No differences were found between the results obtained with the eyepiece graticule microscale and those with the electronic writing tablet methods. These results indicate that PMN spreading measured as the proportion of adherent cells showing spread morphology differs with respect to the concentration of gamma globulin from spreading calculated as the mean area of the adherent cells. This difference appears to be explicable in terms of the aspects of cell morphology used for the assay. Measurements of the proportion of cells spread on the membrane are based on changes in the appearance of the cells from spherical or polar (both counted as unspread) to spread, irrespective of the degree of spreading. This change occurs in the range between 0.005% gamma globulin and 0.05% gamma globulin. In contrast, PMN spreading measured as the mean area of cells gives an estimate which includes the degree of spreading. At the lower concentrations, mean area does not change sufficiently to be statistically significant, i.e., the simple conversion of polar cells to spread cells involves little change in area. However, at higher concentrations of gamma globulin, the degree of spreading and hence the mean area of all cells increases accordingly. From the findings in this study, investigators should be aware that the method chosen for the measurement of P M N spreading can affect the results of the study.
References Bignold, L.P. (1987) A novel polycarbonate (Nnclepore) membrane demonstrates chemotaxis unaffected by chemokinesis in the Boyden chamber. J. Immunol. Methods 105, 275. Bignold, L.P. (1988) Measurement of chemotaxis of polymorphonuclear leukocytes in vitro. The problems of control of gradients of chemotactic factors, of the control of the cells and of the separation of chemotaxis from chemokinesis (review article) J. Immunol. Methods 108, 1. Bignold, L.P. and Ferrante, A. (1987) Mechanism of separation of polymorphonuclear leukocytes from whole blood by the one-step Hypaque-Ficoll method. J. Immunol. Methods 96, 29. Bignold, L.P., Rogers, S.D. and Harkin, D.G. (1990) Effects of plasma proteins on adhesion, spreading, polarisation in suspension, random motility and chemotaxis of neutrophil
284 leukocytes on polycarbonate (Nuclepore) filtration membrane. Eur. J. Cell Biol., in press. Buescher, E.S., Gaither, T., Nath, J. and GaUin, I. (1985) Abnormal adherence-related functions of neutrophils, monocytes, and Epstein-Barr virus-transformed B cells in a patient with C3bi receptor deficiency. Blood 65, 1382. Burgaleta, C., Sofia, H. and Martinez, F. (1985) Stimulation effects of carbenicillin and ticarcillin on the adhesion and spreading of human polymorphonuclear leukocytes. J. Antimicrob. Chemother. 15, 729. Crowley, C.A., Curnutte, J.T., Rosin, R.E., Andre-Schwartz, J., Gallin, J.I., Klempner, M., Snyderman, R., Southwick, F.S., Stossel, T.P. and Babior, B.M. (1980) An inherited abnormality of neutrophil adhesion. Its genetic transmission and its association with a missing protein. New Engl. J. Med. 302, 1163. Ferrante, A. and Thong, Y.H. (1978) A rapid one-step procedure for purification of mononuclear and polymorphonuclear leukocytes from human blood using a modification of the Hypaque-Ficoll technique. J. Immunol. Methods 24, 389. Gallin, J.l., Seligmarm, B.E., Cramer, E.B., Schiffmann, E. and Fletcher, M.P. (1982) Effects of vitamin K on human neutrophil function. J. Immunol. 128, 1399. Hafeman, D.G., Smith, L.M., Fearon, D.T. and McConnell, H.M. (1982) Lipid monolayer coated solid surfaces do not perturb the lateral motion and distribution of C3b receptors on neutrophils. J. Cell Biol. 94, 224. Hook, G.R. and Rasband, W. (1987) Macmeasure: a low-cost, easy to operate quantitative morphometrics system for the Macintosh computer. In: G.W. Bailey (Ed.), Proceedings of the 45th annual meeting of the Electron Microscopy Society of America. San Francisco Press, San Francisco, CA, p. 920. Keller, H.-U., Barandun, S., Kistler, P. and Ploem, J.S. (1979)
Locomotion and adhesion of neutrophil granulocytes. Effects of albumin, fibrinogen and gamma globulins studied by refection contrast microscopy. Exp. Cell Res. 122, 351. Keller, H.-U., Zimmermann, A. and Cottier, H. (1983) Crawling-like movements, adhesion to solid substrata and chemokinesis of neutrophil granulocytes. J. Cell Sci. 64, 89. Kruskal, B.A., Shak, S. and Maxfield, F.R. (1986) Spreading of human neutrophils is immediately preceded by a large increase in cytoplasmic free calcium. Proc. Natl. Acad. Sci. U.S.A. 83, 2919. Roberts, R.L., Nath, J., Friedman, M.M. and Gallin, J.l. (1982) Effects of taxol on human neutrophils. J. Imrnunol. 129, 2134. Sullivan, G.W., Carper, H.T., Sullivan, J.A., Murata, T. and Mandell, G.L. (1989) Both recombinant interleukin-1 (beta) and purified human monocyte interleukin-1 prime human neutrophils for increased oxidative activity and promote neutrophil spreading. J. Leukocyte Biol. 45, 389. Valerius, N.H. (1983) Chemotaxis, spreading and oxidative metabolism of neutrophils: influence of albumin in vitro. Acta Pathol. Microbiol. Scand. Sect. C 91, 43. Vasiliev, J.M. (1982) Spreading and locomotion of tissue cells: factors controlling the distribution of pseudopodia. Phil. Trans. R. Soc. Lond. B299, 159. Vasiliev, J.M. (1985) Spreading of non-transformed and transformed cells. Biochima Biophysica Acta 780: 21. Wallis, W.J., Hickstein, D.D., Schwartz, B.R., June, C.H., Ochs, H.D., Beatty, P.G., Klebanoff, S.J. and Harlan, J.M. (1986) Monoclonal antibody-defined functional epitopes on the adhesion-promoting glycoprotein complex (CDw18) of human neutrophils. Blood 67, 1007. Wilkinson, P.C. (1982) Chemotaxis in inflammation. 2nd edn, Churchill-Livingstone, Edinburgh.