Printed in Sweden Copyright @ 1979by Academic Press, Inc. All rights of reproduction in any form reserved ool4-4827/79/osO325-05~Z.OO/O
Experimental Cell Research 121 (1979) 325-329 ANALYSIS
BY THE LEADING-FRONT Random Locomotion
U.-B. SODERSTROM, G. SIMMINGSKijLD, B. NORBERG, 0. BACK and L. RYDGREN Department
of Internal Medicine, University of Lund, S-221 85 Lund, Sweden and Department of Dermatology. University of Umeii, S-901 87 VmeB, Sweden
SUMMARY The present study was designed to elucidate the contribution of non-stimulated random movement, stimulated random movement, antitubulin-resistant chemotaxis and antitubulin-sensitive chemotaxis to the casein-induced PMN migration into a micropore filter, evaluated by the leadingfront technique. This analysis was conducted by a simplified test design including PMN migration, (a) without casein; (b) in a gradient of casein; and (c) in casein without gradient. Treatment with the antitubulin SPI (a podophyllotoxin derivative) inhibited PMN migration within a casein gradient down to the level of the stimulated PMN random movement induced by casein. Thg casein-induced PMN chemotaxis measured by the leading-front technique is thus composed of stimulated random movement and antitubulin-sensitive chemotaxis without evidence of antitubulin-resistant chemotaxis. It is suggested that the anti-inflammatory effects of the antitubulins (colchicine, podophyllotoxin, Vinca alcaloids, griseofulvin) are due to an inhibition of the antitubulin-sensitive chemotaxis.
The PMN chemotaxis is a complex process which is dependent on cell motility, cell adherence and direction-finding. Several factors must be considered in trials for the quantification of PMN chemotaxis [ 11: (1) non-stimulated random movement; (2) stimulated random movement; and (3) chemotaxis, both antitubulin-resistant and antitubulin-sensitive. The PMN chemotaxis can be quantified by (1) time-lapse filming [2, 3, 41; (2) micropore filter techniques-leading front and cell counts [5,6,7]; and (3) PMN migration under agarose [8,9]. Since the migration of PMNs into a filter is influenced by many factors, different filter assay methods may not produce comparable results. Thus we found in a previous study [l] that antitubulin inhibition of PMN chemotaxis was more pronounced
when evaluated by means of accumulated cells, than when evaluated by means of the leading-front method (cf fig. 2). The aim of the present study was to analyse the contribution of non-stimulated random movement, stimulated random movement, antitubulin-resistant chemotaxis and antitubulin-sensitive chemotaxis to the casein-induced PMN migration in a micropore filter, evaluated by the distance from the upper surface to the level of the leading front. In order to optimize the experimental conditions, the time course of the antitubulin inhibition of the casein-induced PMN migration into the filter was first studied. MATERIAL
Venous blood (10 ml) from the antecubital fossa was obtained from 14 healthy male donors. The leukocytes were isolated by sedimentation with 1.0% methylExp Cell Res 121 (1979)
Siiderstriim et al.
Table 1. The contribution of non-stimulated random locomotion (a), stimulated random locomotion (c, d) and the antitubulin-inhibited chemotaxis (e, fl to the PMN migration into the filter of the Wilkinson chamber towards a casein gradient (b) G, Gey’s solution; C, casein solution 5 mg/ml. 1.Oand 0.1 = 1.0 and 0.1 pg/ml of SPI. h’= 14
Above filter Below filter Median Qi-Qz
G G 16.8 10.0-20.5 K&25.0
G G+C 52.0 38.0-60.0 19.5-81s
c+c G+C 35.0 22.5-50.0 13.0-68.0
G+C+l.O G+C 33.5 27.0-43.5 17.0-44.5 p