J. Cell Set, 48, 315-331 (1981)
315
Printed in Great Britain
INHIBITION OF LEUKOCYTE LOCOMOTION BY HYALURONIC ACID J. V. FORRESTER* AND P. C. WILKINSONf • Departments of Ophthalmology and of f Bacteriology and Immunology, University of Glasgow {Western Infirmary), Glasgow, Scotland
SUMMARY The effect of hyaluronate on neutrophil motility in vitro was studied by the micropore filter technique and by direct visual analysis of the locomotion of neutrophils on glass. Both directed and random locomotion of neutrophils were inhibited by physiological concentrations (0-5-6-0 m g m l ' ^ o f hyaluronate in a dose-and molecular weight-dependent manner. Inhibition of cell movement was more pronounced for high molecular weight chemoattractants such as casein than for small chemotactic peptides such as f-Met-Leu-Phe. Chemotactic factor gradient formation in filter chambers was profoundly retarded by hyaluronate, which may partly explain the inhibitory effects of hyaluronate on directed neutrophil locomotion. In addition, hyaluronate inhibited the binding of chemotactic factor to the neutrophil surface. This effect, together with a reduction in cell-to-substratum adhesion, may provide an additional explanation for hyaluronate-induced inhibition of random neutrophil locomotion. Inhibition of locomotion by hyaluronate was easily reversed by washing the cells free of hyaluronate; thus competition by hyaluronate for cell-surface binding sites is unlikely, and physical effects such as steric exclusion or molecular sieving by the large hyaluronate polymer provide the most probable explanations of its inhibitory effect on cell locomotion. Since hyaluronate is a major constituent of tissue matrices, these results draw attention to the importance of the extracellular environment in regulating inflammatory cell movement in vivo. INTRODUCTION
Inflammatory cells accumulate at sites of tissue injury by migrating through the extracellular matrix from the blood stream in response to gradients of locally produced chemotactic factors (Wilkinson, 1974). Changes in the content and type of extracellular matrix components are therefore likely to modulate the chemotactic response. The extracellular matrix consists of several species of macromolecules, including the glycosaminoglycans, of which hyaluronic acid is the most widely distributed. In loose connective tissue hyaluronic acid occurs in concentrations ranging from 0-05 to 3'O mg ml" 1 (Comper & Laurent, 1978); in compact matrices and healing tissue (Ogston, 1970; Shetlar et al. 1973) concentrations between 4 and 10 mg ml" 1 seem likely on the basis of tissue osmotic pressure studies while, in developing tissues, estimates of hyaluronic acid concentrations varying between 12 and 20 mg ml" 1 have been made from microspectrophotometric studies (Pratt, Larsen & Johnston, 1975). In some tissues or fluids, such as the vitreous of the eye (Balazs et al. 1959) and the synovial joint fluid (Sundblad, 1965) hyaluronic acid is virtually the sole glycosaminoglycan. Hyaluronic acid in solution at physiological concentrations has a very large
316
J. V. Forrester and P. C. Wilkinson
hydrodynamic volume, approximately io 3 times its volume in the dry state (Ogston & Stainier, 1951; Laurent & Gergely, 1955) and it forms a continuous polymer network which can be shown experimentally to exclude and to impede the diffusion of macromolecules such as proteins (Corriper & Laurent, 1978). Hyaluronate has also been shown to influence many cellular activities (Forrester & Balazs, 1980) including the inhibition of macrophage migration from capillary tubes (Balazs & Darzynkiewicz, 1973), an effect which was correlated with the increased viscosity of the medium. A similar study, using agarose, suggested that increasing the viscosity of the medium reduced macrophage migration by preventing cellular adhesions (Folger et al. 1978). Since leukocyte chemotaxis depends on diffusion of an attractant from a source to form a gradient, and since leukocytes respond to the gradient by crawling through tissues which in vivo are rich in hyaluronate, it 19 clearly important to establish the effect of hyaluronate both on the formation of gradients of chemotactic factors and on the locomotion of cells towards them. Such effects of hyaluronate have not been studied previously. The present study examines the effect of hyaluronate on neutrophil locomotion induced by chemotactic factors. The effect of hyaluronates of varying molecular sizes on both random and directed neutrophil locomotion was studied by filter and visual assays. Since the large hyaluronate molecule may sterically impede binding of chemotactic factors to the neutrophil surface, binding studies were also undertaken. These investigations revealed that hyaluronic acid can impede the locomotory responses of leukocytes by actions at several levels.
MATERIALS AND METHODS
Chemotactic factors Casein (Merck, Darmstadt) was solubilized with NaOH, pH 12 and readjusted to pH 7-2 with HC1 in Gey's solution. Human serum albumin (HSA) (Behringwerke, Marburg) was rendered chemotactic by denaturation with NaOH, pH 12 at 20 °C as described previously (Wilkinson & Allan, 1978). Formyl methionyl-leucyl-phenylalanine (f-Met-Leu-Phe) (Miles, Slough, U.K.) was used in the dose range io~* M to 5 x io~' M in the presence of HSA, 1 mg ml"1.
Polysaccharides Several samples of hyaluronate of varying molecular weight were used in this study. Details are given in Table 1. Commercial hyaluronic acid (intrinsic viscosity [rf\ 59—67; molecular weight approx. 1 x 10*) (Cleland & Wang, 1970) was from Miles, Slough. It was prepared from human umbilical cords as a potassium salt and contained 2-3 % protein and 3 % chondroitin sulphate. Samples of high molecular weight hyaluronic acid were a gift from Dr E. A. Balazs, Columbia University, New York. They were prepared as sodium salts from rooster combs by repeated precipitation with cetyl-pyridinium chloride (Chakrabarti & Balazs, J973). followed by heat treatment and fractionation by ion-exchange adsorption chromatography on a (diethylamino)ethylcellulose (DEAE-cellulose) column according to the method of Cleland, Cleland, Lipsky & Lynn (1968). Samples of various weight average molecular weights were eluted with 02 M NaCl, exhuastively dialysed against several volumes of glass-distilled water to remove excess salts and other impurities, and lyophilized. The samples were stored at 4 CC in a desiccator before use. Protein concentrations of the samples are shown in Table 1. Tetrasaccharides of sodium hyaluronate were prepared by exhaustive
Leukocyte locomotion and hyaluronic acid
317
digestion of hyaluronate solutions with testicular hyaluronidase (Worthington Biochemical, Freehold, N.J.) and elution of the digested material on a DEAE-cellulose column with 0-2 M NaCl. Chondroitin-6-sulphate was obtained from Sigma Chemical Co., London. Dextrans (molecular weights 60-90x10* and 500x10* daltons) and dextran sulphate (500 x io 3 daltons) were from Pharmacia AB, Uppsala, Sweden. Table 1. Hyaluronic acid preparations Sample
Intrinsic viscosity, cm'/g
i*
59-67
2
600
3 4 5 6
1600 2620 3620
Weight average mol. wt 10 x 2-9 x 7-0 x 1-5 x 2-4 x
io* io 6 io 6 io' io 6
Protein concentration, 0/
/o
2-3 30
S-o 1 25 2-40 300
597° 375 x 10' • Sample 1 was a potassium salt (Miles); all others were sodium salts of hyaluronic acid. Other materials D-glucuronic acid and iV-acetyl-D-glucosamine were from Sigma. Crude neuraminidase (specific activity 0-5-1-0/*g mg"1) was from Worthington Biochemicals, Freehold, N.J. The calcium ionopnore A23187 was supplied by Eli-Lilly (Windlesham, U.K.).
Cells Human neutrophils were obtained from the peripheral blood of healthy adult donors. Blood was collected in heparin (20 /tg/ml) without preservative (Evans Medical, Ltd, U.K.), and separated by dextran sedimentation followed by centrifugation on Ficoll-Triosil (Pharmacia, Uppsala and Nyegaard, Oslo). The cells were washed 3 times in Gey's solution for use in assays of cell locomotion. Rabbit neutrophils were obtained from the peritoneal cavity of white New Zealand rabbits 4 h after the intraperitoneal injection of 400 ml of 0-15 M NaCl containing o-i % oyster glycogen (Sigma). Monodisperse populations were prepared by passing the exudate fluid through a Nitex (Begg and Cousland, Glasgow) filter (pore size 10 /tm). Cell suspensions from both sources contained greater than 95 % neutrophils. Cell viability as determined by trypan blue exclusion was > 95 %, and was unaffected by the presence of polysaccharides.
Quantitation of cell migration Microporefilterassay. Cell migration was measured in modified Boyden chambers (Wilkinson, 1974) using the leading-front method (Zigmond & Hirsch, 1973). Duplicate filters were used for each test and 5 readings were taken at random from each filter. Cells (2-5-3-0 x io' neutrophils/ml) were placed in the upper chamber and a chemotactic agent in the lower chamber in Gey's solution, separated by a Millipore filter (3 /im pore diamter). The effects of hyaluronate and other polysaccharides on polymorphonuclear leukocyte (PMN) migration into the filter, were tested by adding the sugars to the lower, upper or both compartments of the test system. In the presence of viscous solutions of polysaccharides, where phase separation between aqueous solutions was likely to occur, care was taken to ensure thorough mixing of the sugar with the chemotactic agents on a rotomixer. Various modifications of the basic test system were employed, such as preincubation of the cells or chemotactic agent with the sugarg and addition of sugars to either chamber in the presence of uniform concentrations
318
J. V. Forrester and P. C. Wilkinson
of chemoattractant above and below the filter. Neutrophil migration was expressed as a locomotion index: distance migrated (fim) in the presence of sugar and chemotactic factor distance migrated (fim) in the presence of chemotactic factor alone The effect of hyaluronate on directed neutrophil migration was also tested by checkerboard analysis (Zigmond & Hirsch, 1973) where increasing concentrations of chemoattractant were placed above and below the filter. A chemotactic increment was calculated from the difference between the observed distance migrated by the cells into the filter, and the expected distance estimated on the basis of a chemokinetic response alone (for definitions of chemotaxis and chemokinesis, see Keller et al. 1977). This was derived as follows: . . observed distance (wm) — expected distance (/im) chemotactic increment = . expected distance (Jim) Visual assay using time-lapse cinematography This technique has been described in full previously (Allan & Wilkinson, 1978). Human neutrophils in Gey's solution were allowed to adhere to glass coverslips in a moist chamber at 37 °C for 5 min. Non-adherent cells and erythrocytes were removed by rinsing. Casein (1 mg/ml"1) or HSA (1 mg/ml"1) in Gey's solution or in solutions of hyaluronate were added to the chambers which were then sealed. The cells were filmed on a warm stage of an inverted microscope over a period of 20 min. The paths taken by each cell were tracked as described previously (Allan & Wilkinson, 1978) and the mean speed of the cell population determined. The percentage of non-locomoting cells in a given population was also recorded. Studies of chemotactic gradients in filter assays The effect of hyaluronate on the development of a chemotactic gradient was assessed by direct measurement. HSA (40 mg/ml"1) was labelled with 2 millicurie of 1U I using the chJoramine T iodination method (Hunter & Greenwood, 1962) and then denatured by treatment with alkali (Wilkinson & Allan, 1978). lu I-denatured HSA (dHSA) (1 mg ml"1) was placed in the lower chamber of a filter apparatus in the presence or absence of hyaluronate (total volume 36 ml) and mixed well. It was allowed to diffuse through the filter for varying times. Radioactivity in the upper chamber (total volume 0-2 ml) was measured on separate samples, at intervals of 30 min for 2 h, by removing the entire volume of fluid in the upper chamber. Care was taken not to contaminate the samples with fluid still adherent to the lower surface of the filter. The test was performed in triplicate for each time period, and the radioactivity per ml in the upper wells was expressed as a percentage of the total radioactivity added to the lower well. Binding of chemoattroctants to substrata The effect of hyaluronate on the binding of chemoattractants to micropore filters was studied by 2 methods. Micropore filters were preincubated with hyaluronate for 1 h at 20 °C and then used immediately or washed x 3 in Gey's solution before use in the micropore filter assay with casein (1 mg/ml"1) as the chemoattractant. In the second method, micropore filters were incubated with 1MI-dHSA (1 mg/ml"1) in the presence or absence of hyaluronate for 60 min at 20 °C, after which they were washed x 3 in Gey's solution and the filter-bound radioactivity measured in a gamma-counter. The tests were performed in triplicate.
Binding of ^I-dHSA
to rabbit PMN
The effect of hyaluronate on the binding of lu I-dHSA to rabbit PMN was studied by a modification of the method of Leslie & Cohen (1974), as described previously (Wilkinson & Allan, 1978). Serial dilutions of lu I-dHSA were added to glass tubes to give concentrations
Leukocyte locomotion and hyaluronic acid of 2-250 fig per tube in 0-5 ml Gey's solution. The total radioactivity of each tube was then measured. Rabbit neutrophils (5 x 10') in 0-5 ml Gey's solution or 0-5 ml hyaluronate (mol. wt 1 X io 4 ; 6 mg ml"1) were then added to each tube and the cells incubated for 60 min at 20 °C. The cell suspensions were centrifuged and the tubes inverted on absorbent paper to drain. At 15-min intervals the cells were washed and the radioactivity of the cells counted after each wash. A total of 5 separate washes was performed. This provided an estimate of the binding of m I-dHSA to the cells at equilibrium, i.e. before washing (Wilkinson & Allan, 1978). A graph of the cell-bound m I-dHSA against the total lu I-dHSA added to the cells was plotted for samples of cells in Gey's solution and in hyaluronate. Scatchard analysis allowed an estimate of the Ka (association constant) for the binding of m I-dHSA to rabbit PMN in the presence and absence of hyaluronate to be made. Viscosity determinations Viscosity measurements of polysaccharide solutions were made in an Ostwald capillary viscometer, BS/U/M, size M2 at 25 °C in a water bath. RESULTS Effect of hyaluronate on neutrophil migration in micropore filter assay Various samples of hyaluronate at different molecular weights were tested for their effect on neutrophil migration in the Boyden filter assay as described. In the absence of chemoattractant, hyaluronate had no effect on cell migration in this assay. However,
1-20 -
1-0
20
6-0
Hyaluronate, mg ml"'
Fig. 1. Effect of hyaluronates of varying molecular weight on casein-induced (1 mg ml" 1 ) migration of human neutrophils into micropore filters. Hyaluronate was mixed with the chemotactic factor below the filter. Each curve represents the activity of a different hyaluronate sample, identified by its molecular weight. Curve A, tetrasaccharide; B, 2-9 x io 5 hyaluronate; C, i-o x io 4 ; D, 7 0 x 10'; E, 1-5 x 10'; F, 2-4 x io°; G, 3-75 x io 6 . Control value (in absence of hyaluronate) = i-o (see Materials and methods). Bar = S.E.M.
320
J. V. Forrester and P. C. Wilkinson
hyaluronate inhibited the migration of neutrophils into micropore filters in response to casein. Inhibition was observed only when the polymer was placed below the filter with the casein and not when placed above with the cells, and was dosedependent within a concentration range from 0-05 to r o mg ml" 1 (Fig. 1). High molecular weight hyaluronates were more inhibitory than low molecular weight polymers. Hyaluronate tetrasaccharides and the monosaccharide /?-D-glucuronic acid (6 mg ml"1) were ineffective, while iV-acetyl-D-glucosamine (6 mg ml"1) enhanced the neutrophil response to casein (distance migrated in ./V-acetyl-D-glucosamine, 94-2 ± 9-6 fim, compared to 66-6 + 2-1 /tm without it).
i-2r S
10
•8
I 0-8
10
c 0-8 o
o o 0-6
I 0'6 -•»-
o X 0-4
o 8 0-4
1
0-2
0-2 0-5
10
1-5
Hyaluronate, mg ml" 1
20
0-5 1-5 10 Hyaluronate, mg ml" 1
20
Fig. 2. Effect of hyaluronates of various molecular weights on neutrophil migration x); dHSA, 1 mg ml" 1 ( O — O ) ; and f-Met-Leu-Phe, due to casein, 1 mg ml" 1 (x 1 5 x i o " M ( • — • ) . In A, hyaluronate of mol. wt 2-40 x 10" was used, and in B, hyaluronate of mol. wt 1 x 10*; values on scale are mg ml" 1 . Control value in absence of hyaluronate = i-o. Bar = S.E.M.
Hyaluronate also inhibited the neutrophil response to chemotactic factors other than casein (Figs. 2 A, B). High molecular weight hyaluronate (2-4 x io 8 daltons) at concentrations higher than 0-5 mg ml" 1 caused approximately similar reductions in neutrophil migration to casein, alkali-denatured HSA (dHSA) and f-Met-Leu-Phe. However, at o-i mg ml""1 the response to dHSA and casein were reduced, while that to f-Met-Leu-Phe was unaltered. In contrast, low molecular weight hyaluronate ( r o x 10* daltons) inhibited casein-induced neutrophil locomotion at concentrations above 0-5 mg ml- 1 , and dHSA-induced locomotion above i-o mg ml- 1 , but had little effect on the f-Met-Leu-Phe-induced response at concentrations up to 1-5 mg ml" 1 (Fig. 2B). Effect of hyaluronate on the formation of a chemotactic gradient
Boyden chambers were set up with 1MI-dHSA below the filter in the presence or absence of hyaluronate and samples were taken from the upper chambers for radioactivity counts at 30-min intervals as described in Materials and methods. A linear increase in radioactivity in the upper well with time was observed when 12*I-dHSA
Leukocyte locomotion and hyaluronic acid
321
was present alone in the lower well. When hyaluronate was mixed with 126I-dHSA, the rate of appearance of radioactivity in the upper well was markedly reduced and was non-linear (Fig. 3), indicating that gradient formation between the 2 wells was inhibited by the hyaluronate. Preincubation of filters with m I-dHSA and hyaluronate apparently had no effect on the total amount of filter-bound chemoattractant (Table 2). Furthermore, neutrophils migrated equally well into micropore filters which had been preincubated
0-5
10
1-5
Time, h Fig. 3. Effect of hyaluronate (mol.wt 1 x io 4 ; 6 mg ml"1) on the diffusion of *"I-dHSA from lower to upper well of Boyden apparatus. Control (x x); hyaluronate (•
•)•
Table 2. Effect of hyaluronate on binding of luI-denatured HSA to micropore filters in Boyden assay* Filter-bound radioactivity as percentage of total radioactivity added Hyaluronate
Mean
Control (buffer)
Below filter
Above filter
0-87 0-91 083 087
0-79 097 089 088
1 23 0-92 113
A
i-oo
Both sides of filter I'22
086 083 007
• Triplicate filters were incubated in Boyden chambers with ltBI-dHSA (1 mg ml"1) in the lower well, while hyaluronate (mol.wt 1 x io 4 ; 6 mg ml"1) was placed above, below, or on both sides of the filter. The chambers were incubated for 60 min at 20 °C. The filters were removed, washed x 5 in Gey's solution and the filter-bound radioactivity counted.
322
J. V. Forrester and P. C. Wilkinson 1
with casein (i mg ml" ) in the presence or absence of hyaluronate and then washed (data not shown). These results suggested that hyaluronate exerted a greater inhibitory effect on neutrophil migration to fluid-phase chemoattractant diffusing from below the filter than to substratum-bound chemoattractant. In the former case, the cells may never be exposed to an effective attractant concentration, whereas in the latter case, the cells commence locomotion at the top of the filter on a surface bearing an effective concentration. The ability of neutrophils to respond by chemotaxis in fluid-phase gradients was also tested by checkerboard analysis (Zigmond & Hirsch, 1973) (see Materials and methods). In the absence of hyaluronate the mean chemotactic increment for positive gradients of casein was + 8-5 % and for negative gradients - 7-8 %. When hyaluronate (mol. wt i-o x io 4 ; 6 mg ml"1) was incorporated into the lower wells of each Boyden chamber, the mean chemotactic increment was +0-8% for positive gradients and + 6-3 % for negative gradients. These results showed that the chemotactic response of the cells was abolished in the presence of hyaluronate, and for negative gradients the response even appeared to be reversed. Such effects are consistent with an inhibition of chemotactic factor gradient formation by hyaluronate as shown above. However, other mechanisms are possible to explain the effect of hyaluronate in a checkerboard study; for instance, hyaluronate may prevent access of the chemotactic factor to the cell surface by steric hindrance in spite of the presence of a gradient. Furthermore, checkerboard assessment of cell locomotion is based on an estimated chemokinetic response of the cells to the chemotactic factor (Zigmond & Hirsch, 1973). However, the assumption that the cells are moving in a uniform concentration of attractant in the chemokinetic chambers cannot be made when hyaluronate is present in the lower chamber only, since excluded volume effects (Comper & Laurent, 1978) of the polymer may effectively reduce the available attractant concentration in the lower chamber. It was therefore necessary to explore the effects of hyaluronate on chemokinesis directly, using a visual assay. Effect of hyaluronate on chemokinesis
Stimulated random locomotion of neutrophils (Keller et al. 1977) was measured by direct visual analysis using fluid-phase casein as chemoattractant (1 mg ml"1) in uniform concentration throughout the system (see Materials and methods). Cells moving randomly on glass in the presence of high molecular weight hyaluronate (1-5 x io6 and 2-4 x io8 daltons) were considerably slowed down (Table 3). Moreover, fewer cells showed a locomotory response in the presence of hyaluronate. Cells in low molecular weight hyaluronate ( I - O X I O 4 daltons) showed some reduction in random locomotion at concentrations of 6 mg ml" 1 but the effect was less marked (Table 3). These results showed that hyaluronate inhibited neutrophil locomotion in the absence of a gradient of chemoattractant and clearly indicated that hyaluronate was acting by mechanisms other than on gradient formation. Several studies have suggested that stimulated random locomotion by neutrophils is dependent upon changes in cell-substratum adhesiveness (Keller, Hess & Cottier, 1977; Weiss & Glaves, 1975; Smith, Lackie & Wilkinson, 1979; O'Flaherty, Kreutzer & Ward,
Leukocyte locomotion and hyaluronic acid
323
1977). These changes are induced in the cell on contact with chemotactic factors which are specifically bound by the cell membrane. The possibility that hyaluronate interfered with the binding of chemotactic factor to the neutrophil was therefore explored. Table 3. Effect of hyaluronate on random migration of PMN in presence of casein* using the visual assay No. of neutrophils observed
Speed, /im/min
Non-locomoting cells, %
Expt. 1 Control Hyaluronate 1-5 mg ml"1, mol. wt 2 4 x 10°
26 26
11-92 ±0-7! 6-73 ±0-5
19-23 30-76
Expt. 2 Control Hyaluronate 1 -5 mg ml" 1 , mol. wt 1.5 x 10" Hyaluronate 6-o mg ml" 1 , mol. wt 1-o x 10*
31 30 31
8-4210-40 5-4210-37 7'34i°'59
22-6 300 22 '6
• Casein: 1 mg ml"1. f Results expressed: meanls.E.M.
Effect of hyaluronate on cell-surface binding of chemotactic factor,
dHSA
It has been shown that denatured HSA (dHSA) has a greater binding affinity for the neutrophil cell surface than non-chemotactic native HSA (Wilkinson & Allan, 1978). Rabbit neutrophils (5 x io6 ml"1) were incubated with m I-dHSA in the presence or absence of hyaluronate (mol. wt 1 x io 4 ; 6 mg ml"1) for 60 min at 20 °C and the radioactivity of the cells counted after each wash for a series of 5 washes at 15-min intervals (see Materials and methods). Fig. 4 shows the relationship between the amount of 125I-dHSA bound to the cells and the total amount of protein added to the cells. It is clear that less protein became cell-bound when hyaluronate was present in the cell suspension. Scatchard analysis (Fig. 4, inset) of dHSA binding affinity indicated that, in the absence of hyaluronate, dHSA had an association constant (Ka) of 10 x io 6 1. mol"1 and that the number of binding sites (n) was 2-57 x io6 cell"1. The correlation coefficient was —0-804. Data at higher binding affinities only were considered in determining these values (see Fig. 4 for explanation). In the presence of hyaluronate the scatter of values for binding affinity was such that a reliable estimate of Ka could not be made. However, the data confirmed the general reduction in dHSA binding to neutrophils and suggested a marked heterogeneity of binding affinity in the presence of hyaluronate. Evidence of competition between hyaluronate and chemotactic factor for the neutrophil binding site was sought by indirect means. Cells preincubated in hyaluronate (mol. wt 1 x io 4 ; 6 mg ml"1) for 30 min at 20°C, and then washed once with Gey's solution, showed no reduction in casein-induced, random locomotion, compared to similarly treated buffer control cells migrating in the presence of
J. V. Forrester and P. C. Wilkinson
324
300
8
o
I- 200 U5
I
•o
100
12
16
20
dHSA added to cells, pg per 10' cells
Fig. 4. Effect of hyaluronate (mol. wt 1 x io 4 ; 6 mg ml"1) on binding of m I-dHSA to rabbit neutrophils. Inset shows Scatchard plot of binding affinities: ordinate, % dHSA bound/10' cells; abscissa, ng dHSA bound/10' cells, x, Control values of binding affinity; # , binding in presence of hyaluronate, The regression line for the control values in the Scatchard plot was obtained by considering binding at high affinities only (i.e. excluding 2 furthermost points to the right) since binding at low affinities did not vary significantly with total protein added to the cells (see Wilkinson & Allan, 1978). No reliable regression line could be obtained for binding in presence of hyaluronate.
hyaluronate (Table 4). This removal of inhibition following a single wash makes competition by hyaluronate for a chemotaxis receptor highly unlikely. Inhibition of neutrophil locomotion was also reversed by incorporating hyaluronidase (150/igml- 1 ) into the assay system. Under these conditions, the hyaluronidase could have been affecting cell receptors rather than exogenous hyaluronic acid. However, this was made less likely by the observation that cells pretreated with hyaluronidase (^o/tgml" 1 for 30 min at 37 °C) and then washed,
Leukocyte locomotion and hyaluronic acid
325
showed the same hyaluronate-induced reduction in locomotion as untreated control cells (data not shown). Similar results were obtained with neuraminidase-treated cells (100 fig ml" 1 for 30 min, 37 °C). Table 4. Effect of washing on locomotion of hyaluronate-treated neutrophils in micropore filter chambers Distance migrated by PMN into micropore filter, /im
Normal cells Cells migrating in presence of hyaluronatet Cells preincubated in hyaluronate followed by a single washj
Control
Casein*
35-2 ±i-8
89-013-1
—
63-012-0
—
946 ± 3-7
1
• Casein: 1 mg ml" , both sides of filter. f Hyaluronate: mol. w t i x io 4 ; 6 mg ml" 1 both sides of filter. X Cells preincubated in hyaluronate, 6 mg ml"1 for 30 min at 20 °C, then washed x 1 Gey's medium. Results expressed: mean±s.E.M. Table 5. Effect of chondroitin sulphate and dextran sulphate on casein-induced PMN migration Distance migrated by PMN into Millipore filter, /im Control None Chondroitin sulphatet Dextran sulphate § • t t §
19-9 ±0-87! 23-211-91 2i-o±o-o8 Results expressed: mean ± S.E.M.
Casein* 66-6±2-o8 66-614-89 66-8 ±2-8
Casein: 1 mg ml" 1 below filter. Values represent mean ± S.E.M. Chondroitin sulphate: 6 mg ml" 1 present below filter. Dextran sulphate: 5 mg ml" 1 present below filter.
Comparison of effects of hyaluronate and other charged polysaccharides on cell migration
The possibility that mutual repulsion between the high-density charges on the cell surface and the hyaluronate molecule may have affected migration of neutrophils in hyaluronate-containing media was explored by comparing the effects of hyaluronate with those of chondroitin sulphate and dextran sulphate used within the same concentration range. Chondroitin sulphate contains twice the number of charged moieties per disaccharide residue (Comper & Laurent, 1978) and is thus more highly charged than hyaluronic acid on a weight for weight basis. However, neither dextran sulphate nor chondroitin sulphate had any effect on neutrophil migration at concentrations up to 6 mg ml" 1 when placed in the lower well with the chemoattractant (Table 5).
326
J. V. Forrester and P. C. Wilkinson
The role of calcium binding on cell migration in the presence of hyaluronic acid
Since hyaluronic acid is a weak chelator of calcium ions (Comper & Laurent, 1978) and adopts a specific structure in their presence (Winter & Arnott, 1977), it is possible that the inhibitory effects of hyaluronate on neutrophil migration were mediated by a relative depletion of calcium ions. Although neutrophils respond chemotactically in calcium-free medium, the magnitude of the response is reduced (Becker & Showell, 1972). This effect can be reversed by the calcium ionophore A23187, presumably by mobilizing stores of intracellular calcium ions (Wilkinson, Table 6. Effect of hyaluronate of casein-induced PMN migration in the presence of the calcium ionophore A2318J Distance migrated by PMN into Millipore filter, fim
Normal cells Cells in medium containing A23187J
Control
Casein*
32-6 ± i-o —
62-7 ±2-4 62-8 ±2-7
Hyaluronatef +casein* 49'5±2 - 3 48-512-1
Results expressed: mean±s.E.M. • Casein: 1 mg ml" 1 below filter. f Hyaluronate: (mol. wt 2-4 x 10'), 0-5 mg ml"1. X A23187: io~' M.
1976). Thus, if hyaluronate acted by chelating extracellular calcium, this effect should be abrogated by adding A23187 to the system. However, Table 6 shows that the inhibition by hyaluronate of casein-induced neutrophil migration was not reversed by A23187. This suggested that the inhibitory effects of hyaluronate on cell locomotion were not due to a hyaluronate-induced depletion of extracellular calcium ions available to the cell. DISCUSSION
This study has shown that hyaluronate in physiological concentrations reduced the locomotion of neutrophils to chemoattractants when the polymer was mixed with the chemotactic factor in the lower chamber of the Boyden apparatus. High molecular weight hyaluronate was considerably more effective than low molecular weight hyaluronate. In addition, studies on the rate of diffusion of 126I-dHSA between the lower and upper chambers of the Boyden apparatus showed that hyaluronate prevented the formation of a gradient of chemotactic factor between the 2 chambers. It is likely that this accounted for its effects on directed locomotion. However, visual analysis of cells in chemokinetic conditions suggested that hyaluronate also had a direct effect on neutrophil locomotion. A reduction in binding of chemotactic factors to the cell surface in the presence of hyaluronate, as demonstrated in the present study, may partly explain its inhibitory effect on random cell locomotion. Experiments in progress (Forrester & Lackie, unpublished data) also
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indicate that hyaluronate may alter cell locomotion directly by inhibiting neutrophil adhesion. Recent studies have shown that hyaluronate inhibits fibroblast adhesion (Underhill & Dorfman, 1978) and it is likely that its effects on neutrophil adhesion operate by a similar mechanism. Thus hyaluronate inhibits locomotion of neutrophils to chemotactic factors in at least 3 ways: namely, inhibition of chemotactic factor gradient formation; prevention of binding of chemoattractants to cells; and reduction of cellular adhesion to substrata. A reduction in the rate of diffusion of chemoattractant between the lower and upper chambers of the Boyden apparatus is wholly consistent with the known structure and properties of hyaluronic acid. Dry-fibre studies of hyaluronates have shown that the molecule exists as a single helical chain of repeat disaccharide units of /?-./V-acetyl-D-glucosamine and /?-r>glucuronic acid with a 3-fold symmetry (3 disaccharide units per twist of chain) when calcium is present as the counterion (Winter & Arnott, 1977). In solution, hyaluronic acid probably occurs as a relatively stiff random coil (Scott & Tigwell, 1978) and has a very large hydrodynamic volume, approx. io 3 x its volume in the dry state (Ogston & Stanier, 1951; Laurent & Gergely, 1955). Consequently at low concentrations the molecule adopts an extended chain configuration (Cleland, 1977) but at higher concentrations (> 1 mg ml"1) there is a considerable overlap between individual molecules (Ogston & Stanier, 1951; Cleland & Wang, 1970), which results in a continuous polymer network (Comper & Laurent, 1978). Several studies have shown that the transport of macromolecules through such networks is considerably retarded (for review, Comper & Laurent, 1978). The rate of diffusion depends not oniy on the concentration and molecular size of the hyaluronate but also on the size and shape of the diffusant molecule. Generally, large molecules diffuse at slower rates than small molecules; in addition, in dilute solutions of hyaluronate, globular molecules such as albumin are retarded more than linear molecules such as polyadenylate (Laurent et al. 1975), which is consistent with a molecular-sieving mechanism of transport rather than a frictional resistance effect (Preston, Obrink & Laurent, 1973). The diffusion of small uncharged particles such as monosaccharides is apparently unaffected by hyaluronate solutions (1-2 mg ml"1) (Preston & Snowden, 1972), but charged particles such as micro-ions are considerably retarded, in this case probably as a result of electrostatic interaction (Comper & Preston, 1975). Such phenomena may explain the effects of hyaluronate on directed migration of neutrophils using chemoattractants of different molecular weights. Thus, high concentrations of high molecular weight hyaluronate (mol. wt 2-4 x io 8 daltons) inhibited neutrophil locomotion to chemoattractants of various molecular sizes, but dilute solutions of the same hyaluronate inhibited locomotion only to high molecular weight chemotactic factors and not to synthetic tripeptides. In contrast, low molecular weight hyaluronate inhibited locomotion to chemotactic factors in a graded fashion, i.e. casein > denatured HSA > f-Met-Leu-Phe, according to molecular size of the diffusant molecules. It is unlikely that hyaluronate inhibited locomotion by inducing direct conformational changes in the chemotactic factors. Such effects occur only for certain protein-glycosaminoglycan interactions which depend on specific factors such as
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the length of the polypeptide side-chains, the position of the anionic group and the degree of glycosaminoglycan sulphation (Gelman & Blackwell, 1974). The variety of protein molecules used as chemotactic factors in this study, all of which elicited reduced neutrophil responsiveness in the presence of hyaluronate, would preclude any specific interactions of this nature. Rather, the more general physical effects of the hyaluronate molecule such as molecular sieving, are likely to have caused the inhibition of directed neutrophil migration. The inhibition of binding of chemotactic factor to the neutrophil surface induced by hyaluronate probably resulted from similar physical effects of the polymer, rather than from specific binding of hyaluronate to the cell membrane. Ligand binding by cells coated in polysaccharide is likely to be altered as a result of both molecularsieving and steric-exclusion effects. Molecular sieving by intrinsic cell-surface polysaccharides or by extrinsic polysaccharide loosely attached to the cell may produce marked heterogeneity in binding affinity of proteins, as was observed in the present study, since access of the protein to the cell membrane will depend upon the siting and frequency of 'holes' in the polymer network. Steric-exclusion effects of a polysaccharide cell-coat, whereby molecules are physically excluded from the domain of other molecules (Laurent, 1964), will cause a general reduction in protein binding. In the case of hyaluronate the molecular domain of the hydrated molecule is very large (see above). The considerably greater effect of high molecular weight hyaluronate compared to the low molecular weight species on stimulated random locomotion of neutrophils on glass would support a steric exclusion effect. Furthermore, the inhibitory effect of hyaluronate on the locomotor response was reversed by a single wash of the cells, which makes it very unlikely that hyaluronate could act by competing for cell-surface chemotaxis receptors. It is also true that many chemotactic factors are hydrophobic (Wilkinson & McKay, 1971) and likely to interact with hydrophobic membrane sites. Thus cells surrounded by hyaluronate might bind less chemoattractant because of mutual repulsion between the hydrophilic polysaccharide and the hydrophobic protein or peptide. Similar physical factors are probably important in relation to neutrophil adhesion in the presence of hyaluronate. Weiss (1959) has shown that cells adhered poorly to surfaces coated with polysaccharide while Folger et al. (1978) showed that both macrophage migration and adhesion to glass were reduced in the presence of viscous solutions of agarose. They attributed the former effect to a vertical viscous 'drag' on the moving cell which prevented formation of adhesion plaques between the cell and the substratum. Our preliminary observations on neutrophil adhesion in the presence of hyaluronate support these findings and will be reported in a further communication. Other mechanisms for the effect of hyaluronic acid locomotion were explored. A possible calcium-binding effect of hyaluronate (Winter & Arnott, 1977) was excluded by the use of the calcium ionophore A23187, which is known to reverse the effects of calcium-depletion on normal cell migration (Wilkinson, 1976), but failed to reverse the inhibitory effects of hyaluronate. Possible alteration in the zeta potential of migrating neutrophils (Gallin, Durocher & Kaplan, 1975) induced by
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negatively charged hyaluronate was also excluded by the lack of effect of dextran sulphate and chondroitin sulphate at equivalent concentrations. The effect of hyaluronate on neutrophil migration has important implications for processes such as invasiveness and wound-healing where chemotaxis is known to occur. The rise in tissue concentrations of hyaluronic acid during the later stages of wound-healing (Shetlar et al. 1973) occurs at a time when the migration of inflammatory cells, particularly neutrophils, into the wound is rapidly declining. It is possible that these events are closely linked in a negative feedback mechanism, thus suggesting a regulatory role for the molecule in the control of the inflammatory response. In other circumstances, the presence of a hyaluronic acid matrix may be less beneficial to the organism. Certain tumour cells are known to synthesize and secrete higher than normal quantities of hyaluronate in vitro (Satoh, Duff, Rapp & Davidson, 1973; Glimelius, Norling, Westermark & Wasteson, 1979). Furthermore, the presence of a cell-surface layer of hyaluronic acid has been shown to protect tumour cells in vitro from attack by cytolytic immune lymphocytes (McBride & Bard, 1979). Such effects in vivo may impede the cellular defence mechanism of the body and permit the growth and metastasis of tumours. REFERENCES R. B. & WILKINSON, P. C. (1978). A visual analysis of chemotactic and chemokinetic locomotion of human neutrophil leukocytes. Expl Cell Res. 111, 191-203. BALAZS, E. A. & DARZYNKIEWICZ, Z. (1973). The effect of hyaluronic acid on fibroblasts, mononuclear phagocytes and lymphocytes. In The Biology of the Fibroblast (ed. E. Kulonen & J. Pikkarainen), pp. 237-252. London, New York: Academic Press.
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