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Platelet activating factor is a mediator of equine neutrophil and eosinophil migration in vitro
Research in Veterinary Science 1992, 53, 223-229
Platelet activating factor is a mediator of equine neutrophil and eosinophil migration in vitro A. P. FOSTER, P. LEES, F. M. CUNNINGHAM, Department of Veterinary Basic Sciences,
The Royal Veterinary College, Hawkshead Lane, North Mymms, Hertfordshire, AL9 7TA
Platelet activating factor (PAF) is known to be a chemoattractant for equine neutrophils in vivo and in vitro. In this study the in vitro migratory response of equine eosinophils and neutrophils to PAF has been examined and compared with that to leukotriene (LT)B4. PAF (10- s to 10- s M), but not lyso-eAF (10 -6 M), caused dose related migration of both equine cosinophils and neutrophils, maximal responses occurring at 10 -6 M. Responses to PAF were inhibited by the receptor antagonist WEB 2086. LTa4 (10 -s to 10 -6 M) also induced migration of both cell types, although the maximum effect was observed with a 10-fold lower concentration. Moreover, the m a x i m u m response of equine eosinophils to LTB4 was significantly greater than to PAF. It is concluded that LTB4 and PAF, if released in vivo at sites of allergic or inflammatory reactions, could mediate the recruitment of leucocytes to the involved tissue.
Moreover, the concentrations that elicit maximal responses are similar to those required for human neutrophils. The chemotactic responses of equine eosinophils have not been extensively studied. Potter et al (1985) reported in vitro eosinophil migration towards a range of arachidonic acid derived monohydroxyfatty acids (HETEs)and LTB4 using cell populations of between 30 and 70 per cent purity. Only modest chemotactic responses were obtained with LTB4,a finding which agrees with results obtained by Wardlaw et al (1986) using semi-pure eosinophil populations prepared from human patients with eosinophilia. In contrast, in the same study, PAF elicited a time- and dosedependent directional migration of human eosinophils and it was suggested that PAF is a potent chemotactic factor for these cells. As has been demonstrated in man (Archer et al 1985), intradermal injection of PAF in normal THE in vitro and in vivo chemoattractant prop- horses causes neutrophil, but not eosinophil, erties ofplatelet activating factor (PAF)for inflam- recruitment to the skin (Foster et al 1990). Prematory cells such as the neutrophil have been liminary evidence has, however, been obtained well documented (Barnes et a11988). Early studies to show that PAF injection into the skin of horses suggested that equine neutrophils might differ with the allergic skin disease sweet itch does from those of other species in their migratory induce an accumulation of eosinophils (Foster responses to chemotactic factors since the bac- et al 1992a). A similar finding was reported in terial tripeptide, formyl-methionyl-leucyl-phenyl- atopic human subjects after injection OfPAF,antialanine (FMLP), either did not cause movement gen or FMLP (Henocq and Vargaftig 1988). of equine cells, or produced a response only at In this study the in vitro chemoattractant activvery high concentrations (Snyderman and Pike ity of PAF for equine neutrophils has been exam1980, Camp and Leid 1982, Zinkl and Brown ined in microchemotaxis chambers. PAF-induced 1982). Any difference does, however, appear to migratory responses have been compared to those be restricted to FMLP receptor-mediated events of LTB4 using neutrophils from the same donor since equine neutrophils respond to zymosan acti- horses since the relative potency of these comvated serum or plasma, PAF and the arachidonic pounds for neutrophil movement has not been acid lipoxygenase product, leukotriene (LT)B4 in established using cells from the same horses. The vitro (Lees et al 1986, Dawson et al 1988). effect of PAF on equine eosinophil migration has 223
224
A. P. Foster, P. Lees, F. M. Cunningham
also been studied for the first time and compared to that of LTB4,which has previously been reported to be a weak stimulant of equine eosinophil migration (Potter et al 1985). Materials and methods
Materials C16:0 PAF and C16:0 lyso-eAF were obtained from Bachem UK. Stock solutions (1 mg m1-1) were prepared in ethanol and stored at -20°C. LTB4 (100 ~tg m1-1 in methanol) was kindly supplied by Dr A. W. Ford-Hutchinson, Merck Frosst Canada Inc and stored at -70°C. Serial dilutions of both PAF and LTB4 were prepared by evaporation of the solvent under nitrogen and resuspension of the residue in Eagle's minimal essential medium containing 0.4 per cent equine serum albumin (MEM/ESA). Zymosan activated equine plasma (zM') was prepared by incubation of freshly prepared heparinised plasma with 1 mg m1-1 zymosan A for 30 minutes at 37°C. The plasma was then inactivated by heating to 56°C for 45 minutes, the zymosan removed by centrifugation at 1400 g for 10 minutes and samples stored at -20°C until required. Dilutions of ZAP were prepared in MEM/ESA.WEB 2086 was kindly supplied by Boehringer Ingelheim Vetmedica, Germany. Hank's balanced salt solution (HBSS) was obtained from Life Technologies, uK and Percoll from Pharmacia, uK. All other chemicals and solvents were obtained from Sigma Chemical, UK or BDH, UI(, and were of reagent grade.
minutes at 4°C and the neutrophils recovered in a band between the 70 per cent and 85 per cent Percoll solutions. The cells were washed twice in MEMcontaining 0-003 per cent deoxyribonuclease (DNase) I and finally resuspended at a concentration of 5 x 106 ml-1 in M~M containing 0'4 per cent chick albumin (CA). The purity and viability of the cells were at least 99 per cent (n = 11 determinations). Viability was assessed using trypan blue dye exclusion.
Purification of equine eosinophils Leucocytes were separated from 100 ml jugular venous blood samples as described above. After resuspension in 10 ml leucocyte poor plasma the cells were layered on to a 70:85:100 per cent Percoll gradient. Following centrifugation at 400 g for 15 minutes at 4°C, the eosinophils and erythrocytes were found at the 85:100 per cent Percoll interface and dispersed through the 100 per cent Percoll layer. The appropriate layers containing the eosinophils and erythrocytes were recovered, washed in MEM containing 0.003 per cent DNase I, the erythrocytes lysed by hypotonic shock (0.2 per cent saline [10 ml] for 30 seconds, followed by the addition of an equal volume of 1-6 per cent saline) and the eosinophils washed again in MEM containing DNase I. The cells Were finally resuspended in MEM/CA at a concentration of 5 x 106 ml-1. The purity of the cells was at least 99 per cent and the viability was greater than 93 per cent (n = 6 determinations).
Chemotaxis assay Separation of equine neutrophils Stock solutions of Percoll were prepared by mixing nine parts Percoll with one part 10 ti=,'_'~es concentrated, calcium and magnesium free HBSS. Further dilutions of this stock were made with HBSS to give Percoll solutions of the required density and the pH adjusted to pH 7-4. Blood samples (40 ml) were then collected from the jugular veins of normal horses into 0-4 M EDTA (1:39). The blood was allowed to sediment for 20 minutes at room temperature and the leucocyte rich plasma harvested. After centrifugation (200 g, 4°C, five minutes) the cell pellet was resuspended in 5 ml of leucocyte poor plasma and layered on to a 70:85 per cent Percoll gradient. The gradients were centrifuged at 400 g for 15
MEM/ESA or chemoattractants dissolved in MEM/ESA (26.5 ~d) were placed in the bottom wells of 48-well microchemotaxis chambers (Falk et al 1980, Harvath et al 1980). Cellulose nitrate filters (100 pm thick; 8 prn pore size) which had been soaked for five minutes in MEM were placed over the top of the wells. A silicon gasket was laid over the filter and the top of the chamber secured in place. Fifty ~d of cell suspension was next added to each of the top wells and the chambers placed in a humidified box within an incubator at 37°C. After incubation for 1.5 hours (neutrophils) or three hours (eosinophils) the filters were removed, the upper surface wiped free of non-adherent cells and fixed in 10 per cent formalin for five minutes.
eAr-induced equine leucocyte The filters were then stained with haemalum, rinsed in distilled water and blued in 1 per cent a m m o n i u m hydroxide. After d e h y d r a t i o n in alcohol the filters were cleared in xylene and mounted in immersion oil on glass slides with the lower surface uppermost. The numbers of cells that had migrated to the lower surface of the filter in two (neutrophils) or five (eosinophils) fields of view at a magnification of × 400 were counted. The mean of triplicate determinations for each concentration of chemoattractant or medium was then calculated, and the results expressed as n u m b e r o f cells per 0-1 m m 2 or 0.3 mm2 for neutrophils and eosinophils, respectively.
Agonist-induced leucocyte migration In preliminary experiments the effects of PAF (10-9 to 10-5 M) on equine neutrophil migration were examined in the 48-well microchemotaxis assembly. ZAe, which had previously been shown to cause a marked neutrophil migration in the Boyden chamber system (Dawson et al 1988), was used as a positive control. The effect of the biologically inactive precursor and metabolite of PAF, lyso-PAF, was also examined at a concentration that, for eAF, had been found to cause maximal migration. To show that neutrophil migration to PAF was a specific receptor mediated event, the selective receptor antagonist WEB 2086 (6.6 x 10-8 to 6.6 x 10-6 M) in MEM/CA was admixed with the cell population and migration responses compared to those obtained in the absence of the antagonist. The specificity of the inhibitory activity was determined by comparing the effects of WEB 2086 on PAF with those on ZAP-induced responses. In a further series of experiments eosinophils and neutrophils were prepared from the same donor horses and the effects of PAF (10 8 to 104 M) compared to those of LTB4 (10-8 to 10-6 M).
were dose related. A 5 per cent significance level was chosen.
Results PAF induced a dose related migration of equine neutrophils in the microchemotaxis chamber over the concentration range 10-8 to 10-s M, maximal responses being observed at 10 -6 M (Fig 1). The somewhat smaller response to 10-5 M PAF suggests a bell-shaped concentration-response relationship, although higher concentrations of PAF were not used to confirm this. Lyso-PAF (10 -6 M) was without effect upon neutrophil migration (49 _+ 8 compared with 46 _+ 11 neutrophils/0.1 m m 2 for lyso-PAF and MEM/ESA, respectively, mean + SEM, n = 6). Inhibition of the migratory responses of equine neutrophils to PAY in the presence of WEB 2086 was investigated in two experiments. In the first, WEB 2086 (6"6 × 10-6 M) abolished the neutrophil migration response to PAF (10 6 M). Lower concentrations of WEB 2086
220"
18
14
In each experiment the mean + SEM results were calculated from values obtained using different horses. Results were analysed using a twotailed Student's paired t test, although analysis of variance followed by a studentised range test was used to establish that migratory responses
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20
Statistical analysis
225
,
,
10-9
10-8
,
,
10-7
10 6
!04
PAF [M]
FIG 1: Effects of PAF ( [ ~ ) and 25 per cent ZAP ( A ) on neutrophil migration in the 48-well microchemotaxis chamber. (Q) Random migration to MEM/ESA (control). Results are expressed as means ± SEM, n = 6 neutrophil preparations from different horses. *P<0-05; ** P<0-01 compared with MEM/ESA
226
A. P. Foster, P. Lees, F. M. Cunningham 240
E E ,"7 O oo
240
(a)
180
180
120
120
60
60
(b)
§ Z
# ,
i
MEM/ 10 -8 ESA
10-7
, 10 -6
10-5
0 MEM/ ESA
PAF [M]
10--8
10-7
10--6
LTB4[M]
FIG 2: The effects of PAF(a) and LTB4 (b) on neutrophil migration in the 48-well microchemotaxis chamber. ( 0 ) Random migration in response to MEM/ESA (control). Results are expressed as means + SEM, n = 6 preparations from different horses. ** P<0.01 compared with MEM/ESA
were either less effective (6.6 x 10-7 M) or without effect (6.6 x 10-s M) (Table 1). In the second study the selectivity of the action of WEB 2086 was investigated by comparing the migratory responses of neutrophils to ZAP and PAF, both in the presence and in the absence of WEB 2086. The antagonist, at a concentration of 6.6 x 10-6 M, markedly reduced the response to PAF but failed to modify ZAP-induced migration (Table 2). As illustrated in Fig 2a and b LTB4, as well as PAY, caused migration of equine neutrophils, although the maximum response to LTB 4 w a s observed at a 10-fold lower concentration than PAF. In contrast to earlier reports using human and equine eosinophils (Potter et al 1985, Wardlaw et al 1986) La'B4,at similar concentrations to those used to stimulate equine neutrophil
migration, also induced a marked migratory response in highly purified equine eosinophils. Furthermore, the maximum response to LTB 4 w a s significantly greater than that to PAF (P<0-05; Student's paired t test; Fig 3a and b). Discussion In this study of in vitro neutrophil and eosinophil chemotaxis a 48-well microchemotaxis assembly has been used for the first time to measure the response of equine leucocytes to the potent chemoattractants PAF and LTB4. There are several advantages of this system over the Boyden chamber apparatus which has been more commonly used in previous studies with equine cells. First, both the volume of chemoattractant and the number of cells required are low, and this TABLE 2: Effect of wEs 2086 on PAF-induced neutrophil migration
TAB LE 1 : Effect of WEB 2086 on PAF-induced neutrophil migration
Lower chamber
Neutrophils (/0.1 mm 2)
Lower chamber
Neutrophils (/0.1 mm2)
Control (MEM/ESA) 10-6 M PAF 10-8 M PAF + 6"6 x 10-6 M WEB 2086 10 6 M PAF + 6"6 x 10 7 M WEB 2086 10-6 M PAF + 6"6 x 10-8 M WEB 2086
37 -+ 15 158 _+30 183 _+33 115+24 49 -+13
Control (MEM/ESA) 6.6 x 10-6 M WEB 2086 25% ZAP 25% ZAP + 6"6 x 10 6 M WEB 2086 10-8 M PAF 10 6 M PAF + 6"6 x 10-8 M WEB 2086
39 -+ 12 41 _+12 152 _+46 145 _+48 167 _+31 *'57_+15
Results are expressed as means _+SEM, n = 4 neutrophil preparations from different horses
Results are expressed as means + SEM, n = 6 neutrophil preparations from different horses ** P<0.01 compared with PAF alone
PAF-induced equine leucocyte 200
227
(b)
(a) 200
150 160
E E o
100 100
kLl
50 50
O i
MEM/10 -8
i
i
i
10 7
10 6
10 s
ESA
0
0 i
MEM/ 10 8
10 7
10-6
ESA
PAF[M]
LTB4[M]
FIG 3: Effects of PAF (a) and LTB4 (b) on eosinophil migration in the 48-well microchemotaxis chamber. (O) Random migration in response to MEM/ESA. Results are expressed as mean + SEN, n = 6 preparations from different horses. * P<0-05 and ** P<0"01 compared with MEM/ESA
facilitates studies of cell types like the eosinophil that are difficult to isolate in large numbers. Moreover, the characterisation of small quantities ofchemoattractants obtained, for example, from biological fluids, is made possible. In addition, it has been suggested that the migration responses obtained using the microchemotaxis assembly are more reproducible since the small size ensures that the conditions are uniform throughout the chamber during the incubation period (Falk et al 1980). Dawson et al (1988) previously reported that, although concentrations of PAF between 2 x 10-7 M and 2 x 104 M induced migration of equine neutrophils in a modified Boyden chamber, the responses were not dose related. In addition, at 2 x 10 8 M and 2 × 10-9 M, the distance moved by leading front cells was significantly reduced when compared to random migration. In contrast, in the present study, in which migration responses have been assessed as the number of cells migrating to the lower surface of the filter, PAF was found to cause dose related responses of neutrophils over the range 10-8 to 10.6 M and no inhibition of migration occurred with low concentrations (10 -9 and 10 -s M) o f PAF. Furthermore, the dose response curve was bellshaped since 10-5 M PAF produced a response that was smaller than the maximum response to
10 -6 M PAF. Although the microchemotaxis chamber does not distinguish between chemotactic and chemokinetic responses, PAF has been shown to induce neutrophil migration in an agarose microdroplet chemokinesis assay (Dawson et al 1988). Hence, in part, the effects of PAF obtained in experiments using the microchemotaxis assembly are likely to be attributable to its chemokinetic activity. The present findings extend those of Dawson et al (1988) in demonstrating that PAy-induced migration of equine neutrophils is mediated by activation of a specific receptor, since responses to PAF, but not ZAP, were reduced in the presence of the competitive and specific PAF receptor antagonist WEB 2086. This confirms in vivo data demonstrating that local administration of the antagonist inhibited neutrophil accumulation in equine skin (Foster et al 1992b). The effects Of PAF and LTB4 have not previously been compared directly using the same population of equine neutrophils. When, in this study, such a comparison was carried out, no significant difference was seen between the m a x i m u m responses to the two chemoattractants, although LTB4 induced its maximal effect at a 10-fold lower concentration. Similar observations have been made with human neutrophils (Wardlaw et al 1986).
228
A. P. Foster, P. Lees, F. M. Cunningham
Enhanced migration of equine eosinophils in response to PAFhas not previously been described, although it has been reported that these cells responded to a range of HETES. Moreover, only weak migratory responses to LTB 4 have been observed (Potter et al 1985). In the present study, however, not only did LTB4 cause significant equine eosinophil migration, a maximal effect being obtained at 10-7 M, but the maximal response was significantly greater than that obtained with a 10-fold higher concentration of PAF. A higher chemotactic index (ratio between the number of cells migrating through a filter in the presence of the agonist and the number migrating in medium alone) was obtained for LTB4 than for PAF when the migration of guinea pig peritoneal eosinophils was examined, the response to PAF being maximal at 10-8 M (Coeffier et al 1991). Similar chemotactic indices have also been reported for PAF and LTB4 when human eosinophils were studied, but the concentration of LTB4 required to produce the same response was 100-fold lower (Coeffier et al 1990). In contrast to these findings, Wardlaw et al (1986) have found that LTB4only induced a modest migratory response of human eosinophils, whereas PAF was a potent chemoattractant for these cells. Despite the apparent differences in the potencies and stimulatory capacities of PAF and LTB4that have been observed in in vitro chemotaxis assays using cells from different species, the results obtained in the present study demonstrate that both chemoattractants cause the migration of equine neutrophils and eosinophils. Moreover, the concentration of PAF required to elicit a maximal response (10 -6 M) was the same for neutrophils and eosinophils. It is of interest, therefore, that injection of PAF into normal equine skin led to neutrophil, but not eosinophil, accumulation in vivo (Foster et al 1990, 1992b). Possible explanations for the difference between in vitro and in vivo studies include the inability to detect a response histologically because of the low cell numbers in the circulation, the state of activation of the eosinophils or perhaps the failure of PAF to induce eosinophil-endothelial cell interactions via the expression of adhesion molecules. Significant eosin0Phil recruitment has, on the other hand, been observed in the skin of allergic horses and atopic human subjects following the injection of PAF (Henocq and Vargaftig 1988, Foster et al 1992a). Thus, the release of PAF or LTB4 in vivo
during inflammatory or allergic reactions could be involved in the recruitment of leucocytes to the site of damage. To date, both LTB4 and lysoPAF have been identified in exudates arising from the subcutaneous implantation of irritant-soaked polyester sponges in horses (Higgins and Lees 1984, Lees et al 1990).
Acknowledgements A.P.F. is a Horserace Betting Levy Board training scholar. We are also grateful to Boehringer Ingelheim Vetmedica for financial support.
References ARCHER, C. B., PAGE, C. P., MORLEY, J. & MACDONALD, D. M. (1985) Accumulation of inflammatory cells in response to intracutaneous platelet activating factor (Paf-acether) in man. British Journal of Dermatology 112, 285-290 BARNES, P. J., CHUNG, K. F. & PAGE, C. P. (1988) Inflammatory mediators and asthma. Pharmacological Reviews 40, 49-84 CAMP, C. J. & LEID, R. J. (1982) Chemotaxis of radiolabelled equine neutrophils. American Journal of Veterinary Research 43, 397-401 COEFFIER, E., BARAU, E., JOSEPH, D., DUPONT, C. & VARG A F T I G , B. B. (1990) PAF-aeether and LTB4, two potent chemotactic factors for guinea-pig and human eosinophils in vitro. Journal of Lipid Mediators 2, 237 COEFFIER, E., JOSEPH, D. & VARGAFTIG, B. B. (1991) LTB4, a potent chemotactic factor for purified guinea pig eosinophils: interference of Pav-acether antagonists. International Journal of Immunopharmaeology 13, 273-280 DAWSON, J., LEES, P. & SEDGWICK, A. D. (1988) Platelet activating factor as a mediator of equine cell locomotion. Veterinary Research Communications 12, 101-107 FALK, W., GOODWIN, R. H. & LEONARD, E. J. (1980) A 48well microchemotaxis assembly for rapid and accurate measurement of leukocyte migration. Journal of Immunological Methods 33, 239247 FOSTER, A. P., CUNNINGHAM, F. M. & LEES, P. (1990) The inflammatory effects of PAF in equine skin. British Journal of Pharmacology 99, 86P FOSTER, A., CUNNINGHAM, F. & LEES, P. (1992a) PAF:A putative mediator of allergic skin disease in the horse. Clinical and Experimental Allergy 22, 129 FOSTER, A. P., LEES, P., ANDREWS, M. A. & CUNNINGHAM, F. M. (1992b) Effects of WEn 2086, an antagonist to the receptor for platelet-activating factor (PAF), on PAz-induced responses in the horse. Equine Veterinary Journal 24, 203-207 HARVATH, L., FALK, W. & LEONARD, E. J. (1980) Rapid quantitation of neutrophil chemotaxis: Use of a polyvinylpyrrolidonefree polycarbonate membrane in a multiwell assembly. Journal of Immunological Methods 37, 39-45 HENOCQ, E. & VARGAFTIG, B. B. (1988) Skin eosinophilia in atopic patients. Journal of Allergy and Clinical Immunology 81, 691-695 HIGGINS, A. J. & LEES, P. (1984) Detection of leukotriene B4 in equine inflammatory exudate. Veterinary Record 115, 275 LEES, P., DAWSON, J. & SEDGWICK, A. D. (1986) Eicosanoids and equine leucocyte locomotion in vitro. Equine Veterinary Journal 18, 493-497 LEES, P., MAY, S. A., CAMBRIDGE, H., HOOKE, R. E., RUSSELL, C. S., DAWSON, J. & ANDREWS, M. J. (1990) Pathophysiology of inflammation and joint diseases in the horse: Mechanisms, mediators and medicines. In Veterinary Pharmacology
•PAF-induced equine leucocyte Toxicology and Therapy in Food Producing Animals, Proceedings of the 4th Congress of the European Association for Veterinary Pharmacology and Toxicology Ed F. Simon, P. Lees & G. Semjen. Budapest: Unipharma. pp197-204 POTTER, K. A., LEID, R. W., KOLATTUKUDY, P. E. & ESPELIE, K. E. (1985) Stimulation of equine eosinophil migration by hydroxyacid metabolites of arachidonic acid. American Journal of Pathology 121, 361-368 SNYDERMAN, R. & PIKE, M. C. (1980) N-Formylmethionyl peptide receptors on equine leukocytes initiate secretion but not chemotaxis. Science 209, 493-494
229
WARDLAW, A. J., MOQBEL, R., CROMWELL, O. & KAY, A. B. (1986)Platelet-activating factor. A potent chemotactic and chemokinetic factor for human eosinophils. Journal of Clinical Investigation 78, 1701-1706 ZINKL, J. G. & BROWN, P. D. (1982) Chemotaxis of horse polymorphonuclear leucocytes to N-formyl-L-methionyl-L-leucyl-Lphenylalanine. American Journal of Veterinary Reserach 43, 613-616 Received October 24, 1991 Accepted March 10, 1992
Platelet activating factor is a mediator of equine neutrophil and eosinophil migration in vitro A. P. FOSTER, P. LEES, F. M. CUNNINGHAM, Department of Veterinary Basic Sciences,
The Royal Veterinary College, Hawkshead Lane, North Mymms, Hertfordshire, AL9 7TA
Platelet activating factor (PAF) is known to be a chemoattractant for equine neutrophils in vivo and in vitro. In this study the in vitro migratory response of equine eosinophils and neutrophils to PAF has been examined and compared with that to leukotriene (LT)B4. PAF (10- s to 10- s M), but not lyso-eAF (10 -6 M), caused dose related migration of both equine cosinophils and neutrophils, maximal responses occurring at 10 -6 M. Responses to PAF were inhibited by the receptor antagonist WEB 2086. LTa4 (10 -s to 10 -6 M) also induced migration of both cell types, although the maximum effect was observed with a 10-fold lower concentration. Moreover, the m a x i m u m response of equine eosinophils to LTB4 was significantly greater than to PAF. It is concluded that LTB4 and PAF, if released in vivo at sites of allergic or inflammatory reactions, could mediate the recruitment of leucocytes to the involved tissue.
Moreover, the concentrations that elicit maximal responses are similar to those required for human neutrophils. The chemotactic responses of equine eosinophils have not been extensively studied. Potter et al (1985) reported in vitro eosinophil migration towards a range of arachidonic acid derived monohydroxyfatty acids (HETEs)and LTB4 using cell populations of between 30 and 70 per cent purity. Only modest chemotactic responses were obtained with LTB4,a finding which agrees with results obtained by Wardlaw et al (1986) using semi-pure eosinophil populations prepared from human patients with eosinophilia. In contrast, in the same study, PAF elicited a time- and dosedependent directional migration of human eosinophils and it was suggested that PAF is a potent chemotactic factor for these cells. As has been demonstrated in man (Archer et al 1985), intradermal injection of PAF in normal THE in vitro and in vivo chemoattractant prop- horses causes neutrophil, but not eosinophil, erties ofplatelet activating factor (PAF)for inflam- recruitment to the skin (Foster et al 1990). Prematory cells such as the neutrophil have been liminary evidence has, however, been obtained well documented (Barnes et a11988). Early studies to show that PAF injection into the skin of horses suggested that equine neutrophils might differ with the allergic skin disease sweet itch does from those of other species in their migratory induce an accumulation of eosinophils (Foster responses to chemotactic factors since the bac- et al 1992a). A similar finding was reported in terial tripeptide, formyl-methionyl-leucyl-phenyl- atopic human subjects after injection OfPAF,antialanine (FMLP), either did not cause movement gen or FMLP (Henocq and Vargaftig 1988). of equine cells, or produced a response only at In this study the in vitro chemoattractant activvery high concentrations (Snyderman and Pike ity of PAF for equine neutrophils has been exam1980, Camp and Leid 1982, Zinkl and Brown ined in microchemotaxis chambers. PAF-induced 1982). Any difference does, however, appear to migratory responses have been compared to those be restricted to FMLP receptor-mediated events of LTB4 using neutrophils from the same donor since equine neutrophils respond to zymosan acti- horses since the relative potency of these comvated serum or plasma, PAF and the arachidonic pounds for neutrophil movement has not been acid lipoxygenase product, leukotriene (LT)B4 in established using cells from the same horses. The vitro (Lees et al 1986, Dawson et al 1988). effect of PAF on equine eosinophil migration has 223
224
A. P. Foster, P. Lees, F. M. Cunningham
also been studied for the first time and compared to that of LTB4,which has previously been reported to be a weak stimulant of equine eosinophil migration (Potter et al 1985). Materials and methods
Materials C16:0 PAF and C16:0 lyso-eAF were obtained from Bachem UK. Stock solutions (1 mg m1-1) were prepared in ethanol and stored at -20°C. LTB4 (100 ~tg m1-1 in methanol) was kindly supplied by Dr A. W. Ford-Hutchinson, Merck Frosst Canada Inc and stored at -70°C. Serial dilutions of both PAF and LTB4 were prepared by evaporation of the solvent under nitrogen and resuspension of the residue in Eagle's minimal essential medium containing 0.4 per cent equine serum albumin (MEM/ESA). Zymosan activated equine plasma (zM') was prepared by incubation of freshly prepared heparinised plasma with 1 mg m1-1 zymosan A for 30 minutes at 37°C. The plasma was then inactivated by heating to 56°C for 45 minutes, the zymosan removed by centrifugation at 1400 g for 10 minutes and samples stored at -20°C until required. Dilutions of ZAP were prepared in MEM/ESA.WEB 2086 was kindly supplied by Boehringer Ingelheim Vetmedica, Germany. Hank's balanced salt solution (HBSS) was obtained from Life Technologies, uK and Percoll from Pharmacia, uK. All other chemicals and solvents were obtained from Sigma Chemical, UK or BDH, UI(, and were of reagent grade.
minutes at 4°C and the neutrophils recovered in a band between the 70 per cent and 85 per cent Percoll solutions. The cells were washed twice in MEMcontaining 0-003 per cent deoxyribonuclease (DNase) I and finally resuspended at a concentration of 5 x 106 ml-1 in M~M containing 0'4 per cent chick albumin (CA). The purity and viability of the cells were at least 99 per cent (n = 11 determinations). Viability was assessed using trypan blue dye exclusion.
Purification of equine eosinophils Leucocytes were separated from 100 ml jugular venous blood samples as described above. After resuspension in 10 ml leucocyte poor plasma the cells were layered on to a 70:85:100 per cent Percoll gradient. Following centrifugation at 400 g for 15 minutes at 4°C, the eosinophils and erythrocytes were found at the 85:100 per cent Percoll interface and dispersed through the 100 per cent Percoll layer. The appropriate layers containing the eosinophils and erythrocytes were recovered, washed in MEM containing 0.003 per cent DNase I, the erythrocytes lysed by hypotonic shock (0.2 per cent saline [10 ml] for 30 seconds, followed by the addition of an equal volume of 1-6 per cent saline) and the eosinophils washed again in MEM containing DNase I. The cells Were finally resuspended in MEM/CA at a concentration of 5 x 106 ml-1. The purity of the cells was at least 99 per cent and the viability was greater than 93 per cent (n = 6 determinations).
Chemotaxis assay Separation of equine neutrophils Stock solutions of Percoll were prepared by mixing nine parts Percoll with one part 10 ti=,'_'~es concentrated, calcium and magnesium free HBSS. Further dilutions of this stock were made with HBSS to give Percoll solutions of the required density and the pH adjusted to pH 7-4. Blood samples (40 ml) were then collected from the jugular veins of normal horses into 0-4 M EDTA (1:39). The blood was allowed to sediment for 20 minutes at room temperature and the leucocyte rich plasma harvested. After centrifugation (200 g, 4°C, five minutes) the cell pellet was resuspended in 5 ml of leucocyte poor plasma and layered on to a 70:85 per cent Percoll gradient. The gradients were centrifuged at 400 g for 15
MEM/ESA or chemoattractants dissolved in MEM/ESA (26.5 ~d) were placed in the bottom wells of 48-well microchemotaxis chambers (Falk et al 1980, Harvath et al 1980). Cellulose nitrate filters (100 pm thick; 8 prn pore size) which had been soaked for five minutes in MEM were placed over the top of the wells. A silicon gasket was laid over the filter and the top of the chamber secured in place. Fifty ~d of cell suspension was next added to each of the top wells and the chambers placed in a humidified box within an incubator at 37°C. After incubation for 1.5 hours (neutrophils) or three hours (eosinophils) the filters were removed, the upper surface wiped free of non-adherent cells and fixed in 10 per cent formalin for five minutes.
eAr-induced equine leucocyte The filters were then stained with haemalum, rinsed in distilled water and blued in 1 per cent a m m o n i u m hydroxide. After d e h y d r a t i o n in alcohol the filters were cleared in xylene and mounted in immersion oil on glass slides with the lower surface uppermost. The numbers of cells that had migrated to the lower surface of the filter in two (neutrophils) or five (eosinophils) fields of view at a magnification of × 400 were counted. The mean of triplicate determinations for each concentration of chemoattractant or medium was then calculated, and the results expressed as n u m b e r o f cells per 0-1 m m 2 or 0.3 mm2 for neutrophils and eosinophils, respectively.
Agonist-induced leucocyte migration In preliminary experiments the effects of PAF (10-9 to 10-5 M) on equine neutrophil migration were examined in the 48-well microchemotaxis assembly. ZAe, which had previously been shown to cause a marked neutrophil migration in the Boyden chamber system (Dawson et al 1988), was used as a positive control. The effect of the biologically inactive precursor and metabolite of PAF, lyso-PAF, was also examined at a concentration that, for eAF, had been found to cause maximal migration. To show that neutrophil migration to PAF was a specific receptor mediated event, the selective receptor antagonist WEB 2086 (6.6 x 10-8 to 6.6 x 10-6 M) in MEM/CA was admixed with the cell population and migration responses compared to those obtained in the absence of the antagonist. The specificity of the inhibitory activity was determined by comparing the effects of WEB 2086 on PAF with those on ZAP-induced responses. In a further series of experiments eosinophils and neutrophils were prepared from the same donor horses and the effects of PAF (10 8 to 104 M) compared to those of LTB4 (10-8 to 10-6 M).
were dose related. A 5 per cent significance level was chosen.
Results PAF induced a dose related migration of equine neutrophils in the microchemotaxis chamber over the concentration range 10-8 to 10-s M, maximal responses being observed at 10 -6 M (Fig 1). The somewhat smaller response to 10-5 M PAF suggests a bell-shaped concentration-response relationship, although higher concentrations of PAF were not used to confirm this. Lyso-PAF (10 -6 M) was without effect upon neutrophil migration (49 _+ 8 compared with 46 _+ 11 neutrophils/0.1 m m 2 for lyso-PAF and MEM/ESA, respectively, mean + SEM, n = 6). Inhibition of the migratory responses of equine neutrophils to PAY in the presence of WEB 2086 was investigated in two experiments. In the first, WEB 2086 (6"6 × 10-6 M) abolished the neutrophil migration response to PAF (10 6 M). Lower concentrations of WEB 2086
220"
18
14
In each experiment the mean + SEM results were calculated from values obtained using different horses. Results were analysed using a twotailed Student's paired t test, although analysis of variance followed by a studentised range test was used to establish that migratory responses
"i I,
6 03
20
Statistical analysis
225
,
,
10-9
10-8
,
,
10-7
10 6
!04
PAF [M]
FIG 1: Effects of PAF ( [ ~ ) and 25 per cent ZAP ( A ) on neutrophil migration in the 48-well microchemotaxis chamber. (Q) Random migration to MEM/ESA (control). Results are expressed as means ± SEM, n = 6 neutrophil preparations from different horses. *P<0-05; ** P<0-01 compared with MEM/ESA
226
A. P. Foster, P. Lees, F. M. Cunningham 240
E E ,"7 O oo
240
(a)
180
180
120
120
60
60
(b)
§ Z
# ,
i
MEM/ 10 -8 ESA
10-7
, 10 -6
10-5
0 MEM/ ESA
PAF [M]
10--8
10-7
10--6
LTB4[M]
FIG 2: The effects of PAF(a) and LTB4 (b) on neutrophil migration in the 48-well microchemotaxis chamber. ( 0 ) Random migration in response to MEM/ESA (control). Results are expressed as means + SEM, n = 6 preparations from different horses. ** P<0.01 compared with MEM/ESA
were either less effective (6.6 x 10-7 M) or without effect (6.6 x 10-s M) (Table 1). In the second study the selectivity of the action of WEB 2086 was investigated by comparing the migratory responses of neutrophils to ZAP and PAF, both in the presence and in the absence of WEB 2086. The antagonist, at a concentration of 6.6 x 10-6 M, markedly reduced the response to PAF but failed to modify ZAP-induced migration (Table 2). As illustrated in Fig 2a and b LTB4, as well as PAY, caused migration of equine neutrophils, although the maximum response to LTB 4 w a s observed at a 10-fold lower concentration than PAF. In contrast to earlier reports using human and equine eosinophils (Potter et al 1985, Wardlaw et al 1986) La'B4,at similar concentrations to those used to stimulate equine neutrophil
migration, also induced a marked migratory response in highly purified equine eosinophils. Furthermore, the maximum response to LTB 4 w a s significantly greater than that to PAF (P<0-05; Student's paired t test; Fig 3a and b). Discussion In this study of in vitro neutrophil and eosinophil chemotaxis a 48-well microchemotaxis assembly has been used for the first time to measure the response of equine leucocytes to the potent chemoattractants PAF and LTB4. There are several advantages of this system over the Boyden chamber apparatus which has been more commonly used in previous studies with equine cells. First, both the volume of chemoattractant and the number of cells required are low, and this TABLE 2: Effect of wEs 2086 on PAF-induced neutrophil migration
TAB LE 1 : Effect of WEB 2086 on PAF-induced neutrophil migration
Lower chamber
Neutrophils (/0.1 mm 2)
Lower chamber
Neutrophils (/0.1 mm2)
Control (MEM/ESA) 10-6 M PAF 10-8 M PAF + 6"6 x 10-6 M WEB 2086 10 6 M PAF + 6"6 x 10 7 M WEB 2086 10-6 M PAF + 6"6 x 10-8 M WEB 2086
37 -+ 15 158 _+30 183 _+33 115+24 49 -+13
Control (MEM/ESA) 6.6 x 10-6 M WEB 2086 25% ZAP 25% ZAP + 6"6 x 10 6 M WEB 2086 10-8 M PAF 10 6 M PAF + 6"6 x 10-8 M WEB 2086
39 -+ 12 41 _+12 152 _+46 145 _+48 167 _+31 *'57_+15
Results are expressed as means _+SEM, n = 4 neutrophil preparations from different horses
Results are expressed as means + SEM, n = 6 neutrophil preparations from different horses ** P<0.01 compared with PAF alone
PAF-induced equine leucocyte 200
227
(b)
(a) 200
150 160
E E o
100 100
kLl
50 50
O i
MEM/10 -8
i
i
i
10 7
10 6
10 s
ESA
0
0 i
MEM/ 10 8
10 7
10-6
ESA
PAF[M]
LTB4[M]
FIG 3: Effects of PAF (a) and LTB4 (b) on eosinophil migration in the 48-well microchemotaxis chamber. (O) Random migration in response to MEM/ESA. Results are expressed as mean + SEN, n = 6 preparations from different horses. * P<0-05 and ** P<0"01 compared with MEM/ESA
facilitates studies of cell types like the eosinophil that are difficult to isolate in large numbers. Moreover, the characterisation of small quantities ofchemoattractants obtained, for example, from biological fluids, is made possible. In addition, it has been suggested that the migration responses obtained using the microchemotaxis assembly are more reproducible since the small size ensures that the conditions are uniform throughout the chamber during the incubation period (Falk et al 1980). Dawson et al (1988) previously reported that, although concentrations of PAF between 2 x 10-7 M and 2 x 104 M induced migration of equine neutrophils in a modified Boyden chamber, the responses were not dose related. In addition, at 2 x 10 8 M and 2 × 10-9 M, the distance moved by leading front cells was significantly reduced when compared to random migration. In contrast, in the present study, in which migration responses have been assessed as the number of cells migrating to the lower surface of the filter, PAF was found to cause dose related responses of neutrophils over the range 10-8 to 10.6 M and no inhibition of migration occurred with low concentrations (10 -9 and 10 -s M) o f PAF. Furthermore, the dose response curve was bellshaped since 10-5 M PAF produced a response that was smaller than the maximum response to
10 -6 M PAF. Although the microchemotaxis chamber does not distinguish between chemotactic and chemokinetic responses, PAF has been shown to induce neutrophil migration in an agarose microdroplet chemokinesis assay (Dawson et al 1988). Hence, in part, the effects of PAF obtained in experiments using the microchemotaxis assembly are likely to be attributable to its chemokinetic activity. The present findings extend those of Dawson et al (1988) in demonstrating that PAy-induced migration of equine neutrophils is mediated by activation of a specific receptor, since responses to PAF, but not ZAP, were reduced in the presence of the competitive and specific PAF receptor antagonist WEB 2086. This confirms in vivo data demonstrating that local administration of the antagonist inhibited neutrophil accumulation in equine skin (Foster et al 1992b). The effects Of PAF and LTB4 have not previously been compared directly using the same population of equine neutrophils. When, in this study, such a comparison was carried out, no significant difference was seen between the m a x i m u m responses to the two chemoattractants, although LTB4 induced its maximal effect at a 10-fold lower concentration. Similar observations have been made with human neutrophils (Wardlaw et al 1986).
228
A. P. Foster, P. Lees, F. M. Cunningham
Enhanced migration of equine eosinophils in response to PAFhas not previously been described, although it has been reported that these cells responded to a range of HETES. Moreover, only weak migratory responses to LTB 4 have been observed (Potter et al 1985). In the present study, however, not only did LTB4 cause significant equine eosinophil migration, a maximal effect being obtained at 10-7 M, but the maximal response was significantly greater than that obtained with a 10-fold higher concentration of PAF. A higher chemotactic index (ratio between the number of cells migrating through a filter in the presence of the agonist and the number migrating in medium alone) was obtained for LTB4 than for PAF when the migration of guinea pig peritoneal eosinophils was examined, the response to PAF being maximal at 10-8 M (Coeffier et al 1991). Similar chemotactic indices have also been reported for PAF and LTB4 when human eosinophils were studied, but the concentration of LTB4 required to produce the same response was 100-fold lower (Coeffier et al 1990). In contrast to these findings, Wardlaw et al (1986) have found that LTB4only induced a modest migratory response of human eosinophils, whereas PAF was a potent chemoattractant for these cells. Despite the apparent differences in the potencies and stimulatory capacities of PAF and LTB4that have been observed in in vitro chemotaxis assays using cells from different species, the results obtained in the present study demonstrate that both chemoattractants cause the migration of equine neutrophils and eosinophils. Moreover, the concentration of PAF required to elicit a maximal response (10 -6 M) was the same for neutrophils and eosinophils. It is of interest, therefore, that injection of PAF into normal equine skin led to neutrophil, but not eosinophil, accumulation in vivo (Foster et al 1990, 1992b). Possible explanations for the difference between in vitro and in vivo studies include the inability to detect a response histologically because of the low cell numbers in the circulation, the state of activation of the eosinophils or perhaps the failure of PAF to induce eosinophil-endothelial cell interactions via the expression of adhesion molecules. Significant eosin0Phil recruitment has, on the other hand, been observed in the skin of allergic horses and atopic human subjects following the injection of PAF (Henocq and Vargaftig 1988, Foster et al 1992a). Thus, the release of PAF or LTB4 in vivo
during inflammatory or allergic reactions could be involved in the recruitment of leucocytes to the site of damage. To date, both LTB4 and lysoPAF have been identified in exudates arising from the subcutaneous implantation of irritant-soaked polyester sponges in horses (Higgins and Lees 1984, Lees et al 1990).
Acknowledgements A.P.F. is a Horserace Betting Levy Board training scholar. We are also grateful to Boehringer Ingelheim Vetmedica for financial support.
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•PAF-induced equine leucocyte Toxicology and Therapy in Food Producing Animals, Proceedings of the 4th Congress of the European Association for Veterinary Pharmacology and Toxicology Ed F. Simon, P. Lees & G. Semjen. Budapest: Unipharma. pp197-204 POTTER, K. A., LEID, R. W., KOLATTUKUDY, P. E. & ESPELIE, K. E. (1985) Stimulation of equine eosinophil migration by hydroxyacid metabolites of arachidonic acid. American Journal of Pathology 121, 361-368 SNYDERMAN, R. & PIKE, M. C. (1980) N-Formylmethionyl peptide receptors on equine leukocytes initiate secretion but not chemotaxis. Science 209, 493-494
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WARDLAW, A. J., MOQBEL, R., CROMWELL, O. & KAY, A. B. (1986)Platelet-activating factor. A potent chemotactic and chemokinetic factor for human eosinophils. Journal of Clinical Investigation 78, 1701-1706 ZINKL, J. G. & BROWN, P. D. (1982) Chemotaxis of horse polymorphonuclear leucocytes to N-formyl-L-methionyl-L-leucyl-Lphenylalanine. American Journal of Veterinary Reserach 43, 613-616 Received October 24, 1991 Accepted March 10, 1992