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Chemokinetic and chemotactic activity of various prostaglandins for neutrophil granulocytes
Chemokinetic and Chemotactic Activity of Various Prostaglandins for Neutrophil Granulocytes GERD TILL,
ECKHARD
KOWNATZKI,
DIETHARD Institute
of Immunoloyy.
University
Received
MICHAEL
Szrrz,
AND
GEMSA
August
of Heidelberg.
Germany
3 I, 1978
Prostaglandins (PGs) of the E, A. and F type were assayed for chemokinetic and chemotactic activity in vitro. In the absence of bovine serum albumin (BSA) which is chemokinetically active, PGs of the F type (lo-’ to lo-” M) induced a chemokinetic and chemotactic response in rabbit peritoneal neutrophils. Under the same conditions, PGs of the E and A type had no significant effect on cell locomotion. However, in the presence of BSA (1 mg/ml), PGE and PGA induced a chemotactic response, thus indicating that they lack chemokinetic but possess chemotactic activity. In the absence of BSA, PGE, caused an increase in cyclic AMP, which was not observed when BSA was present. This supports previous findings that an increase of cyclic AMP is associated with inhibition of chemotactic locomotion. Since PGs are released by phagocytosing leukocytes and since albumin is generally available in inflamed tissues, the results suggest that PGs may contribute to the inflammatory accumulation of neutrophils
INTRODUCTION
During the past years, prostaglandins (PGs) of various types have been implicated to participate in acute inflammatory responses. They were found to be increased in inflamed tissues (l-4) and their presence has been correlated with leukocyte infiltration (5, 6). Furthermore, particular endocytic stimuli have recently been recognized which induced a pronounced release of PGs of the E and F type from neutrophils and macrophages (7-9). However, the effect of various PGs on the development of an inflammatory response is far from clear. On the one hand, proinflammatory properties, such as vasodilation, hyperemia, pain, and accumulation of leukocytes have been described (10, 11). On the other hand, antiinflammatory qualities have been attributed, particularly to PGE, and PGE*, via generation of cyclic AMP, which is considered to be an inhibitory signal leading to the reduction of various metabolic and functional activities of leukocytes (12-15). Recently, we proposed that PGE, and PGE, may be mediators which are responsible for the maintenance of homeostasis in inflammation by exerting in concert pro- as well as antiinflammatory properties (16). The present study was undertaken to solve a long-lasting controversy, whether PGs possess chemotactic activity or inhibit leukocyte migration. In this report we present evidence that PGs are chemotactically active but differ in their chemokinetic properties. MATERIALS Rabbit neutrophils. A neutrophil-rich
AND METHODS
peritoneal exudate was obtained 4 hr after an ip injection of 100 mg of glycogen suspended in 100 ml of sterile saline. The cells were washed twice in Hanks’ balanced salt solution (HBSS), buffered at pH 111 0090-1229/79/010111-08$01.00/O Copyright All rights
0 1979 by Academic Press. Inc. of reproduction in any form reserved.
112
~1‘11.1. E7 Al
7.4 with 26 mM tris(hydroxymethyl)aminomethane. resuspended in HBSS, and adjusted to 2.5 x 10” cells/ml. The cell preparations contained more than 90% neutrophils as determined by staining with May-Griinwald-Giemsa. Chenzofcuis rrssay. Random movement. chemokinesis (increased random locomotion in the presence of chemical agents). and chemotaxis (directed migration toward a chemotactic factor) of rabbit peritoneal neutrophils were assayed by the filter technique ( 17) as modified by Zigmond and Hirsch ( 18). Chemotaxis chambers were set up by adding I ml of test solution and 1 ml of cell suspension (3.5 x 10” cells) to the lower and upper compartments. respectively. Following a 90-min incubation period in humidified air at 37°C the filters were removed, and the distance between the leading front of migrated neutrophils and the nonmigrated cells on top of the filter was measured by light microscopy ( X400) in stained and xylene-cleared cellulose nitrate filters (Schleicher and Schiill, Dassel, Germany) of 3-pm pore size. embedded in Canada balsam (E. Merck. Darmstadt, Germany) between a slide and a coverslip. Chambers were set up in triplicate and the migration distance of neutrophils was determined at five different filter sites. In general. all measured values of triplicate filters differed by no more than 10%. Throughout all experiments casein at a concentration of I mgiml (E. Merck) was used as a positive migration control. Assqfor cyclic AMP. Neutrophils ( 10 x IO”) were suspended in I ml of HBSS or HBSS plus BSA ( I mg/ml) in glass centrifuge tubes. PGE, or PGF,, was added at the start of the incubation which was carried out at 37°C in a rotating water bath set at 100 rpm. After 10 min, the incubation was terminated by centrifuging the tubes for 20 set at 10,000~. The cell pellet was processed for cyclic adenosine 3’, S’-monophosphate (cyclic AMP) determination by the method of Gilman ( 19) with modifications as previously described (10). Reagenrs. HBSS was always freshly prepared, contained 1.8 x IO-” M Ca”‘. 0.8 x 10 mRM Mg’&, 0.22% glucose (w/v), and 76 mM tris(hydroxymethyl)aminomethane, and was adjusted to pH 7.4. Crystalline bovine serum albumin (BSA) was dissolved in HBSS and adjusted to pH 7.4. Prostaglandins (PGs) were kindly provided by Dr. J. Pike. Upjohn, Company (Kalamazoo, Mich.). Prior to the experiments, the PGs were dissolved in ethanol and then diluted in HBSS. The stability of the employed PGs of the E type was confirmed by thin-layer chromatography. The concentration of ethanol in the incubation medium did not exceed 0.4% and did not affect motility or cell viability as tested by trypan blue exclusion. Control incubations without PGs received a similar concentration of ethanol. RESULTS
Ej’ject of PGs on Nerttrophil Locomotior~ When various concentrations of different PGs were added to the lower compartment of a chemotaxis chamber, a dose-dependent increase of neutrophil migration toward PGF,, and PGF.,, was found (Fig. 1). PGs of the E or A type, however, exhibited no significant effect on cell locomotion. The maximal cell response toward PGF occurred at a concentration of lo-> M. Higher concentrations led to a decrease of cell migration which was shown to be an effect of ethanol serving as a solvent for the PGs.
PROSTAGLANDINS
I
AS
CHEMOATTRACTANTS
113
L,, 0
108
,a7
lo6
,05
PG(M) FIL. 1. Effect of prostaglandins (PC&) on neutrophil locomotion. Rabbit peritoneal neutrophils (2.5 x 10”) were suspended in I ml of HBSS and added to the upper compartment of a chemotaxis chamber. Various concentrations of different PGs, diluted in HBSS. were added to the lower compartment and the distance of neutrophil migration was measured after 90 min of incubation. Distance of migration for random locomotion t HBSS) and directional locomotion (casein. I mgiml) were 26 and 160 pm. respectively. Shown is a representative experiment. performed in triplicate. in which the values obtained differed by no more than 10%.
Compared with the distance of migration ( 160 pm) toward casein (1 mg/ml), the PGF-induced neutrophil migration was rather weak. Since serum albumin is known to increase neutrophil movement by its chemokinetic activity (21, 22), we studied its effect on cell migration toward PGs. When BSA at a concentration of 1 mg/ml was added to the cell suspension in the upper compartment. random locomotion of neutrophils increased by approximately 100% as did migration toward PGF (Fig. 2). Surprisingly, however, the PGs of the E and A type also induced a pronounced migration of neutrophils in the presence of BSA (Fig. 2). The maximum migration obtained with all three types of PGs was observed at a concentration of lo-” M. Similar effects were obtained when rabbit or human instead of bovine serum albumin was used. Evidence for Chemotactic
Activity
of PGs
To investigate in more detail whether the observed increase in neutrophil migration was caused by chemotactic attraction of PGs or whether it merely reflected increased random locomotion (chemokinesis), various concentrations of PGEl or PGF, were added to the lower or upper compartment of the chemotaxis chamber and neutrophil migration was tested in the presence or absence of BSA. By using such a system, the influence of both the absolute concentrations and the concentration gradient can be studied, i.e., migration in a positive gradient, in a negative gradient, and in the absence of a gradient. Table 1 shows a checkerboard assay in which neutrophils migrated in various concentrations or gradients of PGF,,. The numbers in the diagonal drawn from upper left to lower right represent migration distances in various absolute concentrations of PGF, in the absence of a gradient. As can be seen, the locomotion increased with the PGF, concentration, i.e., the neutrophils showed a chemokinetic response to PGF,,. Furthermore, it became evident that cells moving in a positive PGF, gradient (below the diagonal) were penetrating deeper into the filter than in the absence of a gradient (diagonal) or in a negative gradient (above the diagonal). These findings clearly show that PGF, can induce both a chemokinetic and a chemotactic response in neutrophils.
114
'IILl
E7‘.4L.
with BSA
E=PEY wlthout
BSA
Et-.
Ii7
d
TO5
PG (MI
wlthout
BSA b------=0-
Ga
0
---d
Id6
Ii7
Iti5
PG IM)
1507
c
with BSA 5 100. s ; PE (I 2 so-‘ L8
f z"
.-<,10,
without +--+-'oco
BSA -P------Y
I
0
FIG,. 2. Effect mgiml). Incubation
of various conditions
PGs on neutrophil locomotion as described in Fig. I legend.
in the presence
or absence
of BSA
PROSTAGLANDINS
AS
TABLE CHECKERBOARD PRESENCE
ASSAY OF VARIOUS
TO STUDY
THE
ABSOLUTE
CONCENTRATIONS
Concentration (M) of PGF,, in the lower compartment
115
CHEMOATTRACTANTS
MIGRATION
1 OF NEUTROPHILS AKD
SUSPENDED
COIUCENTRATION
IN
GRADIENTS
HBSS IK THE OF PGF,;
Concentration
(M) of PGF,, in the cell compartment ,0-” 10-R IO-’ 10-e
0
” Migration (in micrometers) was determined after 90 min of incubation. Values shown are means of four separate experiments in which the values obtained differed by no more than 10%.
A similar checkerboard experiment was performed with PGE,, except that neutrophils were suspended in HBSS plus BSA (I mg/ml) to facilitate migration (Table 2). When compared to the data presented in Table 1, it is evident that BSA enhanced random locomotion and, furthermore, that with increasing concentrations of PGE, in both chemotaxis chambers (diagonal) no increase but rather a slight depression of migration occurred, i.e., PGE, failed to induce a chemokinetic response. However, a completely different picture evolved when neutrophils were allowed to migrate in a positive concentration gradient (below the diagonal) in which case the cells were attracted by PGE, in a dose-dependent manner. These data demonstrate that PGE, was able to cause only a chemotactic response of neutrophils. Not shown are checkerboard assays demonstrating that PGF,. PGE,, PGAI, and PGA, were also chemotactically active but they differed in their ability to induce chemokinesis which was exclusively found in the PGs of the F class. Effect of PGs on Intracellular
Concentrations of Cyclic AMP
PGs of the E type have repeatedly been described as inhibitors of chemotaxis (14, 23, 15) because of the generation of an inhibitory signal, cyclic AMP, via a TABLE
Z
CHECKERBOARD ASSAY TO STUDY THE MIGRATIOX OF NEUTROPHILS SUSPENDED IN HBSS CONTAINI~VG BSA IN THE PRESENCE OF VARIOUS ABSOLUTE CONCENTRATIONS AND COI\‘CENTRATION GRADIENTS OF PGE,”
Concentration (M) of PGE, in the lower compartment
Concentration
(M) of PGE, in the cell compartment
0
10-7
10-e
,O-”
0 10-7 10-C 10-s a Migration (in micrometers) was determined after 90 min of incubation. Values shown are the means of four separate experiments in which the values obtained differed by no more than 10%. The concentration of BSA was 1 mgiml.
III.1. ET ./if-.
116
potent stimulation of the adenylate cyclase. Experiments were therefore performed to study changes of cyclic AMP in neutrophils exposed to PGE, in the presence or absence of BSA in order to correlate the generation of cyclic AMP with the induction of chemotaxis. Figure 3 shows that PGEl increased cyclic AMP only in the absence of BSA, i.e., under a condition in which no chemotaxis and a depression of random locomotion occurred, whereas no significant cyclic AMP generation to PGE, was found in the presence of BSA. A similar finding was obtained by employing PGE2, PGA,, or PGA, as cyclic AMP-increasing agents. It should be noted that BSA itself caused a slight increase of cyclic AMP: the explanation for this effect is unclear at present. Neither PGF,, nor PGF, caused a change of cyclic AMP over a wide concentration range. whether added to neutrophils in the presence or absence of BSA. DISCUSSION The chemotactic activity of PGs has been a matter of controversy for several years. On the one hand, their chemotactic properties for leukocytes could be demonstrated (3, 24) which stood, on the other hand, in clear contrast to a lack or even suppression of chemotaxis ( 14, IS, 23 . 35). An explanation for these discrepant results has not been forwarded previously. Our data demonstrate that all PGs tested were chemotactically active for rabbit peritoneal neutrophils. The expression of the chemotactic activity. however. was found to be fundamentally distinct between the two main groups of PGs. Whereas PGF,, and PGF, possessed both chemokinetic and chemotactic activity, PGs of the E or A type were able to induce chemotaxis only in the presence of albumin which is known to be an inducer of chemokinesis (21, 21). The mechanism by which albumin enables PGs of the E or A type to express chemotactic activity is unknown at present. Three possibilities may be considered. (a) PGs of the E or A type may bind to albumin which may facilitate their presentation to cell membrane receptors in such a way that a sufftcient distur-
HBSS
121
0
sd
HESS + BSA
5a-7
0
54d6
5wJ-7
PGE, (Ml FIG.. 3. PCE,-Induced generation of cyclic AMP in neutrophils Rabbit peritoneal neutrophils ( IO * 10’9. suspended in HBSS exposed for IO min to PGE, at concentrations indicated. Bars cyclic AMP of four separate incubations.
in the presence or absence of BSA. with or without BSA (I mgiml). were represent the mean t i- SD) content of
PROSTAGLANDINS
AS
117
CHEMOATTRACTANTS
bance occurs to set the signal for directional movement. Such a mechanism has been discussed for the action of low molecular weight leukocyte chemotactic factors such as fatty acids, formyl-methionyl peptides, and plasma peptides (26). (b) As we have shown, albumin prevents the cyclic AMP generation by PGs of the E or A type which is usually observed in neutrophils. Since an increase of cyclic AMP has been established as an inhibitory signal for various leukocyte functions (12- 14, 27), prevention of cyclic AMP production may uncover the additional chemotactic property of PGs of the E or A type. Some support for this possibility may be drawn from the finding presented here that PGF, and PGF, did not increase cyclic AMP in neutrophils and therefore albumin was not required to restrain stimulation of the adenylate cyclase. (c) Neutrophils may possess different receptors for chemokinesisand chemotaxis-inducing substances. Binding to chemokinesis receptors may only increase random movement whereas binding to chemotaxis receptors may serve for gradient sensing and orientation of the cell toward higher concentrations of a chemotactic factor. Our findings indicate that neutrophils are only capable of responding to a chemotactic stimulus, provided a preceding or a simultaneous chemokinetic stimulation, i.e., promotion of cell motility, has taken place. Thus, PGs of the E or A type appear to require albumin as an important chemokinesisinducing cofactor when compared to PGs of the F type which by themselves possess chemokinetic and chemotactic properties. On the basis of our in vitro findings, an important function of various PGs as mediators of acute inflammatory reactions may become apparent. Production and release of PGs is clearly an event associated with phagocytosis (7-9, 28) and possibly with other membrane-perturbing incidents. On the one hand, PGs of the E type may serve to regulate homeostasis of inflammation by controlling macrophage and lymphocyte function via generation of cyclic AMP (16). On the other hand, PGs of the E and F type may attract additional leukocytes which serve to amplify the pool of leukocytes involved in the control of inflammation. ACKNOWLEDGMENTS The authors gratefully Miss Wibke Kramer.
acknowledge
the
technical
assistance
of Miss
Helga
Braun
and
REFERENCES I. 2. 3. 4. 5. 6.
7. 8. 9. .O. I I.
Willis. A. L.. .I. Pharm. Pharmud. 21, 126, 1969. Piper, P.. and Vane, J., Ann. N.Y. Acad. Sci. 180, 363. 1971. Kaley, G.. and Weiner, R.. Nature New Bid. 234, 114. 1971. Robinson, D. R.. McGuire. M. B., and Levine, L., Ann. N. Y. Acad. Sci. 256, 318, Sondergaard, J., and Wolf-Jtirgensen, P. Acra Dermato-Venertd. 52, 361, 1972. Willoughby, D. A., Giroud, J. P., Di Rosa, M., and Velo, G. P.. In “Prostaglandins AMP. Biological Actions and Clinical Applications” (R. H. Kahn and W. E. M. Lands, 187-206, Academic Press, New York. 1973. Higgs, G. A., and Youlten. C. J. F., Brit. J. Phurmrrcol. 44, 330, 1972. Humes, J. L., Bonney, R. J., Pelus, L.. Dahlgreen, M. E., Sadowski. S. J., Kuhl, Davies, P. Nature (London) 269, 149, 1977. Gemsa, D., Seitz. M., Kramer, W., Till, G.. and Resch, K., J. Immunol. 120, 1187, Morley, J., Prostcrgltrndins 8, 315, 1974. Williams, 1. J., and Peck, M. J., Nrrture (LO&ON) 270, 530. 1977.
1975. and Cyclic Eds.), pp.
F. A.. and 1978.
118 I?. 13. 14. 15. 16. 17. 18. 19. 10. 7 I. 22. 23. 14. 25. 26. 27 28.
IILI
ET AL
Bourne. H. R., Lichtenstein. L. M., Melmon. K. L.. Henney. C. S.. Weinstein. Y.. and Shearer. G. M.. Scier~cr 184, 19. 1974. Gemsa. D.. Woo, C. H.. Webb. D.. Fudenberg. H. H.. and Schmid, R.. C‘ell. Iw,rrlr/ro/. 15. ‘1, 197.5. Rivkin. I.. Rosenblatt. J.. and Becker. E. L.. J. Inrr?rrr/r~~/. 115, 1126. 1975. Hatch. G. E.. Nichols. W. K.. and Hill. H. R.. .I. Immune/. 119, 450. 1977. Gemsa, D.. Z. I~~~r~~~~/?if~~r.s~~~.s(.h. Imnrurrohiol. Srrpp/. 2, 72, 1977. Boyden. S. V.. J. Erp. Med. 115, 453, 1962. Zigmond, S. H.. and Hirsch, J. G.. J. E.rp. Med. 137. 387. 1973. Gilman. A. G.. Proc. Ntrt. Ac.d. Sci. USA 67, 305. 1970. Gemsa. D., Steggemann. L.. Till, G.. and Resch. K.. J. Irnrrlurt~~l. 119. 524. 1977. Wilkinson. P. C.. Parrott. D. M. V.. Russell. R. J.. and Sless. F.. J. El-p. Med. 145, 1158. 1977. Keller. H. U.. Wissler. J. H., Hess, M. W.. and Cottier. H.. ENT. J. Immunol. 8, I. 1978. Diaz-Perez. J. L.. Goldyne. M. E.. and Winkelmann. R. K.. J. I~z~~e,st. Dermrrtol. 66, 149, 1976. Ford-Hutchinson, A. W.. Smith. M. J. H.. and Walker, J. R.. J. Phnrmacol. 57. 467. 1976. Bray. M. A.. and France. M., Int. Arch. Allergy Appl. In~murrol. 56, 500, 1978. Wilkinson. P. C.. Erp. C‘rll Res. 103, 415. 1976. Estensen. R. D.. Hill. H. R., Quie. P. G.. Hogan. N., and Goldberg. N. D.. lVrrturr fLo,~dorr) 245, 458. 1973. Zurier. R. B.. f/r “Advances in Prostaglandin and Thromboxane Research” iB. Samuelsson and R. Paoletti. Eds.). pp. 815-818, Raven Press. New York, 1976.
ECKHARD
KOWNATZKI,
DIETHARD Institute
of Immunoloyy.
University
Received
MICHAEL
Szrrz,
AND
GEMSA
August
of Heidelberg.
Germany
3 I, 1978
Prostaglandins (PGs) of the E, A. and F type were assayed for chemokinetic and chemotactic activity in vitro. In the absence of bovine serum albumin (BSA) which is chemokinetically active, PGs of the F type (lo-’ to lo-” M) induced a chemokinetic and chemotactic response in rabbit peritoneal neutrophils. Under the same conditions, PGs of the E and A type had no significant effect on cell locomotion. However, in the presence of BSA (1 mg/ml), PGE and PGA induced a chemotactic response, thus indicating that they lack chemokinetic but possess chemotactic activity. In the absence of BSA, PGE, caused an increase in cyclic AMP, which was not observed when BSA was present. This supports previous findings that an increase of cyclic AMP is associated with inhibition of chemotactic locomotion. Since PGs are released by phagocytosing leukocytes and since albumin is generally available in inflamed tissues, the results suggest that PGs may contribute to the inflammatory accumulation of neutrophils
INTRODUCTION
During the past years, prostaglandins (PGs) of various types have been implicated to participate in acute inflammatory responses. They were found to be increased in inflamed tissues (l-4) and their presence has been correlated with leukocyte infiltration (5, 6). Furthermore, particular endocytic stimuli have recently been recognized which induced a pronounced release of PGs of the E and F type from neutrophils and macrophages (7-9). However, the effect of various PGs on the development of an inflammatory response is far from clear. On the one hand, proinflammatory properties, such as vasodilation, hyperemia, pain, and accumulation of leukocytes have been described (10, 11). On the other hand, antiinflammatory qualities have been attributed, particularly to PGE, and PGE*, via generation of cyclic AMP, which is considered to be an inhibitory signal leading to the reduction of various metabolic and functional activities of leukocytes (12-15). Recently, we proposed that PGE, and PGE, may be mediators which are responsible for the maintenance of homeostasis in inflammation by exerting in concert pro- as well as antiinflammatory properties (16). The present study was undertaken to solve a long-lasting controversy, whether PGs possess chemotactic activity or inhibit leukocyte migration. In this report we present evidence that PGs are chemotactically active but differ in their chemokinetic properties. MATERIALS Rabbit neutrophils. A neutrophil-rich
AND METHODS
peritoneal exudate was obtained 4 hr after an ip injection of 100 mg of glycogen suspended in 100 ml of sterile saline. The cells were washed twice in Hanks’ balanced salt solution (HBSS), buffered at pH 111 0090-1229/79/010111-08$01.00/O Copyright All rights
0 1979 by Academic Press. Inc. of reproduction in any form reserved.
112
~1‘11.1. E7 Al
7.4 with 26 mM tris(hydroxymethyl)aminomethane. resuspended in HBSS, and adjusted to 2.5 x 10” cells/ml. The cell preparations contained more than 90% neutrophils as determined by staining with May-Griinwald-Giemsa. Chenzofcuis rrssay. Random movement. chemokinesis (increased random locomotion in the presence of chemical agents). and chemotaxis (directed migration toward a chemotactic factor) of rabbit peritoneal neutrophils were assayed by the filter technique ( 17) as modified by Zigmond and Hirsch ( 18). Chemotaxis chambers were set up by adding I ml of test solution and 1 ml of cell suspension (3.5 x 10” cells) to the lower and upper compartments. respectively. Following a 90-min incubation period in humidified air at 37°C the filters were removed, and the distance between the leading front of migrated neutrophils and the nonmigrated cells on top of the filter was measured by light microscopy ( X400) in stained and xylene-cleared cellulose nitrate filters (Schleicher and Schiill, Dassel, Germany) of 3-pm pore size. embedded in Canada balsam (E. Merck. Darmstadt, Germany) between a slide and a coverslip. Chambers were set up in triplicate and the migration distance of neutrophils was determined at five different filter sites. In general. all measured values of triplicate filters differed by no more than 10%. Throughout all experiments casein at a concentration of I mgiml (E. Merck) was used as a positive migration control. Assqfor cyclic AMP. Neutrophils ( 10 x IO”) were suspended in I ml of HBSS or HBSS plus BSA ( I mg/ml) in glass centrifuge tubes. PGE, or PGF,, was added at the start of the incubation which was carried out at 37°C in a rotating water bath set at 100 rpm. After 10 min, the incubation was terminated by centrifuging the tubes for 20 set at 10,000~. The cell pellet was processed for cyclic adenosine 3’, S’-monophosphate (cyclic AMP) determination by the method of Gilman ( 19) with modifications as previously described (10). Reagenrs. HBSS was always freshly prepared, contained 1.8 x IO-” M Ca”‘. 0.8 x 10 mRM Mg’&, 0.22% glucose (w/v), and 76 mM tris(hydroxymethyl)aminomethane, and was adjusted to pH 7.4. Crystalline bovine serum albumin (BSA) was dissolved in HBSS and adjusted to pH 7.4. Prostaglandins (PGs) were kindly provided by Dr. J. Pike. Upjohn, Company (Kalamazoo, Mich.). Prior to the experiments, the PGs were dissolved in ethanol and then diluted in HBSS. The stability of the employed PGs of the E type was confirmed by thin-layer chromatography. The concentration of ethanol in the incubation medium did not exceed 0.4% and did not affect motility or cell viability as tested by trypan blue exclusion. Control incubations without PGs received a similar concentration of ethanol. RESULTS
Ej’ject of PGs on Nerttrophil Locomotior~ When various concentrations of different PGs were added to the lower compartment of a chemotaxis chamber, a dose-dependent increase of neutrophil migration toward PGF,, and PGF.,, was found (Fig. 1). PGs of the E or A type, however, exhibited no significant effect on cell locomotion. The maximal cell response toward PGF occurred at a concentration of lo-> M. Higher concentrations led to a decrease of cell migration which was shown to be an effect of ethanol serving as a solvent for the PGs.
PROSTAGLANDINS
I
AS
CHEMOATTRACTANTS
113
L,, 0
108
,a7
lo6
,05
PG(M) FIL. 1. Effect of prostaglandins (PC&) on neutrophil locomotion. Rabbit peritoneal neutrophils (2.5 x 10”) were suspended in I ml of HBSS and added to the upper compartment of a chemotaxis chamber. Various concentrations of different PGs, diluted in HBSS. were added to the lower compartment and the distance of neutrophil migration was measured after 90 min of incubation. Distance of migration for random locomotion t HBSS) and directional locomotion (casein. I mgiml) were 26 and 160 pm. respectively. Shown is a representative experiment. performed in triplicate. in which the values obtained differed by no more than 10%.
Compared with the distance of migration ( 160 pm) toward casein (1 mg/ml), the PGF-induced neutrophil migration was rather weak. Since serum albumin is known to increase neutrophil movement by its chemokinetic activity (21, 22), we studied its effect on cell migration toward PGs. When BSA at a concentration of 1 mg/ml was added to the cell suspension in the upper compartment. random locomotion of neutrophils increased by approximately 100% as did migration toward PGF (Fig. 2). Surprisingly, however, the PGs of the E and A type also induced a pronounced migration of neutrophils in the presence of BSA (Fig. 2). The maximum migration obtained with all three types of PGs was observed at a concentration of lo-” M. Similar effects were obtained when rabbit or human instead of bovine serum albumin was used. Evidence for Chemotactic
Activity
of PGs
To investigate in more detail whether the observed increase in neutrophil migration was caused by chemotactic attraction of PGs or whether it merely reflected increased random locomotion (chemokinesis), various concentrations of PGEl or PGF, were added to the lower or upper compartment of the chemotaxis chamber and neutrophil migration was tested in the presence or absence of BSA. By using such a system, the influence of both the absolute concentrations and the concentration gradient can be studied, i.e., migration in a positive gradient, in a negative gradient, and in the absence of a gradient. Table 1 shows a checkerboard assay in which neutrophils migrated in various concentrations or gradients of PGF,,. The numbers in the diagonal drawn from upper left to lower right represent migration distances in various absolute concentrations of PGF, in the absence of a gradient. As can be seen, the locomotion increased with the PGF, concentration, i.e., the neutrophils showed a chemokinetic response to PGF,,. Furthermore, it became evident that cells moving in a positive PGF, gradient (below the diagonal) were penetrating deeper into the filter than in the absence of a gradient (diagonal) or in a negative gradient (above the diagonal). These findings clearly show that PGF, can induce both a chemokinetic and a chemotactic response in neutrophils.
114
'IILl
E7‘.4L.
with BSA
E=PEY wlthout
BSA
Et-.
Ii7
d
TO5
PG (MI
wlthout
BSA b------=0-
Ga
0
---d
Id6
Ii7
Iti5
PG IM)
1507
c
with BSA 5 100. s ; PE (I 2 so-‘ L8
f z"
.-<,10,
without +--+-'oco
BSA -P------Y
I
0
FIG,. 2. Effect mgiml). Incubation
of various conditions
PGs on neutrophil locomotion as described in Fig. I legend.
in the presence
or absence
of BSA
PROSTAGLANDINS
AS
TABLE CHECKERBOARD PRESENCE
ASSAY OF VARIOUS
TO STUDY
THE
ABSOLUTE
CONCENTRATIONS
Concentration (M) of PGF,, in the lower compartment
115
CHEMOATTRACTANTS
MIGRATION
1 OF NEUTROPHILS AKD
SUSPENDED
COIUCENTRATION
IN
GRADIENTS
HBSS IK THE OF PGF,;
Concentration
(M) of PGF,, in the cell compartment ,0-” 10-R IO-’ 10-e
0
” Migration (in micrometers) was determined after 90 min of incubation. Values shown are means of four separate experiments in which the values obtained differed by no more than 10%.
A similar checkerboard experiment was performed with PGE,, except that neutrophils were suspended in HBSS plus BSA (I mg/ml) to facilitate migration (Table 2). When compared to the data presented in Table 1, it is evident that BSA enhanced random locomotion and, furthermore, that with increasing concentrations of PGE, in both chemotaxis chambers (diagonal) no increase but rather a slight depression of migration occurred, i.e., PGE, failed to induce a chemokinetic response. However, a completely different picture evolved when neutrophils were allowed to migrate in a positive concentration gradient (below the diagonal) in which case the cells were attracted by PGE, in a dose-dependent manner. These data demonstrate that PGE, was able to cause only a chemotactic response of neutrophils. Not shown are checkerboard assays demonstrating that PGF,. PGE,, PGAI, and PGA, were also chemotactically active but they differed in their ability to induce chemokinesis which was exclusively found in the PGs of the F class. Effect of PGs on Intracellular
Concentrations of Cyclic AMP
PGs of the E type have repeatedly been described as inhibitors of chemotaxis (14, 23, 15) because of the generation of an inhibitory signal, cyclic AMP, via a TABLE
Z
CHECKERBOARD ASSAY TO STUDY THE MIGRATIOX OF NEUTROPHILS SUSPENDED IN HBSS CONTAINI~VG BSA IN THE PRESENCE OF VARIOUS ABSOLUTE CONCENTRATIONS AND COI\‘CENTRATION GRADIENTS OF PGE,”
Concentration (M) of PGE, in the lower compartment
Concentration
(M) of PGE, in the cell compartment
0
10-7
10-e
,O-”
0 10-7 10-C 10-s a Migration (in micrometers) was determined after 90 min of incubation. Values shown are the means of four separate experiments in which the values obtained differed by no more than 10%. The concentration of BSA was 1 mgiml.
III.1. ET ./if-.
116
potent stimulation of the adenylate cyclase. Experiments were therefore performed to study changes of cyclic AMP in neutrophils exposed to PGE, in the presence or absence of BSA in order to correlate the generation of cyclic AMP with the induction of chemotaxis. Figure 3 shows that PGEl increased cyclic AMP only in the absence of BSA, i.e., under a condition in which no chemotaxis and a depression of random locomotion occurred, whereas no significant cyclic AMP generation to PGE, was found in the presence of BSA. A similar finding was obtained by employing PGE2, PGA,, or PGA, as cyclic AMP-increasing agents. It should be noted that BSA itself caused a slight increase of cyclic AMP: the explanation for this effect is unclear at present. Neither PGF,, nor PGF, caused a change of cyclic AMP over a wide concentration range. whether added to neutrophils in the presence or absence of BSA. DISCUSSION The chemotactic activity of PGs has been a matter of controversy for several years. On the one hand, their chemotactic properties for leukocytes could be demonstrated (3, 24) which stood, on the other hand, in clear contrast to a lack or even suppression of chemotaxis ( 14, IS, 23 . 35). An explanation for these discrepant results has not been forwarded previously. Our data demonstrate that all PGs tested were chemotactically active for rabbit peritoneal neutrophils. The expression of the chemotactic activity. however. was found to be fundamentally distinct between the two main groups of PGs. Whereas PGF,, and PGF, possessed both chemokinetic and chemotactic activity, PGs of the E or A type were able to induce chemotaxis only in the presence of albumin which is known to be an inducer of chemokinesis (21, 21). The mechanism by which albumin enables PGs of the E or A type to express chemotactic activity is unknown at present. Three possibilities may be considered. (a) PGs of the E or A type may bind to albumin which may facilitate their presentation to cell membrane receptors in such a way that a sufftcient distur-
HBSS
121
0
sd
HESS + BSA
5a-7
0
54d6
5wJ-7
PGE, (Ml FIG.. 3. PCE,-Induced generation of cyclic AMP in neutrophils Rabbit peritoneal neutrophils ( IO * 10’9. suspended in HBSS exposed for IO min to PGE, at concentrations indicated. Bars cyclic AMP of four separate incubations.
in the presence or absence of BSA. with or without BSA (I mgiml). were represent the mean t i- SD) content of
PROSTAGLANDINS
AS
117
CHEMOATTRACTANTS
bance occurs to set the signal for directional movement. Such a mechanism has been discussed for the action of low molecular weight leukocyte chemotactic factors such as fatty acids, formyl-methionyl peptides, and plasma peptides (26). (b) As we have shown, albumin prevents the cyclic AMP generation by PGs of the E or A type which is usually observed in neutrophils. Since an increase of cyclic AMP has been established as an inhibitory signal for various leukocyte functions (12- 14, 27), prevention of cyclic AMP production may uncover the additional chemotactic property of PGs of the E or A type. Some support for this possibility may be drawn from the finding presented here that PGF, and PGF, did not increase cyclic AMP in neutrophils and therefore albumin was not required to restrain stimulation of the adenylate cyclase. (c) Neutrophils may possess different receptors for chemokinesisand chemotaxis-inducing substances. Binding to chemokinesis receptors may only increase random movement whereas binding to chemotaxis receptors may serve for gradient sensing and orientation of the cell toward higher concentrations of a chemotactic factor. Our findings indicate that neutrophils are only capable of responding to a chemotactic stimulus, provided a preceding or a simultaneous chemokinetic stimulation, i.e., promotion of cell motility, has taken place. Thus, PGs of the E or A type appear to require albumin as an important chemokinesisinducing cofactor when compared to PGs of the F type which by themselves possess chemokinetic and chemotactic properties. On the basis of our in vitro findings, an important function of various PGs as mediators of acute inflammatory reactions may become apparent. Production and release of PGs is clearly an event associated with phagocytosis (7-9, 28) and possibly with other membrane-perturbing incidents. On the one hand, PGs of the E type may serve to regulate homeostasis of inflammation by controlling macrophage and lymphocyte function via generation of cyclic AMP (16). On the other hand, PGs of the E and F type may attract additional leukocytes which serve to amplify the pool of leukocytes involved in the control of inflammation. ACKNOWLEDGMENTS The authors gratefully Miss Wibke Kramer.
acknowledge
the
technical
assistance
of Miss
Helga
Braun
and
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