CELLULAR
IMMUNOLOGY
Stimulation
43, 103- 112 (1979)
of Human T-Lymphocyte Arachidonic Acid JANET MCCARTY
AND EDWARD
Chemokinesis
by
J. GOETZL
Departments of Medicine, Harvard Medical School, Boston, Massachusetts 02115, and the Robert B. Brigham Hospital Division of the AfJliated Hospitals Center, Inc., Boston, Massachusetts 02120 Received August 28, 1978
The migration of human T lymphocytes, assessed in modified Boyden chambers, was chemokinetically stimulated by arachidonic acid in a dose-related manner that achieved a peak level of 127 2 34% enhancement (mean ? SD) at 8 pM arachidonic acid. The chemokinetic effect was dependent on the metabolism of the arachidonic acid by the T lymphocytes as derivatives of arachidonic acid that do not serve as prostaglandin and thromboxane precursors were without effect, while the cycle-oxygenase inhibitors indomethacin (IDS0 = 10 PM) and 5,8,11,14-eicosatetraynoic acid (ETYA) (ID,, = 20 PM) suppressed the stimulation of migration by arachidonic acid. The cycle-oxygenase product 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) reproduced part of the chemokinetic effect of arachidonic acid, but the lipoxygenase product 12-L-hydroxy-5,8,10,14eicosatetraenoic acid (HETE) as well as PGE,, PGF 2a, and thromboxane Bz had no stimulatory activity. The ability of ETYA, but not indomethacin, to suppress the migration of unstimulated T lymphocytes suggested that a lipoxygenase metabolite of endogenous arachidonic acid contributes to the maintenance of their normal levels of spontaneous migration.
INTRODUCTION The factors which regulate the complex patterns of migration, homing, and accumulation of lymphocytes in vivo (l-5) have recently been investigated by in vitro techniques that utilize defined populations of lymphocytes and permit a distinction between principles which evoke increased random locomotion, termed chemokinesis, and those which stimulate directed migration, termed chemotaxis (6). Initial studies of the stimulation of mouse lymphocyte motility were based on quantitating increases in the fraction of splenic B cells exhibiting morphological features suggestive of movement, and revealed a significant enhancement by bivalent specific anti-IgG (7). The stimulatory effect of anti-IgG on motility was dependent on the metabolic integrity of the B cells and was inhibited by cytochalasin B, diisopropyl fluorophosphate, and elevations of intracellular levels of cyclic 3’,5’-adenosine monophosphate, none of which influenced the capping of IgG-anti-IgG complexes (8,9). The observation that mouse and rat B lymphocytes migrated into micropore filters of modified Boyden chambers led to the documentation of the chemokinetic activity of maximally stimulatory concentrations of anti-IgG (7, 10) and revealed a chemotactic effect of lower concentrations 103 0008-8749/79/030103-10$02.00/O Copyright All rights
0 1979 by Academic Press, of reproduction in any form
Inc. reserved.
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MCCARTY
AND GOETZL
(10). That blast transformation facilitates the migration of B lymphocytes was confirmed by the demonstration of especially active chemotactic and chemokinetic responses of human B lymphoblasts to both casein and endotoxin-activated serum (11) and by the greater mobility of mouse B cells following exposure to mitogens (10). Early analyses of the determinants of optimal chemokinetic and chemotactic migration of rodent and human T lymphocytes revealed uniquely specific stimulation by lymphocyte products (10) and some mitogens (12), and suggested a dependence on prior blast transformation which was similar to that of B lymphocytes (11, 12). The potential heterogeneity of chemotactic receptors on mitogen-transformed human T lymphoblasts was illustrated by the selective inhibition of responses to casein and endotoxin-activated plasma by prior treatment of the lymphoblasts with phospholipase C or sphingomyelinase D, while the responses to submitogenic levels of phytohemagglutinin and staphylococcal protein A were blocked with a comparable selectivity by pretreatment with trypsin or a-mannosidase (12). In contrast to B lymphoblasts, the enhanced mobility of the T lymphoblasts was not predominantly a function of the mitogen-induced transformation, but rather was attributable to the period of preincubation in medium containing fetal calf serum. The spontaneous mobility of human T lymphocytes obtained from peripheral blood and tonsillar tissue and their chemokinetic responses to casein and endotoxin-activated serum were enhanced markedly following such preincubation and were not increased further in the presence of mitogenic concentrations of concanavalin A (13, 14). The explanation for the stimulatory effect of preincubation was revealed by studies of the migration of human T-lymphocyte subsets (14). The spontaneous and chemokinetic migration of T lymphocytes possessing Fcp receptors increased with preincubation in parallel with increases in the capacity to form EA-IgM rosettes, while T lymphocytes with Fe-y receptors exhibited little mobility under any conditions. The current demonstration that resting human T lymphocytes are chemokinetitally stimulated by arachidonic acid in the absence of any preincubation suggests the involvement of a mechanism not available to previously described stimuli. Further, as indomethacin blocks the enhancing effect of arachidonic acid on the migration of platelet-free T lymphocytes, some resting T lymphocytes appear to possess a cycle-oxygenase-like enzyme that is capable of generating metabolites of arachidonic acid which enhance lymphocyte migration in a manner analogous to the stimulation of PMN leukocyte chemokinesis by the cycle-oxygenase product 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) (15). MATERIALS
AND METHODS
Blind-ended acrylic migration chambers with Teflon cell compartments (Neuroprobe, Inc., Bethesda, Md.), Ficoll-Paque and Sephadex G-10 (Pharmacia Fine Chemicals, Piscataway, N.J.), cytochalasin B, indomethacin, arachidonic acid, arachidonic acid methyl- and ethyl-esters, and arachidonyl alcohol (Sigma Chemical Co., St. Louis, MO.), Hanks’ balanced salt solution and Eagle’s medium (Microbiological Associates, Bethesda, Md.), five times crystallized ovalbumin (Miles Laboratories, Inc., Elkhart, Ind.), cr-thioglycerol (K & K Laboratories, Inc., Hollywood, Calif.), Sartorius nitrocellulose micropore filters (Beckman Instruments, Mountainside, N.J.), Nucleopore polycarbonate micropore filters (Arthur
LYMPHOCYTE
CHEMOKINETIC
EFFECT
OF ARACHIDONATE
105
H. Thomas Co., Philadelphia, Pa.), 4-mm diameter solid spherical glass beads (Fisher Scientific Co., Medford, Mass.), 2-iodoacetamide (Eastman Kodak Co., Rochester, N.Y.), and nylon wool (Leuko-Pak Leukocyte Filter, Fenwall Laboratories, Deer-field, Ill.) were obtained as noted. A supply of purified 5,8,11,14-eicosatetraynoic acid (ETYA) was kindly provided by Dr. James Hamilton of the Hoffman-La Roche Company (Nutley, N.J.). Colchicine was agift from the Eli Lilly Company (Indianapolis, Ind.), and PGEz and PGFI, were gifts from the Upjohn Company (Kalamazoo, Mich.). Preparation and purification of lipid factors. The source of 12-L-hydroxy5,8,10,14-eicosatetraenoic acid (HETE) was a mixture of human platelets and arachidonic acid that was incubated in the presence of 0.10 mM indomethacin to block cycle-oxygenase activity as described (15, 16). The HETE was extracted and purified by repeated silicic acid chromatography, and the purity of the final product exceeded 98% as assessed by thin-layer chromatography and gas-liquid chromatography/mass spectrometry (16, 17). 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) was prepared by incubating arachidonic acid with a suspension of crude microsomal powder that had been obtained by acetone-pentane treatment of sheep seminal vesicles and which served as the source of cycle-oxygenase (15, 18). The HHT was extracted and purified by repeated silicic acid column chromatography (15); the purity of the final product exceeded 90% as assessedby gas-liquid chromatography/mass spectrometry (15,19). Thromboxane Bz was produced by total organic synthesis and purified as described (20). Puri$cation of human lymphocytes migration. Thirty-milliliter portions
and measurement
of in vitro lymphocyte
of blood from normal volunteers were defibrinated by continuous agitation for 10 min in 40-ml capped plastic tubes with 15-20 glass beads to prevent coagulation and remove platelets. The defibrinated blood was diluted with an equal volume of Hanks’ solution made 0.005 M in Tris-HCl (pH 7.4) (Hanks’-Tris) and 30-ml portions were layered on 30-ml cushions of Ficoll-Paque and centrifuged at 400g for 30 min at room temperature. The mixed mononuclear leukocytes at the top of the Ficoll-Paque cushions (21) were recovered by gentle aspiration, pooled, washed twice with Hanks’-Tris buffer (pH 7.4), suspended in the same buffer containing 0.4 g/100 ml of ovalbumin, and filtered on a Sephadex G-10 column of IO-ml bed volume that was supported on a Ito 2-ml pad of nylon wool. The lymphocyte population that filtered through the column contained fewer than 1 platelet and 1 monocyte per 500 lymphocytes according to phase-contrast microscopic enumeration and differential counts of Wright-Giemsa stained smears. Analyses of the cell content of the purified lymphocyte suspensions utilizing the techniques of sheep erythrocyte rosetting, C3d receptor rosetting, and assessment of surface immunoglobulins (22-24) indicated a composition of 74-85% T lymphocytes and fewer than 2-6% B lymphocytes. Migration was assessed in modified Boyden chambers using Hanks’-Tris buffer made 0.2 g/100 ml in ovalbumin for both compartments. One-half milliliter of a suspension containing 3.5-4.0 x lo6 lymphocytes was pipetted into the top compartment of each chamber which was separated from the 0.2-ml buffer compartment below by a layer of two filters consisting of an 8-pm pore Nucleopore filter over an 8-pm pore Sartorius filter. After incubation of the chambers for 4 hr at 37°C in an atmosphere of 5% COz in air, the lower (Sartorius) filter was removed,
106
MCCARTY
AND GOETZL
washed in Hanks’ -Tris, and fixed and stained in hematoxylin as described for PMN leukocytes (15,25). The lymphocytes which migrated in the front at 60-80 pm from the cell source were enumerated in 20 high power fields (hpf) for each of duplicate or triplicate chambers. Lymphocyte migration is expressed as mean lymphocytes/5 hpf, while alterations induced by the prior addition of lymphocyte agonists to one or both compartments is expressed as % inhibition or % enhancement. RESULTS Initial experiments were designed to determine the optimal conditions for human T-lymphocyte spontaneous migration into a layer of two micropore filters. The incubation of triplicate chambers at 22°C and 4°C resulted in a mean suppression of migration of 77% and 96%, respectively, from a level of 49 lymphocytes/5 hpf at 37°C in a room air environment, while migration was enhanced by 64% in an environment of 5% CO, in air at 37°C. The use of calcium- and magnesium-free Hanks’ solution in two experiments inhibited migration by a mean of 50% and the further addition of 1 mM ethylenediamine tetraacetate increased the level of inhibitionto 60%, where the control levels of migration in complete Hanks’ solution were 38 and 35 lymphocytes/5 hpf. The effect on migration of the ovalbumin content of the buffer was assessedat concentrations of 0.05,O. 1,0.2,0.4, and 0.8 g/100 ml of ovalbumin, which resulted in mean levels of migration + 1 SD of 29 + 11, 33 of 8, 41 f 15, 38 ~fi 10, and 35 f 19 lymphocytes/5 hpf. All subsequent migration studies thus were carried out with complete Hanks’ solution containing 0.2 g of ovalbumin/lOO ml and with incubations at 37°C in 5% COz in air. T-lymphocyte migration was suppressed by 2-iodoacetamide, which blocks the glycolytic pathway (26), and by cytochalasin B, which interferes with microfilament function (27) (Fig. 1). Fifty percent inhibition of migration was achieved by 10m4M 2-iodoacetamide and approximately lows M cytochalasin B. Lymphocyte migration was increased in a dose-related fashion by colchicine, an effect that has been documented previously to be a result of the action of colchicine on microtubules (11, 28, 29), and by a-thioglycerol, at concentrations of 10-6-10-5 M which stimulate other lymphocyte functions (30) (Fig. 1). Stimulation
of T Lymphocyte
Migration
by Arachidonic
Acid
Arachidonic acid enhanced the migration of T lymphocytes in a dose-related manner at concentrations which exhibited no cytotoxic effect as assessedby trypan blue dye exclusion (Table 1). Maximal stimulation of migration was achieved at 8 and 16 pM concentrations of arachidonic acid irrespective of whether it was added to the buffer or cell compartment. When added to both compartments, the arachidonic acid gave peak stimulation at a level of 8 pM with less effect at 16 PM. As the enhancement of migration observed was a function of net concentration irrespective of a gradient, arachidonic acid appeared to act solely by a chemokinetic action. Arachidonic acid thus was added to both compartments in all subsequent protocols. Preincubation of the purified T lymphocytes in buffer containing fetal calf serum increased the level of spontaneous migration relative to that seen with fresh cells obtained from the same donors (Table 2). However, the peak enhancement by arachidonic acid at a concentration of 8 pM in both compartments was actually
LYMPHOCYTE
CHEMOKINETIC
107
EFFECT OF ARACHIDONATE
l 120 +100 +50 s s e 9 E
+40-
P >r
+20-
]/j~j[
mi
+50-
11
o----------~------------1 P
ii 23 .E:
-2o-
!3
1
$8 -40-5o-
I
i
-------I
I
I -so!, -100 h I lo-5lo-4m-3 lo-9104lo-'lo-@ 1
I
Concentmtim
of agonist (Ml
FIG. 1. Effects on T-lymphocyte migration of known inhibitors and enhancers of lymphocyte function. Each point and bracket depicts the mean + 1 SD for n experiments with each agonist. The spontaneous migration values in buffer alone (0% change) for the experiments with each agonist were: 85.63, and 27 lymphocytes/S hpf with 2Godoacetamide (n = 3); 35,32,63, and 85 with cytochalasin B (n = 4); 25.27, and 19 with a-thioglycerol (n = 3); and 36, 41, and 28 with colchicine (n = 3). All agonists were present at an equal concentration in the buffer and cell compartments.
decreased from 118% without preincubation and 107% with preincubation in buffer alone to 56% by preincubation in the presence of fetal calf serum. Arachidonyl alcohol and the methyl- and ethyl-esters of arachidonic acid, which are not metabolized by the cycle-oxygenase of platelets (31) and other cells, did not stimulate T-lymphocyte migration, whereas comparable concentrations of TABLE 1 Modified Checkerboard Analysis of Arachidonate Stimulation of T-Lymphocyte Migration Concentration of arachidonate (fl)
Buffer compartment only Cell compartment only Both compartments Enhancement (%)*
0
2
4
8
46” -
39 56 57 55 + 25
67 53 61 91 + 38
88 62 96 127 + 34
16
32
89 79 62 80~31
57 38 18 -
a Each value is the mean for triplicate chambers in one experiment expressed as lymphocytes/5 hpf. * Mean percentage enhancement + 1 SD for six experiments using different cell donors and with arachidonate in both compartments.
MCCARTY AND GOETZL TABLE 2 Effects of Preincubation of T Lymphocytes on Response to Arachidonic Acid Concentration of arachidonate (&I)
No preincubation Preincubation in Eagle’s medium without protein Preincubation in Eagle’s medium with fetal calf serum
0
2
4
8
16
28 lr 7”
462 11
55 zk 13
61 ? 10
47 ” 12
30 f 4
51 * 12
56 f 15
62 k 8
44 “_ 10
43 ” 2
52k
59 f 6
67 2 17
56 f 9
11
(1Each value is the mean 2 SD for three experiments performed in duplicate and is expressed as lymphocyte45 hpf. Arachidonate was present at the stated concentrations in both compartments.
arachidonic acid produced the characteristic effect, exhibiting a peak enhancement of 132% at the 8 pM level (Fig. 2). The Mechanism of Chemokinetic Stimulation of T Lymphocytes by Arachidonic Acid That the metabolism of arachidonic acid by lymphocyte enzymes, which are analogous to the cycle-oxygenase and lipoxygenase of platelets, might be involved in the stimulation of migration of platelet-free lymphocytes was investigated by using known enzymatic inhibitors. Indomethacin, which selectively inhibits the activity of cycle-oxygenase (19,32), significantly suppressed the stimulatory effect of 16 PM arachidonic acid, exhibiting a 50% inhibitory level of approximately 10 PM, but did not influence spontaneous migration (Table 3). ETYA, which inhibits the activity of both cycle-oxygenase and lipoxygenase (19), suppressed both spontaneous migration and the stimulatory effect of arachidonic acid with 50%
-30'
4
6
16
I 32
Concentmtion of amchidonic acid OTderivative ( pM)
FIG. 2. Chemokinetic effects on T lymphocytes of arachidonic acid and arachidonate derivatives. Each bar and bracket depicts the mean and range of two experiments performed in duplicate. The level of spontaneous migration (0% enhancement) for the two experiments were 28 and 32 lymphocytes/5 hpf.
LYMPHOCYTE
CHEMOKINETIC
109
EFFECT OF ARACHIDONATE
TABLE 3 Inhibition by Indomethacin and ETYA of the Spontaneous and Arachidonate-Stimulated Migration of T Lymphocytes Arachidonate” (VW
5
ETYA ETYA
0 16
14 f 8b 26+21
Indomethacin lndomethacin
0 16
Inhibitor
Concentration of indomethacin or ETYA (fl)
-
20
40
80
160
23 2 16 56 k 4*
53 iz 20 71 k 12*
69t 31 94 k 19*
91 2 17* 98 + 8*
10 4226 55 L 18**
-
2k31 66 + 14*
-
21 2 33 74 2 19*
a Arachidonate and the inhibitors were present in both compartments without a gradient. b Each value is the mean percentage inhibition k 1 SD for three experiments performed in duplicate. Levels of statistical significance are depicted as follows: *P < 0.01; **P < 0.05. The percentage enhancement k 1 SD by 16 fl arachidonate was 148 2 20% for the ETYA experiments and 117 k 32% for the indomethacin experiments.
inhibition at concentrations of 40 ph4 and 20 pM, respectively. In order to determine which type of metabolite of arachidonic acid might be responsible for the observed stimulation of T-lymphocyte migration, a range of purified and synthetic lipids were examined for their lymphocyte chemokinetic activity (Table 4). HHT, a platelet cycle-oxygenase product of arachidonic acid (19), stimulated lymphocyte migration by up to 90% at a concentration of 16pM, but only achieved 56-70% of the peak enhancing effect of comparable concentrations of arachidonic acid in the same experiments. In contrast, the lipoxygenase product HETE as well as PGEz inhibited spontaneous migration at the highest concentrations, while PGFza exhibited no substantial effects on migration and thromboxane B, was stimulatory only at a concentration of 32 pM. DISCUSSION The quantitation of human T-lymphocyte mobility by a technique employing a layer of two micropore filters provided uniform levels of background and stimulated migration for cell preparations from diverse donors (Figs. 1, 2; Table 2). Presumably, this was due to an even delivery of a functionally more homogenous population of lymphocytes to the bottom filter as a result of prior migration through the thin top filter. That the movement of the T lymphocytes into the bottom filter was a function of cellular migration was suggested by their transformation to elongated motile-appearing forms, which has been described (11, 13), and was confirmed by the inhibitory effects of iodoacetamide and cytochalasin B, at concentrations of the latter agent known to interfere with the integrity of microtilaments (27) (Fig. 1). Crystallized ovalbumin did not have a striking influence on T-lymphocyte migration, as the variations from the overall mean level of migration were within 20 + 2% for individual values obtained at ovalbumin concentrations ranging from 0.05-0.8 g/100 ml. Since the generally marked stimulation of PMN-leukocyte or monocyte migration by native albumins (33) has been attributed to a modulation of surface contact between the leukocytes and the filter matrix, it is possible that T-lymphocyte migration is less dependent on
110
MCCARTY AND GOETZL TABLE 4 Influence of Synthetic Lipids on T-Lymphocyte Migration Concentration of lipid factor @IV)”
Arachidonate HHT HETE TBzC PGEI PGFa,
4
8
16
32
1036 41 11 -12 4 2
151 84 7 0 -
129 90 2 16 -18 9
91 53 -25 73 -57 5
a The lipid factors were present in both compartments at an equal concentration. b Each value is mean percentage enhancement for two experiments performed in duplicate; values in the absence of any lipid (0% enhancement) were 26 and 32 lymphocytes/5 hpf. e Thromboxane B,.
interactions of the cells with the filter material at the pores. In contrast to the inhibition of PMN-leukocyte migration by colchicine (34), this and other microtubule-disaggregating agents enhance the migration of lymphocytes which may reflect a different regulatory function for lymphocyte microtubules (Fig. 1; 11, 28,29). T-lymphocyte migration also was stimulated by up to 100%by 10W6-10e5M cr-thioglycerol, a reducing principle known to stimulate other lymphocyte functions (30). Varying the concentration of each of the enhancing factors in the lymphocyte and buffer compartments of replicate chambers led to the conclusion that the effects of arachidonate (Table 1) and the other stimuli were virtually entirely chemokinetic. Thus the results suggested that human T-lymphocyte migration in vitro is modulated by microfilaments and microtubules, and is enhanced by previously recognized lymphocyte stimuli. The demonstration that arachidonic acid significantly enhanced the in vitro migration of the T lymphocytes both led to studies of the cellular requirements for the stimulatory effect and provided an opportunity to probe some of the pathways by which lymphocytes elaborate products of arachidonic acid which exhibit feed-back regulatory functions (35). Arachidonic acid stimulated lymphocyte migration by 93-161% (Table 1) and a modified checkerboard analysis (6) confirmed the fact that chemokinesis was the predominant mode of stimulation. The chemokinetic effect on T lymphocytes was diminished at higher concentrations of arachidonic acid, which also is a characteristic of low molecular weight factors that are chemokinetic and chemotactic for PMN leukocytes (36, 37). Prior incubation of T-lymphocyte suspensions in 20% fetal calf serum increased the level of spontaneous migration, but had no significant effect on the extent of chemokinetic stimulation by arachidonic acid (Table 2). This clearly distinguished the mechanism of stimulation by arachidonic acid from those of other lymphocyte migration-enhancing factors such as casein, endotoxin-activated serum, staphylococcal protein A, and phytohemagglutinin, whose activities are dependent on at least prior incubation of the lymphocytes in media containing fetal calf serum (13, 14). Several lines of evidence together suggested that the chemokinetic activity of
LYMPHOCYTE
CHEMOKINETIC
EFFECT OF ARACHIDONATE
111
arachidonic acid was dependent on its metabolic conversion by the platelet-free T lymphocytes. Esters of arachidonic acid, which are not metabolized by the cycle-oxygenases of platelets (31) or other cells, did not stimulate T-lymphocyte migration at concentrations in the maximally effective range for arachidonic acid (Fig. 2). The addition of indomethacin to the lymphocytes prevented the expression of arachidonic acid chemokinetic activity with an IDso of 10 PM (Table 3), a value that is comparable to the ID,, for the inhibition by indomethacin of platelet cycle-oxygenase activity (19,32). ETYA, which inhibits both the cycle-oxygenase and lipoxygenase of platelets (19), not only prevented the chemokinetic effect of arachidonic acid but also suppressed the spontaneous migration of lymphocytes (Table 3). In order to assess the possible nature of the lymphocyte-generated chemokinetic metabolite(s) of arachidonic acid, a series of defined platelet metabolites of arachidonic acid were studied in the lymphocyte migration assay. Only the cycle-oxygenase product HHT was capable of substantially stimulating T-lymphocyte migration in a dose-related fashion (Table 4), which suggests that the chemokinetic product of arachidonic acid produced by the indomethacininhibitable lymphocyte pathway may be an unsaturated hydroxy-fatty acid. One or more subpopulations of human suppressor T lymphocytes influence the functions of other lymphocytes by elaborating inhibitory concentrations of prostaglandins of the E series (35). The suppressor activity of such lymphocytes can be attenuated or eliminated by indomethacin in circumstances as diverse as the altered immunocompetence of pregnancy and of Hodgkin’s disease (35, 38). That resting lymphocytes appear to convert arachidonic acid into products which also are capable of enhancing lymphocyte migration suggests that endogenously elaborated lipids may represent a feed-back system for the bidirectional modulation of lymphocyte function. ACKNOWLEDGMENTS Dr. Goetzl is Director of the Laboratories for the Study of Immunological Diseases of the Howard Hughes Medical Institute. The authors are grateful to Dr. David Glass for his analyses of the constituents of the purified populations of lymphocytes.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Zatz, M. M., and Lance, E. M., .I. Exp. Med. 143, 224, 1971. Cahill, R. N. P., Frost, H., and Trnka, Z., J. Exp. Med. 143, 870, 1976. McCluskey, R. T., Benacerraf, B., and McCluskey, J. W., J. Immunol. 98, 466, 1963. Prendergast, R. A., J. Exp. Med. 111, 377, 1964. Emeson, E. E., J. Exp. Med. 147, 13, 1978. Zigmond, S. H., and Hirsch, J. G., J. Exp. Med. 137, 387, 1973. Schreiner, Cl. F., and Unanue, E. R., J. Immunol. 114, 809, 1975. Unanue, E. R., Ault, K. A., and Karnovsky, M. J., J. Exp. Med. 139, 295, 1974. Schreiner, G. F., and Unanue, E. R., J. Immunol. 114, 802, 1975. Ward, P. A., Unanue, E. R., Goralnick, S. J., and Schreiner, G. F., J. Immunol. 119,416, 1977. Russell, R. J., Wilkinson, P. C., Sless, F., and Parrott, D. M. V., Nature (London) 256,646, 1975. Wilkinson, P. C., Roberts, J. A., Russell, R. J., and McLaughlin, M., Clin. Exp. Immunol. 25,280, 1976. 13. O’Neill, G. J., and Parrott, D. M. V., Cell Zmmunol. 33, 257, 1977. 14. Parrott, D. M. V., Good, R. A., O’Neill, G. J., and Gupta, S., hoc. Nat. Acad. Sci. U.S.A. 75,2392, 1978.
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15. Goetzl, E. J., and Got-man, R. R., J. Zmmunol. 120, 526, 1978. 16. McGuire, J. C., Kelley, R. C., Gorman, R. R., and Sun, F. F., Prep. Biochem. 8, 147, 1978. 17. Goetzl, E. J., Woods, J. M., and Got-man, R. R., J. Clin. Znvesf. 59, 179, 1977. 18. Wallach, D. P., and Daniels, E. G., Biochim, Biophys. Acta. 231, 445. 1971. 19. Hamberg, M., and Samuelsson, B., Proc. Nat. Acad. Sci. U.S.A. 71, 3400, 1974. 20. Nelson, N. A., and Jackson, R. W., Tetrahedron Lert., 3275, 1976. 21. Boyum, A.,Scnnd. .Z. Lab. Clin. Invest. 21(Suppl. 97), 31, 1%8. 22. Hoffman, T., and Kunkel, H. G., In “In Vitro Methods in Cell-Mediated and Tumor Immunity” (B. R. Bloom, and P. R. David, Eds.), pp. 71-82. Academic Press, New York, 1976. 23. Ehlenberger, A. G., and Nussenzweig, V., In “In Vitro Methods in Cell-Mediated and Tumor Immunity” (B. R. BloomandP. R. David, Eds.),pp. 113-121. AcademicPress, New York, 1976. 24. Preud-Homme, J. L., and Labaume, S., In “In Vitro Methods in Cell-Mediated and Tumor Immunity” (B. R. BloomandP. R. David, Eds.), pp. 155-169. Academic Press, New York, 1976. 25. Goetzl, E. J., and Austen, K. F., J. Exp. Med. 136, 1564, 1972. 26. Kamovsky, M. L., Physiol. Rev. 42, 143, 1962. 27. Wessells, N. K., Spooner, B. S., Ash, J. F., Bradley, M. O., Luduena, M. A., Taylor, E. L., Wrenn, J. T., and Yamada, K. M., Science 171, 135, 1971. 28. Fram, R. J., Sidmar, C. L., and Unanue, E. R., J. Zmmunol. 117, 1456, 1976. 29. Center, D. M., Wasserman, S. I., and Austen, K. F., Cell. Zmmunol. 39, 325, 1978. 30. Goodman, M. G., and Weigle, W. 0.. J. Exp. Med. 154, 473, 1977. 31. Van Dorp, D., In “Prostaglandins” (P. Ramwell and J. E. Shaw, Eds.), pp. 181-199. N.Y. Acad. Sci., New York, 1971. 32. Samuelsson, B., In “Advances in Prostaglandin and Thromboxane Research” (B. Samuelsson and R. Paoletti, Eds.), Vol 1, pp. l-6. Raven Press, New York, 1976. 33. Wilkinson, P. C., Exp. Cell Res. 103, 415, 1976. 34. Caner, J. E. Z., Arthr. Rheum. 8, 757, 1965. 35. Goodwin, J. S., Bankhurst, A. D., and Messner, R. P., J. Exp. Med. 146, 1719, 1977. 36. Goetzl, E. J., and Austen, K. F., J. Exp. Med. 144, 1424, 1976, 37. Showell, H. J., Freer, R. J., Zigmond, S. H., Schiffman, E., Aswanikumar, S., Corcoran, B., and Becker, E. L., J. Exp. Med. 143, 1154, 1976. 38. Goodwin, J. S., Messner, R. P., Bankhurst, A. D., Peake, G. T., Saiki, J. H., and Williams, R. C., Jr., N. Engl. J. Med. 297, 963, 1977.
IMMUNOLOGY
Stimulation
43, 103- 112 (1979)
of Human T-Lymphocyte Arachidonic Acid JANET MCCARTY
AND EDWARD
Chemokinesis
by
J. GOETZL
Departments of Medicine, Harvard Medical School, Boston, Massachusetts 02115, and the Robert B. Brigham Hospital Division of the AfJliated Hospitals Center, Inc., Boston, Massachusetts 02120 Received August 28, 1978
The migration of human T lymphocytes, assessed in modified Boyden chambers, was chemokinetically stimulated by arachidonic acid in a dose-related manner that achieved a peak level of 127 2 34% enhancement (mean ? SD) at 8 pM arachidonic acid. The chemokinetic effect was dependent on the metabolism of the arachidonic acid by the T lymphocytes as derivatives of arachidonic acid that do not serve as prostaglandin and thromboxane precursors were without effect, while the cycle-oxygenase inhibitors indomethacin (IDS0 = 10 PM) and 5,8,11,14-eicosatetraynoic acid (ETYA) (ID,, = 20 PM) suppressed the stimulation of migration by arachidonic acid. The cycle-oxygenase product 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) reproduced part of the chemokinetic effect of arachidonic acid, but the lipoxygenase product 12-L-hydroxy-5,8,10,14eicosatetraenoic acid (HETE) as well as PGE,, PGF 2a, and thromboxane Bz had no stimulatory activity. The ability of ETYA, but not indomethacin, to suppress the migration of unstimulated T lymphocytes suggested that a lipoxygenase metabolite of endogenous arachidonic acid contributes to the maintenance of their normal levels of spontaneous migration.
INTRODUCTION The factors which regulate the complex patterns of migration, homing, and accumulation of lymphocytes in vivo (l-5) have recently been investigated by in vitro techniques that utilize defined populations of lymphocytes and permit a distinction between principles which evoke increased random locomotion, termed chemokinesis, and those which stimulate directed migration, termed chemotaxis (6). Initial studies of the stimulation of mouse lymphocyte motility were based on quantitating increases in the fraction of splenic B cells exhibiting morphological features suggestive of movement, and revealed a significant enhancement by bivalent specific anti-IgG (7). The stimulatory effect of anti-IgG on motility was dependent on the metabolic integrity of the B cells and was inhibited by cytochalasin B, diisopropyl fluorophosphate, and elevations of intracellular levels of cyclic 3’,5’-adenosine monophosphate, none of which influenced the capping of IgG-anti-IgG complexes (8,9). The observation that mouse and rat B lymphocytes migrated into micropore filters of modified Boyden chambers led to the documentation of the chemokinetic activity of maximally stimulatory concentrations of anti-IgG (7, 10) and revealed a chemotactic effect of lower concentrations 103 0008-8749/79/030103-10$02.00/O Copyright All rights
0 1979 by Academic Press, of reproduction in any form
Inc. reserved.
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(10). That blast transformation facilitates the migration of B lymphocytes was confirmed by the demonstration of especially active chemotactic and chemokinetic responses of human B lymphoblasts to both casein and endotoxin-activated serum (11) and by the greater mobility of mouse B cells following exposure to mitogens (10). Early analyses of the determinants of optimal chemokinetic and chemotactic migration of rodent and human T lymphocytes revealed uniquely specific stimulation by lymphocyte products (10) and some mitogens (12), and suggested a dependence on prior blast transformation which was similar to that of B lymphocytes (11, 12). The potential heterogeneity of chemotactic receptors on mitogen-transformed human T lymphoblasts was illustrated by the selective inhibition of responses to casein and endotoxin-activated plasma by prior treatment of the lymphoblasts with phospholipase C or sphingomyelinase D, while the responses to submitogenic levels of phytohemagglutinin and staphylococcal protein A were blocked with a comparable selectivity by pretreatment with trypsin or a-mannosidase (12). In contrast to B lymphoblasts, the enhanced mobility of the T lymphoblasts was not predominantly a function of the mitogen-induced transformation, but rather was attributable to the period of preincubation in medium containing fetal calf serum. The spontaneous mobility of human T lymphocytes obtained from peripheral blood and tonsillar tissue and their chemokinetic responses to casein and endotoxin-activated serum were enhanced markedly following such preincubation and were not increased further in the presence of mitogenic concentrations of concanavalin A (13, 14). The explanation for the stimulatory effect of preincubation was revealed by studies of the migration of human T-lymphocyte subsets (14). The spontaneous and chemokinetic migration of T lymphocytes possessing Fcp receptors increased with preincubation in parallel with increases in the capacity to form EA-IgM rosettes, while T lymphocytes with Fe-y receptors exhibited little mobility under any conditions. The current demonstration that resting human T lymphocytes are chemokinetitally stimulated by arachidonic acid in the absence of any preincubation suggests the involvement of a mechanism not available to previously described stimuli. Further, as indomethacin blocks the enhancing effect of arachidonic acid on the migration of platelet-free T lymphocytes, some resting T lymphocytes appear to possess a cycle-oxygenase-like enzyme that is capable of generating metabolites of arachidonic acid which enhance lymphocyte migration in a manner analogous to the stimulation of PMN leukocyte chemokinesis by the cycle-oxygenase product 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) (15). MATERIALS
AND METHODS
Blind-ended acrylic migration chambers with Teflon cell compartments (Neuroprobe, Inc., Bethesda, Md.), Ficoll-Paque and Sephadex G-10 (Pharmacia Fine Chemicals, Piscataway, N.J.), cytochalasin B, indomethacin, arachidonic acid, arachidonic acid methyl- and ethyl-esters, and arachidonyl alcohol (Sigma Chemical Co., St. Louis, MO.), Hanks’ balanced salt solution and Eagle’s medium (Microbiological Associates, Bethesda, Md.), five times crystallized ovalbumin (Miles Laboratories, Inc., Elkhart, Ind.), cr-thioglycerol (K & K Laboratories, Inc., Hollywood, Calif.), Sartorius nitrocellulose micropore filters (Beckman Instruments, Mountainside, N.J.), Nucleopore polycarbonate micropore filters (Arthur
LYMPHOCYTE
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105
H. Thomas Co., Philadelphia, Pa.), 4-mm diameter solid spherical glass beads (Fisher Scientific Co., Medford, Mass.), 2-iodoacetamide (Eastman Kodak Co., Rochester, N.Y.), and nylon wool (Leuko-Pak Leukocyte Filter, Fenwall Laboratories, Deer-field, Ill.) were obtained as noted. A supply of purified 5,8,11,14-eicosatetraynoic acid (ETYA) was kindly provided by Dr. James Hamilton of the Hoffman-La Roche Company (Nutley, N.J.). Colchicine was agift from the Eli Lilly Company (Indianapolis, Ind.), and PGEz and PGFI, were gifts from the Upjohn Company (Kalamazoo, Mich.). Preparation and purification of lipid factors. The source of 12-L-hydroxy5,8,10,14-eicosatetraenoic acid (HETE) was a mixture of human platelets and arachidonic acid that was incubated in the presence of 0.10 mM indomethacin to block cycle-oxygenase activity as described (15, 16). The HETE was extracted and purified by repeated silicic acid chromatography, and the purity of the final product exceeded 98% as assessed by thin-layer chromatography and gas-liquid chromatography/mass spectrometry (16, 17). 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) was prepared by incubating arachidonic acid with a suspension of crude microsomal powder that had been obtained by acetone-pentane treatment of sheep seminal vesicles and which served as the source of cycle-oxygenase (15, 18). The HHT was extracted and purified by repeated silicic acid column chromatography (15); the purity of the final product exceeded 90% as assessedby gas-liquid chromatography/mass spectrometry (15,19). Thromboxane Bz was produced by total organic synthesis and purified as described (20). Puri$cation of human lymphocytes migration. Thirty-milliliter portions
and measurement
of in vitro lymphocyte
of blood from normal volunteers were defibrinated by continuous agitation for 10 min in 40-ml capped plastic tubes with 15-20 glass beads to prevent coagulation and remove platelets. The defibrinated blood was diluted with an equal volume of Hanks’ solution made 0.005 M in Tris-HCl (pH 7.4) (Hanks’-Tris) and 30-ml portions were layered on 30-ml cushions of Ficoll-Paque and centrifuged at 400g for 30 min at room temperature. The mixed mononuclear leukocytes at the top of the Ficoll-Paque cushions (21) were recovered by gentle aspiration, pooled, washed twice with Hanks’-Tris buffer (pH 7.4), suspended in the same buffer containing 0.4 g/100 ml of ovalbumin, and filtered on a Sephadex G-10 column of IO-ml bed volume that was supported on a Ito 2-ml pad of nylon wool. The lymphocyte population that filtered through the column contained fewer than 1 platelet and 1 monocyte per 500 lymphocytes according to phase-contrast microscopic enumeration and differential counts of Wright-Giemsa stained smears. Analyses of the cell content of the purified lymphocyte suspensions utilizing the techniques of sheep erythrocyte rosetting, C3d receptor rosetting, and assessment of surface immunoglobulins (22-24) indicated a composition of 74-85% T lymphocytes and fewer than 2-6% B lymphocytes. Migration was assessed in modified Boyden chambers using Hanks’-Tris buffer made 0.2 g/100 ml in ovalbumin for both compartments. One-half milliliter of a suspension containing 3.5-4.0 x lo6 lymphocytes was pipetted into the top compartment of each chamber which was separated from the 0.2-ml buffer compartment below by a layer of two filters consisting of an 8-pm pore Nucleopore filter over an 8-pm pore Sartorius filter. After incubation of the chambers for 4 hr at 37°C in an atmosphere of 5% COz in air, the lower (Sartorius) filter was removed,
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washed in Hanks’ -Tris, and fixed and stained in hematoxylin as described for PMN leukocytes (15,25). The lymphocytes which migrated in the front at 60-80 pm from the cell source were enumerated in 20 high power fields (hpf) for each of duplicate or triplicate chambers. Lymphocyte migration is expressed as mean lymphocytes/5 hpf, while alterations induced by the prior addition of lymphocyte agonists to one or both compartments is expressed as % inhibition or % enhancement. RESULTS Initial experiments were designed to determine the optimal conditions for human T-lymphocyte spontaneous migration into a layer of two micropore filters. The incubation of triplicate chambers at 22°C and 4°C resulted in a mean suppression of migration of 77% and 96%, respectively, from a level of 49 lymphocytes/5 hpf at 37°C in a room air environment, while migration was enhanced by 64% in an environment of 5% CO, in air at 37°C. The use of calcium- and magnesium-free Hanks’ solution in two experiments inhibited migration by a mean of 50% and the further addition of 1 mM ethylenediamine tetraacetate increased the level of inhibitionto 60%, where the control levels of migration in complete Hanks’ solution were 38 and 35 lymphocytes/5 hpf. The effect on migration of the ovalbumin content of the buffer was assessedat concentrations of 0.05,O. 1,0.2,0.4, and 0.8 g/100 ml of ovalbumin, which resulted in mean levels of migration + 1 SD of 29 + 11, 33 of 8, 41 f 15, 38 ~fi 10, and 35 f 19 lymphocytes/5 hpf. All subsequent migration studies thus were carried out with complete Hanks’ solution containing 0.2 g of ovalbumin/lOO ml and with incubations at 37°C in 5% COz in air. T-lymphocyte migration was suppressed by 2-iodoacetamide, which blocks the glycolytic pathway (26), and by cytochalasin B, which interferes with microfilament function (27) (Fig. 1). Fifty percent inhibition of migration was achieved by 10m4M 2-iodoacetamide and approximately lows M cytochalasin B. Lymphocyte migration was increased in a dose-related fashion by colchicine, an effect that has been documented previously to be a result of the action of colchicine on microtubules (11, 28, 29), and by a-thioglycerol, at concentrations of 10-6-10-5 M which stimulate other lymphocyte functions (30) (Fig. 1). Stimulation
of T Lymphocyte
Migration
by Arachidonic
Acid
Arachidonic acid enhanced the migration of T lymphocytes in a dose-related manner at concentrations which exhibited no cytotoxic effect as assessedby trypan blue dye exclusion (Table 1). Maximal stimulation of migration was achieved at 8 and 16 pM concentrations of arachidonic acid irrespective of whether it was added to the buffer or cell compartment. When added to both compartments, the arachidonic acid gave peak stimulation at a level of 8 pM with less effect at 16 PM. As the enhancement of migration observed was a function of net concentration irrespective of a gradient, arachidonic acid appeared to act solely by a chemokinetic action. Arachidonic acid thus was added to both compartments in all subsequent protocols. Preincubation of the purified T lymphocytes in buffer containing fetal calf serum increased the level of spontaneous migration relative to that seen with fresh cells obtained from the same donors (Table 2). However, the peak enhancement by arachidonic acid at a concentration of 8 pM in both compartments was actually
LYMPHOCYTE
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EFFECT OF ARACHIDONATE
l 120 +100 +50 s s e 9 E
+40-
P >r
+20-
]/j~j[
mi
+50-
11
o----------~------------1 P
ii 23 .E:
-2o-
!3
1
$8 -40-5o-
I
i
-------I
I
I -so!, -100 h I lo-5lo-4m-3 lo-9104lo-'lo-@ 1
I
Concentmtim
of agonist (Ml
FIG. 1. Effects on T-lymphocyte migration of known inhibitors and enhancers of lymphocyte function. Each point and bracket depicts the mean + 1 SD for n experiments with each agonist. The spontaneous migration values in buffer alone (0% change) for the experiments with each agonist were: 85.63, and 27 lymphocytes/S hpf with 2Godoacetamide (n = 3); 35,32,63, and 85 with cytochalasin B (n = 4); 25.27, and 19 with a-thioglycerol (n = 3); and 36, 41, and 28 with colchicine (n = 3). All agonists were present at an equal concentration in the buffer and cell compartments.
decreased from 118% without preincubation and 107% with preincubation in buffer alone to 56% by preincubation in the presence of fetal calf serum. Arachidonyl alcohol and the methyl- and ethyl-esters of arachidonic acid, which are not metabolized by the cycle-oxygenase of platelets (31) and other cells, did not stimulate T-lymphocyte migration, whereas comparable concentrations of TABLE 1 Modified Checkerboard Analysis of Arachidonate Stimulation of T-Lymphocyte Migration Concentration of arachidonate (fl)
Buffer compartment only Cell compartment only Both compartments Enhancement (%)*
0
2
4
8
46” -
39 56 57 55 + 25
67 53 61 91 + 38
88 62 96 127 + 34
16
32
89 79 62 80~31
57 38 18 -
a Each value is the mean for triplicate chambers in one experiment expressed as lymphocytes/5 hpf. * Mean percentage enhancement + 1 SD for six experiments using different cell donors and with arachidonate in both compartments.
MCCARTY AND GOETZL TABLE 2 Effects of Preincubation of T Lymphocytes on Response to Arachidonic Acid Concentration of arachidonate (&I)
No preincubation Preincubation in Eagle’s medium without protein Preincubation in Eagle’s medium with fetal calf serum
0
2
4
8
16
28 lr 7”
462 11
55 zk 13
61 ? 10
47 ” 12
30 f 4
51 * 12
56 f 15
62 k 8
44 “_ 10
43 ” 2
52k
59 f 6
67 2 17
56 f 9
11
(1Each value is the mean 2 SD for three experiments performed in duplicate and is expressed as lymphocyte45 hpf. Arachidonate was present at the stated concentrations in both compartments.
arachidonic acid produced the characteristic effect, exhibiting a peak enhancement of 132% at the 8 pM level (Fig. 2). The Mechanism of Chemokinetic Stimulation of T Lymphocytes by Arachidonic Acid That the metabolism of arachidonic acid by lymphocyte enzymes, which are analogous to the cycle-oxygenase and lipoxygenase of platelets, might be involved in the stimulation of migration of platelet-free lymphocytes was investigated by using known enzymatic inhibitors. Indomethacin, which selectively inhibits the activity of cycle-oxygenase (19,32), significantly suppressed the stimulatory effect of 16 PM arachidonic acid, exhibiting a 50% inhibitory level of approximately 10 PM, but did not influence spontaneous migration (Table 3). ETYA, which inhibits the activity of both cycle-oxygenase and lipoxygenase (19), suppressed both spontaneous migration and the stimulatory effect of arachidonic acid with 50%
-30'
4
6
16
I 32
Concentmtion of amchidonic acid OTderivative ( pM)
FIG. 2. Chemokinetic effects on T lymphocytes of arachidonic acid and arachidonate derivatives. Each bar and bracket depicts the mean and range of two experiments performed in duplicate. The level of spontaneous migration (0% enhancement) for the two experiments were 28 and 32 lymphocytes/5 hpf.
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EFFECT OF ARACHIDONATE
TABLE 3 Inhibition by Indomethacin and ETYA of the Spontaneous and Arachidonate-Stimulated Migration of T Lymphocytes Arachidonate” (VW
5
ETYA ETYA
0 16
14 f 8b 26+21
Indomethacin lndomethacin
0 16
Inhibitor
Concentration of indomethacin or ETYA (fl)
-
20
40
80
160
23 2 16 56 k 4*
53 iz 20 71 k 12*
69t 31 94 k 19*
91 2 17* 98 + 8*
10 4226 55 L 18**
-
2k31 66 + 14*
-
21 2 33 74 2 19*
a Arachidonate and the inhibitors were present in both compartments without a gradient. b Each value is the mean percentage inhibition k 1 SD for three experiments performed in duplicate. Levels of statistical significance are depicted as follows: *P < 0.01; **P < 0.05. The percentage enhancement k 1 SD by 16 fl arachidonate was 148 2 20% for the ETYA experiments and 117 k 32% for the indomethacin experiments.
inhibition at concentrations of 40 ph4 and 20 pM, respectively. In order to determine which type of metabolite of arachidonic acid might be responsible for the observed stimulation of T-lymphocyte migration, a range of purified and synthetic lipids were examined for their lymphocyte chemokinetic activity (Table 4). HHT, a platelet cycle-oxygenase product of arachidonic acid (19), stimulated lymphocyte migration by up to 90% at a concentration of 16pM, but only achieved 56-70% of the peak enhancing effect of comparable concentrations of arachidonic acid in the same experiments. In contrast, the lipoxygenase product HETE as well as PGEz inhibited spontaneous migration at the highest concentrations, while PGFza exhibited no substantial effects on migration and thromboxane B, was stimulatory only at a concentration of 32 pM. DISCUSSION The quantitation of human T-lymphocyte mobility by a technique employing a layer of two micropore filters provided uniform levels of background and stimulated migration for cell preparations from diverse donors (Figs. 1, 2; Table 2). Presumably, this was due to an even delivery of a functionally more homogenous population of lymphocytes to the bottom filter as a result of prior migration through the thin top filter. That the movement of the T lymphocytes into the bottom filter was a function of cellular migration was suggested by their transformation to elongated motile-appearing forms, which has been described (11, 13), and was confirmed by the inhibitory effects of iodoacetamide and cytochalasin B, at concentrations of the latter agent known to interfere with the integrity of microtilaments (27) (Fig. 1). Crystallized ovalbumin did not have a striking influence on T-lymphocyte migration, as the variations from the overall mean level of migration were within 20 + 2% for individual values obtained at ovalbumin concentrations ranging from 0.05-0.8 g/100 ml. Since the generally marked stimulation of PMN-leukocyte or monocyte migration by native albumins (33) has been attributed to a modulation of surface contact between the leukocytes and the filter matrix, it is possible that T-lymphocyte migration is less dependent on
110
MCCARTY AND GOETZL TABLE 4 Influence of Synthetic Lipids on T-Lymphocyte Migration Concentration of lipid factor @IV)”
Arachidonate HHT HETE TBzC PGEI PGFa,
4
8
16
32
1036 41 11 -12 4 2
151 84 7 0 -
129 90 2 16 -18 9
91 53 -25 73 -57 5
a The lipid factors were present in both compartments at an equal concentration. b Each value is mean percentage enhancement for two experiments performed in duplicate; values in the absence of any lipid (0% enhancement) were 26 and 32 lymphocytes/5 hpf. e Thromboxane B,.
interactions of the cells with the filter material at the pores. In contrast to the inhibition of PMN-leukocyte migration by colchicine (34), this and other microtubule-disaggregating agents enhance the migration of lymphocytes which may reflect a different regulatory function for lymphocyte microtubules (Fig. 1; 11, 28,29). T-lymphocyte migration also was stimulated by up to 100%by 10W6-10e5M cr-thioglycerol, a reducing principle known to stimulate other lymphocyte functions (30). Varying the concentration of each of the enhancing factors in the lymphocyte and buffer compartments of replicate chambers led to the conclusion that the effects of arachidonate (Table 1) and the other stimuli were virtually entirely chemokinetic. Thus the results suggested that human T-lymphocyte migration in vitro is modulated by microfilaments and microtubules, and is enhanced by previously recognized lymphocyte stimuli. The demonstration that arachidonic acid significantly enhanced the in vitro migration of the T lymphocytes both led to studies of the cellular requirements for the stimulatory effect and provided an opportunity to probe some of the pathways by which lymphocytes elaborate products of arachidonic acid which exhibit feed-back regulatory functions (35). Arachidonic acid stimulated lymphocyte migration by 93-161% (Table 1) and a modified checkerboard analysis (6) confirmed the fact that chemokinesis was the predominant mode of stimulation. The chemokinetic effect on T lymphocytes was diminished at higher concentrations of arachidonic acid, which also is a characteristic of low molecular weight factors that are chemokinetic and chemotactic for PMN leukocytes (36, 37). Prior incubation of T-lymphocyte suspensions in 20% fetal calf serum increased the level of spontaneous migration, but had no significant effect on the extent of chemokinetic stimulation by arachidonic acid (Table 2). This clearly distinguished the mechanism of stimulation by arachidonic acid from those of other lymphocyte migration-enhancing factors such as casein, endotoxin-activated serum, staphylococcal protein A, and phytohemagglutinin, whose activities are dependent on at least prior incubation of the lymphocytes in media containing fetal calf serum (13, 14). Several lines of evidence together suggested that the chemokinetic activity of
LYMPHOCYTE
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EFFECT OF ARACHIDONATE
111
arachidonic acid was dependent on its metabolic conversion by the platelet-free T lymphocytes. Esters of arachidonic acid, which are not metabolized by the cycle-oxygenases of platelets (31) or other cells, did not stimulate T-lymphocyte migration at concentrations in the maximally effective range for arachidonic acid (Fig. 2). The addition of indomethacin to the lymphocytes prevented the expression of arachidonic acid chemokinetic activity with an IDso of 10 PM (Table 3), a value that is comparable to the ID,, for the inhibition by indomethacin of platelet cycle-oxygenase activity (19,32). ETYA, which inhibits both the cycle-oxygenase and lipoxygenase of platelets (19), not only prevented the chemokinetic effect of arachidonic acid but also suppressed the spontaneous migration of lymphocytes (Table 3). In order to assess the possible nature of the lymphocyte-generated chemokinetic metabolite(s) of arachidonic acid, a series of defined platelet metabolites of arachidonic acid were studied in the lymphocyte migration assay. Only the cycle-oxygenase product HHT was capable of substantially stimulating T-lymphocyte migration in a dose-related fashion (Table 4), which suggests that the chemokinetic product of arachidonic acid produced by the indomethacininhibitable lymphocyte pathway may be an unsaturated hydroxy-fatty acid. One or more subpopulations of human suppressor T lymphocytes influence the functions of other lymphocytes by elaborating inhibitory concentrations of prostaglandins of the E series (35). The suppressor activity of such lymphocytes can be attenuated or eliminated by indomethacin in circumstances as diverse as the altered immunocompetence of pregnancy and of Hodgkin’s disease (35, 38). That resting lymphocytes appear to convert arachidonic acid into products which also are capable of enhancing lymphocyte migration suggests that endogenously elaborated lipids may represent a feed-back system for the bidirectional modulation of lymphocyte function. ACKNOWLEDGMENTS Dr. Goetzl is Director of the Laboratories for the Study of Immunological Diseases of the Howard Hughes Medical Institute. The authors are grateful to Dr. David Glass for his analyses of the constituents of the purified populations of lymphocytes.
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15. Goetzl, E. J., and Got-man, R. R., J. Zmmunol. 120, 526, 1978. 16. McGuire, J. C., Kelley, R. C., Gorman, R. R., and Sun, F. F., Prep. Biochem. 8, 147, 1978. 17. Goetzl, E. J., Woods, J. M., and Got-man, R. R., J. Clin. Znvesf. 59, 179, 1977. 18. Wallach, D. P., and Daniels, E. G., Biochim, Biophys. Acta. 231, 445. 1971. 19. Hamberg, M., and Samuelsson, B., Proc. Nat. Acad. Sci. U.S.A. 71, 3400, 1974. 20. Nelson, N. A., and Jackson, R. W., Tetrahedron Lert., 3275, 1976. 21. Boyum, A.,Scnnd. .Z. Lab. Clin. Invest. 21(Suppl. 97), 31, 1%8. 22. Hoffman, T., and Kunkel, H. G., In “In Vitro Methods in Cell-Mediated and Tumor Immunity” (B. R. Bloom, and P. R. David, Eds.), pp. 71-82. Academic Press, New York, 1976. 23. Ehlenberger, A. G., and Nussenzweig, V., In “In Vitro Methods in Cell-Mediated and Tumor Immunity” (B. R. BloomandP. R. David, Eds.),pp. 113-121. AcademicPress, New York, 1976. 24. Preud-Homme, J. L., and Labaume, S., In “In Vitro Methods in Cell-Mediated and Tumor Immunity” (B. R. BloomandP. R. David, Eds.), pp. 155-169. Academic Press, New York, 1976. 25. Goetzl, E. J., and Austen, K. F., J. Exp. Med. 136, 1564, 1972. 26. Kamovsky, M. L., Physiol. Rev. 42, 143, 1962. 27. Wessells, N. K., Spooner, B. S., Ash, J. F., Bradley, M. O., Luduena, M. A., Taylor, E. L., Wrenn, J. T., and Yamada, K. M., Science 171, 135, 1971. 28. Fram, R. J., Sidmar, C. L., and Unanue, E. R., J. Zmmunol. 117, 1456, 1976. 29. Center, D. M., Wasserman, S. I., and Austen, K. F., Cell. Zmmunol. 39, 325, 1978. 30. Goodman, M. G., and Weigle, W. 0.. J. Exp. Med. 154, 473, 1977. 31. Van Dorp, D., In “Prostaglandins” (P. Ramwell and J. E. Shaw, Eds.), pp. 181-199. N.Y. Acad. Sci., New York, 1971. 32. Samuelsson, B., In “Advances in Prostaglandin and Thromboxane Research” (B. Samuelsson and R. Paoletti, Eds.), Vol 1, pp. l-6. Raven Press, New York, 1976. 33. Wilkinson, P. C., Exp. Cell Res. 103, 415, 1976. 34. Caner, J. E. Z., Arthr. Rheum. 8, 757, 1965. 35. Goodwin, J. S., Bankhurst, A. D., and Messner, R. P., J. Exp. Med. 146, 1719, 1977. 36. Goetzl, E. J., and Austen, K. F., J. Exp. Med. 144, 1424, 1976, 37. Showell, H. J., Freer, R. J., Zigmond, S. H., Schiffman, E., Aswanikumar, S., Corcoran, B., and Becker, E. L., J. Exp. Med. 143, 1154, 1976. 38. Goodwin, J. S., Messner, R. P., Bankhurst, A. D., Peake, G. T., Saiki, J. H., and Williams, R. C., Jr., N. Engl. J. Med. 297, 963, 1977.
