Eur. J. Biochem. 196, 395-400 (1991) 0FEBS 1991 0014295691001566
Effects of monocyte-lymphocyte interaction on the synthesis of leukotriene B4 Per-Johan JAKOBSSON, Bjorn ODLANDER and Hans-Erik CLAESSON Department of Physiological Chemistry, Karolinska Institutet, Stockholm, Sweden (Received August 21/December 3, 1990) - EJB 90 1006
Human monocytes in monolayers were challenged with the calcium ionophore A23187. Methanol trapping of the products in the cell-free supernatants, followed by analysis on HPLC and by ultraviolet spectroscopy, revealed the presence of two compounds, which exhibited a conjugated-triene spectrum and chromatographed with the compounds formed when synthetic leukotriene (LT) A, was added to warm acidified methanol. Furthermore, addition of purified LTA4 hydrolase to the cell-free supernatant of monocytes, stimulated with the ionophore A23187, resulted in increased levels of LTB4. These results indicate that monocytes release LTA4 extracellularly after activation with the calcium ionophore. Incubation of monocytes together with monoclonal lymphocytic cells, of both B and T cell lineage, yielded increased levels of LTB4 whereas the non-enzymatic isomers of this compound, i. e. d6-trans-LTB4 and 12-epi-A6trans-LTB,, declined. In addition, the sum of LTB4 and its non-enzymatically formed isomers increased in mixed cultures of monocytes and monoclonal lymphocytic cells as compared to monocytes alone. The present study indicates that activated monocytes release LTA,, which is converted into LTB4 by monoclonal lymphocytic cells. Furthermore, the increase of the total amounts of leukotrienes on incubation of monocytes with lymphocytic cells, suggests the presence of an additional mechanism leading to activation of the 5-lipoxygenase pathway in monocytes.
Monocytes and macrophages possess 5-lipoxygenase activity and convert arachidonic acid to leukotrienes (LT) [l]. Several stimuli such as calcium ionophore A23187 [2], zymosan [2] and aggregated immunoglobulins [3, 41, can induce formation of leukotrienes in these cell types. Human tonsil B lymphocytes and peripheral B and T lymphocytes are devoid of 5-lipoxygenase activity [5 - lo], but express LTA4 hydrolase activity [9, 111. Monoclonal lymphoblastoid cells possess higher LTA4 hydrolase activity than normal lymphocytes [9]. Furthermore, the expression of LTA, hydrolase but not of 5-lipoxygenase was demonstrated at both transcriptional and translational levels in Raji cells [8], an established Burkitt’s lymphoma B cell line. Recently, LTA4 hydrolase was isolated and purified from Raji cells [121. It has been demonstrated that LTA, is released from activated polymorphonuclear leukocytes [13] and studies on cell/ cell interactions have revealed that this compound can be
utilized by erythrocytes [14] and endothelial cells [15] for synthesis of LTB,. Several cell types, including platelets [16], mast cells [13] and endothelial cells [15,17] can convert extracellular LTA, into cysteine-containing leukotrienes. Leukotrienes influence several lymphocyte functions. Specific high-affinity binding sites for LTB4 have been described on these cells [18], and it has been reported that this compound induces natural killer and suppressor/cytotoxic cell activity [19]. LTB4 stimulates, in synergy with Blymphotrophic factors, expression of the activation-associated surface antigen CD23 on resting B lymphocytes [20]. Furthermore, initiation of DNA synthesis, cell growth and immunoglobulin production are enhanced in B cells by LTB, [20]. LTB, has also been reported to influence the formation and effects of several cytokines [21-241. The present study deals with the release of LTA, from activated monocytes and effects of monocytes monoclonal lymphoblastoid cell interactions on LTB4 synthesis.
Correspondence to P.-J. Jakobsson, Department of Physiological Chemistry 11, Karolinska Institutet, Box 60400, S-10401 Stockholm, MATERIALS AND METHODS Sweden. Abbreviations. Leukotriene (LT) A,, (5S)-trans-5,6-oxido-7,9-Materials truns-11,14-cis-eicosatetraenoic acid; LTB,, (5S, 12R)-5,12Synthetic leukotriene A4 was a generous gift from Dr A. dihydroxy-6,14-cis-8,10trans-eicosatetraenoic acid ; LTC4, 5(S)Ford-Hutchinson (Merck Frosst, Canada). Alkaline hydrolyhydroxy-6(R)-S-glutathionyl-7.9-trans-l1 ,I 4-cis-eicosatetraenoic acid ; 5-HETE, 5 (S)-5-hydroxy-6-truns-8,I 1,14-cis-eicosatetraenoic sis of leukotriene A4 methyl ester was carried out as described acid; DHETE, dihydroxy-eicosatetraenoic acid; RP-HPLC, reverse- in [25]. Medium M199, penicillin-streptomycin, FungizoneR, trypsin and plastic Petri dishes were from Flow Laboratories phase HPLC. Enzymes. Arachidonate 5-lipoxygenase (EC 1 .I 3.11.34); leu- (Rockville, MD). The calcium ionophore A23187 was obtained from Calbiochem-Behring (La Jolla, CA). Carkotriene-A, hydrolase (EC 3.3.2.6).
396 water, 1 : 1. Analysis was performed on reversed-phase HPLC (RP-HPLC). Incubation of the supernatantjrom activatedmonocytes with purified LTA4 hyeolase. LTA4 hydrolase was isolated and Isolation and cultivation o f c d l s purified from Raji cells [12]. Monocytes, still attached to the Human monocytes were isolated from leukocyte concen- Petri dish after extensive washing, were scraped off with a trates obtained from healthy donors at the Karolinska Hospi- plastic cell lifter, centrifuged and suspended in buffer A contal (Stockholm, Sweden). The cell suspension was diluted 1: 2 taining 0.5% albumin. Cell concentration was adjusted to with Dulbecco's phosphate-buffered saline, pH 7.4, sup- 4 x lo6 cells/ml and viability was > 90% as determined by plemented with Ca2+and Mg2 (buffer A). Erythrocytes were means of trypan blue dye exclusion. 4 ml cell suspension was sedimented with 0.5% (mass/vol.) Dextran. Platelets were re- poured into Petri dishes (2 ml/dish) and equilibrated for 5 min moved by centrifuging the leukocyte-rich plasma at 300 x g in the incubator. The cells were stimulated with 5 pM for 10 min. The pellet was washed in buffer A and centrifuged ionophore A23187 for 4min. The incubation fluids were at 100 x g for three cycles. After the final centrifugation the pooled and immediately centrifuged until 1000 x g was platelet-poor leukocyte fraction was applied on a discontinu- reached (total time 90 s). The cell-free supernatant (4 ml) was ous density gradient (Ficoll-Isopaque) and centrifuged at divided into two parts, one of which received LTA4 hydrolase and one of which received buffer A only. The enzymatic 600 x g for 30 min [26]. The monomorphonuclear cells were collected and diluted reaction was allowed to occur for 20 min at room temperawith buffer A, centrifuged and further washed twice with ture, and thereafter stopped with 3 vol. methanol, centrifuged buffer A at 75 x g and then suspended in medium M199 sup- and evaporated to dryness under reduced pressure. The resiplemented with 20% human serum, penicillin (100 IU/ml), dues were resolved in methanol, dried under argon and finally streptomycin (0.1 mg/ml), FungizoneR (2.5 pg/ml) and L-glu- resolved in methanol/water (1 : 1) before RP-HPLC tamine (0.15 mg/ml). The cell concentration was adjusted to analysis. lo7 cells/ml and layered onto Petri dishes (63 cm'), 5 ml/dish. The cells were incubated for 1 h at 37°C in an atmosphere Incubation of monocytes with monoclonal lymphocytic cell lines of 5% CO,. Non-adherent cells were removed by vigorous Lymphocytic cells were washed twice in buffer A, suswashing with buffer A . Attached cells were either used directly in experiments or cultured overnight in medium. The purity of pended in buffer A and added to adherent monocytes. The the cells was estimated by morphological examination (May- mixed cell population was incubated for 10 min in the incuGriinwald/Giemsa staining) and by presence of non-specific bator prior to the addition of calcium ionophore A23187 (5 pM, 10 min). Incubations were stopped by addition of esterase [27]. The range of monocyte purity was 75-95%, with lymphocytes as the major remaining cell type. Granulo- 1 vol. methanol. Prior to RP-HPLC analysis, the samples were cytes were always less than 1% (mainly basophil granulo- centrifuged at 15000 x g for 20 min. cytes). Cell lines used in this study were Raji [28], a B-lymphocytic Analysis of leukotrienes Burkitt lymphoma and MP-6 [29], a T4-T hybridoma. The Analysis of LTA4-trapping products. Trapping products Raji and MP-6 lymphoblastoid cell lines were cultivated in medium RPMI 1640 supplemented with fetal calf serum (So/,), were analyzed by RP-HPLC, using a Nucleosil C I 8 column L-glutamine (0.3 mg/ml, penicillin 100 IU/ml and strepto- (3 mm x 100 mm, 120 - 3 pm particles), and eluted at a flow rate of 0.4 ml/min with methanol/water/acetic acid mycin 0.1 mg/ml. (68: 32: 0.01, by vol.). Eluted compounds were detected and quantified utilizing a programable Hewlett-Packard 8451A Detection of extracellular LTA4 diode-array spectrophotometer, on-line with the HPLC sysTrapping of LTA4 with methanol. After extensive washing tem. The identification of eluted compounds was based on of adherent blood cells, 2 ml buffer A, supplemented with 1% chromatography with standards and on ultraviolet specalbumin, in order to stabilize leukotriene A4 [30], were added troscopy. Material in peaks eluting with standards were colto each Petri dish. The cells were allowed to equilibrate in the lected and treated with diazomethane, giving methyl esters of cell incubator (37 C, 5% CO,, 15 min) before challenging the compounds. The compounds were further analyzed either with the calcium ionophore A23187 (15 pM) for 6 min. Since in a straight-phase HPLC system, using a 250 mm nucleosil albumin diminishes the effect of ionophore stimulation [31], 5 pm column with hexane/isopropanol(98 :2, by vol.) as mothis concentration was essential for a proper stimulation of bile phase, or in a RP-HPLC system, using methanol/water the monocytes in the presence of albumin. Ionophore A23187 (75 : 25, by vol.). Quantification o j leukotrienes. Leukotrienes were quanin a concentration of 15 pM was toxic for cells in the absence of albumin, but in the presence of albumin (1% mass/vol.), tified by RP-HPLC, as described in [34], with the exception toxicicity as assessed by trypan blue dye exclusion was that no pre-column was utilized. Briefly, HPLC was performed elminated ( > 97% viable cells). The incubation fluid from on a Constametric I11 equipment with a CI-10 integrator from several Petri dishes was collected and centrifuged for 1 min at LDC/Milton Roy with a Nucleosil c18 column (pore size, 800 x g . Thereafter the supernatant was immediately poured 12 nm; 3 pm particles, Skand. Genetec, Sweden). The mobile into 10 vol. warm acidified methanol (40"C, pH 3) [32]. Pre- phase was acetonitrile/methanol /water (29.5: 19.5: 51) supcipitated material was removed by centrifugation and the re- plemented with 1% acetic acid (by vol.) and adjusted to pH maining fluid was evaporated under reduced pressure. The 5.6 with 30% NaOH (by vol.). Prior to analysis, the integrator sample was dissolved in distilled H,O and applied to a Sep- was calibrated with known amounts of standards. Ultraviolet Pak cartridge, which was washed with 10 ml water fol- monitoring was carried out at 270 nm, and quantitative analylowed by 10 in1 hexane [33]. Finally, leukotrienes were eluted sis was performed by integration of the elution profile. Qualiwith 10 nil methanol, evaporated and dissolved in methanol/ tative analysis was performed by comparison with retention
tridges containing octadecylsilyl silica (Sep-PakR) were purchased from Waters Associates (Milford, MA).
+
397
I
Ill
1
IV
v
0
I
0
I
I
15
30
1
45
Tirne(min1
Fig. 1. RP-HPLC chromatogram ofthe products released by monocytes after stimulation with the calcium ionophore A23187. Adherent human monocytes were stimulated with the ionophore A23187 (15 FM) for 6 rnin in the presence of 1 % albumin. The cell-free supernatant was mixed with 10 vol. warm acidified methanol, purified and subjected to RP-HPLC. Peaks 1-111 coeluted with standards of A6-trans-LTB,, 12-epi-A6-trans-LTB, and LTB4, respectively. Peaks IV and V coeluted with the products formed after addition of synthetic LTA, to warm acidified methanol. Insert: ultraviolet spectra of peaks IV and V
volumes of known standards and analysis of ultraviolet spectral properties of eluted compounds on a Hewlett-Packard 8450 computerized diode-array spectrophotometer.
15 Time (rnin)
20
Fig. 2. RP-HPLC chromatogram of the methyl esters of the material in peaks ZV and V. The upper chromatogram show the RP-HPLCprofile of the methyl esters of the products formed after addition of synthetic LTA4 to warm acidified methanol, i.e. the methyl esters of 5(S)-hydroxy-l2(R)-methoxy-d6-trans-LTB4and 5(S)-hydroxy-12epi-methoxy-d6-trans-LTB4.The lower trace shows the elution profile in this system of the methyl esters of the material in peaks IV and V
Table 1 . Detection of LTA, in the supernatants of stimulated monocytes by incubation with LTA4 hydrolase Monocytes were stimulated with ionophore A23187 (5 pM) for 4 min in the presence of 0.5% albumin. Cell-free supernatants were prepared and either plain buffer or purified LTA, hydrolase was added. The samples were incubated for 20 min at room temperature and subsequently analyzed by RP-HPLC. Each value represents the mean & range of two independent experiments LTA4 detected A6-trans-LTB, 12-epi-A6trans-LTB,
RESULTS
LTB4
total
Release of LTA4from activated monocytes Monocytes attached to Petri dishes were incubated with the ionophore A23187 (15 pM) for 6 min in the presence of 1% albumin. The cell-free incubation fluids were mixed with warm acidified methanol and subsequently analyzed on RPHPLC. Fig. 1 shows the RP-HPLC chromatogram of products released from monocytes stimulated with the calcium ionophore. The retention times of peaks I and I1 corresponded to those of the non-enzymatically formed isomers of LTB4, A 6-trans-LTB4 and 12-epi-A6-trans-LTB4, respectively. Peak I11 eluted as synthetic LTB4. Peaks IV and V comigrated with the products formed, when synthetic LTA4 was treated with warm acidified methanol. Ultraviolet spectral analysis of peaks IV and V revealed maxima at 270 nm and shoulders at 260 nm and 280 nm (Fig. 1, insert). The retention times of these peaks were different from those of the two epimers of 5,6-dihydroxyeicosatetraenoicacid. Furthermore, they did not appear if the incubations were stopped with 3 vol. ethanol. The material eluting in peaks IV and V was collected, treated with diazomethane, and subsequently submitted to further analysis by RP-HPLC (Fig. 2). Two main peaks were detected, which comigrated with the two products generated when synthetic LTA4 was treated identically as the sample. The ultraviolet spectra of these peaks were in agreement with those reported for leukotrienes. In addition, if the methyl esters of the material in peaks IV and V were analyzed on straightphase HPLC, this also revealed two peaks coeluting with the
pmo1/lO7 monocytes Monocytes/buffer Monocytes/ LTA4 hydrolase
k 15
222
2
313 k 13
88k 4
485
87
573 _+ 83
91
two products formed nonenzymatically from synthetic LTA4 (data not shown). Taken together, these data show that peaks IV and V represent the trapping products in methanol of LTA4, i. e. 5(S)-hydroxy-12(R)-methoxy-d6-trans-LTB4 and 5(S)-hydroxy-l2-epi-methoxy-d6-trans-LTB4 [32]. Detection of monocyte-released LTA4 using purified LTA4 hydrolase
The experimental procedure was as described for methanol trapping of LTA4, with the exception that instead of adding warm acidified methanol to the samples, either purified LTA4 hydrolase in buffer or buffer alone was added to the cell-free monocyte supernatants. The addition of LTA4 hydrolase led to an approximate twofold increase in LTB, levels compared to buffer control (Table 1). No difference in the levels of nonenzymatically formed LTB4 isomers could be detected in the albumin-stabilized incubations. The same pattern was ob-
398
LTB,
E
I
LTB,
I
0
. N t
c
a
a, U
C
: n
Q
0
15
30 Time (min)
0
L5
15 30 Tirne(min)
45
Fig. 3. RP-HPLC chromatograms o j t h e products formed by monocytes alone ( A ) and monocytes incuhated together with MP-6 cells ( B ) after stimulation with the ionophore A23187. Human monocytes were incubated with the calcium ionophore A231 87 ( 5 pM) for 10 min in the absence (A) or in the presence of MP-6 cells ( B ) . The ratio of monocytes/MP-6 cells was 1 :4. The retention volumes of synthetic standards are indicated. LTC4, leukotricnc C4: LTB4, leukotriene B4; I and TI, 12-epimers of 6-trans-LTB4
served in control experiments, when purified LTA4 hydrolase or buffer was added to albumin-containing buffer A supplemented with synthetic LTA4 (data not shown).
T
E,
Effects of monoc~te/monoclonallymphocytic cell interactions on the metabolism of leukotrienes
Monocytes alone or together with either Raji or MP-6 cells were stimulated with ionophore A23187 ( 5 pM) for 10 min. Fig. 3A shows an RP-HPLC chromatogram of the products formed by 12 x l o 6 adherent monocytes. When the same number of monocytes were incubated together with MP-6 cells (48 x lo6 cells), this led to an alteration of the leukotriene pattern (Fig. 3B). The amounts of LTB4 produced were increased, whereas the levels of A‘-trans-LTB4 and 12-epi-A6trans-LTB4 declined. The significance and magnitude of the observed changes in leukotriene formation by interaction of monocytes with Raji cells was estimated by statistical evaluation. Data obtained from five separate experiments, at a ratio of monocytes/Raji cells of 1 : 4, was analyzed by Student’s paired t test (Fig. 4). In samples with monocytes alone, the level of LTB4 was 255 41 pmol/107 monocytes (mean SE, n = 17). In the presence of Raji cells, the level of LTB4 was 459 f 67 pmol/107 monocytes (mean f SE, n = 17), corresponding to an 80% increase of LTB4 levels. In addition, the non-enzymatic isomers of LTB4 declined from 115 20 pmol/ lo7 monocytes to 20 5 7 pmol/107 monocytes in the mix cultures (mean SE, n = 17). The sum of LTB4 and its nonenzymatic isomers increased from 370 f 59 pmol/107 monocytes to 479 74 pmol/107 monocytes in the presence of Raji cells. This increment was statistically significant (P< 0,001).
. s
500
5 400 E 300 ln a,
&-
200
c L
100 3
J
O
1+11
LTBq
I+II+LTB4
Fig. 4. Effect of the interaction of monocytes with Raji cells on the synthesis of LTB4 and its non-enzymatic isomers. Monocytes were incubated in the presence or absence of Raji cells with the ionophore A23187 ( 5 pM) for 10 min. The products were quantified by RPHPLC; each bar represents mean SE, n = 17. Open bars depict the products formed by monocytes alone and hatched bars the compounds formed by monocytes with Raji cells. The ratio of monocytes/Raji cells was 1 :4. Statistical significance of differences in leukotriene formation was estimated by using Student’s paired t test. ***, P < 0.001
+
*
DISCUSSION
Monocytes have the capacity to metabolize arachidonic acid to leukotriene B4 [ I , 21, a compound which influences certain lymphocyte functions [18 -24, 351.
The present study indicates, as demonstrated by two independent methods, that monocytes release the unstable intermediate LTA4 upon stimulation with the calcium ionophore A23187. First, trapping experiments showed that two products, exhibiting a conjugated-triene spectrum, appeared when the cell-free supernatant of stimulated monocytes was mixed with warm acidified methanol. These compounds chromatographed on RP-HPLC with the products formed when synthetic LTA4 was added to warm acidified methanol (Figs 1 and 2). Second, incubation of cell-free supernatants, derived from stimulated monocyte cultures, with purified LTA4 hydrolase led to an increase in the levels of LTB4 (Table 1). The enzyme could apparently utilize monocyte-released LTA4 which was stabilized by albumin, as demonstrated by the increased formation of LTB4. The increase in the levels of LTB4 was not paralleled by a decrease in its non-enzymatically
399 formed isomers (Table 1).Apparently, the detected non-enzy- be part of an autoregulatory function with impact on inflammatic isomers are rapidly formed after release from the cells. matory and immunological processes. The LTA4 which becomes associated with albumin hydrolyzes This work was supported by grants from King Gustaf V’s 80only slowly [30], and non-metabolized LTA4 in the buffer incubations was presumably precipitated together with the year fund, The Swedish Medical Research Council (03X-7235), Magn protein when incubations were terminated. This hypothesis Bergvalls stiftelse, and The Swedish Cancer Society (2801-B91was supported by control incubations, showing an increase in 02XBB). the total amounts of LTB4 and its isomers after addition of purified LTA4 hydrolase to albumin-stabilized synthetic LTA4 REFERENCES in buffer (data not shown). 1. Samuelsson, B. (1983) Science 220, 568 - 575. Extracellular release of LTA4 by monocytes has earlier 2. Lewis, R. A. & Austen, K. 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38. Goodwin, J. S., Atluru, D., Sierakowski, S. & Lianos, E. A. (1 986) J. Clin. Invest. 77, 1244- 1250. 39. Schulam, P. G. & Shearer, W. T. (1990) J . Immunol. 144,26962701. 40. Goldyne, M. E. & Stobo, J. D. (1982) Prostaglandins 24, 623630. 41. Goppelt-Struebe, M., Kyas. U. & Resch, K. (1986) FEBS Lett. 202.45 - 48. 42. Tsai, Jaw-Ji., Cromwell, O., Maestrelli, P., O’Hehir, R. E., Moqbel, R. & Kay, A. B. (1989) J . Immunol. 142, 16611668. 43. Schroder, J. M. (1989) J . Exp. Med. 170, 847-863.
Effects of monocyte-lymphocyte interaction on the synthesis of leukotriene B4 Per-Johan JAKOBSSON, Bjorn ODLANDER and Hans-Erik CLAESSON Department of Physiological Chemistry, Karolinska Institutet, Stockholm, Sweden (Received August 21/December 3, 1990) - EJB 90 1006
Human monocytes in monolayers were challenged with the calcium ionophore A23187. Methanol trapping of the products in the cell-free supernatants, followed by analysis on HPLC and by ultraviolet spectroscopy, revealed the presence of two compounds, which exhibited a conjugated-triene spectrum and chromatographed with the compounds formed when synthetic leukotriene (LT) A, was added to warm acidified methanol. Furthermore, addition of purified LTA4 hydrolase to the cell-free supernatant of monocytes, stimulated with the ionophore A23187, resulted in increased levels of LTB4. These results indicate that monocytes release LTA4 extracellularly after activation with the calcium ionophore. Incubation of monocytes together with monoclonal lymphocytic cells, of both B and T cell lineage, yielded increased levels of LTB4 whereas the non-enzymatic isomers of this compound, i. e. d6-trans-LTB4 and 12-epi-A6trans-LTB,, declined. In addition, the sum of LTB4 and its non-enzymatically formed isomers increased in mixed cultures of monocytes and monoclonal lymphocytic cells as compared to monocytes alone. The present study indicates that activated monocytes release LTA,, which is converted into LTB4 by monoclonal lymphocytic cells. Furthermore, the increase of the total amounts of leukotrienes on incubation of monocytes with lymphocytic cells, suggests the presence of an additional mechanism leading to activation of the 5-lipoxygenase pathway in monocytes.
Monocytes and macrophages possess 5-lipoxygenase activity and convert arachidonic acid to leukotrienes (LT) [l]. Several stimuli such as calcium ionophore A23187 [2], zymosan [2] and aggregated immunoglobulins [3, 41, can induce formation of leukotrienes in these cell types. Human tonsil B lymphocytes and peripheral B and T lymphocytes are devoid of 5-lipoxygenase activity [5 - lo], but express LTA4 hydrolase activity [9, 111. Monoclonal lymphoblastoid cells possess higher LTA4 hydrolase activity than normal lymphocytes [9]. Furthermore, the expression of LTA, hydrolase but not of 5-lipoxygenase was demonstrated at both transcriptional and translational levels in Raji cells [8], an established Burkitt’s lymphoma B cell line. Recently, LTA4 hydrolase was isolated and purified from Raji cells [121. It has been demonstrated that LTA, is released from activated polymorphonuclear leukocytes [13] and studies on cell/ cell interactions have revealed that this compound can be
utilized by erythrocytes [14] and endothelial cells [15] for synthesis of LTB,. Several cell types, including platelets [16], mast cells [13] and endothelial cells [15,17] can convert extracellular LTA, into cysteine-containing leukotrienes. Leukotrienes influence several lymphocyte functions. Specific high-affinity binding sites for LTB4 have been described on these cells [18], and it has been reported that this compound induces natural killer and suppressor/cytotoxic cell activity [19]. LTB4 stimulates, in synergy with Blymphotrophic factors, expression of the activation-associated surface antigen CD23 on resting B lymphocytes [20]. Furthermore, initiation of DNA synthesis, cell growth and immunoglobulin production are enhanced in B cells by LTB, [20]. LTB, has also been reported to influence the formation and effects of several cytokines [21-241. The present study deals with the release of LTA, from activated monocytes and effects of monocytes monoclonal lymphoblastoid cell interactions on LTB4 synthesis.
Correspondence to P.-J. Jakobsson, Department of Physiological Chemistry 11, Karolinska Institutet, Box 60400, S-10401 Stockholm, MATERIALS AND METHODS Sweden. Abbreviations. Leukotriene (LT) A,, (5S)-trans-5,6-oxido-7,9-Materials truns-11,14-cis-eicosatetraenoic acid; LTB,, (5S, 12R)-5,12Synthetic leukotriene A4 was a generous gift from Dr A. dihydroxy-6,14-cis-8,10trans-eicosatetraenoic acid ; LTC4, 5(S)Ford-Hutchinson (Merck Frosst, Canada). Alkaline hydrolyhydroxy-6(R)-S-glutathionyl-7.9-trans-l1 ,I 4-cis-eicosatetraenoic acid ; 5-HETE, 5 (S)-5-hydroxy-6-truns-8,I 1,14-cis-eicosatetraenoic sis of leukotriene A4 methyl ester was carried out as described acid; DHETE, dihydroxy-eicosatetraenoic acid; RP-HPLC, reverse- in [25]. Medium M199, penicillin-streptomycin, FungizoneR, trypsin and plastic Petri dishes were from Flow Laboratories phase HPLC. Enzymes. Arachidonate 5-lipoxygenase (EC 1 .I 3.11.34); leu- (Rockville, MD). The calcium ionophore A23187 was obtained from Calbiochem-Behring (La Jolla, CA). Carkotriene-A, hydrolase (EC 3.3.2.6).
396 water, 1 : 1. Analysis was performed on reversed-phase HPLC (RP-HPLC). Incubation of the supernatantjrom activatedmonocytes with purified LTA4 hyeolase. LTA4 hydrolase was isolated and Isolation and cultivation o f c d l s purified from Raji cells [12]. Monocytes, still attached to the Human monocytes were isolated from leukocyte concen- Petri dish after extensive washing, were scraped off with a trates obtained from healthy donors at the Karolinska Hospi- plastic cell lifter, centrifuged and suspended in buffer A contal (Stockholm, Sweden). The cell suspension was diluted 1: 2 taining 0.5% albumin. Cell concentration was adjusted to with Dulbecco's phosphate-buffered saline, pH 7.4, sup- 4 x lo6 cells/ml and viability was > 90% as determined by plemented with Ca2+and Mg2 (buffer A). Erythrocytes were means of trypan blue dye exclusion. 4 ml cell suspension was sedimented with 0.5% (mass/vol.) Dextran. Platelets were re- poured into Petri dishes (2 ml/dish) and equilibrated for 5 min moved by centrifuging the leukocyte-rich plasma at 300 x g in the incubator. The cells were stimulated with 5 pM for 10 min. The pellet was washed in buffer A and centrifuged ionophore A23187 for 4min. The incubation fluids were at 100 x g for three cycles. After the final centrifugation the pooled and immediately centrifuged until 1000 x g was platelet-poor leukocyte fraction was applied on a discontinu- reached (total time 90 s). The cell-free supernatant (4 ml) was ous density gradient (Ficoll-Isopaque) and centrifuged at divided into two parts, one of which received LTA4 hydrolase and one of which received buffer A only. The enzymatic 600 x g for 30 min [26]. The monomorphonuclear cells were collected and diluted reaction was allowed to occur for 20 min at room temperawith buffer A, centrifuged and further washed twice with ture, and thereafter stopped with 3 vol. methanol, centrifuged buffer A at 75 x g and then suspended in medium M199 sup- and evaporated to dryness under reduced pressure. The resiplemented with 20% human serum, penicillin (100 IU/ml), dues were resolved in methanol, dried under argon and finally streptomycin (0.1 mg/ml), FungizoneR (2.5 pg/ml) and L-glu- resolved in methanol/water (1 : 1) before RP-HPLC tamine (0.15 mg/ml). The cell concentration was adjusted to analysis. lo7 cells/ml and layered onto Petri dishes (63 cm'), 5 ml/dish. The cells were incubated for 1 h at 37°C in an atmosphere Incubation of monocytes with monoclonal lymphocytic cell lines of 5% CO,. Non-adherent cells were removed by vigorous Lymphocytic cells were washed twice in buffer A, suswashing with buffer A . Attached cells were either used directly in experiments or cultured overnight in medium. The purity of pended in buffer A and added to adherent monocytes. The the cells was estimated by morphological examination (May- mixed cell population was incubated for 10 min in the incuGriinwald/Giemsa staining) and by presence of non-specific bator prior to the addition of calcium ionophore A23187 (5 pM, 10 min). Incubations were stopped by addition of esterase [27]. The range of monocyte purity was 75-95%, with lymphocytes as the major remaining cell type. Granulo- 1 vol. methanol. Prior to RP-HPLC analysis, the samples were cytes were always less than 1% (mainly basophil granulo- centrifuged at 15000 x g for 20 min. cytes). Cell lines used in this study were Raji [28], a B-lymphocytic Analysis of leukotrienes Burkitt lymphoma and MP-6 [29], a T4-T hybridoma. The Analysis of LTA4-trapping products. Trapping products Raji and MP-6 lymphoblastoid cell lines were cultivated in medium RPMI 1640 supplemented with fetal calf serum (So/,), were analyzed by RP-HPLC, using a Nucleosil C I 8 column L-glutamine (0.3 mg/ml, penicillin 100 IU/ml and strepto- (3 mm x 100 mm, 120 - 3 pm particles), and eluted at a flow rate of 0.4 ml/min with methanol/water/acetic acid mycin 0.1 mg/ml. (68: 32: 0.01, by vol.). Eluted compounds were detected and quantified utilizing a programable Hewlett-Packard 8451A Detection of extracellular LTA4 diode-array spectrophotometer, on-line with the HPLC sysTrapping of LTA4 with methanol. After extensive washing tem. The identification of eluted compounds was based on of adherent blood cells, 2 ml buffer A, supplemented with 1% chromatography with standards and on ultraviolet specalbumin, in order to stabilize leukotriene A4 [30], were added troscopy. Material in peaks eluting with standards were colto each Petri dish. The cells were allowed to equilibrate in the lected and treated with diazomethane, giving methyl esters of cell incubator (37 C, 5% CO,, 15 min) before challenging the compounds. The compounds were further analyzed either with the calcium ionophore A23187 (15 pM) for 6 min. Since in a straight-phase HPLC system, using a 250 mm nucleosil albumin diminishes the effect of ionophore stimulation [31], 5 pm column with hexane/isopropanol(98 :2, by vol.) as mothis concentration was essential for a proper stimulation of bile phase, or in a RP-HPLC system, using methanol/water the monocytes in the presence of albumin. Ionophore A23187 (75 : 25, by vol.). Quantification o j leukotrienes. Leukotrienes were quanin a concentration of 15 pM was toxic for cells in the absence of albumin, but in the presence of albumin (1% mass/vol.), tified by RP-HPLC, as described in [34], with the exception toxicicity as assessed by trypan blue dye exclusion was that no pre-column was utilized. Briefly, HPLC was performed elminated ( > 97% viable cells). The incubation fluid from on a Constametric I11 equipment with a CI-10 integrator from several Petri dishes was collected and centrifuged for 1 min at LDC/Milton Roy with a Nucleosil c18 column (pore size, 800 x g . Thereafter the supernatant was immediately poured 12 nm; 3 pm particles, Skand. Genetec, Sweden). The mobile into 10 vol. warm acidified methanol (40"C, pH 3) [32]. Pre- phase was acetonitrile/methanol /water (29.5: 19.5: 51) supcipitated material was removed by centrifugation and the re- plemented with 1% acetic acid (by vol.) and adjusted to pH maining fluid was evaporated under reduced pressure. The 5.6 with 30% NaOH (by vol.). Prior to analysis, the integrator sample was dissolved in distilled H,O and applied to a Sep- was calibrated with known amounts of standards. Ultraviolet Pak cartridge, which was washed with 10 ml water fol- monitoring was carried out at 270 nm, and quantitative analylowed by 10 in1 hexane [33]. Finally, leukotrienes were eluted sis was performed by integration of the elution profile. Qualiwith 10 nil methanol, evaporated and dissolved in methanol/ tative analysis was performed by comparison with retention
tridges containing octadecylsilyl silica (Sep-PakR) were purchased from Waters Associates (Milford, MA).
+
397
I
Ill
1
IV
v
0
I
0
I
I
15
30
1
45
Tirne(min1
Fig. 1. RP-HPLC chromatogram ofthe products released by monocytes after stimulation with the calcium ionophore A23187. Adherent human monocytes were stimulated with the ionophore A23187 (15 FM) for 6 rnin in the presence of 1 % albumin. The cell-free supernatant was mixed with 10 vol. warm acidified methanol, purified and subjected to RP-HPLC. Peaks 1-111 coeluted with standards of A6-trans-LTB,, 12-epi-A6-trans-LTB, and LTB4, respectively. Peaks IV and V coeluted with the products formed after addition of synthetic LTA, to warm acidified methanol. Insert: ultraviolet spectra of peaks IV and V
volumes of known standards and analysis of ultraviolet spectral properties of eluted compounds on a Hewlett-Packard 8450 computerized diode-array spectrophotometer.
15 Time (rnin)
20
Fig. 2. RP-HPLC chromatogram of the methyl esters of the material in peaks ZV and V. The upper chromatogram show the RP-HPLCprofile of the methyl esters of the products formed after addition of synthetic LTA4 to warm acidified methanol, i.e. the methyl esters of 5(S)-hydroxy-l2(R)-methoxy-d6-trans-LTB4and 5(S)-hydroxy-12epi-methoxy-d6-trans-LTB4.The lower trace shows the elution profile in this system of the methyl esters of the material in peaks IV and V
Table 1 . Detection of LTA, in the supernatants of stimulated monocytes by incubation with LTA4 hydrolase Monocytes were stimulated with ionophore A23187 (5 pM) for 4 min in the presence of 0.5% albumin. Cell-free supernatants were prepared and either plain buffer or purified LTA, hydrolase was added. The samples were incubated for 20 min at room temperature and subsequently analyzed by RP-HPLC. Each value represents the mean & range of two independent experiments LTA4 detected A6-trans-LTB, 12-epi-A6trans-LTB,
RESULTS
LTB4
total
Release of LTA4from activated monocytes Monocytes attached to Petri dishes were incubated with the ionophore A23187 (15 pM) for 6 min in the presence of 1% albumin. The cell-free incubation fluids were mixed with warm acidified methanol and subsequently analyzed on RPHPLC. Fig. 1 shows the RP-HPLC chromatogram of products released from monocytes stimulated with the calcium ionophore. The retention times of peaks I and I1 corresponded to those of the non-enzymatically formed isomers of LTB4, A 6-trans-LTB4 and 12-epi-A6-trans-LTB4, respectively. Peak I11 eluted as synthetic LTB4. Peaks IV and V comigrated with the products formed, when synthetic LTA4 was treated with warm acidified methanol. Ultraviolet spectral analysis of peaks IV and V revealed maxima at 270 nm and shoulders at 260 nm and 280 nm (Fig. 1, insert). The retention times of these peaks were different from those of the two epimers of 5,6-dihydroxyeicosatetraenoicacid. Furthermore, they did not appear if the incubations were stopped with 3 vol. ethanol. The material eluting in peaks IV and V was collected, treated with diazomethane, and subsequently submitted to further analysis by RP-HPLC (Fig. 2). Two main peaks were detected, which comigrated with the two products generated when synthetic LTA4 was treated identically as the sample. The ultraviolet spectra of these peaks were in agreement with those reported for leukotrienes. In addition, if the methyl esters of the material in peaks IV and V were analyzed on straightphase HPLC, this also revealed two peaks coeluting with the
pmo1/lO7 monocytes Monocytes/buffer Monocytes/ LTA4 hydrolase
k 15
222
2
313 k 13
88k 4
485
87
573 _+ 83
91
two products formed nonenzymatically from synthetic LTA4 (data not shown). Taken together, these data show that peaks IV and V represent the trapping products in methanol of LTA4, i. e. 5(S)-hydroxy-12(R)-methoxy-d6-trans-LTB4 and 5(S)-hydroxy-l2-epi-methoxy-d6-trans-LTB4 [32]. Detection of monocyte-released LTA4 using purified LTA4 hydrolase
The experimental procedure was as described for methanol trapping of LTA4, with the exception that instead of adding warm acidified methanol to the samples, either purified LTA4 hydrolase in buffer or buffer alone was added to the cell-free monocyte supernatants. The addition of LTA4 hydrolase led to an approximate twofold increase in LTB, levels compared to buffer control (Table 1). No difference in the levels of nonenzymatically formed LTB4 isomers could be detected in the albumin-stabilized incubations. The same pattern was ob-
398
LTB,
E
I
LTB,
I
0
. N t
c
a
a, U
C
: n
Q
0
15
30 Time (min)
0
L5
15 30 Tirne(min)
45
Fig. 3. RP-HPLC chromatograms o j t h e products formed by monocytes alone ( A ) and monocytes incuhated together with MP-6 cells ( B ) after stimulation with the ionophore A23187. Human monocytes were incubated with the calcium ionophore A231 87 ( 5 pM) for 10 min in the absence (A) or in the presence of MP-6 cells ( B ) . The ratio of monocytes/MP-6 cells was 1 :4. The retention volumes of synthetic standards are indicated. LTC4, leukotricnc C4: LTB4, leukotriene B4; I and TI, 12-epimers of 6-trans-LTB4
served in control experiments, when purified LTA4 hydrolase or buffer was added to albumin-containing buffer A supplemented with synthetic LTA4 (data not shown).
T
E,
Effects of monoc~te/monoclonallymphocytic cell interactions on the metabolism of leukotrienes
Monocytes alone or together with either Raji or MP-6 cells were stimulated with ionophore A23187 ( 5 pM) for 10 min. Fig. 3A shows an RP-HPLC chromatogram of the products formed by 12 x l o 6 adherent monocytes. When the same number of monocytes were incubated together with MP-6 cells (48 x lo6 cells), this led to an alteration of the leukotriene pattern (Fig. 3B). The amounts of LTB4 produced were increased, whereas the levels of A‘-trans-LTB4 and 12-epi-A6trans-LTB4 declined. The significance and magnitude of the observed changes in leukotriene formation by interaction of monocytes with Raji cells was estimated by statistical evaluation. Data obtained from five separate experiments, at a ratio of monocytes/Raji cells of 1 : 4, was analyzed by Student’s paired t test (Fig. 4). In samples with monocytes alone, the level of LTB4 was 255 41 pmol/107 monocytes (mean SE, n = 17). In the presence of Raji cells, the level of LTB4 was 459 f 67 pmol/107 monocytes (mean f SE, n = 17), corresponding to an 80% increase of LTB4 levels. In addition, the non-enzymatic isomers of LTB4 declined from 115 20 pmol/ lo7 monocytes to 20 5 7 pmol/107 monocytes in the mix cultures (mean SE, n = 17). The sum of LTB4 and its nonenzymatic isomers increased from 370 f 59 pmol/107 monocytes to 479 74 pmol/107 monocytes in the presence of Raji cells. This increment was statistically significant (P< 0,001).
. s
500
5 400 E 300 ln a,
&-
200
c L
100 3
J
O
1+11
LTBq
I+II+LTB4
Fig. 4. Effect of the interaction of monocytes with Raji cells on the synthesis of LTB4 and its non-enzymatic isomers. Monocytes were incubated in the presence or absence of Raji cells with the ionophore A23187 ( 5 pM) for 10 min. The products were quantified by RPHPLC; each bar represents mean SE, n = 17. Open bars depict the products formed by monocytes alone and hatched bars the compounds formed by monocytes with Raji cells. The ratio of monocytes/Raji cells was 1 :4. Statistical significance of differences in leukotriene formation was estimated by using Student’s paired t test. ***, P < 0.001
+
*
DISCUSSION
Monocytes have the capacity to metabolize arachidonic acid to leukotriene B4 [ I , 21, a compound which influences certain lymphocyte functions [18 -24, 351.
The present study indicates, as demonstrated by two independent methods, that monocytes release the unstable intermediate LTA4 upon stimulation with the calcium ionophore A23187. First, trapping experiments showed that two products, exhibiting a conjugated-triene spectrum, appeared when the cell-free supernatant of stimulated monocytes was mixed with warm acidified methanol. These compounds chromatographed on RP-HPLC with the products formed when synthetic LTA4 was added to warm acidified methanol (Figs 1 and 2). Second, incubation of cell-free supernatants, derived from stimulated monocyte cultures, with purified LTA4 hydrolase led to an increase in the levels of LTB4 (Table 1). The enzyme could apparently utilize monocyte-released LTA4 which was stabilized by albumin, as demonstrated by the increased formation of LTB4. The increase in the levels of LTB4 was not paralleled by a decrease in its non-enzymatically
399 formed isomers (Table 1).Apparently, the detected non-enzy- be part of an autoregulatory function with impact on inflammatic isomers are rapidly formed after release from the cells. matory and immunological processes. The LTA4 which becomes associated with albumin hydrolyzes This work was supported by grants from King Gustaf V’s 80only slowly [30], and non-metabolized LTA4 in the buffer incubations was presumably precipitated together with the year fund, The Swedish Medical Research Council (03X-7235), Magn protein when incubations were terminated. This hypothesis Bergvalls stiftelse, and The Swedish Cancer Society (2801-B91was supported by control incubations, showing an increase in 02XBB). the total amounts of LTB4 and its isomers after addition of purified LTA4 hydrolase to albumin-stabilized synthetic LTA4 REFERENCES in buffer (data not shown). 1. Samuelsson, B. (1983) Science 220, 568 - 575. Extracellular release of LTA4 by monocytes has earlier 2. Lewis, R. A. & Austen, K. F. (1984) J . Clin. Invest. 73, 889 - 897. been suggested [36], since incubations of monocytes with 3. Rouzer, C. A., Scott, W. A,, Hamill, A. L., Liu, F.-T., Katz, D. platelets led to an increased production of LTC4, although the H. & Cohn, Z. A. (1982) J . Exp. Med. 156, 1077- 1086. actual release of LTA4 was not documented. 4. Ferreri, N . R., Howland, W. C. & Spiegelberg, H. L. (1986) J . Several studies have demonstrated that lymphocytes can Immunol. 136,4188-4198. not synthesize leukotrienes de novo from arachidonic acid, due 5. Goldyne, M. E., Burrish, G. F., Poubelle, P. & Borgeat, P. (1984) to lack of an active 5-lipoxygenase [5 -91. In contrast to these J . Biol. Chem. 259,8815-8819. studies, other investigators have reported that lymphocytes 6. Poubelle, P. A,, Borgeat, P. & Rola-Pleszczynski, M. (1987) J . have the capacity to synthesize 5-hydroxyeicosatetraenoic acid Immunol. 139,1273- 1277. I . Goldyne, M. E. & Rea, L. (1987) Prostaglandins 34, 783-795. and leukotrienes from arachidonic acid [37,38]. However, one 8. Medina, J. F., Odlander, B., Funk, C. D., Fu, Ji-Yi., Claesson, of these groups has not been able to confirm their initial H.-E. & RBdmark, 0. (1989) Biochem. Biophys. Res. Commun. findings [lo]. Recently, the formation of 5-hydroxy161,140-745. eicosatetraenoic acid without concomitant synthesis of LTA4 9. Odlander, B., Jakobsson, P.-J., Rosen, A. & Claesson, H.-E. was reported in several B-lymphocytic cell lines [39]. The (1988) Biochem. Biophys. Res. Commun. 153, 203-208. biosynthetic pathway, however, responsible for this latter for- 10. Behrens, T. W., Lum, L. G., Lianos, E. A. & Goodwin, J. S. mation is not clarified and the chemical characterization of (1989) J . Immunol. 143,2285-2294. the product formed is incomplete. 11. Fu, Ji-Yi., Medina, J. F., Funk, C. D., Wetterholm, A. & Normal human lymphocytes and especially monoclonal RBdmark, 0. (1988) Prostaglandins 36. 241 -248. lymphocytic cells possess an active LTA4 hydrolase [9, 111, 12. Odlander, B., Haeggstrom, J., Bergman, T., RBdmark, O., Wetterholm, A. & Claesson, H.-E. (1990) in Advances inprostawhich enables the synthesis of LTB4 from exogenous LTA4. glandin, thromhoxane and leukotrienyresearch (Samuelsson, B., Since monocytes and lymphocytes interact closely at several Ramwell, P. W., Paoletti, R., Folco, G. & Granstrom, E., eds) levels of the immunological regulatory process, we investivol. 21, pp. 133-136, Raven Press, New York. gated whether activated monocytes could supply lymphocytes 13. Dahinden, C. A,, Clancy, R. M., Gross, M., Chiller, J. M. & with LTA4. Hugli, T. E. (1985) Proc. Natl Acad. Sci. USA 82, 6632The levels of LTB4 in incubations of monocytes with 6636. monoclonal lymphocytic cells, stimulated with ionophore 14. McGee, J. E. & Fitzpatrick, F. A. (1986) Proc. Natl Acad. Sci. A23187, increased while those of the non-enzymatic isomers USA 83, 1349-1353. of LTB4 decreased in comparison to the amounts produced 15. Claesson, H.-E. & Haeggstrom, J. (1988) Eur. J . Biochem. 173, 93 - 100. by monocytes alone (Figs 3 and 4). Due to the possible interference of albumin with leukotriene synthesis, mixed cell incu- 16. Edenius, C., Heidvall, K. & Lindgren, J. A. (1988) Eur. J . Biochem. 178, 81 - 86. bations were performed in the absence of this protein. Thus, 17. Feinmark, S. J. & Cannon, P. J. (1986) J. Biol. Chem. 261, LTA4 released extracellularly from monocytes may either be 16466-16471. non-enzymatically degraded or further metabolized by neigh- 18. Payan, D. G., Missirian-Bastian, A. & Goetzl, E. J. (1984) Proc. bouring lymphocytes. The current results indicate that transNatl Acad. Sci. USA 81, 3501 -3505. fer of LTA4 from stimulated monocytes to MP-6 or Raji cells 19. Rola-Pleszczynski, M. (1985) Immunol. Today 6, 302- 307. is likely to be, at least in part, responsible for the increased 20. Yamaoka, K. A,, Claesson, H.-E. & Rosen, A. (1989) J . Immunol. 143, 1996-2000. biosynthesis of LTB4 observed in incubations of these cells. However, the sum of LTB4 and its non-enzymatically formed 21. Rola-Pleszczynski, M. & Lemaire, I. (1985) J . Immunol. 135, 3958 - 3961. isomers increased in mixed incubations (Fig. 4), suggesting Rola-Pleszczynski, M., Chavaillaz, P.-A. & Lemaire, I. (1986) 22. that another mechanism(s) also operates, which causes actiProstaglandins Leukotrienes Med. 23, 207 - 210. vation of the 5-lipoxygenase pathway in monocytes. The 23. Rola-Pleszczynski, M., Bouvrette, L., Gingras, D. & Girard, M. transfer of arachidonic acid from activated monoclonal (1987) J . Immunol. 139, 513-517. lymphocytic cells to monocytes might be one factor which 24. Dubois, C. M., Bissonnette, E. & Rola-Pleszczynski, M. (1989) contributes to the increased synthesis of LTB4 in mixed-cell Am. Rev. Respir. Dis. 139, 1251- 1264. incubations [40, 411. Lymphocyte derived factors, which 25. Haeggstrom, J., Ridmark, 0.& Fitzpatrick, F. A. (1985) Biochim. Biophys. Acta 835, 318 - 384. amplify leukotriene release from granulocytes [42, 431, might also contribute to the activation of the 5-lipoxygenase path- 26. Beryum, A. (1 974) Tissue Antigens 4 , 269 -274. 27. Sun, T., Li, C. Y. &Yam, L. T. (1985) in Atlas ofcytochemistry and way in monocytes. immunochernistry qf hematologic neoplasms, p. 97, American In summary, the present study shows that monocytes reSociety of Clinical Pathologists Press, Chicago. lease LTA4 extracellularly, which can be further metabolized 28. Pulvertaft, R. J. V. (1965) J . Clin. Pathol. (Lond.) 18, 261by lymphocytes into LTB4. Furthermore, it is demonstrated 273. that lymphocytic cells have the capacity to amplify the syn- 29. RosCn, A., Uggla, C., Szigeti, R., Kallin, B., Lindquist, C. & thesis of LTB4 in incubations with monocytes. 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400 31. Hoffman, T. & Lizzio, E. F. (1988) J . Immunol. Methods 112, 9-14. 32. Borgeat, P. & Samuelsson. B. (1979) Proc. Natl Acad. Sci. U S A 76, 3213-3217. 33. Powell, W. S . (1982) Methods Enzymol. 86, 467-471. 34. Odlander, B. & Claesson, H.-E. (1987) Biomed. Chromatogr 2, 145-147. 35. Atluru, D. & Goodwin, J . S. (1984) J . Clin. Invest. 74, 14441450. 36. Bigby, T. D. & Meslier, N. (1989) J. Immunol. 143, 1948-1954. 37. Goetzl, E. (1981) Biochem. Biophys. Res. Commun. 101, 344350.
38. Goodwin, J. S., Atluru, D., Sierakowski, S. & Lianos, E. A. (1 986) J. Clin. Invest. 77, 1244- 1250. 39. Schulam, P. G. & Shearer, W. T. (1990) J . Immunol. 144,26962701. 40. Goldyne, M. E. & Stobo, J. D. (1982) Prostaglandins 24, 623630. 41. Goppelt-Struebe, M., Kyas. U. & Resch, K. (1986) FEBS Lett. 202.45 - 48. 42. Tsai, Jaw-Ji., Cromwell, O., Maestrelli, P., O’Hehir, R. E., Moqbel, R. & Kay, A. B. (1989) J . Immunol. 142, 16611668. 43. Schroder, J. M. (1989) J . Exp. Med. 170, 847-863.
