0192-0561/92 $5.00 + .00 Pergamon Press plc. International Society for lmmunopharmacology.
Int. J. lmmunopharmac., Vol. 14, No. 3, pp. 441-449, 1992. Printed in Great Britain.
L E U K O T R I E N E B4 IN THE I M M U N E SYSTEM HANS-ERIK CLAESSON, BJORN ODLANDER and PER-JOHAN JAKOBSSON Department of Physiological Chemistry, Karolinska Institutet, Stockholm S-104 01, Sweden
- - Leukotriene (LT) B4 is a biologically active molecule derived from arachidonic acid via the 5-1ipoxygenasepathway. It mediates certain inflammatory and immunological reactions. The role of LTB4 in the immune system has been questioned since lymphocytes have been regarded to lack the enzymes involved in LTB4 formation. This review focuses on the recently described biosynthesis of LTB4 in B-lymphocytes and the effects of this compound on lymphocyte functions. Abstract
Biosynthesis of leukotriene (LT) B4 from arachidonic acid was initially described in polymorphonuclear leukocytes (PMNL) and monocytes (Samuelsson, 1983). The key enzyme in leukotriene biosynthesis is 5-1ipoxygenase which possesses two catalytic activities: (i) conversion of arachidonic acid to 5hydroperoxy-eicosatetraenoic acid (5-HPETE), which can be degraded to 5-HETE; and (ii) the subsequent formation of leukotriene (LT) A4 (Rouzer, Matsumoto & Samuelsson, 1986). The 5lipoxygenase is a complex enzyme requiring calcium, ATP, F L A P (5- lipoxygenase activating protein) and certain unidentified factors for maximal activity (Samueisson & Funk, 1989; Reid et al., 1990; Denis, Falgueyret, Riendeau & Abramovitz, 1991). Leukotriene A4 can be hydrolyzed either enzymatically to LTB4, catalyzed by the enzyme LTA4 hydrolase, or non-enzymaticaily to the C12 epimers of /x6-trans LTB4 or the two isomers of 5,6-DHETE (Fig. 1).
acid by highly purified B- and T-lymphocytes or monoclonal lymphoblastoid cell lines could not be observed by us or by other investigators (Goldyne, Burrish, Poubelle & Borgeat, 1984; Goldyne & Rea, 1987; Poubelle, Borgeat & Rola-Pleszczynski, 1987; Odlander, Jakobsson, Ros6n & Claesson, 1988; Fu, Medina, Funk, Wetterholm & R~dmark, 1988). Later, one group retracted their original reports concerning the production of LTB4 in T-cells since they were unable to reproduce their initial findings (Goodwin & Behrens, 1988; Behrens, Lum, Lianos & Goodwin, 1989). Evidence for the synthesis of 5-HETE in certain established B-cell lines without concomitant formation of LTA4 was reported recently (Schulam & Shearer, 1990; Schulam, Kuruvilla, Putcha, Mangus, Franklin-Johnson & Shearer, 1991). As a result of these studies the general concensus was that neither human T- or B-cells can produce LTB4.
CRYPTIC 5-LIPOXYGENASE ACTIVITY IN
Metabolism o f arachidonic acid in sonicates o f B-lymphocytes
B-LYMPHOCYTES
In contrast to previous studies we investigated the metabolism of arachidonic acid in sonicated B-lymphocytes (Jakobsson, Odlander, Steinhilber, Ros6n & Claesson, 1991b). Incubation of sonicated BL41-E95-A cells (an Epstein-Barr (EBV) virus transformed Burkitt's lymphoma cell line) with arachidonic acid in the presence of ATP and calcium led to the formation of LTB4. Cell sonicates of several EBV negative B-cell lines and their EBV transformed counterparts converted arachidonic acid to LTB4 (Fig. 2). In contrast, cell sonicates of several T-cell lines did not produce detectable
One of the most controversial matters in the leukotriene research field has been whether or not human lymphocytes possess the enzymatic machinery to convert arachidonic acid to LTB4. During the last decade, several reports suggested that human T-cells produce and release LTB4 (Goetzl, 1981; Goodwin, Atluru, Sierakowski & Lianos, 1986; Atluru, Lianos & Goodwin, 1986). These reports initiated studies in many laboratories. However, the formation of LTB4 from arachidonic 441
442
H.-E. CLAESSONet al.
BIOTRANSFORMATIONOF ARACHIDONICACID Prostaglandin Endoperoxide OOH Synthase Arachidonic
I-
acid
1
~~
5-1ipoxygenase
C O O H
~ O O H 5-HETE
5-HPETE
I
Prostaglandins Thr omboxanes
i
HO~COOH
~
6-trans-LTB4 and t2-epi-6-t rans-LTB~
~-~/--C5Hll Leukotriene
LTA4 hvdrolase
A/, (LTA/)
5,6- EXHETE
[
~
COOH
Leukotriene
B~ I L I B ~ I
Fig. 1. Metabolism of arachidonic acid.
O O
E M
O
E v I-
tu
Fig. 2. Biosynthesis of LTB~ in sonicates of various monoclonal B-cells and PMNL upon stimulation with arachidonic acid in the presence of ATP and calcium. amounts of LTB~ or 5-HETE under similar conditions (Jakobsson et al., 1991b). The ability of freshly isolated B-lymphocytes to synthesize LTB~ was also investigated (Jakobsson et al., 1991b). Human B-cells of high density, i.e. resting cells, were isolated from tonsils and half of the cells were immediately sonicated and incubated with arachidonic acid. The second half was activated with the polyclonal B-cell mitogen Staphylococcus aureus Cowan I (SAC) and a mixture of recombinant
interleukin-2 (rIL-2)/rIL-6 and B-cell stimulatory factor from T-helper cell MP6 supernatants. As demonstrated in Fig. 3, activated B-cells possessed a higher capacity to generate LTB4 and 5-HETE than non-activated B-cells. The B-cell preparation used contained more than 98°70 B-lymphocytes and less than 0.2% monocytes. These results indicate that the tonsillar B-cells and not the monocytes are the principal source of LTB4 and 5-HETE in these preparations. Recently, we have also demonstrated the expression of the 5-1ipoxygenase and FLAP genes in human tonsillar B-cells and in lymphoblastoid B-cell lines by reversed transcription (RT)-PCR analysis (Jakobsson, Steinhilber, Odlander, R~dmark, Claesson & Samuelsson, 1992). Effects o f glutathione depleting agents on L TB4 synthesis in intact B-cells
The observation that sonicates of B-cells have the capacity to synthesize LTB4 led us to reexamine the synthesis of that compound in intact B-lymphocytes. Incubation of a large number of BL41-E95-A cells (200 x 106) with arachidonic acid and calcium ionophore A23187 resulted in barely detectable but yet significant amounts of LTB4. These results initiated studies on the mechanism of activation of the cryptic 5-1ipoxygenase activity in B-lymphocytes.
Leukotriene B4
Activated B Lymphocytes
Resting B Lymphocytes
PGB 1
LT84
443
PGB 1 [-LTB 4
5-HETE
5-HETE
0.05~~E.o~co
< 0 0.05
g
r.D
0 10
20
30
Time (min)
;0
2O '
30
Time (min)
Fig. 3. RP-HPLC chromatograms of the products formed in sonicates of resting (left panes) and activated (right panes) tonsillar B-lymphocytes after stimulation with arachidonic acid, ATP and calcium, u.v.-monitoring was carried out at 270 nm for detection of LTB~ (upper panels) or at 236 nm for detection of 5-HETE (lower panels). Since the glutathione depleting agents 1-chloro-2,4dinitrobenzene and diamide have been shown to activate the 5-1ipoxygenase in polymorphonuclear leukocytes (Hatzelmann & Ullrich, 1987; Hatzelmann, Schatz & Ullrich, 1989), we investigated the effect of these drugs on the activity of 5-1ipoxygenase in intact BL41-E95-A cells (Jakobsson et al., 1992). In the presence of these drugs, intact BL41-E95-A cells, stimulated with both arachidonic acid and ionophore A23187, produced similar amounts of LTB4 as those formed by sonicated cells (Fig. 4), indicating that the glutathione status might be of importance for the activity of 5-1ipoxygenase in B-lymphocytes. Why has it taken so long to demonstrate that B-lymphocytes have the enzymatic potential to convert arachidonic acid to LTB4? First, intact lymphocytic cells produce very low amounts of LTB4 upon stimulation with arachidonic acid and the calcium ionophore in comparison to the amounts produced by intact PMNL. However, by manipulating the glutathione status, the 5-1ipoxygenase of
intact cells was shifted into an active state, and thus, converted exogenously added arachidonic acid to 5-HETE and LTB4 upon stimulation with the ionophore A23187. Second, cell preparations of activated and differentiated B-cells are often contaminated with monocytes, while resting cells are possible to obtain with higher purity. Since resting B-cells have a relatively low 5-1ipoxygenase activity, investigators paying careful attention to the purity of their cell preparations have, in order to remove contaminating monocytes, also removed the activated 5-1ipoxygenase-expressing lymphocytes.
LEUKOTRIENE A4 HYDROLASE IN LYMPHOCYTES The second enzyme involved in the biosynthesis of LTB4 from arachidonic acid, LTA4 hydrolase, was discovered four years ago in human B- and T-lymphocytes (Odlander et al., 1988; Fu et al., 1988).
H.-E. CLAESSON et al.
444 A .2., ~
g~
30'
÷ - -o
20. m
It.
10' ÷,.J
Int. J. lmmunopharmac., Vol. 14, No. 3, pp. 441-449, 1992. Printed in Great Britain.
L E U K O T R I E N E B4 IN THE I M M U N E SYSTEM HANS-ERIK CLAESSON, BJORN ODLANDER and PER-JOHAN JAKOBSSON Department of Physiological Chemistry, Karolinska Institutet, Stockholm S-104 01, Sweden
- - Leukotriene (LT) B4 is a biologically active molecule derived from arachidonic acid via the 5-1ipoxygenasepathway. It mediates certain inflammatory and immunological reactions. The role of LTB4 in the immune system has been questioned since lymphocytes have been regarded to lack the enzymes involved in LTB4 formation. This review focuses on the recently described biosynthesis of LTB4 in B-lymphocytes and the effects of this compound on lymphocyte functions. Abstract
Biosynthesis of leukotriene (LT) B4 from arachidonic acid was initially described in polymorphonuclear leukocytes (PMNL) and monocytes (Samuelsson, 1983). The key enzyme in leukotriene biosynthesis is 5-1ipoxygenase which possesses two catalytic activities: (i) conversion of arachidonic acid to 5hydroperoxy-eicosatetraenoic acid (5-HPETE), which can be degraded to 5-HETE; and (ii) the subsequent formation of leukotriene (LT) A4 (Rouzer, Matsumoto & Samuelsson, 1986). The 5lipoxygenase is a complex enzyme requiring calcium, ATP, F L A P (5- lipoxygenase activating protein) and certain unidentified factors for maximal activity (Samueisson & Funk, 1989; Reid et al., 1990; Denis, Falgueyret, Riendeau & Abramovitz, 1991). Leukotriene A4 can be hydrolyzed either enzymatically to LTB4, catalyzed by the enzyme LTA4 hydrolase, or non-enzymaticaily to the C12 epimers of /x6-trans LTB4 or the two isomers of 5,6-DHETE (Fig. 1).
acid by highly purified B- and T-lymphocytes or monoclonal lymphoblastoid cell lines could not be observed by us or by other investigators (Goldyne, Burrish, Poubelle & Borgeat, 1984; Goldyne & Rea, 1987; Poubelle, Borgeat & Rola-Pleszczynski, 1987; Odlander, Jakobsson, Ros6n & Claesson, 1988; Fu, Medina, Funk, Wetterholm & R~dmark, 1988). Later, one group retracted their original reports concerning the production of LTB4 in T-cells since they were unable to reproduce their initial findings (Goodwin & Behrens, 1988; Behrens, Lum, Lianos & Goodwin, 1989). Evidence for the synthesis of 5-HETE in certain established B-cell lines without concomitant formation of LTA4 was reported recently (Schulam & Shearer, 1990; Schulam, Kuruvilla, Putcha, Mangus, Franklin-Johnson & Shearer, 1991). As a result of these studies the general concensus was that neither human T- or B-cells can produce LTB4.
CRYPTIC 5-LIPOXYGENASE ACTIVITY IN
Metabolism o f arachidonic acid in sonicates o f B-lymphocytes
B-LYMPHOCYTES
In contrast to previous studies we investigated the metabolism of arachidonic acid in sonicated B-lymphocytes (Jakobsson, Odlander, Steinhilber, Ros6n & Claesson, 1991b). Incubation of sonicated BL41-E95-A cells (an Epstein-Barr (EBV) virus transformed Burkitt's lymphoma cell line) with arachidonic acid in the presence of ATP and calcium led to the formation of LTB4. Cell sonicates of several EBV negative B-cell lines and their EBV transformed counterparts converted arachidonic acid to LTB4 (Fig. 2). In contrast, cell sonicates of several T-cell lines did not produce detectable
One of the most controversial matters in the leukotriene research field has been whether or not human lymphocytes possess the enzymatic machinery to convert arachidonic acid to LTB4. During the last decade, several reports suggested that human T-cells produce and release LTB4 (Goetzl, 1981; Goodwin, Atluru, Sierakowski & Lianos, 1986; Atluru, Lianos & Goodwin, 1986). These reports initiated studies in many laboratories. However, the formation of LTB4 from arachidonic 441
442
H.-E. CLAESSONet al.
BIOTRANSFORMATIONOF ARACHIDONICACID Prostaglandin Endoperoxide OOH Synthase Arachidonic
I-
acid
1
~~
5-1ipoxygenase
C O O H
~ O O H 5-HETE
5-HPETE
I
Prostaglandins Thr omboxanes
i
HO~COOH
~
6-trans-LTB4 and t2-epi-6-t rans-LTB~
~-~/--C5Hll Leukotriene
LTA4 hvdrolase
A/, (LTA/)
5,6- EXHETE
[
~
COOH
Leukotriene
B~ I L I B ~ I
Fig. 1. Metabolism of arachidonic acid.
O O
E M
O
E v I-
tu
Fig. 2. Biosynthesis of LTB~ in sonicates of various monoclonal B-cells and PMNL upon stimulation with arachidonic acid in the presence of ATP and calcium. amounts of LTB~ or 5-HETE under similar conditions (Jakobsson et al., 1991b). The ability of freshly isolated B-lymphocytes to synthesize LTB~ was also investigated (Jakobsson et al., 1991b). Human B-cells of high density, i.e. resting cells, were isolated from tonsils and half of the cells were immediately sonicated and incubated with arachidonic acid. The second half was activated with the polyclonal B-cell mitogen Staphylococcus aureus Cowan I (SAC) and a mixture of recombinant
interleukin-2 (rIL-2)/rIL-6 and B-cell stimulatory factor from T-helper cell MP6 supernatants. As demonstrated in Fig. 3, activated B-cells possessed a higher capacity to generate LTB4 and 5-HETE than non-activated B-cells. The B-cell preparation used contained more than 98°70 B-lymphocytes and less than 0.2% monocytes. These results indicate that the tonsillar B-cells and not the monocytes are the principal source of LTB4 and 5-HETE in these preparations. Recently, we have also demonstrated the expression of the 5-1ipoxygenase and FLAP genes in human tonsillar B-cells and in lymphoblastoid B-cell lines by reversed transcription (RT)-PCR analysis (Jakobsson, Steinhilber, Odlander, R~dmark, Claesson & Samuelsson, 1992). Effects o f glutathione depleting agents on L TB4 synthesis in intact B-cells
The observation that sonicates of B-cells have the capacity to synthesize LTB4 led us to reexamine the synthesis of that compound in intact B-lymphocytes. Incubation of a large number of BL41-E95-A cells (200 x 106) with arachidonic acid and calcium ionophore A23187 resulted in barely detectable but yet significant amounts of LTB4. These results initiated studies on the mechanism of activation of the cryptic 5-1ipoxygenase activity in B-lymphocytes.
Leukotriene B4
Activated B Lymphocytes
Resting B Lymphocytes
PGB 1
LT84
443
PGB 1 [-LTB 4
5-HETE
5-HETE
0.05~~E.o~co
< 0 0.05
g
r.D
0 10
20
30
Time (min)
;0
2O '
30
Time (min)
Fig. 3. RP-HPLC chromatograms of the products formed in sonicates of resting (left panes) and activated (right panes) tonsillar B-lymphocytes after stimulation with arachidonic acid, ATP and calcium, u.v.-monitoring was carried out at 270 nm for detection of LTB~ (upper panels) or at 236 nm for detection of 5-HETE (lower panels). Since the glutathione depleting agents 1-chloro-2,4dinitrobenzene and diamide have been shown to activate the 5-1ipoxygenase in polymorphonuclear leukocytes (Hatzelmann & Ullrich, 1987; Hatzelmann, Schatz & Ullrich, 1989), we investigated the effect of these drugs on the activity of 5-1ipoxygenase in intact BL41-E95-A cells (Jakobsson et al., 1992). In the presence of these drugs, intact BL41-E95-A cells, stimulated with both arachidonic acid and ionophore A23187, produced similar amounts of LTB4 as those formed by sonicated cells (Fig. 4), indicating that the glutathione status might be of importance for the activity of 5-1ipoxygenase in B-lymphocytes. Why has it taken so long to demonstrate that B-lymphocytes have the enzymatic potential to convert arachidonic acid to LTB4? First, intact lymphocytic cells produce very low amounts of LTB4 upon stimulation with arachidonic acid and the calcium ionophore in comparison to the amounts produced by intact PMNL. However, by manipulating the glutathione status, the 5-1ipoxygenase of
intact cells was shifted into an active state, and thus, converted exogenously added arachidonic acid to 5-HETE and LTB4 upon stimulation with the ionophore A23187. Second, cell preparations of activated and differentiated B-cells are often contaminated with monocytes, while resting cells are possible to obtain with higher purity. Since resting B-cells have a relatively low 5-1ipoxygenase activity, investigators paying careful attention to the purity of their cell preparations have, in order to remove contaminating monocytes, also removed the activated 5-1ipoxygenase-expressing lymphocytes.
LEUKOTRIENE A4 HYDROLASE IN LYMPHOCYTES The second enzyme involved in the biosynthesis of LTB4 from arachidonic acid, LTA4 hydrolase, was discovered four years ago in human B- and T-lymphocytes (Odlander et al., 1988; Fu et al., 1988).
H.-E. CLAESSON et al.
444 A .2., ~
g~
30'
÷ - -o
20. m
It.
10' ÷,.J
