PROSTAGLANDINSLEUKOTRIENES ANDESSENTIALFATTYACIDS Prostaglandlns Leukotrienes and Essentnl Q Lonpman Group UK Ltd 1992
Farty Aads
(I*?)
46. 167-173
Review
Prostaglandin Synthetase, Interleukin 1 and Inflammation in the Skin H. A. Bull and P. M. Dowd Department of Dermatology, (Reprint requests to HAB)
UCMSM, The Middlesex Hospital, Mortimer Street, London WIN 844,
UK
human recombinant interleukin 1-alpha (hrIL- la) is characterised by a delayed onset of 4-6 h but is then continuous up to at least 48 h, as long as the agonist is present ( 11). IL-l induced release of PGIl does not exhibit tachyphylaxis and is not inhibited by either cycloheximide or actinomycin D. IL-2 (12) and TNF-a (13) also induce slow release of PGI? from HUVEC, however the amount of PGI? released over 24 h is lOOfold less than that released by IL- 1. The actions of IL-l and histamine on PGIJ release from HUVEC indicate the existence of one pathway involving rapid stimulation of pre-existing enzymes and release of PGIz and another characterized by slow activation and sustained PGI, release. That quantitatively different responses in this pathway depend on the nature of the agonist is illustrated by the experiments with IL- 1, IL-2 and TNF-a. There are at least three possible rate limiting reactions in the agonist-/receptor-mediated synthesis of prostaglandins (Fig. 1). Firstly, synthesis may be regulated at the level of the membrane receptor (17). Secondly, there is a requirement for free arachidonic acid which is regulated by: (i) phospholipases predominantly phospholipase A, (PLA2) which mobilizes arachidonic acid from membrane stores (18) and (ii) acyl transferases, specifically acyl-CoA-lysolecithin acyltransferase (LAT) which facilitates re-incorporation of AA into membrane phospholipids (19). The third point of regulation is the conversion of AA into prostaglandin endoperoxides which is catalysed by the cyclooxygenaselperoxidase enzyme complex, prostaglandin synthetase (PG synthetase) (EC 1.14.99.1) (20).
In our current understanding of the pathophysiology of inflammatory reactions prostaglandin (PG) synthesis initiated by a variety of cytokines and pepiides is thought to be an important mechanism mediating cellular responses at sites of inflammation and tissue repair. In human skin cells, including human epidermal keratinocytes (HEK) (1, 2). human dermal fibroblasts (HDF) (3, 4) and human dermal microvascular endothelial cells (HDMEC) (5,6), in which arachidonic acid (AA) is converted into prostaglandins, the predominant metabolite appears to be prostaglandin Ez (PGE,). In contrast, large vessel endothelial cells, including human umbilical vein endothelial cells (HUVEC) release prostacyclin (PGI,) as the major prostaglandin metabolite of AA in response to a variety of agonists such as histamine (7), thrombin (S), the calcium ionophore A23187 (9) and the cytokines interleukin- 1 (IL- 1) ( 10,ll) interleukin-2 (IL-2) ( 12) and tumour necrosis factor-a (TNF-a) (13).
REGULATION OF PROSTAGLANDIN SYNTHESIS AND RELEASE Investigation of the responses of HUVEC to proinflammatory and vasoactive agonists such as IL-l and histamine has indicated that the release of PGI, is controlled by at least two different mechanisms of receptormediated prostaglandin synthesis. Histamine induces a rapid release of PGI, which is maximal within 30 min (14). HUVEC are reportedly tachyphylactic to repeated stimulation with histamine, the return of PG12 synthesizing capacity being dependent on the metabolism and clearance of histamine by serum-derived diamine oxidases (15). In the absence of serum and continued presence of histamine, release of PGI, is biphasic (16). The initial rapid release of PG12 is followed by a lagphase of 2-4 h, after which time release of PG12 begins again and continues up to 24 h. In contrast to histamine, release of PGI, from HUVEC incubated with
PROSTAGLANDIN
SYNTHETASE
(EC 1.14.99.1)
Conversion of arachidonic acid into prostaglandin endoperoxides is thought to be the rate limiting step in the synthesis of prostaglandins in large vessel endo167
168
Prostaglaodins
Leukotrienes
and Essential Fatty Acids CONVERSION
-
SIGNAL TRANSDUCTION
ENZYME ACTIVATION
I
------+
-
-SIT,, PIP2 &&+
RECEPTOR (S)@
L3------+
Ca2+mobilization
PHOSPHOLIPID
diacylglycerol
_I-
li
//
I
Ca2+ channels ’
/ lHISTAMINE RECEPTOR(s)
_
+
rt
Lyso_phos~
protein kinase C activation , ARACHIDONIC ACID
monoacyl glycerol
cycle-oxygenase I +??? -----_-) CYTOKINE RECEPTOR(S)???,
PG synthetase
1
prostaglandin =+
peroxidase
Gp
1
n prostaglandin
1 PROSTAGLANDINS
I
Fie. 1 Mechanisms of activation Abbreviations as in the text.
H2
of enzvmes mediating
thelium. Evidence for this comes from two areas; first. only about 5510% of AA released from membrane phospholipids by the action of PLA, is converted into prostaglandins, predominantly PGI, (2 1) and secondly, the cyclooxygenase activity of the PG synthetase enzyme is rapidly and irreversibly inactivated by an intrinsic destructive intermediate generated during the reduction of PGG, to PGH, (22,23). These data have originated from studies utilizing agonists such as thrombin and histamine which induce rapid activation of PLA, and release of PGI, and although PG synthetase activity may be rate limiting in PGI, release induced by such vasoactive peptides, the slow protracted release of PGI, from HUVEC incubated with hrIL-la indicates the existence of rate limiting mechanisms other than the rapid inactivation of PG synthetase. Large vessel endothelial cells appear to be the only cell type that release PGI, as their major prostaglandin metabolite of AA and the rapid release of large quantities of PGIz from HUVEC may be indicative of the role of this prostaglandin as a vasodilator and inhibitor of platelet adhesion in large blood vessels. However, PGI, is not the principal AA metabolite in microvascular endothelial cells nor is it released in any significant amount by cells at sites of inflammation which argues against its having an important role as a pro-inflammatory mediator. PGE, appears to be the major prostaglandin synthesized at sites of tissue inflammation and by cells involved in cutaneous inflammatory processes (with the exception of mast cells which release PGD, (24) and there is evidence that PGE, is a regulator of cell proliferation (25. 26).
prostaglandin
synthesis.
cytokine synthesis (27, 28) and immune responses (29) as well as being a vasodilator (30. 3 1). PGE, is released slowly over 24 h from a variety of human skin cells including HEK (32) and HDF (33. 34) incubated with IL-l and HDMEC incubated with either IL- I (6) or histamine (35). Pro-inflammatory vasoactive peptides such as histamine and bradykinin. are poor agonists of prostaglandin synthesis and release in HEK (32) and HDF (34).
INTERLEUKIN 1 INDUCED PG SYNTHESIS AND PG SYNTHETASE ACTIVATION Preliminary investigation of the mechanisms of IL-I induced PG synthesis in HDF indicated that prior exposure of the cells to the supematant from stimulated monocytes, which contains IL-l activity, potentiated release of PGE, induced by either exogenous AA. bradykinin or the mitogen, platelet derived growth factor (PDGF)(34). With all of these agonists, the potentiating effect of IL-I was blocked by cycle-heximide and actinomycin D. That both AA conversion to PGE, and receptor-mediated PGE, release were potentiated indicates that the action of IL-I was on PG synthetase. Subsequently, it was shown that IL-I increased incorporation of 9 methionine into PG synthetase protein as well as increasing PG synthetase enzyme activity indicating that IL-l induced release of PGE? was mediated primarily. if not solely, via induction of PG synthetase synthesis, in a manner that was concentration- and time-
Prostaglandin
dependent (36). In separate experiments, the interaction between bradykinin and IL-1 has been shown to be associated with increased PLA, activity (37), however, an increase in free AA could not be demonstrated in HDF incubated with IL-l alone (38) thus indicating that bradykinin was responsible for increased PLA, activity in these cells. There is also evidence that IL-l has a ‘priming’ effect on PG synthesis in HEK and in both microvascular and large vessel endothelium. Vasoactive peptides such as histamine and bradykinin are poor agonists for PG synthesis in HEK. However, following incubation with IL-I, HEK release PGE, in response to histamine (39). Similarly, pre-incubation of HDMEC with IL-1 potentiates histamine induced release of PGE2 in a concentration-dependent manner (40) (Fig. 2). The action of IL-l is associated with increased PG synthetase activity in HDMEC and cycloheximide partially reduces the increased enzyme activity while significantly attenuating release of PGE, (40). In HUVEC, IL-l potentiates both histamine-and thrombin-induced release of PGI, (4 1, 42). Examination of the timecourse of the action of IL-i
Synthetase,
Interleukin
1 and Inflammation
A
.
.
.
lU/ml IL-1
10pM
’
100
.
1mM 10kM
~PM B
0.1
0.5
_
ImM
,
IL-1 + Histamine
Histamine
=u) $
Hist.
_
100
1.0
0.1
-
T
0.5
1.0
I
t I L -1 (U/ml)
IL-l
+ Histamine
Fig. 2 IL-l potentiation of histamine induced release of prostaglandins from human endothelial cells. (A) PGE, release from HDMEC incubatedwith 1 U/ml IL-I for 24 h (0). with histamine (10 PM-1 mM) for 30 min (I) or ‘primed’ with 1 U/ml IL- 1 for 24 h and then stimulated with histamine ( IO PM1mM) for 30 min @.). (p
Farty Aads
(I*?)
46. 167-173
Review
Prostaglandin Synthetase, Interleukin 1 and Inflammation in the Skin H. A. Bull and P. M. Dowd Department of Dermatology, (Reprint requests to HAB)
UCMSM, The Middlesex Hospital, Mortimer Street, London WIN 844,
UK
human recombinant interleukin 1-alpha (hrIL- la) is characterised by a delayed onset of 4-6 h but is then continuous up to at least 48 h, as long as the agonist is present ( 11). IL-l induced release of PGIl does not exhibit tachyphylaxis and is not inhibited by either cycloheximide or actinomycin D. IL-2 (12) and TNF-a (13) also induce slow release of PGI? from HUVEC, however the amount of PGI? released over 24 h is lOOfold less than that released by IL- 1. The actions of IL-l and histamine on PGIJ release from HUVEC indicate the existence of one pathway involving rapid stimulation of pre-existing enzymes and release of PGIz and another characterized by slow activation and sustained PGI, release. That quantitatively different responses in this pathway depend on the nature of the agonist is illustrated by the experiments with IL- 1, IL-2 and TNF-a. There are at least three possible rate limiting reactions in the agonist-/receptor-mediated synthesis of prostaglandins (Fig. 1). Firstly, synthesis may be regulated at the level of the membrane receptor (17). Secondly, there is a requirement for free arachidonic acid which is regulated by: (i) phospholipases predominantly phospholipase A, (PLA2) which mobilizes arachidonic acid from membrane stores (18) and (ii) acyl transferases, specifically acyl-CoA-lysolecithin acyltransferase (LAT) which facilitates re-incorporation of AA into membrane phospholipids (19). The third point of regulation is the conversion of AA into prostaglandin endoperoxides which is catalysed by the cyclooxygenaselperoxidase enzyme complex, prostaglandin synthetase (PG synthetase) (EC 1.14.99.1) (20).
In our current understanding of the pathophysiology of inflammatory reactions prostaglandin (PG) synthesis initiated by a variety of cytokines and pepiides is thought to be an important mechanism mediating cellular responses at sites of inflammation and tissue repair. In human skin cells, including human epidermal keratinocytes (HEK) (1, 2). human dermal fibroblasts (HDF) (3, 4) and human dermal microvascular endothelial cells (HDMEC) (5,6), in which arachidonic acid (AA) is converted into prostaglandins, the predominant metabolite appears to be prostaglandin Ez (PGE,). In contrast, large vessel endothelial cells, including human umbilical vein endothelial cells (HUVEC) release prostacyclin (PGI,) as the major prostaglandin metabolite of AA in response to a variety of agonists such as histamine (7), thrombin (S), the calcium ionophore A23187 (9) and the cytokines interleukin- 1 (IL- 1) ( 10,ll) interleukin-2 (IL-2) ( 12) and tumour necrosis factor-a (TNF-a) (13).
REGULATION OF PROSTAGLANDIN SYNTHESIS AND RELEASE Investigation of the responses of HUVEC to proinflammatory and vasoactive agonists such as IL-l and histamine has indicated that the release of PGI, is controlled by at least two different mechanisms of receptormediated prostaglandin synthesis. Histamine induces a rapid release of PGI, which is maximal within 30 min (14). HUVEC are reportedly tachyphylactic to repeated stimulation with histamine, the return of PG12 synthesizing capacity being dependent on the metabolism and clearance of histamine by serum-derived diamine oxidases (15). In the absence of serum and continued presence of histamine, release of PGI, is biphasic (16). The initial rapid release of PG12 is followed by a lagphase of 2-4 h, after which time release of PG12 begins again and continues up to 24 h. In contrast to histamine, release of PGI, from HUVEC incubated with
PROSTAGLANDIN
SYNTHETASE
(EC 1.14.99.1)
Conversion of arachidonic acid into prostaglandin endoperoxides is thought to be the rate limiting step in the synthesis of prostaglandins in large vessel endo167
168
Prostaglaodins
Leukotrienes
and Essential Fatty Acids CONVERSION
-
SIGNAL TRANSDUCTION
ENZYME ACTIVATION
I
------+
-
-SIT,, PIP2 &&+
RECEPTOR (S)@
L3------+
Ca2+mobilization
PHOSPHOLIPID
diacylglycerol
_I-
li
//
I
Ca2+ channels ’
/ lHISTAMINE RECEPTOR(s)
_
+
rt
Lyso_phos~
protein kinase C activation , ARACHIDONIC ACID
monoacyl glycerol
cycle-oxygenase I +??? -----_-) CYTOKINE RECEPTOR(S)???,
PG synthetase
1
prostaglandin =+
peroxidase
Gp
1
n prostaglandin
1 PROSTAGLANDINS
I
Fie. 1 Mechanisms of activation Abbreviations as in the text.
H2
of enzvmes mediating
thelium. Evidence for this comes from two areas; first. only about 5510% of AA released from membrane phospholipids by the action of PLA, is converted into prostaglandins, predominantly PGI, (2 1) and secondly, the cyclooxygenase activity of the PG synthetase enzyme is rapidly and irreversibly inactivated by an intrinsic destructive intermediate generated during the reduction of PGG, to PGH, (22,23). These data have originated from studies utilizing agonists such as thrombin and histamine which induce rapid activation of PLA, and release of PGI, and although PG synthetase activity may be rate limiting in PGI, release induced by such vasoactive peptides, the slow protracted release of PGI, from HUVEC incubated with hrIL-la indicates the existence of rate limiting mechanisms other than the rapid inactivation of PG synthetase. Large vessel endothelial cells appear to be the only cell type that release PGI, as their major prostaglandin metabolite of AA and the rapid release of large quantities of PGIz from HUVEC may be indicative of the role of this prostaglandin as a vasodilator and inhibitor of platelet adhesion in large blood vessels. However, PGI, is not the principal AA metabolite in microvascular endothelial cells nor is it released in any significant amount by cells at sites of inflammation which argues against its having an important role as a pro-inflammatory mediator. PGE, appears to be the major prostaglandin synthesized at sites of tissue inflammation and by cells involved in cutaneous inflammatory processes (with the exception of mast cells which release PGD, (24) and there is evidence that PGE, is a regulator of cell proliferation (25. 26).
prostaglandin
synthesis.
cytokine synthesis (27, 28) and immune responses (29) as well as being a vasodilator (30. 3 1). PGE, is released slowly over 24 h from a variety of human skin cells including HEK (32) and HDF (33. 34) incubated with IL-l and HDMEC incubated with either IL- I (6) or histamine (35). Pro-inflammatory vasoactive peptides such as histamine and bradykinin. are poor agonists of prostaglandin synthesis and release in HEK (32) and HDF (34).
INTERLEUKIN 1 INDUCED PG SYNTHESIS AND PG SYNTHETASE ACTIVATION Preliminary investigation of the mechanisms of IL-I induced PG synthesis in HDF indicated that prior exposure of the cells to the supematant from stimulated monocytes, which contains IL-l activity, potentiated release of PGE, induced by either exogenous AA. bradykinin or the mitogen, platelet derived growth factor (PDGF)(34). With all of these agonists, the potentiating effect of IL-I was blocked by cycle-heximide and actinomycin D. That both AA conversion to PGE, and receptor-mediated PGE, release were potentiated indicates that the action of IL-I was on PG synthetase. Subsequently, it was shown that IL-I increased incorporation of 9 methionine into PG synthetase protein as well as increasing PG synthetase enzyme activity indicating that IL-l induced release of PGE? was mediated primarily. if not solely, via induction of PG synthetase synthesis, in a manner that was concentration- and time-
Prostaglandin
dependent (36). In separate experiments, the interaction between bradykinin and IL-1 has been shown to be associated with increased PLA, activity (37), however, an increase in free AA could not be demonstrated in HDF incubated with IL-l alone (38) thus indicating that bradykinin was responsible for increased PLA, activity in these cells. There is also evidence that IL-l has a ‘priming’ effect on PG synthesis in HEK and in both microvascular and large vessel endothelium. Vasoactive peptides such as histamine and bradykinin are poor agonists for PG synthesis in HEK. However, following incubation with IL-I, HEK release PGE, in response to histamine (39). Similarly, pre-incubation of HDMEC with IL-1 potentiates histamine induced release of PGE2 in a concentration-dependent manner (40) (Fig. 2). The action of IL-l is associated with increased PG synthetase activity in HDMEC and cycloheximide partially reduces the increased enzyme activity while significantly attenuating release of PGE, (40). In HUVEC, IL-l potentiates both histamine-and thrombin-induced release of PGI, (4 1, 42). Examination of the timecourse of the action of IL-i
Synthetase,
Interleukin
1 and Inflammation
A
.
.
.
lU/ml IL-1
10pM
’
100
.
1mM 10kM
~PM B
0.1
0.5
_
ImM
,
IL-1 + Histamine
Histamine
=u) $
Hist.
_
100
1.0
0.1
-
T
0.5
1.0
I
t I L -1 (U/ml)
IL-l
+ Histamine
Fig. 2 IL-l potentiation of histamine induced release of prostaglandins from human endothelial cells. (A) PGE, release from HDMEC incubatedwith 1 U/ml IL-I for 24 h (0). with histamine (10 PM-1 mM) for 30 min (I) or ‘primed’ with 1 U/ml IL- 1 for 24 h and then stimulated with histamine ( IO PM1mM) for 30 min @.). (p
