Br. J. Pharmacol.

(1991), 102, 7-9

SPECIAL REPORT

Macmillan Press Ltd, 1991

Antipyretic actions of human recombinant lipocortin- 1

1J. Davidson, *R.J. Flower, A.S. Milton, *S.H. Peers & 2D. Rotondo
Division of Pharmacology, Marischal College, University of Aberdeen, Aberdeen AB9 lAS and *Department of Biochemical Pharmacology, The Medical College of Saint Bartholomew's Hospital, Charterhouse Square, London EC1M 6BQ The effect of human recombinant lipocortin-1 (hrLC-1) on the pyrogenic actions of the synthetic polyribonucleotide polyinosinic: polycytidylic acid (poly I: C) has been studied in conscious rabbits. Poly I: C (2.5 pg kg- ) given i.v. produced a biphasic fever with a first peak after 90-105 min and a second peak between 225-240 min. hrLC-1 (50ougkg- ) given i.v. simultaneously with the poly I: C produced a significant reduction in the febrile response but without complete suppression. The thermal response index over 5 h (TRI5) was 4.69 + 0.51 for poly I: C given with saline and the TRI5 for poly I: C given with hrLC-1 was 2.66 + 0.45 (values are for n = 5 + s.e.mean, P < 0.05). hrLC-1 administered alone had no effect on body temperature and its antipyretic activity was lost on heating. In a separate series of experiments 1 h pretreatment with dexamethasone (1 mgkg -) given i.v. reduced the pyrogenic response (TRI5) to poly I: C (2.5 ug kg-1) from 4.87 + 0.54 without dexamethasone to 2.00 + 0.25 (n = 5, P < 0.05) and dexamethasone given alone had no effect on body temperature. These data demonstrate that LC- 1 possesses antipyretic actions and raises the possibility that the antipyretic actions of dexamethasone are mediated through the induction of LC-1.

Introduction Lipocortin-1 (LC-1, also called annexin-1 or calpactin-2; Crumpton & Dedman, 1990) is a member of a diverse group of proteins which exhibit calcium and phospholipid binding properties. A common structural feature of this group of proteins is a 70amino acid repeat unit containing a highly conserved consensus region of 17 amino acids which is thought to be important for calcium/phospholipid binding (Pepinsky et al., 1988). Apart from a core sequence, members of this family of proteins have structurally different N-termini which possibly confer different biological properties on each member of the family. LC-1 applied externally to cells can inhibit eicosanoid generation (Cirino et al., 1989) and has potent anti-inflammatory actions when injected locally (Cirino et al., 1989) or intravenously (Browning et al., 1990) into the rat. The mechanism of action by which LC-1 inhibits eicosanoid biosynthesis could occur by inhibition of phospholipase A2 but this is not clear and has recently been disputed. Systemic treatment of man with glucocorticoids results in an induction of LC-1 synthesis and the appearance of LC-1 on the cell-surface of monocytes/macrophages (Goulding et al., 1990). This evidence suggests that the reduction of eicosanoid release in response to glucocorticoids may be mediated through the induction of LC-1. Recently it has been shown that pretreatment of rabbits with dexamethasone attenuates the febrile response to exogenous immunomodulatory agents such as polyinosinic: polycytidylic acid (poly I: C) and lipopolysaccharide (Milton et al., 1989). Both these pyrogenic agents appear to produce a febrile response by increasing blood levels of prostaglandin E2(PGE2), dexamethasone attenuates both the fever and the increase in circulating PGE2 in response to these pyrogens (Milton et al., 1989). The attenuation of both the fever and increase in blood levels of PGE2 by dexamethasone is only observed if animals are pretreated for between 30 and 60 min, indicating that this action of dexamethasone may require the induction of a mediator, possibly lipocortin. In the present investigation, therefore, the effect of human recombinant LC-1 (hrLC-1) on poly I: C-induced fever has been studied.
1 Author for correspondence. 2 Present address: Division of Biochemical Sciences, Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB.

Methods Male Dutch rabbits, weighing 1.7-2.0 kg were lightly restrained in conventional stocks throughout each experiment. To minimize any error in body temperature measurements due to restraint stress, all rabbits were accustomed to the stocks over a period of 5 days before beginning this series of experiments. All experiments were conducted at the same time of day (10h 00min-16h 00min) and carried out at an ambient temperature of 22 + 1C. Body temperature was measured continuously with rectal thermistor probes (Yellow Springs Instruments-401 series), inserted to a depth of 9cm, which were connected to a Jacquet chart recorder. Rabbits were left unhandled after insertion of the probes until body temperature was stable for at least 1 h before any drugs were administered. hrLC-1 produced by transfected CHO cells was obtained from Biogen, Boston, MA, U.S.A. The protein was stored as a sterile solution at a concentration of 1 mg ml1- and repetitive freeze-thawing was avoided as this leads to denaturation of the protein. The purity of the preparation was greater than 97% as ascertained by SDS/polyacrylamide gel electrophoresis and reverse phase high performance liquid chromatography (h.p.l.c.). hrLC-1, polyinosinic: polycytidylic acid obtained from Sigma Chemicals (Dorset) and dexamethasone sodium-21-phosphate provided as a gift from Merck Sharp and Dohme (Herts) were prepared using aseptic techniques, and were dissolved in sterile saline (Travenol Laboratories, Norfolk). In experiments where heated hrLC-1 was administered, the required volume of hrLC-1 was transferred to sterile Eppendorf tubes and placed in a water bath at 90C for 30min. All agents were administered i.v. via the marginal ear vein. Poly I: C was administered first and hrLC-1 was given several seconds later. Poly I: C and dexamethasone solutions were passed through Millex-GS 0.22,um filters immediately prior to injection. The amount of agent in each dose was adjusted to give an injection volume of 0.5-1.0ml. The results are expressed as either the change in temperature from basal (AT) in C or as thermal response indexes where the magnitude of the febrile response is determined by integrating the change in temperature (C) against time (h) to give a thermal response index (TRI). Data, expressed as the mean of n experiments + the s.e.mean, were analysed by a paired Student's t test. Treatments were randomised, each rabbit acting as its own control and the differences were considered significant when P < 0.05.

SPECIAL REPORT

Results The dose of poly I: C used (2.5pgkg -1) has previously been shown to give a reproducible fever in rabbits (Rotondo et al., 1987). Poly I: C given i.v. alone produced a biphasic increase in body temperature with a first peak occurring after 90-105min and a second peak between 225240 min. The ATmax for the first peak was 1.14 + 0.13 and 1.29 + 0.16 for the second peak (n = 5). hrLC-1 (50 jig kg- ') given i.v. simultaneously with the poly I: C produced a significant reduction in the febrile response but without complete suppression (Figure 1). The ATmax for poly I: C with hrLC-1 were 0.76 + 0.09'C for the first peak and 0.78 + 0.16'C for the second peak (n = 5). The TRI5 was 4.69 + 0.51 for poly I: C given with saline and the TRI5 for poly I: C given with hrLC-1 was 2.66 + 0.45 (n = 5, P < 0.05). A lower dose of hrLC-1 (25 pgkg -1) did not produce a significant attenuation of the febrile response to poly I: C (TRI5 4.01 + 0.29, n = 3, range 3.444.38). hrLC-I (SOgkg- 1) administered alone had no effect on body temperature, a response similar to that obtained with saline. The antipyretic activity of hrLC-1 was lost if it was heated at 90C for 30min and allowed to cool before being administered. The pyrogenicity of poly I: C when given with heated hrLC-l was similar to its pyrogenicity when given alone. In a separate series of experiments 1 h pretreatment with dexamethasone (1 mg kg- 1) given i.v. reduced the pyrogenic response (TRI5) to 2.5,ugkg-1 poly I: C (Figure 1) from 4.87 + 0.54 without dexamethasone to 2.00 + 0.25 (n = 5, P < 0.05). Dexamethasone given alone had no effect on body temperature. The dose of dexamethasone given (1 mg kg 1) and the pretreatment time have previously been shown to produce a reproducible attenuation of poly I: C fever (Abul et al., 1987). In addition it was shown that if dexamethasone is given with poly I: C then no reduction in the pyrogenic response is observed and 1 h pretreatment appears to be optimal (Abul et al., 1987).
1.5 r
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Figure I The effect of lipocortin-1 and dexamethasone on the pyrogenic action of polyinosinic: polycytidylic acid (poly I: C). Rabbits were injected i.v. with (as indicated by the arrows) either saline (0) or (a) human recombinant lipocortin-1 (5Opgkg-1, 0) and (b) dexamethasone (lmgkg -, 0). Poly I :C (2.5pgkg-') was given i.v. at time zero in both cases. Values are means of n = 5, s.e.means shown by vertical bars.

Discussion This report clearly demonstrates that hrLC-1 attenuates the pyrogenic response of rabbits to poly I: C. A similar magnitude of attenuation of poly I: C pyrogenicity has been observed with dexamethasone (Figure 1). The dose of dexamethasone used was approximately 1.75 .umol kg-1 (1 mgkg- ') and the dose of hrCL-1 used in the present study was approximately 1.39nmolkg-1 (50ogkg-1) therefore, on this molar basis the potency of hrLC-1 in attenuating the pyrogenicity of poly I: C is in the order of 1000 fold greater than dexamethasone. It is most unlikely that this difference in potency could be accounted for by differences in the pharmacokinetic properties of the two agents. LC-1 is a protein and would have a lower ability to penetrate into various compartments than the steroid dexamethasone. The higher potency of LC-1 in comparison to dexamethasone is in agreement with the observations of Cirino et al. (1989) who observed that 20Opg hrLC-1 injected into the rat paw produced a slightly lower response than 1 mg dexamethasone in reducing oedema induced by various inflammatory stimuli. This effect was also observed in adrenalectomized rats indicating that adrenal hormones are not involved (Cirino et al., 1989). LC-1 is found in many tissues (Pepinsky et al., 1988) and is particularly abundant in cells of the monocyte/macrophage lineage and the protein appears to be well conserved in a wide variety of species including man, rat, mouse, hamster and monkey (Pepinsky et al., 1986). Goulding et al. (1990) observed that monocytes contain the largest amount of LC-1 of any cell type in human blood. Furthermore, only monocytes were able to respond to intravenously administered hydrocortisone by producing greater amounts of LC-1 both intracellularly and pericellularly within 2 h. Investigations into LC-1 induction by Browning et al. (1990) also demonstrate that the monocyte/macrophage is a sensitive target for the inducing action of glucocorticoids. Monocytes have also been shown to respond to pyrogenic agents by releasing PGE2, this is attenuated by dexamethasone and appears to occur via the induction of a protein mediator, possibly LC-1 (Milton et al., 1989). Dexamethasone given intravenously may, therefore, induce the biosynthesis of LC-1 from monocytes and many other tissues and increase the level of circulating LC-1. As dexamethasone requires at least a 1 h pretreatment period before it can attenuate the pyrogenic action of poly I: C (Milton et al., 1989) and has been shown in other systems to increase the synthesis of LC-1 this raises the possibility that the suppression of pyrogenic responses by dexamethasone may be mediated in part by a lipocortin, possibly LC-1. The mechanism by which LC-1 attenuates poly I: C pyrogenicity is not clear. Poly I: C appears to exert its pyrogenic action by increasing circulating levels of PGE2 (Rotondo et al., 1988). In the same series of experiments Rotondo et al. (1988) showed that the non-steroidal anti-inflammatory agent, ketoprofen, given i.v. completely abolished the poly I: Cinduced fever and also reduced the blood levels of PGE2 to almost undetectable levels in several animals. In addition it was shown by Milton et al. (1989) that dexamethasone suppressed the increase in circulating levels of PGE2 and the fever in response to poly I: C. The antipyretic action of LC-1 could possibly involve an attenuation of PGE2 biosynthesis as a property which LC-1 possesses is the ability to inhibit eicosanoid generation both in vivo and in vitro (Cirino et al., 1989; Browning et al., 1990). Although it has been suggested that the mechanism of lipocortin action is to inhibit phospholipase A2 therefore inactivating the enzyme, no direct experimental evidence has been cited in the literature. Recently it has been suggested that phospholipase A2 inhibition could be due to lipocortin binding to substrate; however, the exact mechanism of eicosanoid inhibitory action remains to be resolved. Apart from its actions on the arachidonate cascade, little is known of the exact role lipocortins play in immunological processes. This study shows that LC-1 can attenuate an immunological action, fever which is the most easily measured parameter of the acute phase response to infection, in vivo

SPECIAL REPORT

when administered systematically. Although no direct evidence has been presented in the current study this nevertheless raises the possibility that the antipyretic action of dexamethasone is mediated by LC-1.
References
ABUL, H., DAVIDSON, J., MILTON, A.S. & ROTONDO, D. (1987). Dexamethasone pre-treatment is antipyretic toward polyinosinic: polycytidylic acid, lipopolysaccharide and interleukin-1/endogenous pyrogen. Naunyn-Schmiedebergs Arch. Pharmacol., 335, 305-309.
BROWNING, J.L., WARD, M.P., WALLNER, B.P. & PEPINSKY, R.B.

D.R. and S.H.P. were supported by separate grants from the Wellcome Trust and RJ.F. is currently supported by an endowment from Lilly. We also wish to thank Dr J.L. Browning of Biogen for his generous gift of human recombinant lipocortin-1.

PEPINSKY, R.B., SINCLAIR, L.K., BROWNING, J.L., MATTALIANO, RJ., SMART, J.E., CHOW, E.P., FALBEL, T., RIBOLINI, T., GARWIN, J.L.

& WALLNER, B.P. (1986). Purification and partial sequence analysis of a 37-kDa protein that inhibits phospholipase A2 activity from rat peritoneal exudates. J. Biol. Chem., 261, 4239-4246.
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(1990). Studies on the structural properties of lipocortin-1 and the regulation of its synthesis by steroids. In Lipocortins, Cytokines and Inflammation. ed. Parente, L. & Melli, L. New York: Allan R. Liss.
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R.B. (1989). Human recombinant lipocortin-l has acute local antiinflammatory properties in the rat paw oedema test. Proc. Nati. Acad. Sci., U.S.A., 86, 3428-3432. CRUMPTON, M.l. & DEDMAN, J.R. (1990). Protein terminology tangle. Nature, 345, 212.
GOULDING, N.J., GODOLPHIN, J.L., SHARLAND, P.R., PEERS, S.H., SAMPSON, M., MADDISON, P.J. & FLOWER, R.J. (1990). Anti-

inflammatory lipocortin-1 production by peripheral blood leucocytes in response to hydrocortisone. Lancet, 335, 1416-1418. MILTON, A.S., ABUL, H.T., DAVIDSON, J. & ROTONDO, D. (1989). Antipyretic mechanism of action of dexamethasone. In Thermoregulation: Research and Clinical Applications. ed. Lomax, P. & Schonbaum, E. pp. 74-77. Basel: Karger.

WALLNER, B.P. (1988). Five distinct calcium and phospholipid binding proteins share homology with lipocortin-1. J. Biol. Chem., 263, 10799-10811. ROTONDO, D., MILTON, A.S., ABUL, H. & DAVIDSON, J. (1987). The pyrogenic actions of the interferon-inducer polyinosinic polycytidylic acid are antagonised by ketoprofen. Eur. J. Pharmacol., 137,257-260. ROTONDO, D., ABUL, H.T., MILTON, A.S. & DAVIDSON, J. (1988). Pyrogenic immunomodulators increase the level of prostaglandin E2 in the blood simultaneously with the onset of fever. Eur. J. Pharmacol., 154, 145-152.

(Received October 9, 1990 Accepted October 17, 1990)