Gen. Pharmac. Vol. 28, No. 1, pp. 23-26, 1997 Copyright © 1997 Elsevier Science Inc. Printed in the USA.

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REVIEW Antiflammins: Endogenous

Nonapeptides With Regulatory Effect on Inflammation

Juan J. Moreno DEPARTAMENTO ClENCIAS FISlOL6OICAS, UNIDAD FISlOLOGfA, FACULTAD DE FARMACIA,

UNIVERSIDAD DE BARCELONA, AVDA, JOAN XXIII S/N, 08028 BARCELONA, SPAIN [FAx: 343 402 1896]

ABSTRACT. 1. Antiflammins are a new family of peptides that share a common sequence with ute- roglobin and lipocortin-1, which retain the anti-inflammatory action of these proteins.

2. However, it is controversial whether or not the antiflammins have any effect on enzymes involved in arachidonic acid mobilization and/or arachidonic acid metabolism.

3. This review summarizes our current knowledge of the properties and activity of antiflammins. Copyright © 1997 Elsevier Science Inc. GEN PHARMAC 28;1:23--26, 1997.

KEY WORDS. Antiflammins, anti-inflammatory peptides, phospholipase A2, eicosanoids, inflammation

I NTRODUCTION

Inflammatory processes are complex biochemical and cellular phe- nomena that are manifested physiologically in tissues by oedema and leukocyte infiltration and, also, by pain. Now, it is largely recog- nized that phospholipase A2 (PLAz) plays a key role in inflammatory processes. Cellular activation promotes PLA2 translocation to plasma membranes and, in the presence of calcium, PLAz is acti- vated with the consequent hydrolysis of the acyl-ester bonds at the sn-2 position of phosphyglycerides (Clark et al., 1991; Lloret et al., 1995). Hence, PLA2 activation appears as the first rate-limiting step in the eicosanoid cascade generation through the release of the main eicosanoid precursor, arachidonic acid. Thus, time-dependent increases in PLA2 activity and eicosanoid formation have been ob- served in animal and human inflammatory processes (Vadas and Pruzanski, 1986; Lundy et al., 1990; Moreno, 1993).

Glucocorticoids are among the most effective drugs for the treat- ment of inflammatory processes. These drugs are known to produce several biological effects by altering gene expression. As much as 1% of the total genome may be altered by glucocorticoids in their target cells, resulting in changes in the expression of large numbers of en- zymes and other proteins. Thus, numerous studies indicate that their anti-inflammatory effects are mediated, at least in part, by the in- duction of regulatory proteins such as lipocortins (Di Rosa et al., 1984) and uteroglobins (Miele et al., 1987).

Lipocortin-1 is strongly induced by glucocorticoids, which sup- ports the idea that this protein may be mediated by glucocorticoid action. Following cloning, it was recognized that the gene for lipo- cortin-1, at least in rats and humans, contains a number of control factors including at least one glucocorticoid response element (Browning et al., 1990). Hence, steroid regulation is feasible. Fur- thermore, it has been found that lipocortin 1 affects the cellular component of inflammatory responses (Perretti and Flower, 1993). Interestingly, it has also been demonstrated that, in the presence of neutralizing monoclonal antibody anti-lipocortin 1, the anti-

Abbreviations: PLA2, phospholipase A2; UG, uteroglobin; AFs, antiflammins; AF1, antiflammin-1; AF2, antiflammin-2; PLC, phospholipase C; TPA, 12- [3-tetra-decanoylphorbol 13-acetate; AA, arachidonic acid; PGEz, prosta- glandin E2; 6-keto PGFI~, 6-keto prostaglandin FI~; LTB4, leukotriene B 4.

Received 5 February 1996; accepted 6 March 1996.

inflammatory action of dexamethasone is completely suppressed (Perretti and Flower, 1993). Together, these results indicate that at least some of the anti-inflammatory effects of the steroids may be due to the production of lipocortin-1 and its resultant effect.

In a similar way, the synthesis and secretion of uteroglobin (UG) are regulated by different steroid hormones in numerous organs (Miete et al., 1987), which is also involved in the immunomodula- tory/anti-inflammatory action of these hormones.

Although it is now accepted that glucocorticoids act at multiple levels, inhibition of the biosynthesis and release of eicosanoids is an important mechanism of steroid action. It has been proposed that this effect could be the result of the synthesis and/or release of pro- teins with antiPLA2 activity, such as lipocortins (Blackwell et al., 1980). On the other hand, an independent line of investigation has shown that uteroglobin also inhibites PLA2 activity (Levin et al., 1986). Recently, several reviews on lipocortin-1, uteroglobins and lipocortin-derived peptides have attempted to assess the role of these peptides in mediating glucocorticoid-induced effects on in- flammation (Flower and Rothwell, 1994; Miele et al., 1994; Perretti, 1994).

PEPTIDES IDENTIFIED ON THE BASIS OF A SEQUENCE SIMILARITY BETWEEN LIPOCORTINS AND UTEROGLOBINS

At present, the lipocortin/annexin superfamily consists of at least 12 distinct proteins. The six lipocortins most commonly encountered in mammalian cells have been identified and sequenced, each of which contains a common 4-fold repeating domain that is capable of binding negatively charged phospholipids in the presence of cal- cium (Pepinsky etal., 1988). The physiological activity of these pro- teins is doubtful. However, one of these, lipocortin-1 (37 kDa), had been shown to inhibit PLA2 activity and phagocyte chemotaxis in vitro (Davidson et al., 1987; Flower 1988), and to possess inhibitory activity on carrageenan-induced infammation in vivo (Cirino et al., 1989).

On the other hand, uteroglobin is a progesterone-induced (Ran- dall et al., 1991) protein inhibitor of PLA2 that has been identified from rabbit endometrium (Miele et al., 1987). The molecular weight is 15 kDa and it is one of the best characterized proteins, consisting of 2 identical subunits of 70 amino acids, joined in anti-

24 J.J. Moreno

TABLE 1. Antiflammin family

Peptide Parent protein Sequence Reference

AF1 Uteroglobin M Q M K K V L D S Miele et al. (1988) AF2 Lipocortin-1 H D M N K V L D L Miele et al. (1988) AF2a Lipocortin-1 H D A N K V L D L Tetta et al. (1991) AF2n Lipocortin-1 H DNle N K V L D L Tetta et al. (1991) AF2ns Lipocortin- 1 H D N le N K V L D S Tetta et al. (1991) LC5 204-212 Lipocortin-5 S H L R K V F D K Perretti et al. (1991) LC5 206-212 Lipocortin-5 L R K V F D K Perretti et al. (1991)

parallel orientation by two disulfide bonds. Each subunit consists of 4 cx-helices, with a [3 turn between helices 2 and 3. A large hy- drophobic cavity is present at the center of the dimer (Morize et al., 1987). A physiological role for UG has not yet been established. However, during the last few years, uteroglobins have been shown to affect immune and inflammatory processes by at least 3 mechanisms: inhibition of immune recognition of allogenic cells, inhibition of phagocyte chemotaxis and platelet aggregation, or inhibition of the pro inflammatory enzymes, such as PLA2 (Miele et al., 1994). In ad- dition, UG has been shown to inhibit carrageenan-induced acute inflammation in rat foot pad in vivo (Miele et al., 1988). The identi- fication of small portions that may mimic, at least in part, the effect of these full-length proteins represents an attractive and practical way to develop a potential drug with the biological activities of glu- cocorticoids.

On the basis of computer analysis, Miele et al. (1988) identified a region of local sequence similarity between uteroglobin and lipo- cortin-1, corresponding to uteroglobin residues 40-46 and lipocor- tin-1 residues 247-253. In uteroglobin, this sequence corresponds to the C-terminal half of 0~-helix 3 that is not involved in interchain interactions, and is accessible to the solvent (Morize et al., 1987). Considering these facts, Miele et al. (1988) designed several syn- thetic peptides corresponding to the region of highest similarity be- tween uteroglobin and lipocortin-l: the nonapeptides correspond- ing to uteroglobin residues 39-47 (MQMKKVLDS) and lipocortin- 1 residues 246--254 (HDMNKVLDL). Both peptides were shown to be PLAz inhibitors in vitro and were effective in a classic model of acute inflammation in carrageenan-induced rat footpad oedema (Miele et al., 1988). From these results, the peptides were called antiflammins (AFs), AF1 to UG-derived peptide and AF2 to lipo- cortin 1-derived peptide.

The antiflammin family was growing, and the amino acid se- quence of some of these is shown in Table 1. Most of these peptides presented a core tetrapeptide KVLD, which, by itself, was inactive as a PLA2 inhibitor (Miele et al., 1988). However, we can observe that most of these nonapeptides are constituted of a common se- quence: variable amino acid, hydrophilic amino acid, hydrophobic amino acid, Lys, Val, hydrophobic amino acid, Asp and variable amino acid.

BIOLOGICAL AND BIOCHEMICAL PROPERTIES OF ANTIFLAMMINS

In 1988, Mukherjee and coworkers proposed that short peptides de- rived from uteroglobin or lipocortin-1 might have the same spec- trum of activity as these peptides. Thus, they proposed that anti- flammins might directly interact with a type I low-molecular-mass PLA2 such as porcine pancreatic PLA2, and might prevent the activ- ity of this enzyme (Miele et al. , 1988, Facchiano eta/., 1991 ). More- over, they correlated this biochemical action with the anti-inflam-

matory effect of the peptides on rat paw foot acute infammation. In addition to these results, antiflammins inhibited platelet aggrega- tion induced by thrombin and ADP (Vostal et al., 1989) and these nonapeptides also inhibited PLAz from human polymorphonuclear leukocytes and the synthesis of platelet-activating factor (PAF) in these cells (Camussi et al., 1990a,b). In addition, the same authors found that AFs also inhibit the activation of acetyl CoA-lyso PAF acetyltransferase in intact cells (Camussi et al., 1990a,b; Tetta et al.,

1991). However, the ability of antiflammins to inhibit phospholi- pase A2 activity in vitro, as well as their anti-inflammatory activity in vivo has been questioned by several authors. Thus, Masters et al.

(1989) and Hope et al. (1991) observed that antiflammins do not inhibit porcine pancreatic PLA2 activity or carrageenan-induced rat paw oedema (Marki et al., 1990; Marastoni et al., 1991).

Based upon the encouraging preliminary pharmacological results of antiflammins and the later controversy, we assessed the ability of AFs to inhibit PLA2, implicated in pathophysiological processes, and we determined the ability of AFs to mitigate experimental mod- els of inflammation induced by different agents. In our studies, the AFs used were routinely stored below 0°C under anhydrous condi- tions, and dissolved in ice-cold buffer immediately before use. Con- centrated solutions were never stored, and unused portions were dis- carded. Camussi et al. (1990a,b) studied the inactivation of AFs in detail, and observed that freezing these nonapeptides in solution re- suits in complete inactivation and that AFs in general, and AF-1 in particular, are readily inactivated by the oxidation of methionine residues.

In the first results, we did not observe any effect of AFs on porcine pancreatic PLA2 and N . naja naja PLA2, even at peptide concentra- tions as high as 50 IxM (Cabr~ et al., 1992). Our results suggested that there is no interaction of the nonapeptides with either the en- zyme or the substrate, in good agreement with the results obtained by Van Binsbergen et al. (1989). It had been suggested that these peptides could aggregate at micromolar concentrations or that their biological action could be reduced by oxidation. Considering these facts, we, therefore, performed a second series of assays with human synovial fluid PLA2 and porcine pancreatic PLA2, using nanomolar concentrations of AFs and adding reducing agents to the reaction mixture. The experiments were performed with phospholipid-deox- ycholate mixed micelles and E. coli biomembranes as substrate, and no significant inhibitory effect was observed. In contrast with these results in vitro, subplantar administration of AFs in carrageenan-in- duced rat paw oedema produced a significant inhibition of inflam- mation, as had been reported by Miele et al. (1988) . However, when experimental paw oedema was induced directly by PLA2 injection, AFs did not display any anti-inflammatory activity, confirming the data obtained in vitro. Finally, we reported that AFs, applied topi- cally, were able to inhibit inflammatory reactions in other experi- mental models of inflammation, such as murine ear oedema induced

Antiflammins 25

by croton oil or oxazolone in presensitized mice (Cabr6 etal . , 1992). These preliminary results do not seem to involve a direct interaction between peptides and PLA2.

Our subsequent studies were designed to clarify the mechanism of the anti-inflammatory action of AFs. The effect of nonapeptides on platelet aggregation revealed that AFs inhibited the aggregation in- duced by collagen, but not that produced by arachidonic acid, whereas ketoprofen, a classic cyclooxyger~ase inhibitor, was able to reduce the aggregation induced by arachidonic acid and collagen. Moreover, the peptides tested only inhibited TxB2 formation when platelets were stimulated with collagen (Lloret and Moreno, 1992). These results suggested that AFs have no suppressive effects on cyclooxygenase, whereas they could act specifically to inhibit plate- let PLA2 activation, an essential first step in collagen-induced aggre- gation. Similar results were obtained when AFs were used to treat ear oedema induced by arachidonic acid (AA) or croton oil (Cabr~ et al., 1992). Our results confirmed the findings of Perretti et al., (1992), who observed that AFs inhibit contractions of isolated rat stomach strips when elicited by porcine pancreatic PLA2, whereas contractions caused by A A were not affected.

Histamine release and metabolism of arachidonate by mast cells are among the earliest biochemical changes in the inflammatory re- sponse to carrageenan (Vinegar et al., 1987). The effect of AFs dur- ing the early phase of carrageenan-induced paw oedema, in a similar form to that of antihistaminic drugs (Lloret and Moreno, 1994), sug- gested that peptides could modulate mast cell degranulation. In- deed, AFs caused limited but significant inhibition of histamine se- cretion (Lloret and Moreno, 1994). Bruni et al. (1984) indicated that the lysophospharidylserine released by PLA2 activity has a po- tent action on histamine degranulation, and we suggested that the interactive stimulus of lysophosphatidylserine is transmitted through the phosphoinositide-PLC system (Lloret and Moreno, 1995a). Thus, the effect of AFs on histamine degranulation could be explained by an inhibitory interaction with any step of this mechanism.

Acute inflammatory reactions are cha, acterized by changes in vascular permeability and vasodilation, resulting in oedema and cell influx. To understand the effect of AFs on acute inflammation, complete clarification is needed regarding their action on appro- priate cells involved in these pathophysiologic processes. For this purpose, we attempted to measure the effect of AFs on cells and pro- inflammatory mediators involved in the development of acute in- flammation as ear oedema induced by A A or TPA (Lloret and Mor- eno, 1995b). We determined the effect of AFs on vascular permeability and oedema formation, neutrophil and monocyte tis- sue infiltration, and eicosanoids (PGE2, 6-ketoPGFl~ and LTB4) lev- els. All these findings lend strong support to the idea that the anti- oedematous effect and the inhibitory effect on cell influx and eicosanoid formation of AF-2 could be related to an inhibitory ac- tion of nonapeptides on A A mobilization and/or A A metabolism by lipoxygenases in the TPA model. Furthermore, these results pro- vided additional evidence that the effects of AF-2 do not involve inhibition of A A metabolism by cyclooxygenase. There is, however, an alternative mechanism to explain the anti-inflammatory effect of AFs, which may involve an antichemotatic effect. We must con- sider that several authors described this ar~tichemotatic effect by li- pocortins and uteroglobins (Vasanthakumar et al., 1988; Perretti and Flower, 1993). This is not likely to be the result of the suppres- sion of eicosanoid formation because inhib!tors of cyclooxygenase or lipoxygenase do not have the same effect.

Probably, the best way of determining whether a substance is PLA2 inhibitory in vivo is to try to detect inhibition of stimulation

of A A release in whole cells. Recently, we have performed further studies to examine the mechanism of the anti-inflammatory action of AFs in cells involved in inflammatory processes. Preliminary re- suits indicate that AFs were not able to inhibit A A mobilization in cells in which glucocorticoids had this effect, such as fibroblasts. In addition, AFs significantly reduced the migration capability of phagocytes (results not published), in agreement with the previous observations of Camussi et al. (1990a,b), Chan et al. (1991) and Tetta et al. (1991). Taken together, these observations indicated that kFs are potent inhibitors of acute inflammation, not as a result of a reduced release of A A metabolites, as originally proposed, but probably by potent inhibition of phagocyte trafficking and protein activation. Additional experiments should be performed to clarify whether AFs could be acting directly on the phagocytes or whether they were interfering with the expression and/or the activity of ad- hesion molecules on endothelial cells adjacent to the inflammatory lesion.

In conclusion, the studies involving the structure-function rela- tionship of lipocortins and uteroglobins have resulted in the design of nonapeptides, named antiflammins, that show promise as potent therapeutic agents for inflammatory processes. The results of these and other ongoing studies may further our understanding of the mechanism of action of antiflammins and the development of pep- tide-mimetic pharmacological agents for therapy of inflammation.

The author is very grateful to Robin Rycroft for valuable assistance in the prepara- tion of the English manuscript. This work was supported partially by a grant from the Ministerio Espaftol de Educaci6n y Ciencia (DGICYT PB 94-0934).

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