antiflammin peptides in the regulation of inflammatory response

147

Antiflammin Peptides in the Regulation of Inflammatory Response

JUAN J. MORENOa

Department of Physiology, Faculty of Pharmacy, Barcelona University,E-08028 Barcelona, Spain

ABSTRACT: This review focuses on the role of antiflammins in the regulation ofthe inflammatory response, in particular acute inflammation. The results showthat antiflammins were effective on several classical models of inflammation.Preliminary data suggest that antiflammin action may be due to their ability tosuppress leukocyte trafficking to the lesion.

INTRODUCTION

Glucocorticosteroids (GS) are a classic treatment for inflammatory disorders.However, they induce numerous adverse side effects resulting from inadequate se-lectivity of action. These hormones/drugs are known to produce several biologicaleffects by modifying gene expression. As much as 1% of the tools genome may bealtered by GS in their target cells, resulting in changes in the expression of largenumbers of enzymes and other proteins. Several studies indicate that their anti-inflammatory action is mediated, at least in part, by the induction of regulatory pro-teins such as lipocortins and uteroglobins. Thus, in the late 1970s, an important stepin understanding the molecular mechanism of action of GS was the identification ofa protein involved in the glucocorticoid-induced inhibition of the release ofeicosanoids.1 Lipocortin-1 (LC-1) was another protein induced by GS.2 It was rec-ognized that the gene for LC-1 contains a number of control factors, including atleast one GS response element.3

Several biological roles have been attributed to lipocortins. However, most mem-bers of the lipocortin family are known to possess antiphospholipase A2 (PLA2) ac-tivity. This action had been described on the 14-kDa secretory PLA2 (sPLA2) and theproposed mechanism accepted was substrate depletion.4 The discovery of cytosolicPLA2 (cPLA2) and our understanding of its sequence and regulation have permitteda new approach to the lipocortin effect. Using pure cPLA2, Kim et al.5 showed thatthis enzyme was inhibited through a specific interaction by LC-1. Moreover, recom-binant human LC-1 mimics a variety of anti-inflammatory properties of GS both invivo and in vitro.6 Some biological effects of LC-1 related with its anti-inflammatoryaction are summarized in TABLE 1.

aAddress for correspondence: Department of Physiology, Faculty of Pharmacy, BarcelonaUniversity, Avda. Joan XXIII s/n, E-08028 Barcelona, Spain. Voice: 3493 4024505; fax: 34934021896.

[email protected]

148 ANNALS NEW YORK ACADEMY OF SCIENCES

Rabbit blastokinin15 or uteroglobin (UG)16 are other GS-dependent, multi-functional, cytokine-like proteins with potent anti-inflammatory activity.17 UG wasfirst discovered in the rabbit uterus during early pregnancy, but was subsequentlyfound in many other organs, such as thymus, pituitary gland, respiratory and gastro-intestinal tracts, pancreas, mammary gland, prostate, and seminal vesicle.18

UG inhibits low-molecular-weight group I and II sPLA2, enzymes that could playcritical roles in the production of proinflammatory lipid mediators.19,20 This effectwas attributed to the fact that UG sequesters calcium ions, an essential cofactor forsPLA2 activity.21 However, Mukherjee et al.22 observed that mutations in a criticalregion of the UG molecule can abrogate its PLA2 inhibitory activity withoutaffecting sequestration of calcium. Furthermore, UG is a potent inhibitor of neutro-phil and monocyte chemotaxis.23 Its biological activities directly related with itsanti-inflammatory action are summarized in TABLE 2.

ANTIFLAMMINS

Lipocortins constitute a family of at least 13 structurally related cytoplasmic pro-teins widely distributed in mammals. All members have two different regions: the

TABLE 1. Some anti-inflammatory effects of lipocortin-1

Parameter studied Experimental model Reference

Edema Carrageenan paw edema 7

Neutrophil influx Zymosan pleurisy 8

Air-pouch 9

Gel granuloma 10

Monocyte influx Zymosan pleurisy 8

TNF/PGE2 release LPS/IL-1/mononuclear cells 11

NOS levels/activity Lung exposure to LPS 12

NOS activity Synovial macrophages 13

Adhesion neutrophils Intravital microscopic studies 14

NOTE: TNF = tumor necrosis factor; PGE2 = prostaglandin E2; LPS = lipopolysaccharides; IL-1 = interleukin-1; NOS = nitric oxide synthase.

TABLE 2. Some anti-inflammatory effects of uteroglobin

NOTE: PLA2 = phospholipase A2; PGE2 = prostaglandin E2.


Platelet aggregation Thrombin-induced aggregation 24

PLA2 activity Pancreatic PLA2/RAW 264.7 19

Phagocyte chemotaxis Chemotaxis induced by formyl peptides 23

Endometrial PGE2 levels Rabbits treated with progesterone 25

149MORENO: ANTIFLAMMIN PEPTIDES

N-terminal domain, constituted by 32 amino acids of LC-1, and the C-terminaldomain, named the core, which is composed of four repeats of a highly conserved70- to 80-amino-acid sequence.26,27

UG is a homodimeric protein in which the 70 amino acid subunits, in an anti-parallel orientation, are connected by two disulfide bonds.28,29 The dimer structurehas three cavities: cavity C1 could accommodate small molecules such as progester-one or retinol; cavities C2 and C3 are located within each monomer and are formedby the α-helices 1, 2, and 3.29

The identification of small molecules that may mimic the effect of these proteinsis an attractive and practical approach to develop a molecule with the biologicaleffects of LC-1/UG.

On the basis of computer analysis, Miele et al.30 designed several synthetic pep-tides corresponding to the region of highest similarity between UG and LC-1: thenonapeptides corresponding to UG residues 39–47 (MQMKKVLDS) and LC-1 res-idues 246–254 (HDMNKVLDL). Both peptides inhibited pancreatic PLA2 in vitroand were effective on carrageenan-induced rat footpad edema. From these results,these peptides were named antiflammins (AFs): AF-1 for UG-derived peptide andAF-2 for LC-1-derived peptide.

ANTIFLAMMINS IN THE REGULATION OFINFLAMMATORY RESPONSE

Vostal et al.31 observed that AFs inhibited platelet aggregation induced by throm-bin and ADP, and they also inhibited PLA2 from human polymorphonuclear leuko-cytes and the synthesis of platelet-activated factor (PAF).32 However, the ability ofAFs to inhibit PLA2 and their anti-inflammatory activity were questioned. Thus, sev-eral authors observed that AFs do not inhibit porcine pancreatic PLA2 activity invitro or carrageenan-induced rat paw edema.33–35 In an attempt to clarify thesepreliminary results and the later controversy, we studied the effects of AFs on theinflammatory process. It is important to consider that AFs were stored below 0°C inanhydrous conditions and dissolved in ice-cold buffer immediately before use. So-lutions were never stored and unused portions were discarded. AFs are unstable andare also degradable in acidic conditions.36,37 Furthermore, Camussi et al.32 observedthat frozen AFs are readily inactivated by the oxidation of methionine residues.

Our first experiments were performed with phospholipid-deoxycholate mixedmicelles and E. coli biomembranes as substrate, and porcine pancreatic PLA2,N. naja naja PLA2, and human synovial fluid PLA2. Our experimental conditionsavoided the aggregation of AFs and their oxidation. In these conditions, no signifi-cant inhibitory effect on these PLA2’s was observed.38 In contrast, with these resultsin vitro, subplantar administration of AFs inhibited carrageenan-induced rat pawedema during the early and late phases of the process.38,39 These results are in agree-ment with Miele et al.,30 but only partially in accordance with Ialenti et al.,40 whofound that AF-2 was ineffective in the early phase (0–1 h), which is developed main-ly by histamine and serotonin release. However, when experimental inflammationwas induced directly by PLA2 (human synovial fluid PLA2 or N. naja naja PLA2)injection, AFs did not display any significant anti-inflammatory effect, indicatingthat the anti-inflammatory effects of AFs do not involve a direct interaction between


AFs and these PLA2’s. Furthermore, AFs were effective in other models of acuteinflammation induced by different agents. Thus, we reported that AFs were able toinhibit ear edema induced by croton oil or oxazolone in presensitized mice.38

Subsequent studies were performed to clarify the mechanism of the anti-inflammatory activity of AFs. AFs inhibited platelet aggregation and thromboxaneB2 (TxB2) synthesis induced by collagen, but did not affect the aggregation andthromboxane production stimulated by arachidonic acid (AA).41 Similar resultswere obtained when AFs were used to treat ear edema induced by AA or croton oil.Nonapeptides significantly reduced inflammation induced by croton oil.41 These re-sults suggested that AFs have no significant effect on the cyclooxygenase pathwaywhen AA is added exogenously, whereas they could inhibit PLA2 activation and thesubsequent AA release, an essential step in collagen-induced platelet aggregation orcroton oil ear edema.

Acute inflammatory reactions are characterized by changes in vascular perme-ability and vasodilatation, resulting in edema. We examined whether AFs inhibit theincrease in vascular permeability induced by histamine, bradykinin, PAF, and C5a invivo. AFs had no significant effect on the action of these autacoids. Thus, a directantihistaminic effect on the early phase of carrageenan-induced paw edema seemsunlikely, although an indirect action of AFs through inhibition of mast cell degran-ulation should be considered.42

The migration and accumulation of neutrophils and mononuclear cells is anothercharacteristic feature of inflammation. AA application on skin produced a short-lived edema response with rapid onset associated with marked increases in prosta-glandin E2 (PGE2) synthesis and minimal cellular influx, whereas 12-O-tetra-decanoylphorbol 13-acetate (TPA) produced a longer-lasting edema associated withmarked influx of neutrophils and mononuclear cells, as well as predominant forma-tion of leukotriene B4 (LTB4). Thus, AA-induced ear edema appeared to be depen-dent on prostaglandins, whereas TPA-induced edema could be dependent on the cellinflux and the subsequent LTB4 release by these cells. AF-2 dose-dependently re-duced plasma leakage, cell influx, edema, and LTB4 levels in response to TPA, buthad no effect when inflammation was induced by AA.43 These results also suggestthat AFs do not modify the cyclooxygenase pathway, which is in contradiction withCalderano et al.,44 who observed that AF-2 inhibits cyclooxygenase activity in rabbitdistal colonic mucosa. On the other hand, these findings suggest that the anti-edematous effect and the inhibitory effect on cell influx and eicosanoid productionof AF-2 could be related to an inhibitory action of nonapeptides on AA mobilizationand/or AA metabolism by lipoxygenases in the TPA model. However, we must con-sider an alternative mechanism to explain the anti-inflammatory effect of AF-2,which may involve an antichemotactic effect.

Probably, the best way of assessing whether a substance is a PLA2 inhibitor invivo is to determine the effect on AA release in whole cells. Recently, we demon-strated that AF-2 does not significantly reduce AA release stimulated by TPA or cal-cium ionophore A23187 in murine 3T6 fibroblasts or murine resident peritonealmacrophages, whereas dexamethasone was effective.42 However, AFs inhibited AArelease and prostaglandin production induced by thrombin in endothelial cells (per-sonal observation). Recent advances in the understanding of PLA2 have revealedthat, in general, several PLA2’s are involved in cellular regulation and lipid messen-


ger formation. The importance of each PLA2 enzyme depends on the cell type orstimuli used. This could explain these apparent contradictory results.

The accumulation of neutrophils and monocyte/macrophages is a characteristicfeature of the inflammatory response, as mentioned above. This leukocyte extrava-sation into inflamed areas involves a complex interaction of leukocytes with theendothelium through regulated expression of surface adhesion molecules. Recently,we reported that sialidase treatment, which affects the structure of selectins and in-hibits leukocyte influx, significantly reduced eicosanoid and edema in TPA-inducedear edema.45 Our results showed that AF-2 significantly reduced leukocyte adhesionto the endothelium stimulated by N-formyl-Met-Leu-Phe.42 These data were corre-lated with the inhibitory effect of AFs on cell influx and the development of the in-flammatory process. Some effects of AFs related with their anti-inflammatory actionare shown in TABLE 3.

Considering these findings, we proposed that the effects of AFs on the inflamma-tory process may be due to their ability to suppress leukocyte trafficking to the lesion.However, additional experiments should be performed to clarify whether antiflam-mins interfere with the expression or activation/affinity of the adhesion molecules.

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TABLE 3. Some anti-inflammatory effects of antiflammins


Edema Carrageenan rat paw edema 30, 41

Edema TPA ear edema 43

Edema Contact hypersensitivity 38

Cell influx TPA ear edema 43

PGE2/LTB4 levels TPA ear edema 43

Platelet aggregation Collagen-induced platelet aggregation 41

TxB2 release Collagen-induced platelet aggregation 41

Histamine release Mast cell degranulation 39

Erythema Skin irritation 46

NOTE: TPA = 12-O-tetra-decanoylphorbol 13-acetate; PGE2 = prostaglandin E2; LTB4 = leuko-triene B4; TxB2 = thromboxane B2.


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antiflammin peptides in the regulation of inflammatory response

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