Research Paper Mediators of Inflammation, 7, 261–267 (1998)

THIS study intended to characterize pharmacologically the mediator(s) released in the inflammation induced by Soluble Egg Antigen (SEA), the main antigen released from eggs of Schistosoma mansoni, in rat hindpaws. A single intraplantar injection of 0.1-100 microg SEA at day zero induced a dose-dependent increase in the volume of rat hindpaws characterizing an oedema of quick onset (within 15 min) and 4h-duration, which was confirmed by histopathological analysis of the paws. A second injection of SEA in the same paw (1-10 microg) 28 days later induced an increased dose-dependent oedematogenic response. The early oedematogenic response following SEA sensitization was derived from serotonin release and interleukin-1 (IL-1), since treatment with either pizotifen or an antibody against IL-1, reduced the response by 60% and 48%, respectively. The increased oedematogenic response derived from SEA-challenge (10 microg) of rat paws derived from a local rather than systemic reaction, since it was not observed if the sensitization was in the contralateral paw or the peritoneal cavity of the animals. Chronic treatment with inhibitors of IL-2 synthesis/release such as cyclosporin or dexamethasone during the sensitization phase reduced the oedematogenic response due to SEA challenge by 51% and 55%, respectively. These data suggested that SEA-challenge was immune-derived and dependent of IL-2 release. It is discussed the association between cytokine release and the resistance of rats to S. mansoni infection.


Introduction
Schistosomiasis , a chronic dise as e c ause d by infe ction w ith the he lminths Schis to s o m a m a n s o n i, Schis tos o m a h a e m a to biu m or Schis to s o m a ja po n icu m , pre sents a w orldw ide distribution and a varie d degre e of se verity. 1,2 S. m a n s o n i is the pre dominant schistosome found in the We ste rn w orld, apart from be ing also found in the Middle East and Afric a, 2 w hich induc es a disease of moderate se ve rity. 1 The se ve rity of schistosomiasis re lies on the modulation of a tissue granulomatous re sponse derive d from the re lease of adult w orm antigens in the host circ ulation as sociated w ith the slow re lease of larval-e gg c ross-re active antigens and subse que ntly by e gg-spec ific antige ns. 3 ,4 Soluble Egg Antigen or SEA is the te rm used to c ollec tively charac te rize the antigens re lease d by the eggs deposite d by Sch is to s o m a w orms in the tissue s and is composed by nine frac tions of prote ins w ith varie d profile of immunoge nicity. 4 The princ ipal sourc e of SEA is the skin of the embryo inside the egg, w hich at this stage , is calle d miracidium, although it c an also be re leased from the adult w orm and from c ercariae . 3 The host granulomatous re sponse to S. m a n s o n i infec tion in sensitive spec ie s can be dow n-modulate d by enhance d T-suppre ssor ce ll-activity and diminishe d CD4 + T-lymphoc yte re sponsivene ss ex p re sse d by re duction in cytokine re lease and lymphocyte p roliferation. 5 ,6 Curiously, it has be en demonstrate d that rats are re sistant 7 or 'non-p ermissible' 8 hosts to S. m a n s o n i infe ction.
In past years, SEA has be e n use d as a tool to study immune-me diate d inflammatory re actions 9 and the de ve lopme nt of a vacc ine against schistosomiasis mansoni has progressive ly be en appre ciate d. 1 0 -13 In the pre sent study, w e use d a pre paration of eggs and SEA from S. m a n s o n i inje cte d intrap lantarly to e valuate the mechanism of inflammation induce d in rat p aw s.

Animals
Holtzman female rats w e ighing 150-200 g w ere house d at 25± 3°C, under a 12 h light/dark cycle (lights on 07:00 h) w ith w ate r and food a d libitu m . Male albino Sw iss mic e w e re used as a sourc e of S. m a n s o n i eggs. In all ex periments, the numbe r of animals use d varie d be tw een 3 and 16.
Obtaining S. mansoni eggs and SEA S. m a n s o n i eggs w e re obtained from the liver of mic e infec te d w ith ce rc ariae 8 w e eks be fore , as pre viously desc ribe d. 1 4 The purified e ggs w ere fragmented in tissue grinders using phosphate -buffere d saline (PBS, 0.01 M, pH= 7.4). This proce dure de te rmined miracidium re lease thus facilitating the obtainm ent of SEA (Sch is to s o m a m a n s o n i Soluble Egg Antigen). The crude mate rial w as c entrifuged at 100 000 g for 2 h at 4°C. Various sup ernatants (SEA) w e re c ombined and e valuate d for protein conte nt by the Low ry method 1 5 and ke pt at -20°C until assayed. Only one pool of SEA has be e n used throughout the pre se nt studies.

Oedema measurements
Me as ure ments of the volume of rat p aw s w ere c arrie d out using a hydroplethysmomete r Ugo Basile (mode l 7150) be fore and afte r an intra plantar (i.pl.) inje ction of various doses of a suspension of S. m a n s o n i eggs (2000 e ggs/ml in PBS) or SEA dilute d in 0.1 ml of physiological saline at ze ro time . A sec ond inje ction of SEA (in 0.1 ml ste rile physiological saline ) w as also given 28 days later in the same paw w hene ver othe rw is e state d. The contralateral paw re ce ive d the same volume of ste rile physiologic al saline. Re sults are pre se nte d as the mean differe nc e be tw e en volume s (D V, m l) obtaine d from the paw s injecte d w ith eggs or SEA and those injec te d w ith saline ± standard error of the mean (SEM) in each group. In all the ex pe riments the measureme nts w ere obtaine d at 5, 15, 30, 60, 120, 240 min and 24 h follow ing intraplantar (i.pl.) inje ctions (n = 4 -13/group).

Selection of an inhibitory dose of an antibody against interleukin 1b (IL-1b )
To assess an inhibitory dose of an antib ody against human IL-1b , tw o dilutions (1/20 -1/200) of a comme rc ial pre paration of an antibody injecte d by subcutane ous (s.c.) route w ere te ste d in an assay of leukoc yte migration induc ed by intrape ritoneal (i.p.) injection of human IL-1b in rats. The te chnique use d to count leukoc ytes w as desc ribed e lsew here . 1 6 In the as say, it has be e n show n that 1.45 3 10 -1 0 M of human IL-1b incre ase d the rat pe ritoneal le ukoc yte number by 29% (P < 0.05, Anova t-te st, Fig. 1). A dilution of 1/20 of the antib ody re duce d the leukocyte c ounts inc re as ed by human IL-1b to the le ve l of control animals (> 99% re duc tion, Fig. 1), w here as a 1/200 dilution of the antibody did not significantly alter human IL-1b -induc ed e ffec t. The 1/20 dilution of the antibody w as used in rat paw s inje cte d w ith SEA for furthe r studies.

Experimental protocols
Four sets of ex periments w ere e stablishe d to e valuate the partic ipation of know n mediators re lease in the inflammation induc ed by e ggs or SEA from S. m a ns o n i in rat paw s. All antagonists used inhibite d at least 40% of the max imum oedematogenic e ffec t of their re spective agonists injecte d into the rat paw s and te ste d in parallel assays. In the case of HOE 140 (bradykinin antagonist), L-NAME (nitric ox ide synthas e inhibitor), indomethacin and dex amethasone (non-ste roidal and ste roidal anti-inflammatory drugs, re spective ly) the agonist used w as c arragee nin (250 m g/site). For pizotife n, pyrilamine and SR 140333 17 a dose of 5 m g of se rotonin (5-HT), 50 m g of histamine (H) and 50 m g of Substanc e P (SP), re spe ctively, w ere used p er site . In the first set, e ffec tive doses of pizotifen, pyrilamine, indomethacin or intramuscular (i.m.) dex amethasone w ere acute ly admin-FIG. 1. Inhibition of human interleukin-1b -induced leukocyte recruitment to peritoneal cavity of rats by subcutaneous injection of an antibody against human IL-1b . Cell counts were performed 4h after i.p. injections. Control bar (C) represents counts of leukocytes from animals injected i.p. with 1ml of sterile physiological saline. Sterile IL-1b was injected i.p. at 1.45 3 10 -10 M. Dilutions of the antibody were also made in physiological saline. *Represents P < 0.05 (Anova t-test) compared with control animals (n=5-7).**Represents P < 0.01 from comparison with IL-1b -injected animals (Anova t-test).
iste re d 30 min be fore i.pl. injection of SEA (time ze ro), as show n in a pre vious w ork. 1 8 The compound SR 140333 w as i.p . inje cte d 15 min be fore SEA. The inc re as e in paw volume w as obtain ed as de sc ribe d above during sensitization and challenge phase s. In the se cond se t of ex pe riments, pizotifen, dex ame thasone and indome thac in w ere acute ly administe re d by i.pl. route 30 min be fore challenge w ith SEA (10 m g/paw ). In the third se t, dex ame thasone and cyclosporin A (an immunosupp re ssive drug) w ere chronically administe re d by i.m. route from day 0 to day 14 follow ing SEA se nsitization (10 m g/paw ) and oedema me as ure ments w ere obtained after SEA challenge (10 m g/paw ). Finally, a 1/20 dilution of an antib ody against human IL-1b w as also administe re d by i.pl. route 15 min be fore SEA sensitization (10 m g/paw ) and the re sultant effe ct on SEA se nsitization (10 m g/paw ) w as also evaluate d. The dose of all drugs used w e re depic te d in the table s or figure lege nds .

Histopathological studies
Rats injec te d intra plantarly w ith SEA w e re sac rifice d w ith ethe r in differe nt time points and the hindpaw s imme diately c ut at the tibial-tarsic joint. A square of 1 cm w as made w ith a sharp sc alpe l in the pads of each paw (c ontrol and SEA-injec te d) around the site of inje ction to pe rmit easy fix ation of the tissue w hich w as then quickly immerse d in Bouin fix ative overnight. The follow ing day Bouin solution w as ex changed by ethanol 70% and the paw s w ere le ft in this solution until embe dded in parafin and cut in sec tions of 5 m m. Slides c ontainin g the se ctions of paw tissue staine d by haematox ilin-e osin w e re e valuated for oedema and c ell migration in various time points by optical mycrosc op y (1000-fold amplification).

Drugs
Salts used in the pre sent w ork and the follow ing che micals w e re purchased from Sigma: pyrilamine, indomethac in, L-NAME, dex ame thasone , human interleukin 1b and anti-human IL-1b (de ve lope d in rabbit, IgG fraction). Pizotifen and c yclosporin A, SR140333 (a NK 1 -re ce ptor antagonist) and HOE 140 w ere kindly donate d by Flávio J. R. de Aguiar (Sandoz, Brazil), Xavie r Emonds-Alt (Sanofi, Montpellier) and Dr Mauro M. Te ix eira (Departamento de Farmacologia, ICB, UFMG), re spec tively.

Statistical analysis
The inc re ase in volume of rat paw s or pe ritoneal ce llular counts (mean ± standard e rror of the me an) follow ing administration of the agonists w he ther or not under diffe re nt treatments w ere compared w ith a control using Stude nt's t-te st for single c omparisons

Results
Intraplantar administration of S. m a n s o n i e ggs (40 or 200 e ggs/0.1 ml) induc ed a dose -de pendent inc re ase in rat paw volume thus charac te rizing oe de ma formation w hich w as max imal at 120 min follow ing administration (0.27 ± 0.03 ml), re maine d significantly ele vated by 240 min (0.25 ± 0.02 ml; Fig. 2A) and subsided 24 h later (data not show n). A sec ond injection of 200 e ggs in the same paw 28 days late r induc ed an inc re as ed oedematogenic re sponse at 15 min follow ing injection (0.75 ± 0.15 ml; Fig. 2B), w hich w as re produce d if 10 m g SEA w as used inste ad of S. m a n s o n i eggs in the challenge (0.89 ± 0.10; Fig.  2C). Se nsitization of rats w ith SEA also induce d oedema w hich w as max imal 15 -30 min follow ing injection and laste d for 4 h, dep ending on the dose used (Fig. 3). No oedema w as de te c te d follow ing i.pl. injection of SEA afte r 24 h (Fig. 3). How ever, the oedematogenic activity of SEA w as progre ssively decre as ed during the development of the ex perime nts. Such variation is illustrated in the c olumn of control re sults in Table 1. The ac ute oe de ma follow ing SEA sensitization of rats w as 60% re duced by pre vious syste mic administration of pizotife n (2 mg/kg) s.c., but it w as not modified by effe ctive anti-oedemage nic doses of pyrilamine (2 mg/kg) s.c., SR 140333 (1 mg/ kg) i.p ., indome thacin (2 mg/kg) s.c ., L-NAME (0.5 mg/kg) s.c ., or dex ame thasone (1 mg/kg) i.m. (Table 1). In addition, the inc re as ed oe de matoge nic effect pre sented by the latte r animals to SEA challe nge w as not modified 28 days later (data not show n). Challe nge of animals w ith 1 or 10 m g SEA induc ed a dose-depende nt inc re ase of the oe de matoge nic re sponse in animals pre viously se nsitized w ith 10 m g SEA (Table 2). How e ver, this inc re ase w as not see n if Mediators of Inflammation · Vol 7 · 1998   higher dose s of SEA w e re use d in se nsitization, as show n in Table 2. This se condary oedema w as also 57% re duc ed by ac ute local tre atment of the paw s w ith pizotifen (100 m g/site ), but not w ith the same dose of indomethacin or dex amethas one ( Table 3) Chronic treatme nt of rats w ith i.m. 0.1 mg/kg/day dex amethas one or 5 mg/kg/ day c yclosporin from day 0 to day 14 of SEA-sensitize d animals re duce d by 51% and 54%, re spe ctive ly, the oedematogenic re sponse on SEA challenge , i.e . 14 days later ( Table 4). Observation of the animals tre ate d w ith such dose s on sensitization (day 0) did not change the immediate follow ed oe de matoge nic re sponse (data not show n). Ac ute treatme nt of rat paw s w ith an antib ody anti-human IL-1b re duced by 48% the oedematogenic re sponse induce d by SEA on se nsitization of rat paw s (Fig. 4).

Discussion
De velopment of schistosomiasis , a parasitic disease due to the Tre matoda Schis to s o m a m a n s o n i, is highly dep endent on the antigen burde n de rive d either from the adult w orms pre se nt in the c irculation or from the e ggs usually deposite d in the liver of the host. 8 The substanc es ex tracte d from the eggs of S. m a n s o n i are colle ctive ly name d by SEA 4 w hich se ems to be the main induc er of a modulate d re sponse in the chronic phase (granulomatous) of the dise ase , 5, 1 9 In addition, SEA has be en use d as a In fla m m a tio n in du ce d by SEA in ra t pa w s Mediators of Inflammation · Vol 7 · 1998 265   (5) 0.62 ± 0.10 0.34 ± 0.05* 54 a Values were recorded at maximal oedematogenic effect (mean ± SEM). Drugs were administered at the indicated doses from day 0 to 14 of sensitization. *P < 0.05 (Anova t-test).
tool to unde rstand the mechanisms of dise as e involving the immune syste m in ex perime ntal studies. 9 In the pre se nt w ork, w e have show n that S. m a n s o n i eggs induc ed inflammation in rat pads, and that SEA re produc ed the oe de matoge nic e ffec t of the inje ction of S. m a n s o n i e ggs, thus demons tratin g that the main oedematogenic e ffec t derived from eggs is due to SEA. We have also show n that SEA induce d an inc re as ed oe de matoge nic re sponse on a sec ond administration to the animals (challenge), afte r a period of 28 days, suggesting the involvement of the immune syste m in the latter re sponse. A surprising observation, how e ver, w as that the involveme nt of the immune syste m w as loc al rather than syste mic, sinc e the incre as ed effe ct on SEA challenge w as only observe d if the antige n w ere inje cte d in the same loc ation of the se nsitization, i.e. into the ipsilateral paw. This re sult suggeste d that (1) SEA is a w eak antigen, or conve rse ly, rats re strain very e ffec tively the immune re sponse to it, and (2) SEA is very easily cleared out of the animal bodies, unless it is continuously de livere d from the e ggs into the c irculation, as it happens during the c ourse of the diseas e in se nsitive sp ecie s. Irrespe ctive of the inc re ase in polymorphonuclear and mononuclear ce lls (mac rophage s and lymphocytes) into the rat paw s, as show n by histopathological studie s, the cellular sourc e of the 'local' immune re sponse to SEA w as not addre sse d in the pre sent w ork. It w as e stablishe d at le as t 30 ye ars ago by Parrat and West 20 that se rotonin (5-HT) w as the main inflammatory mediator re le as ed in rat pads. In fac t, here w e demonstrate d that serotonin accounte d for 60% and 57% of the oe de matoge nic re sp onse to SEA in se nsitization and challenge of rat paw s, re spec tively, sinc e pizotifen, a 5-HT 2 antagonist, 2 1 blocke d both syste mic ally and loc ally the re sponse . The re lease of other vasoactive substance s by SEA, such as bradykinin, histamine , substanc e P, prostaglandins, le ukotriene s, nitric ox ide or plate let activating factor (PAF) w as discarde d, since pre vious acute administration of their spec ific antagon ists or synthesis inhibitors have not modified the oedematogenic re sponse observe d follow ing SEA se nsitization. There fore , it w as re asonable to suppose that the early serotonin re lease (by 15 min) induc ed vasodilation and inc re ased vasc ular permeability and together w ith IL-1 (see be low ) contribute d to the arrival of circulating leukocyte s to the paw tissue show n in later times by histopathologic al studies. As those treatme nts have not modifie d the oedematogenic re sponse to SEA observe d on challenge , w e could also c onclude that all the me diators above mentione d w ere not important in the pathophysiology of the latte r re sponse . Howe ve r, to our know le dge , it is the first demonstration of an oe de matoge nic effe ct w hich w as not ac utely se nsitive to the pote nt and efficacious ste roidal antiinflammatory drug dex amethas one .
The particip ation of cytokines in the inflammatory re action due to SEA has be en re ported elsew here . 4,1 2 Our data support the notion that the oe de matoge nic effect induc ed by SEA in challe nge is immune -de rive d and depe nds on the re lease of c ytokines in early stages of the re action. Basic ally, thre e arguments favour our hypothesis : (1) a second injection of the antigen induc ed a pote ntiate d oe de matoge nic re sponse; (2) drugs w ith immunosuppre ssive ac tivity, such as dex ame thasone and c yclosp orin chronic ally administe re d during the early stage s of the re action gre atly re duce d (> 50%) the oedematogenic e ffec t observe d in challe nge; and (3) an antib ody spec ific against IL-1b w as e ffec tive in inhibiting by approximately 50% the oe de matoge nic re sponse to SEA. It has be en ex haustive ly show n that dex amethasone and cyclosporin are able to inhibit the synthesis / re lease of various cytokines, spe cially interle ukin-1 and 2, 2 2,2 3 the mainstay c ytokines involve d in the proce ssing of immune re sponse s. 2 4,2 5 As an antibody spe cific to human IL-1b signific antly blocked the oedema from 1 to 4 h of SEA se nsitization of rat paw s by ap prox imately 50%, it w as conclude d that IL-1 w as an important effe ctor of the pathophysiology of this oedema. In such case, IL-1 could e ffec t this re sponse through re c ruitment and activation of leukoc ytes as observe d by the arrival of these c ells to paw tissue from 2 h of SEA inje ctions as confirme d by histopathologic al studies. As dex amethasone treatme nt of rat paw s be fore SEA sens itization did not modify the early oedematogenic re spons e, it w as concluded that the dete cte d re duc tion of the oe de ma by the antibody during sens itization w as , at least partially, due to its binding to a pre formed IL-1, eventually the biologic al/ immunologic al analogous of IL-1b , IL-1a . 2 4 In addition, IL-1 c ould be acting as an important effe ctor of othe r re actions of the immune re sponse to SEA, such as the activation and prolife ration of T-ce ll lines, sinc e the pote ntiated oedematogenic re sponse on SEA challe nge w as observed just in a narrow dose range (1-10 m g). It might be emphasize d that, although w e have used a human IL-1b and a antib ody anti-human IL-1b in rats, the migration of le ukocyte and its blockade by the antib ody w ere ve ry consiste nt (29% inc re as e and > 99% inhibition, re spec tively), suggesting that the site of interac tion be tw een IL-1 and the host is re latively indepe nde nt of its source and w as cons erved in the evolution. Our data have strongly suggeste d that interle ukin 2 is involved in the proce ssing of the immune re sponse to SEA in rats, sinc e e arly treatme nt of the sensitize d animals w ith cyclosporin, as w ith dex ame thasone , gre atly inhibite d the oedematogenic re sponse on challenge (> 50%). Assuming that interle ukin-2 is re lease d by SEA, it is te mpting to sp eculate that the immune re sponse derive d from SEA injec tion in this spec ie s might involve pre dominantly the differe ntiation of the lymphoc ytes into a Th1 subset, rather than a Th2 subse t. 2 6,2 7 If it is so, other c ytokine s such as inte rferon gamma and interle ukin 12 (IL-12) may e ve ntually be dete cte d in this mode l. Rec ent pap ers have show n that ex ogenous IL-12 can inc re ase vaccine-induc ed immunity to S. m a n s o n i in mic e. 1 1,2 8 There fore , the as sociation of IL-12 re le as e w ith a ve ry effective immune re spons e induce d by IL-1 and IL-2, as suggeste d by our re sults, c ould ex plain at least in part, the re sistanc e of rats to S. m a n s o n i infe ction.