ARTICLE IN PRESS

Immunobiology 212 (2007) 603–612

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doi:10.1016/j.im

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Vasoactive intestinal peptide inhibits liver pathology in acute

murine schistosomiasis mansoni and modulates IL-10, IL-12

and TNF-a production

Gamal Allam�

Department of Zoology, Faculty of Science, Beni-Sueif University, Beni-Sueif, Egypt

Received 21 February 2007; received in revised form 6 May 2007; accepted 16 May 2007

Abstract

Vasoactive intestinal peptide (VIP) exerts a broad range of biologic actions that may include modulation of hepaticgranuloma formation. This study aimed to investigate the effect of VIP administration on the course of acute murineschistosomiasis mansoni. Mice were infected each with 40 Schistosoma (S.) mansoni cercariae and injectedintraperitoneally with VIP at a total dose of 1 mg/kg body weight. VIP treatment was very effective in diminishingworm fecundity, hepatic granuloma size and number by about 54%, 75% and 51%, respectively, and reducing livercollagen content. Serum level of interleukin (IL)-10 was increased, while level of IL-12 and tumor necrosis factor(TNF)-a were decreased as a result of VIP administration. Carbohydrate antigen 19.9 (CA 19.9) induced by S. mansoni

infection was decreased with VIP treatment. Activities of hepatic g-glutamyl transferase (g-GT), alanineaminotransferase (ALT), and aspartate aminotransferase (AST) in liver tissue homogenate of infected treated micewere increased. These results indicate that suitable administration of exogenous VIP can be effective in amelioratingimmunopathologic damage associated with schistosomiasis.r 2007 Elsevier GmbH. All rights reserved.

Keywords: Vasoactive intestinal peptide; Murine schistosomiasis mansoni; Hepatic granuloma formation; Cytokine serum levels;

Liver pathology

Introduction

The nervous, endocrine and immune systems arelinked via an elaborate communication system consti-tuted by an array of cytokines and neuropeptides thatinteract to modulate the integrated response of anorganism to disease. One of the potent neuroendocrinemediators is vasoactive intestinal peptide (VIP). VIP is a28 amino acid neuropeptide synthesized by immune cellsand present in the lymphoid microenvironment, which

e front matter r 2007 Elsevier GmbH. All rights reserved.

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elicits a broad spectrum of biological functions, includ-ing the modulation of innate and adaptive immunity,and shows a predominant anti-inflammatory action(Leceta et al., 1996; Martinez et al., 1999; Pozo et al.,2000; Voice et al., 2002; Delgado et al., 2004). VIPmodulates immunoglobulin synthesis (Stanisz et al.,1986), reduces natural killer activity (Sirianni et al.,1992), inhibits nitric oxide (NO) production in murinemacrophages (Xin and Sriram, 1998; Gomariz et al.,2000) and inhibits mobility of lymphocytes (De laFuente et al., 1994; Delgado et al., 1995). Moreover,VIP inhibits degranulation and changes granular con-tent of mast cells (Tuncel et al., 2000), antagonizes the

ARTICLE IN PRESSG. Allam / Immunobiology 212 (2007) 603–612604

effects of inflammatory mediators (e.g. histamine,prostaglandin F2a, neurokinins A and B, leukotrinesC4 and D4, and endothelin) and attenuates platelet-activating factor (PAF)-induced injury and oedema inrat lung (Said, 1991; Pozo et al., 2000).

Schistosomiasis is a major source of morbidity andmortality in developing countries including Africa,South America, the Caribbean and Asia. While anumber of Schistosoma (S.), such as S. haematobium,S. japonicum, S. mekongi and S. intercalatum, can causesystemic human diseases, the main agent of humanschistosomiasis is Schistosoma mansoni (Chitsulo et al.,2000; dos Santos et al., 2007). Schistosomiasis mansoni isa parasitic disease resulting in the deposition of ovapredominantly in the liver and intestines. Parasite eggselicit a CD4+ T-helper (Th) cell-mediated hepaticgranulomatous inflammation, which is the majorpathological consequence of the disease. The granulo-mas are intricate delayed-hypersensitivity reactionsgoverned by numerous cellular and humoral interac-tions (Stavitsky, 2004). In the mouse model, both Th1and Th2 cytokines can orchestrate granuloma develop-ment (Abath et al., 2006). Lacking or neutralization ofinterleukin (IL)-4 reduces granuloma formation andcollagen deposition (Wynn et al., 1993; Cheever et al.,1994; Metwali et al., 1996). However, neutralization orabsence of IL-5 during acute infection prevents eosino-philia but has no effect on granuloma size and fibrosis(Sher et al., 1990). IL-10 is thought to have an importantrole in regulating CD4+ T cell responses induced byS. mansoni. The major role of IL-10 is to controlexcessive Th1 and Th2 polarization during infection(Wynn et al., 1998). It has been shown recently thatIL-10-producing, regulatory T cells, which are derived,in part, from the naturally occurring CD4+, CD25+

and Th2 population, cooperate to suppress the devel-opment of the Th1 response in experimental schistoso-miasis (Hesse et al., 2004; McKee and Pearce, 2004).Tumor necrosis factor (TNF)-a is important for initia-tion of granuloma formation through up-regulation ofintracellular adhesion molecule 1 (ICAM-1), whichmediates cell–cell interactions and migration across theendothelium (Ritter and McKerrow, 1996; Brunet et al.,1998).

It has been shown that granulomas in murineschistosomiasis mansoni contain authentic VIP (Wein-stock and Blum, 1990) and contain VIP-responsivelymphocytes (Weinstock et al., 1991). The granuloma Tcells bear authentic VIP receptors of both type 1 andtype 2 subclasses (Weinstock, 1996). T cells, themselves,can secrete VIP (Gomariz et al., 1994; Delgado andGanea, 2001; Vassiliou et al., 2001) like any other Th2cytokine (Pozo and Delgado, 2004) and in response toVIP show an inhibition of: proliferation, migration(Delgado et al., 2002), and secretion of Th1 cytokinesIL-2 (Metwali et al., 1993) and IFN-g (Delgado et al.,

2000). Moreover, VIP suppresses secretion of IL-4 (Sunand Ganea, 1993; Tang et al., 1995), but enhances IL-5production from granuloma T cells (Mathew et al.,1992). Thus, VIP might regulate immune responses toschistosomiasis by modulating cytokine production. Theaim of this study was to investigate the effect of VIPadministration on IL-10, IL-12, TNF-a production andliver pathology during acute murine schistosomiasismansoni.

Materials and methods

Animals and parasites

Eight-weeks-old male CD1 albino mice were pur-chased from the Schistosome Biological Supply Centre,Theodore Bilharz Research Institute (TBRI, Imbaba,Giza, Egypt). S. mansoni (John Bruce Egyptian strain)cercariae were obtained from infected Biomphalaria

alexandrina snails (TBRI) as previously described bySchubert (1948).

Experimental design

Synthetic porcine VIP (Sigma Chemical Co.,St. Louis, MO, USA) was reconstituted in sterile 0.9%saline–0.1% gelatin containing 0.5% albumin as de-scribed by Karlsson and Ahren (1990). The peptide wasdivided into aliquots and immediately stored at �70 1Cuntil use. Groups of 10 naive (NT) and 10 infected (IT)mice were injected intraperitoneally with VIP at a totaldose of 1 mg/kg body weight divided equally into 24injections (three injections/week for 8 consecutive weeks,starting from the first week of the infection). Controlgroups consisted of both 10 normal untreated (NU) and10 infected untreated (IU) mice were given the sameamount of the vehicle at the same time as treatedgroups.

Infection and worm recovery

Mice were infected with S. mansoni live cercariaeaccording to Stirewalt and Bronson (1955) and Bruceand Radke (1971) with some modifications. Briefly, micewere individually placed in mouse restraining chambers,allowing the tail outside the chamber. After cleaning thetail with dechlorinated tap water, it was inserted into avial containing 40 cercariae in 2ml dechlorinated tapwater and located into an exposure board. Mice wereleft in contact with the infective cercariae for 1 h,and then removed from the chamber and tails wereallowed to dry. Remaining cercariae were counted andmice receiving less than 95% of the cercariae wereexcluded from the experiment. At the end of the 8th

ARTICLE IN PRESSG. Allam / Immunobiology 212 (2007) 603–612 605

week post-infection, all mice were sacrificed and wormburdens were estimated by portal perfusion according toDuvall and DeWitt (1967).

Blood and tissue sampling

Sera were collected from the clotted blood samplesafter centrifugation at 3000 rpm for 15min, then dividedinto aliquots and stored at �70 1C until use. The liverfrom each mouse was excised immediately after perfu-sion and rinsed with ice-cold saline solution. The ventralmedian lobe of the liver was fixed in 10% neutralbuffered formalin for preparation of hematoxylin andeosin-stained sections. Another portion was homoge-nized in 0.9% saline, centrifuged at 3000 rpm for 15minand the supernatant was decanted and stored at �70 1Cuntil used for different biochemical estimations.

Tissue egg load

The number of S. mansoni eggs in the perfused liverand in the intestine (from duodenum to rectum) of micewas estimated after alkali digestion as described byCheever (1968). The mean eggs per gram (g) tissue perpossible worm pairs was calculated using the followingformula:

Eggs=g tissue=worm pairs ¼

ðeggs=g liverþ eggs=g intestineÞ=2� worm pairs:

Histopathology

The diameters of granulomas surrounding a singlemature schistosome egg were measured in the hematox-ylin and eosin-stained liver sections by using an ocularmicrometer (Carl Zeiss, Thornwood, NY, USA).Twenty granulomas per mouse were measured and thelargest diameter and that perpendicular to it wereaveraged (Lichtenberg, 1962). The volume (mm3) ofeach granuloma was calculated assuming a sphericalshape using the following formula: Volume of spher-e ¼ 3pR3 (the radius ‘‘R’’ was obtained by dividing themain diameter of the lesion by two) and the meanvolume for each group was calculated (Mahmoud andWarren, 1974). Single granulomas were counted usingan ocular micrometer in a defined area of 0.5 cm2 ofhematoxylin and eosin-stained liver sections. Thegranuloma mean number per cm2 was determinedaccording to Boros and Warren (1970).

Cytokine serum levels

Sandwich enzyme-linked immunosorbent assay (ELI-SA) was used to measure serum cytokine levels.Cytokine concentrations for mouse IL-10, IL-12 +p40

and TNF-a were determined with commerciallyavailable reagents and ELISA kits purchased fromBioSource International (Camarillo, California, USA).Cytokine concentrations were determined using astandard curve obtained from the known concentrationof cytokine standards included in each assay plateaccording to manufacturer instructions.

Hepatic enzymes activities

Hepatic g-glutamyl transferase (g-GT) activity in theliver tissue homogenate supernatant was determined byusing commercially available kits purchased fromBioSystems S.A. (Spain) according to manufacturerinstructions. Hepatic alanine aminotransferase (ALT)and aspartate aminotransferase (AST) activities in thehomogenate supernatant were estimated according tothe method of Bergmeyer et al. (1978), using reagent kitsobtained from Spinreact Company (Spain).

Hepatic collagen content

The amount of collagen in liver samples wasdetermined from its hydroxyproline content by themethod of Neuman and Logan (1950a, b). Briefly, thedefatted and dried samples were hydrolyzed by auto-claving in 2ml of 6N HCl at 50 pounds pressure. Theprotein hydrolysates were used for the determination ofhydroxyproline and the percentage of collagen of atissue sample was calculated by using the 7.46 factor ofNeuman and Logan (1950a) and Baykal-Erkilic et al.(1995). Meanwhile, a standard curve was constructed inorder to determine the reproducibility of the assay.

Tumor markers

Serum carbohydrate antigen (CA) 19.9 concentrationwas estimated by an immunoradiometric assay kit(Coat-A-Count GI-MA IRMA, DPC, USA) accordingto the method of Frebourg et al. (1988). a-fetoprotein(AFP) in liver homogenate supernatant was determinedby a radioimmunoassay (RIA) kit (Double antibody kit,DPC, USA) according to the methods of Waldmannand McIntire (1974) and Wepsic (1981).

Statistical analysis

The data were analyzed using one-way analysis ofvariance (ANOVA) followed by LSD analysis tocompare various groups with each other (PC-STAT,1985). Results were expressed as mean7standarddeviation (SD) and values of p40.05 were consideredstatistically insignificant, while those of po0.05 and 0.01were considered statistically significant and highly

ARTICLE IN PRESSG. Allam / Immunobiology 212 (2007) 603–612606

significant, respectively. F-probability for each variableexpressed the general effect between groups.

Results

Worm burden

Mice were infected each with 40 S. mansoni cercariaeby tail immersion method. Eight-weeks post-infection,hepatic portal perfusion of IU and IT mice wasperformed and mean worm burden of each group wascalculated. As shown in Fig. 1, VIP treatment ofS. mansoni-infected mice had no significant (p40.05)effect on worm burden.

Tissue egg load

After perfusion has been completed about 0.5 g ofboth liver and intestine for each animal was digested in5ml 5% KOH overnight at 37 1C. Eggs were counted intriplicate in liver and intestine homogenates of eachmouse by using a Mac Master counting slide. Meaneggs per gram liver and intestine was calculated and theneggs per gram tissue per possible worm pairs wasestimated. VIP treatment led to a highly significant(po0.01) decrease in tissue egg load in both liver andintestine. Moreover, eggs per gram tissue per possibleworm pairs were decreased by about 54% in IT mice(Fig. 1).

0

5

10

15

20

25

30

35

40

Worm burden Granulomavolume X

1000

Granuloma

count

Eggs per g

tissue per

worm pairs

/100

IU

IT

** **

**

Fig. 1. Effect of VIP treatment on worm burden (number of

worms), hepatic granuloma volume (mm3), granuloma count

(number/cm2) and eggs per gram tissue (liver and intestine) per

possible worm pairs (number of eggs) of S. mansoni infected

mice. Both infected untreated (IU) and infected treated (IT)

mice were infected each with 40 cercariae and perfused 8-weeks

post-infection. Treated mice received 1mg/kg body weight as a

total dose of VIP, divided into 24 equal injections (three

injections/week along the period of the infection). Values

represented as the mean7SD of six mice. **po0.01 vs.

corresponding IU value.

Hepatic granuloma

Histologic examination of hematoxylin and eosin-stained liver sections of 8 weeks IU and IT mice showedthat VIP treatment highly significantly reduced (po0.01)hepatic granuloma volume and number by about 75% and51%, respectively (Fig. 1). The hepatic granuloma of IUmice was composed of inflammatory cells associated with aloose connective matrix. Necrosis was seen in hepatic tissueand sometimes in the central part of young granulomas,while healing granulomas showed laminated layers offibrous tissue (Fig. 2A). However, granuloma of IT micewas seen as a concentric focus of mononuclear andpolymorphonuclear cells around the egg and the laminatedlayers of fibrous connective tissue disappeared. Minimalmicrovascular changes and no hepatocyte necrosis werenoticed in the liver sections of IT mice (Fig. 2B).

Fig. 2. After perfusion the ventral median lobe of the liver was

fixed in buffered neutral formalin and processed to make

paraffin blocks. The blocks were cut into 4 mm sections.

Sections were stained with hematoxylin and eosin for the

determination of granuloma size and number. (A) Liver

section from infected untreated group showing a healing

granuloma. (B) Liver section from infected VIP-treated group.

ARTICLE IN PRESSG. Allam / Immunobiology 212 (2007) 603–612 607

Cytokine levels in serum

To explore the effect of VIP treatment on cytokineprofile; IL-10, IL-12, and TNF-a in serum of all micegroups were quantified at 8-week post-infection and/orpost-treatment by ELISA. As shown in Fig. 3, IL-10level of both normal treated (NT) and IT mice wassignificantly higher (po0.01) than that of NU and IUmice, respectively. However, the level of IL-12 wassignificantly decreased (po0.01) in NT and IT mice ascompared with NU and IU mice, respectively. Mean-

Table 1. Effect of VIP treatment on liver enzymes activities, liver co

Groups Parameters

Liver tissue

g-GT (mU/g) ALT (mU/100mg) AST (mU/10

NU 70.6377.50a 400.43782.66a 283.83751.65

NT 61.95715.5a 458.107133.33a 413.957100.4

IU 39.7579.27b 165.8773.95c 166.76721.08

IT 63.86710.98a 286.80750.36b 322.50760.03

F-value 8.50 15.94 7.53

F-probabil. po0.01 po0.01 po0.01

LSD at 5% 13.55 95.54 111.52

LSD at 1% 18.48 130.31 152.10

Activities of g-glutamyl transferase (g-GT), alanine aminotransferase (ALT)

homogenate supernatant of naive (NU), normal treated (NT), infection untre

and/or post-treatment. Hepatic collagen was estimated from its hydroxyprol

autoclaving in 6N HCl at 50 pounds pressure. The hydrolysates were used fo

Concentration of a-fetoprotein (AFT) was measured in liver tissue homogena

estimated in serum by immunoradiometric assay. Data are expressed as m

superscripts denote significant differences.

0

50

100

150

200

250

300

350

IL-10 IL-12 TNF-alpha

pg

/ml

NU

NT

IU

IT

d

c

b

a

b

c

a

b

c

c

a

b

Fig. 3. IL-10, IL-12 and TNF-a levels in serum of normal

untreated (NU), normal VIP-treated (NT), infected untreated

(IU) and infected treated (IT) mice. Blood was collected at the

8th week post-infection and/or post-treatment and cytokines

levels were measured by ELISA. Values represented as the

mean7SD of six mice. Columns not sharing common super-

scripts denote significant differences, po0.01.

while, TNF-a level was significantly decreased (po0.01)in IT mice by about 55% as compared with IU mice.However, TNF-a serum level of NU and NT mice wasnot significantly different (p40.05).

Hepatic enzymes activities

To investigate the effect of VIP treatment onS. mansoni-induced liver pathology: g-GT, ALT, andAST activities were measured in liver tissue homogenatesupernatant at 8-weeks post-infection and/or post-treatment. As shown in Table 1, hepatic g-GT andALT activities of NT mice were not significantlychanged (p40.05) with those of NU mice. However,NT mice showed a significant increase (po0.05) inhepatic AST activity only. On the other hand, IU miceshowed a highly significant decrease (po0.01) in g-GTand ALT activities; and a significant decrease (po0.05)in AST activity as compared with NU mice. In contrast,IT mice displayed a high significant increase (po0.01) inthe activities of all tested liver enzymes as comparedwith IU mice. Hepatic g-GT and AST activities of ITmice were not significantly different (p40.05) with thoseof NU mice (Table 1).

Hepatic collagen content

In order to examine the anti-fibrotic effect of VIP onmurine schistosomiasis, collagen was determined inliver tissue of all mice groups at 8-weeks post-infectionand/or post-treatment. As shown in Table 1, NT micehad not any significant difference (p40.05) in hepaticcollagen content in comparison to NU mice. But, IU

llagen and tumor markers of 8 weeks S. mansoni infected mice

Serum

0mg) Collagen (mg/100mg) AFP (U/g) CA 19.9 (U/ml)

b 4.3070.44c 8.5071.88a 5.4370.56b

0a 4.6070.75bc 7.9071.57a 5.1870.64b

c 6.1570.15a 8.7872.47a 6.4070.47a

ab 5.1070.80b 8.0572.75a 5.1070.44b

8.92 0.21 7.79

po0.01 p4 0.05 po0.01

0.76 — 0.64

1.04 — 0.87

, and aspartate aminotransferase (AST) were measured in liver tissue

ated (IU) and infected treated (IT) mice at the 8th week post-infection

ine content after defatting and drying, liver tissues were hydrolyzed by

r the determination of hydroxyproline and the percentage of collagen.

te supernatant by RIA. Carbohydrate antigen 19.9 (CA 19.9) level was

ean7SD of six mice per each group. Values not sharing common

ARTICLE IN PRESSG. Allam / Immunobiology 212 (2007) 603–612608

mice showed a highly significant increase (po0.01) inhepatic collagen content as compared with NU. Treat-ment of S. mansoni-infected mice with VIP led to ahighly significant decrease (po0.01) in hepatic collagencontent of IT mice as compared with IU.

Tumor markers

To test the effect of VIP on tumor markers, the levelof both CA 19.9 and AFP were determined in serum andhepatic tissue homogenate supernatant, respectively.AFP concentration was not significantly changed(p40.05) between different mice groups. However, IUmice showed a highly significant increase (po0.01) inserum level of CA 19.9 as compared with the other micegroups. At the same time, CA 19.9 concentration inserum of IT mice was not significantly different(p40.05) than that of NU or NT mice (Table 1).

Discussion

VIP has emerged as a potent anti-inflammatoryfactor, which exerts its function by regulating theproduction of both anti- and pro-inflammatory media-tors (Delgado et al., 2004). The present study has shownthat serum level of IL-10 highly significantly increases(po0.01) by 382% and 39% with VIP treatment in bothuninfected and S. mansoni-infected mice in comparisonwith NU and IU mice, respectively. This finding iscompatible with previous reports which have shown thatVIP stimulates in vitro and in vivo production of theanti-inflammatory cytokine IL-10 by peritoneal macro-phage in response to LPS injection (Delgado et al.,1999a; Gomariz et al., 2000). The effect of VIP on Th2response seem to be mediated via the VIP/pituitaryadenylate cyclase-activating peptide receptor (VPAC) 2,a type 2 VIP receptor that is increased upon Thstimulation (Voice et al., 2002). More recently, it hasbeen shown that in addition to increasing IL-10production from Th2 cells, VIP treatment can increaseproduction of regulatory T cells, producing high levelsof IL-10 (Chorny et al., 2005; Delgado et al., 2005;Reinke and Fabry, 2006). Regulatory T cells producingIL-10 may cooperate to reduce morbidity and prolongsurvival in schistosomiasis (Hesse et al., 2004). On theother hand, the data of the present study showed thatVIP treatment significantly decreases (po0.01) theserum level of IL-12 by about 31% and 29% in bothuninfected and infected mice respectively, compared tocontrol ones. This result confirms the previous reportswhich stated that VIP inhibits the transcription (Delga-do and Ganea, 1999) and production of pro-inflamma-tory cytokine IL-12 from macrophage (Xin and Sriram,1998; Gomariz et al., 2000) and from Th1-cells (Wang

et al., 1999). Inhibition of Th1 cytokines is mediated byVPAC1, a type 1 VIP receptor, which is expressed on allTh cells (Voice et al., 2002; Grimm et al., 2003).Similarly, the present study clearly showed that TNF-aserum level of infected mice highly significantlydecreased by 55% (po0.01) with VIP treatment incomparison with IU mice. Our observations on TNF-aserum level are in agreement with other reports whichshowed that VIP inhibits TNF-a production in vitro andin vivo from LPS-stimulated macrophage. VIP exertstheir inhibitory action through the binding to VPAC1receptor that leads to activation of the adenylate cyclasesystem and reduction of nuclear factor-kB (NF-kB)binding (Delgado et al., 1998, 1999b, c). Consequently,changes induced with VIP treatment in the levels ofIL-10 and TNF-a are both much larger than the changein IL-12 level.

Granuloma formation is an inflammatory responseagainst parasite eggs trapped in host tissues. Thepresence of VIP within granulomas supports thehypothesis that this neuropeptide has a role in theregulation of granulomatous inflammation (Weinstock,1996; Osman et al., 1997; Pozo et al., 2000). The presentstudy clearly showed that treatment of S. mansoni-infected mice with VIP reduced hepatic granuloma sizeby 75%. This finding confirms the previous findings ofOsman et al. (1997), and is in accordance with previousstudies, which showed that VIP inhibits both IL-2 andIL-4 production by stimulated spleen cells or purifiedstimulated CD4+ T cells (Metwali et al., 1993; Sun andGanea, 1993; Tang et al., 1995). Indeed, both IL-2 andIL-4 play a role in granuloma formation (Wynn et al.,1993; Cheever et al., 1994; Brunet et al., 1998). Also,IL-10 appears to play a role in regulating granulomasize, as mice treated with IL-10 have smaller granulomas(Flores-Villanueva et al., 1996), whereas IL-10-deficientmice make larger granulomas during the acute, but notchronic, period of disease (Wynn et al., 1998). More-over, TNF-a has been shown to play a role in granulomaformation. The pivotal role for this cytokine wasdiscovered in experiments with severe combined im-munodeficient (SCID) mice. After treatment withTNF-a, these mice are able to form discrete (althoughlymphocyte-deficient) granulomas around parasite eggs(Amiri et al., 1992). Further support for a role forTNF-a has come from studies in which the treatment ofimmunocompetent mice with anti-TNF-a serum re-sulted in reduced granuloma size (Joseph and Boros,1993). The present study demonstrated that treatment ofinfected mice with VIP induced the production of IL-10by 39% and inhibited the production of TNF-a by 55%comparing with IU mice. Therefore, the immunomodu-latory activities of VIP on granuloma formation couldbe mediated through its effect on cytokines production.

Another interesting finding in the present study wasthat, despite the insignificant difference (p40.05) in

ARTICLE IN PRESSG. Allam / Immunobiology 212 (2007) 603–612 609

worm burden and possible worm pairs harbored in theportal circulation of infected animals either treated withVIP or not, VIP treatment led to a highly significantdecrease (po0.01) in eggs per gram tissue (liver andintestine) per possible worm pairs by 54%. This in turnled to 51% decrease in hepatic granuloma number.These results are in basic agreement with the finding ofOsman et al. (1997). The main possible explanations forthese results are that VIP may reduce the fecundity ofS. mansoni worm and/or regulate the adhesion mole-cules playing a key role in egg trapping and increase themobilization of eggs from liver and intestine to theexterior of the body in the feces. These observationsindicate that VIP has no anti-helminthic activity, buthas a powerful anti-granulomatous and anti-inflamma-tory action.

In the liver, the parasite-egg antigens induce granu-lomatous inflammatory reactions, involving macro-phages, eosinophils, lymphocytes and hepatic stellatecells. Activated hepatic stellate cells synthesize collagensI and III, and alpha-smooth muscle actin. Thesecollagen subtypes are fibril forming in nature, contribut-ing to a significant part of the scar tissue in liver fibrosis(Schuppan, 1990; Chatterjee et al., 2002). Fibrogenesis isa dynamic process regulated by cytokine produced bymacrophages, lymphocytes and fibrocytes (Wyler, 1996;Chesney et al., 1998). Our data showed that VIPtreatment significantly decreases (po0.01) liver collagencontent in infected mice, but has no significant (p40.05)effect on hepatic collagen in uninfected mice. Thisdecrease in liver collagen content of IT group could beexplained by reduced worm fecundity leading to reducedtissue egg load and consequently reduced liver collagen.Also, VIP could decrease liver collagen content andconsequently liver fibrosis through suppression of IL-4,TGF-b and IL-13 production. VIP has been shown toinhibit IL-4 (Sun and Ganea, 1993; Tang et al., 1995)and TGF-b production (Sun et al., 2000), and modulateIL-13 production (Kimata, 1996). It has been shownthat IL-4, TGF-b and IL-13 promote collagen deposi-tion and enhance liver fibrosis in these circumstances(Czaja et al., 1989; Cheever et al., 1994, 1995; Stavitsky,2004; Kaviratne et al., 2004). Moreover, VIP mayreduce collagen deposition by a direct effect on hepaticstellate cells similar to somatostatin, which has beenshown to modulate collagen I and III synthesis andalpha-smooth muscle actin expression in activatedhepatic stellate cells during schistosomiasis (Chatterjeeet al., 2004). However, the exact mechanism wherebyVIP exerted its antifibrotic effect was unclear and needsfurther investigation.

S. mansoni infection leads to elevation in serum g-GT,ALT and AST (Mansour et al., 1982; Mahmoud et al.,2002), and decreases the activities of these enzymes inliver tissue homogenate (Awadalla et al., 1975; Allamand Ahmed, 2005). The data of the present study

support the previous reports and showed that theactivities of g-GT, ALT and AST in liver homogenateof 8-week infected mice were significantly decreased(po0.01, 0.01 and 0.05, respectively) as a result ofS. mansoni infection compared to those of NT mice.This depletion in liver enzymes activities could be due toleakage of these enzymes from necrotic tissue and thereplacement of normal liver tissue by granulomatouslesions. However, infected mice treated with VIP restorethe hepatic g-GT, ALT, and AST activities that weredecreased by S. mansoni infection. This amelioration inliver enzymes activities could be attributed to thereduction in hepatic granuloma size and number as wellas absence of necrotic liver tissue in IT mice. VIP couldprotect hepatocytes from destructive inflammatoryresponse induced by schistosome eggs via inhibition ofpro-inflammatory cytokines and NO production.

Tumor markers, CA 19.9 and AFP may be elevated inchronic cases of schistosomiasis (Chen et al., 1984;Noeman et al., 1994; Fouad and Khallaf, 1994).Elevated serum CA 19.9 is associated with gastro-intestinal cancer, colo-rectal cancer and gastric ulcera-tion (Fouad and Khallaf, 1994; Chan and Sell, 1996).While, elevated AFP level is associated with hepatocel-lular carcinoma, massive hepatic necrosis, hepatitis andliver cirrhosis (Noeman et al., 1994; Abelev, 2001).Our data showed no significant difference (p40.05) inAFP level in liver tissue homogenate between testedgroups. On the other hand, serum level of CA 19.9significantly increases (po0.01) in IU mice only. Thisresult confirms the previous finding of Allam andAhmed (2005). The more likely explanation for thisresult is that over 50% of the eggs produced byS. mansoni worms remained within the liver andintestine tissues of the host. The lodged eggs in theintestine induce inflammatory response that causesnecrosis and gastric ulceration (Warren, 1982). Gastriculceration is accompanied with elevated level of serumCA 19.9. However, CA 19.9 serum level of infected VIP-treated mice showed no significant difference (p40.05)with either that of NT or uninfected treated mice. Thisfinding indicates that VIP may have ameliorative effecton intestinal tissue in which a large part of S. mansoni

eggs trapped.The most common anti-schistosomiasis drug used in

endemic areas is praziquantel, which is effective againstthe worm stages of the parasite (WHO, 1993), but hasonly some ovicidal activity against mature S. japonicum

eggs (Cheever and Deb, 1989). Moreover, there is nodefinitive treatment available for the egg stages or forthe pathology caused by this stage (Chatterjee et al.,2002). The present study indicates that VIP has potentanti-pathology effect on murine schistosomiasis. There-fore, the combination of both VIP and anti-helminthicdrugs may be the aim for future work to evaluate thetherapeutic possibility of this combination.

ARTICLE IN PRESSG. Allam / Immunobiology 212 (2007) 603–612610

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