34. Shihab FS, Bennett WM, Tanner AM, Andoh TF. Angiotensin IIblockade decreases TGF-beta 1 and matrix proteins in cyclo-sporine nephropathy. Kidney Int 1997; 52: 660.
35. Lee DBN. Cyclosporine and the renin-angiotensin axis. KidneyInt 1997; 52: 248.
36. Tufro-McReddie A, Gomez RA, Norling LL, Omar AA, Moore LC,Kaskel FJ. Effect of CsA on the expression of renin and angio-tensin type 1 receptor genes in the rat kidney. Kidney Int 1993;43: 615.
37. del Moral RG, Andujar M, Ramırez C, et al. Chronic cyclosporinA nephrotoxicity, P-glycoprotein overexpression, and relation-ships with intrarenal angiotensin II deposits. Am J Pathol1997; 151: 1705.
38. Shehata M, Cope GH, Johnson TS, Raftery AT, El Nahas AM.Cyclosporine enhances the expression of TGF-b in the juxta-glomerular cells of the rat kidney. Kidney Int 1995; 48: 1487.
39. Horikoshi S, McCune BK, Ray PE, Kopp JB, Sporn MB, KlotmanPE. Water deprivation stimulates transforming growth fac-tor-b2 accumulation in the juxtaglomerular apparatus ofmouse kidney. J Clin Invest 1990; 88: 2117.
40. Antonipillai I, Le TH, Soceneantu L, Horton R. Transforming
growth factor-beta is a renin secretagogue at picomolar con-centrations. Am J Physiol 1993; 265: F537.
41. Liu A, Ballermann BJ. TGF-b type-2 receptor in rat renal vas-cular development: localization to juxtaglomerular cells. Kid-ney Int 1998; 53: 716.
42. Young B, Burdmann EA, Johnson RJ, et al. Cyclosporine Ainduced arteriolopathy in a rat model of chronic cyclosporinenephropathy. Kidney Int 1995; 48: 431.
43. Meister B, Lippoldt A, Bunnemann B, Inagami T, Ganten D,Fuxe K. Cellular expression of angiotensin type-1 receptormRNA in the kidney. Kidney Int 1993; 44: 331.
44. Yagil Y. Acute effect of cyclosporin on inner medullary blood flowin normal and postischemic rat kidney. Am J Physiol 1990;258: F1139.
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ANTIVIRAL AND IMMUNOMODULATORY EFFECTS OFDESFERRIOXAMINE IN CYTOMEGALOVIRUS-INFECTED RAT
LIVER ALLOGRAFTS WITH REJECTION1
TIMI MARTELIUS,2,3,4 MARTIN SCHOLZ,5 LEENA KROGERUS,6 KRISTER HOCKERSTEDT,2 RAISA LOGINOV,2
CATHRIEN BRUGGEMAN,7 JINDRICH CINATL, JR.,5 HANS W. DOERR,5 AND IRMELI LAUTENSCHLAGER3
Departments of Surgery, Virology, and Pathology, Helsinki University Hospital, Helsinki, Finland;Institute of Medical Virology, J.W. Goethe University, Frankfurt am Main, Germany; and
Department of Medical Microbiology, University of Maastricht, Maastricht, The Netherlands
Background. Cytomegalovirus (CMV) infection is as-sociated with acute and chronic allograft rejection. Wehave recently shown that rat CMV increases portalinflammation and bile duct destruction in a model ofrat liver allograft rejection. Desferrioxamine (DFO),an iron chelator and antioxidant, has recently beendemonstrated to have antiviral as well as immuno-modulatory effects in vitro. We therefore investigated
whether DFO inhibits (a) CMV infection and (b) graftdestruction in our rat model.
Methods. One day after liver transplantation, PVG(RT1c) into BN(RT1n), the rats were infected with ratCMV (RCMV, Maastricht strain; 105 plaque-formingunits i.p.). The effects of 100 mg/kg body weight and200 mg/kg body weight DFO were examined.
Results. In the untreated group, the grafts were uni-formly RCMV culture-positive. In the group receiving200 mg/kg DFO, RCMV replication was effectively in-hibited. Inflammatory response in the graft, and espe-cially the number of macrophages, was significantlyreduced by DFO. Portal inflammation and bile ductdestruction were also significantly reduced. In the un-treated group, the bile duct epithelial cells were foundto be strongly positive for tumor necrosis factor-a andthis expression was clearly decreased by DFO. In ad-dition, DFO significantly inhibited vascular cell adhe-sion molecule-1 expression on sinusoidal endothelialcells.
Conclusions. Our in vivo transplant study stronglysupports the inhibitory effects of metal chelators onCMV infection and their possible usefulness in thetreatment of CMV-induced pathogenic changes.
1 This work was supported by grants from the Mary and Georg C.Ehrnrooth Foundation (T.M.), the Duodecim Foundation (T.M.), Hel-sinki University (T.M.), Sigrid Juselius Foundation (K.H. and I.L.),and Helsinki University Hospital Research Funds (K.H. and I.L.).
2 Transplantation and Liver Surgery Unit, Helsinki UniversityCentral Hospital.
3 Department of Virology, Helsinki University Central Hospital.4 Address correspondence to: Timi Martelius, M.D., Transplanta-
tion and Liver Surgery Unit, Research Lab, Helsinki UniversityHospital, Kasarmikatu 11-13, FIN00130 Helsinki, Finland. E-mail:[email protected].
5 Institute of Medical Virology, J.W. Goethe University.6 Department of Pathology, Helsinki University Central Hospital.7 Department of Medical Microbiology, University Hospital Maastricht.
MARTELIUS ET AL.December 15, 1999 1753
Cytomegalovirus (CMV*) has been suggested to be linkedto kidney and heart allograft rejection (1–4). In liver trans-plantation, the importance of concomitant CMV infectionduring late acute rejections has been reported (5). An asso-ciation between CMV and vanishing bile duct syndrome(VBDS) in hepatic allografts has been found (6–8), and per-sistence of CMV-DNA in hepatocytes (7, 8), and especiallybile ducts and vascular structures (8) of the liver has beendemonstrated in patients with VBDS.
We have previously developed a rat model of prolongedacute liver allograft rejection (9). In this model, the inflam-matory response of acute rejection in the graft can be moni-tored by fine needle aspiration biopsy. The mean survivaltime is 37620 days (9). We have recently shown that the welldefined rat CMV (RCMV) (11) significantly increases intra-graft inflammation and bile duct destruction in this experi-mental model of liver allograft rejection in the rat (11).
The current specific anti-CMV therapy with ganciclovireffectively inhibits viral DNA synthesis and replication andusually demonstrates a good clinical response. However, ithas been suggested that by also inhibiting a feedback mech-anism that down-regulates the expression of the immediateearly (IE) genes of CMV, ganciclovir actually enhances theexpression of the IE genes that are known to trigger manyproinflammatory pathways (12, 13). On the other hand, anattempt to fight the immunopathological phenomena by in-creasing immunosuppression would lead to uncontrolledCMV infection. CMV has been reported to enhance expres-sion of adhesion molecules in vitro (14) and in liver trans-plants (15, 16). CMV has also been demonstrated to up-regulate cytokines such as tumor necrosis factor-a (TNF-a),interleukin (IL)-1, and IL-2 (17–19).
In vitro data indicate that oxidative stress is very impor-tant in the regulation of CMV replication (20–22). The induc-tion of oxidative stress clearly increased CMV immediateearly and late antigen production as well as the release ofinfectious virus (20). In vitro CMV infection of smooth musclecells rapidly leads to generation of reactive oxygen interme-diates (ROIs), and these ROIs are also critical to the trans-activation of the human CMV major immediate early pro-moter (22).
Nontoxic concentrations of desferrioxamine (DFO), an ironchelator and antioxidant, have been demonstrated to haveantiviral (CMV) and immunomodulatory effects in vitro (23,24). DFO is a trihydroxamic acid, which can form a complexwith ferric ion, has antioxidative potency both extra- andintracellularly, and has been used clinically in the treatmentof disorders that involve iron overload (25, 26). It has beenshown to block IL-2 receptor expression on human T lympho-cytes (27) and also to inhibit DNA synthesis due to theinhibition of ribonucleotide reductase (28).
The aim of the present study was to investigate the effectof the iron chelating antioxidant DFO on CMV-induced his-
topathological changes and CMV replication in vivo in our ratmodel of concomitant CMV infection and liver allograft re-jection.
MATERIALS AND METHODS
Rats. A donor-recipient combination of PVG (RT1c) into BN(RT1n) with a previously observed mean survival time of 37 dayswithout immunosuppression was used (9, 29). The rats were fed withregular rat food and tap water ad libitum. The animals receivedhumane care according to the criteria outlined in the Guide for theCare and Use of Laboratory Animals (National Institutes of Health).The study was approved by the committee for experimental researchof the Helsinki University Central Hospital and the regional author-ities.
Transplantation. Liver transplantation under ether anesthesiawas performed using the technique introduced by Kamada (30),supplemented with reconstruction of the hepatic artery. On day 1after transplantation, the animals were infected with rat CMV (seebelow). After transplantation, two groups of eight rats received DFOat 100 and 200 mg/kg body weight/day (divided into two doses),respectively, through the follow-up period. The comparison group of10 rats was infected with RCMV but received no therapeutic agents.No immunosuppressive drugs were given to any of the animals. Inaddition, two uninfected groups of allograft recipients with (n54)and without (n55) DFO treatment (200 mg/kg body weight/day) wereincluded.
All rats included in the study were killed at 4 weeks after trans-plantation, and material for the histologic and immunohistologicstudies was collected (see below).
RCMV infection. The rats were infected by inoculation with 105
plaque-forming units of RCMV (Maastricht strain) intraperitoneally1 day after liver transplantation. The characteristics of rat cytomeg-alovirus and the RCMV infection have been described in detailpreviously (10). The procedure for culturing and inoculating theRCMV was performed as described previously (10, 31).
Demonstration of CMV infection: viral culture. The presence ofRCMV infection in the graft was demonstrated by culturing the virusfrom material obtained by fine needle biopsies (see below). The fineneedle sample was aspirated from the graft into RPMI 1640 culturemedium containing albumin. The virus was cultured in rat embryofibroblasts (REF) under standard virus culture conditions. RCMVinfection of the cells was confirmed by immunofluorescence tech-nique, using monoclonal antibodies against RCMV early and lateantigens and fluorescein isothiocyanate-conjugated anti-mouse anti-body (Organon Teknika Corporation, Durham, UK). The specificityof these RCMV monoclonal antibodies has been described previouslyin detail (32).
Monitoring of inflammation associated with rejection by trans-plant aspiration cytology. Serial fine-needle aspiration biopsies(FNABs) from the grafted liver and corresponding peripheral bloodsamples were obtained from the graft recipients 4 days, 1 week, 2weeks, and 3 weeks after transplantation. In this way, three to foursequential samples were obtained from each of the animals withoutkilling them. A fentanyl-fluanisone (Hypnormr) anesthesia was usedfor the sampling. The processing of the aspirate has been describedin detail previously in experimental and clinical studies of liverallografts (33, 34). The intensity of inflammation associated withrejection was quantified using the increment method as describedpreviously (34). We have previously described the cellular findings inthe FNAB monitoring in acute liver allograft rejection in the ratmodel (9).
Graft histology. The liver tissue specimens taken at 4 weeks aftertransplantation were fixed in 10% buffered formalin for 2 days andembedded in paraffin. Four-micrometer-thick sections were cut andstained with hematoxylin and eosin and Masson’s trichrome.
The histopathological changes were blindly scored by one pathol-ogist (L.K.). The changes looked for were those recommended by the
* Abbreviations: CMV, cytomegalovirus; DFO, desferrioxamine;FNAB, fine-needle aspiration biopsy; ICAM-1, intercellular adhesionmolecule-1; IE, immediate early; IL, interleukin; IL-2-R, interleu-kin-2 receptor; LFA-1, leukocyte function antigen-1; LGL, largegranular lymphocyte; MHC, major histocompatibility complex; NAC,N-acetyl-cysteine; RCMV, rat cytomegalovirus; REF, rat embryo fi-broblast; ROI, reactive oxygen intermediate; TNF-a, tumor necrosisfactor-a; VBDS, vanishing bile duct syndrome; VCAM-1, vascularcell adhesion molecule-1; VLA-4, very late antigen-4.
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International Working Party for Terminology of Hepatic AllograftRejection (35). The scores were derived as follows: no change wasdesignated 0, mild change 1, moderate 2 and severe changes 3. Thefollowing parameters were chosen for further analysis.
(a) The score for the portal inflammation represented both thearea and the density of the portal mononuclear infiltrate. Signifi-cance was also attached to the number of large activated lymphoidcells, as compared to the number of small inactive lymphocytes. (b)The bile duct damage score comprised of the degree of epithelialsloughing, necrosis and the presence of inflammatory cells under andbetween the epithelial cells. When epithelial damage was accompa-nied by periductular fibrosis with narrowing of the ductular lumen,it was considered to be a severe change. (c) The bile duct proliferationscore represented the amount of proliferation of the periportalductules, the degree of which was scored. (d) The arterial damagescore represented damage and edema of the myoepithelial cells andalso by the presence of inflammatory cells within the myoepitheliumor beneath the endothelium causing partial occlusion of the lumen. Ascore of 3 was given when foam cells were seen beneath the endo-thelium in addition to the above changes. (e) The parenchymal ne-crosis score was derived from the estimated percentage of the area ofthe section that was necrotic. Up to 10% was considered mild andwas given a score of 1, between 10 and 30% was given a score of 2,and more than 30% was given a score of 3. (f) The fibrosis scoreconsisted similarly of the estimated percentage of the area of thesection that stained as collagen. Up to 5% was considered mild andwas given a score of 1, between 5 and 20% was given a score of 2, andmore than 20% was given a score of 3.
Immunohistochemistry. For further demonstration of the inflam-matory reaction associated with acute rejection, immune activationmarkers, increase of major histocompatibility complex (MHC) classII antigens in the graft and the appearance of interleukin-2-recep-tors (IL-2-R, CD 25) on the lymphoid cells infiltrating the graft wereinvestigated. The expression of cell surface adhesion molecules, in-tercellular adhesion molecule-1 (ICAM-1, CD 54) and vascular celladhesion molecule-1 (VCAM-1, CD 106), as well as their ligandmolecules, leukocyte function antigen-1 (LFA-1, CD 11a) and verylate antigen-4 (VLA-4, CD 49d), were also studied in frozen sectionsfrom the removed liver grafts.
A three-layer indirect immunoperoxidase technique and monoclo-nal antibodies against the following rat antigens: MHC class II (MAS043, Seralab, Sussex, UK), IL-2-R (MAS 263, Seralab) and ICAM-1(BSA 1, R&D Systems Europe, Abingdon, UK), LFA-1 (BSA 3, R&DSystems Europe, Abingdon, UK), as well as VCAM-1 (gift from Dr. R.Lobb, Biogen, Cambridge, MA) and VLA-4 (PharMingen, San Diego,CA) were used. The frozen liver tissue sections were first incubatedwith the monoclonal mouse antibody, then with peroxidase-conju-gated rabbit anti-mouse antibody (Dako, Copenhagen, Denmark),and thereafter treated with a peroxidase-conjugated goat anti-rabbitantibody (Tago Inc., Burlingame, CA). The reaction was revealed bya 3-amino-9-ethyl carbazole (AEC) solution containing hydrogen per-oxide. Mayer’s hemalum was used as a counterstain. The intensity ofexpression of MHC class II and adhesion molecules in various vas-cular structures and sinusoidal endothelial cells were blindly andsemiquantitatively scored from 2 to 111. The numbers of IL-2receptor- and class II-positive as well as adhesion ligand molecule-expressing graft infiltrating cells were counted per high power visualfield. Three portal fields were examined per graft, and the averagevalue was given.
For the TNF-a staining, a polyclonal rabbit antibody against ratTNF-a (Genzyme, Cambridge, MA) was used, and the secondary andtertiary peroxidase-conjugated antibodies were goat anti-rabbit(Caltag, Burlingame, CA) and swine anti-goat antibody (Caltag,Burlingame, CA), respectively. Similar scoring, from 2 to 111, asdescribed above, was used for the intensity of TNF-a staining. Thestructures that were assessed were the vascular wall and the bileduct epithelial cells. In addition, the inflammatory cells in the portalfields staining positive for TNF-a were counted.
Preparing and administration of DFO. DFO (Novartis PharmaGmbH, Nurnberg) was obtained as dry substance in glass ampoules.It was dissolved in sterile water to a concentration of 100 mg/ml andused fresh. The animals were injected with the solution intraperito-neally twice daily, with a 12-hr interval.
Statistics. The results were expressed as mean6SD. For compar-ison of results between the different groups, Student’s t test was usedwhen data were continuous and normally distributed, and Mann-Whitney U test was used for the ordinal data. The differences wereconsidered significant when the P-value was #0.05.
RESULTS
The effect of DFO on the replication of rat CMV in therejecting allograft. Viral cultures were obtained by fine-nee-dle aspiration from the grafts. The cultures from the un-treated grafts have previously been shown to be uniformlyRCMV-positive from day 5 through 12 after transplantation(11) with a clear cytopathic effect that appeared already after4 days of culture in REF cells. Consistently, also here allcultures from 1 week in the untreated group were positive.
The 100 mg/kg/day dose of DFO could not totally inhibitthe RCMV replication, but the cytopathic effect was weakerand appeared later, after 7–10 days of culture in REF cells(vs. after 4 days in the untreated group of RCMV-infectedrecipients).
In contrast, when the higher dose of 200 mg/kg/day wasapplied, a clear inhibition of RCMV replication was seen. Ofeight animals, only one was culture-positive 9 days aftertransplantation, and even this animal became culture-nega-tive during the second week. All the other recipients demon-strated negative cultures at all time points sampled. Theviral culture results are summarized in Table 1.
The effect of DFO on the rejection-associated inflammationassessed by FNAB. The cellular response of acute rejectionwas significantly influenced by DFO. The total inflammationwas reduced significantly (P,0.05) by both DFO doses at 1and 2 weeks, and also at 3 weeks by the higher dose, al-though at the last time point FNAB was not taken from all ofthe animals. Already on day 4, DFO seemed to reduce thetotal inflammation, but this was not significant. Generallythe effect of the higher dose of DFO was more pronounced(Fig. 1). Of specific inflammatory cell types in the infiltrate,there was no significant reduction in lymphocytes or lym-phoid blasts, the classically most important cells in acuterejection. However, there was a significant decrease in thenumber of macrophages at 1, 2, and 3 weeks. The decrease inmacrophages was the major factor leading to the reduction in
a Table shows presence of the cytopathic effect. Results shown areof individual animals in untreated and DFO-treated (100 and 200mg/kg b.w./day, respectively) groups.
MARTELIUS ET AL.December 15, 1999 1755
the total inflammation value in FNAB (Fig. 2). Also theamount of large granular lymphocytes (LGLs, natural killercells) was significantly reduced at 2 weeks after transplan-tation.
The effect on histological changes. The histologicalchanges at 4 weeks after transplantation are summarized inFigure 3. In the untreated animals, there was moderate tointense portal inflammatory infiltrate. This consisted mainlyof small lymphocytes and macrophages. In addition, the bileduct destruction was severe in most of the untreated grafts.Inflammatory cells infiltrated the ducts, structural integritywas lost, and even necrosis of the duct cells occurred. Some ofthe destroyed ducts were surrounded by collars of fibroblast-
like cells and collagen. Part of the ducts had almost com-pletely disappeared, leaving only these collar-like structuresbehind. Ductular proliferation was also seen. In 4 out of 10untreated grafts, there was arterial damage of some degree,but this varied greatly and in the rest of the grafts thearteries were well preserved. The change most often seen inthe arteries was endothelialitis. Parenchymal necrosis andfibrosis were not prominent findings at this stage, but theywere nevertheless selected for the further analysis as indi-cators of liver parenchymal damage.
In DFO-treated animals, the intensity of the portal inflam-matory infiltrate was only minimally diminished by thelower dose of DFO and this was not significant. A more
FIGURE 2. The numbers of macrophages in the fine needle aspirates from the grafts (untreated and 200 mg/kg body weight/day DFO groups).Figure shows mean number6SD of cells per high power field (*, P,0.05).
FIGURE 1. Total inflammation in fine-needle aspiration biopsy follow-up ofthe CMV-infected liver allografts (un-treated group, 100 mg/kg body weight/day DFO group, and 200 mg/kg bodyweight/day DFO group, respectively).Results are expressed in corrected in-crement units (CIU) (mean6SD;*, P,0.05).
TRANSPLANTATION1756 Vol. 68, No. 11
prominent reduction was seen in the degree of bile ductdestruction, but again this was not statistically significant.
On the contrary, the 200 mg/kg/day dose of DFO wassufficient to significantly reduce the portal inflammation as-sociated with rejection.(1.560.5 in the DFO group vs.2.360.8 in the untreated group, P,0.05). Additionally, thebile duct damage was significantly reduced (P,0.05) by thehigher dose of DFO. The mean score for the bile duct destruc-tion was 1.960.6 in the untreated group and 0.860.8 in theDFO group (Figs. 3 and 4). The bile duct proliferation wassomewhat diminished but not significantly. In addition, thedegree of fibrosis was significantly decreased by DFO(0.060.0 vs. 0.960.6). Obvious arterial damage was notpresent in either of the DFO-treated groups. However, as aresult of great variation in the untreated group, there was nosignificant difference in the arterial damage score. There wasno difference between the groups concerning the amount ofparenchymal necrosis, which was not prominent in any of thegroups.
The effect of DFO on activation markers and on adhesionmolecule expression in the grafts. In immunohistologicalanalyses of adhesion molecules and activation markers fromthe grafts obtained at 4 weeks, the differences were quitesubtle considering the reduction in inflammation. The num-ber of class II antigen- and IL-2 receptor-expressing lym-phoid cells in the inflammatory infiltrate was slightly, butnot significantly, diminished by DFO (Table 2).
The class II expression on the sinusoidal endothelium wasequally intense in both untreated and DFO-treated grafts.Expression of class II was also seen in the vascular endothe-lium and hepatocytes in both groups, and again this was of asimilar intensity. The adhesion molecule ICAM-1 was ex-pressed to a similar degree in both untreated and DFO-treated grafts. The sinusoidal endothelium was intenselyICAM-1-positive, and the vascular endothelium was also
moderately stained (Table 3). Furthermore, the correspond-ing ligand molecule LFA-1 expression on the lymphoid cellsin the DFO-treated grafts was of similar intensity to thatseen in the untreated CMV-infected grafts (Table 2). How-ever, the VCAM-1 expression in the sinusoids was signifi-cantly reduced by DFO (P,0.05). The vascular VCAM-1 ex-pression was also slightly diminished (Table 3). In theexpression of VLA-4, the integrin family ligand for VCAM-1,there was a slight but not significant decrease by DFO(Table 2).
The effect of DFO on TNF-a expression in the graft. At 4weeks after transplantation in the RCMV-infected livergrafts without treatment, there was strong specific stainingof the vascular endothelium and also of the bile duct epithe-lial cells. Both in the endothelial and the bile duct epithelialcells, the staining was cytoplasmic. The vascular and bileduct staining were both scored from moderate to intense. Inmost of the DFO-treated (200 mg/kg/day) grafts, this stainingwas absent. The difference was significant (Table 4). At thisphase, there were only a few positive macrophages in theportal inflammatory infiltrates, mostly only 1–2 positive cellsper portal area. The groups did not differ significantly in thisrespect.
Effects of DFO in the absence of CMV. In noninfectedgroups, there was a slight and not significant decrease intotal inflammation in the FNAB at 1 week (5.061.4 in 200mg/kg/day DFO group vs. 6.360.7 in the untreated group).However, in histology at the end of the follow-up, no clearinhibition of the inflammatory response by DFO could beseen (portal inflammation score 1.960.5 in DFO group vs.2.060.0 in the untreated group) in the uninfected animals.Neither was there a difference in the degree of bile ductdestruction (data not shown). The amount of parenchymalfibrosis was slightly but not significantly decreased (mean
FIGURE 3. Histopathological alterations in the graft 4 weeks after transplantation (mean score from 0 to 36SD; *, P,0.05) (untreated group,100 mg/kg body weight/day DFO group, and 200 mg/kg body weight DFO group, respectively).
MARTELIUS ET AL.December 15, 1999 1757
score 0.360.5 in DFO group vs. 0.860.4 in the untreatedgroup).
In the immunohistological analysis, there were no cleardifferences between the two uninfected groups. The level ofexpression of VCAM-1 in the sinusoids and the TNF-a ex-pression were clearly lower in the uninfected grafts com-pared to CMV-infected grafts. DFO did not decrease theexpression of VCAM-1 or TNF-a in the graft when CMV wasnot present, and neither was there any difference in class II,
ICAM-1, or integrin expression. However, the amount of IL-2receptor-positive cells was slightly decreased by DFO (1463vs. 2065).
DISCUSSION
The present study provides the first results in vivo from atransplantation model to support the suggestion that themetal chelators could be effective in the treatment of CMVinfection and inhibiting its proinflammatory effects. DFO
FIGURE 4. Typical histopathological findings of rat liver allografts with rejection and CMV at 4 weeks (a and b) (paraffin sections;hematoxylin and eosin staining; original magnification, 3200). The untreated grafts (a) display intense portal inflammation and severe bileduct destruction. In grafts treated with DFO (200 mg/kg body weight/day) (b), there is clearly less prominent portal inflammatory infiltrate,and the bile duct cells are well preserved. The expression of VCAM-1 in the sinusoids of the liver grafts (c and d) (frozen sections;immunoperoxidase staining; original magnification, 3400). The sinusoids of the untreated grafts (c) show moderate to intense staining forVCAM-1. In DFO group (200 mg/kg body weight/day) (d), the sinusoids are mainly VCAM-1-negative. TNF-a expression in the structures ofthe liver grafts (e and f) (frozen sections; immunoperoxidase staining; original magnification, 3400). In the untreated group (e), the bile ductsand vascular structures in the portal field are clearly positive in the TNF-a immunostaining, whereas in the DFO group (200 mg/kg bodyweight/day) (f), the staining in the portal field is absent or very mild.
TRANSPLANTATION1758 Vol. 68, No. 11
(200 mg/kg/day) dramatically inhibited RCMV replication inliver allografts in the presence of acute rejection. In addition,the rejection response in the presence of CMV was signifi-cantly decreased both as assessed by transplant aspirationcytology and by histological evaluation. We have previouslydemonstrated that RCMV significantly increases the portalinflammation and bile duct damage in our model of prolongedacute liver allograft rejection (11). In this study, we haveshown that DFO treatment can inhibit these histopatholog-ical changes in the context of RCMV infection.
DFO has previously been shown to possess two kinds ofactivities in vitro that are relevant to our model: antiviraland immunosuppressive (23, 36). In vitro studies have shownthat DFO effectively inhibits CMV replication in human um-bilical vein endothelial cells and also decreases the expres-sion of ICAM-1 and endothelial leukocyte adhesion mole-cule-1 (23). The anti-CMV activity of DFO was completelyabolished by addition of Fe31, and is therefore thought to beassociated with the ability to bind iron (23). The metal ch-elators could possibly act synergistically with drugs inhibit-ing viral DNA synthesis, such as ganciclovir, against CMVbecause of the different mechanisms of action. The immuno-modulatory effect of DFO and another metal chelator dieth-ylenetriamine penta-acetic acid has been shown in vitro inperipheral blood mononuclear cells and endothelial cells (36).There is also some earlier in vivo evidence of the immuno-modulatory effects of DFO. It has been shown to reduceinflammation in a rat arthritis model (37) and inhibit chronicpancreatic islet allograft damage in mice (38). A clear de-crease in the inflammatory response in the grafts infectedwith RCMV was seen in the present study, with a significantdecrease in the total inflammation in transplant aspirationcytology and also significantly milder histological rejectionchanges. However, this clear effect was only seen whenRCMV was present, not in the uninfected grafts. Of specificcell types in the infiltrate, there was a clear decrease in thenumber of LGLs, which is the cell population containing thenatural killer cells (NK-cells), known to be very important inthe defense against CMV. However, most of the effect on theinflammation seen in transplant aspiration cytology was ac-
counted for by the significant decrease in the number ofmacrophages in the graft. Macrophages are important pro-ducers of cytokines, among others, IL-1 and TNF-a.
Many of the proinflammatory effects of CMV, such as theup-regulation of TNF-a, have been reported to be mediated bythe ubiquitous transcription factor NF-kB, a central player inthe human immune response (39). CMV has been reported toincrease NF-kB activity by multiple mechanisms (40, 41). Twoantioxidants, N-acetyl-cysteine (NAC) and pyrrolidine dithio-carbamate, have also been demonstrated to inhibit CMV geneexpression and replication in smooth muscle cells. CMV infec-tion rapidly increased ROI production and NF-kB DNA bindingactivity, and this activity was abolished by NAC (22). The ROI-induced enhancement of CMV replication was suggested to bemainly mediated by the increase in NF-kB activity (22). DFOhas been demonstrated to decrease NF-kB and human immu-nodeficiency virus-1 activation due to oxidative stress in lym-phocytic and promonocytic cell lines (42).
In the present study, the expression of the adhesion mol-ecule VCAM-1 in the sinusoids of the liver grafts was signif-icantly diminished, apparently in quite a specific manner,with no clear differences in ICAM-1 or activation markers. Inthe liver, increased sinusoidal VCAM-1 expression has pre-viously been linked to irreversible and chronic rejection (15).The decrease in macrophage infiltration in our study couldpossibly also be secondary to the decreased VCAM-1 expres-sion. A clear reduction was also seen in the staining of vas-cular structures and bile ducts for TNF-a by DFO. The vas-cular structures and the interlobular bile ducts are thestructures primarily affected in VBDS, the equivalent ofchronic rejection in liver allografts (36). Bile duct destructionwas significantly decreased by DFO in our study. The arte-rial damage was also decreased, although not significantly.Although TNF-a is a soluble cytokine, it was clearly localizedin the cellular structures of the graft. However, in the liver,TNF-a production has previously been found in the endothe-lium of portal and central veins, and bile duct cells have alsobeen demonstrated to produce both TNF-a mRNA and pro-tein in the regenerating liver (44).
TABLE 2. Expression of LFA-1, VLA-4, and the activation markersIL-2 receptor and MHC class II on inflammatory cells in
a Differences between the groups are not significant by t test,a50.05. Table shows untreated and DFO 200 mg/kg b.w. groups at 4weeks, number of positive cells per portal field6SD.
TABLE 3. Expression of ICAM-1, VCAM-1, and MHC class II in the liver grafts at 4 weeks after transplantationa
a Table shows untreated and DFO (200 mg/kg b.w.).b P,0.05, Mann-Whitney U test.
TABLE 4. TNF-a expression in the liver allograftsa
TNF-a
Endothelium Bile ducts Inflammatory cells
RX1CMV 11 11 1–2RX1CMV1DFO 2 to 1b 2 to 1b 1–2
a Table shows untreated and DFO 200 mg/kg b.w./day groups, 4weeks after transplantation, score (endothelium and bile ducts),number of positive inflammatory cells/portal field.
b P,0.05, Mann-Whitney U test.
MARTELIUS ET AL.December 15, 1999 1759
DFO has been reported to decrease IL-2-receptor expression(27) and also to inhibit DNA synthesis via inhibition of ribonu-cleotide reductase (28). These effects could possibly account forthe decrease in inflammation seen in our study but probably notfor the efficient inhibition of CMV replication. In our study, wesaw only minor difference in IL-2-R-expressing lymphocytes inthe portal infiltrate at the end of the follow-up. However, in thisattenuated phase of alloresponse, the level of IL-2-R expressionis not as high as in the early phase of immune activation andinhibition of IL-2-R expression in the earlier stage may be animportant part of the immunomodulatory effect of DFO. On theother hand, the effect on inflammation was only seen whenCMV was present, and the reduction in the amount of macro-phages seemed to be the dominating effect. If decrease of theIL-2-R were the key mechanism, one would expect some effecton the lymphoid blast response.
In conclusion, our in vivo results show that the metalchelator and antioxidant DFO has an inhibitory effect onCMV infection in rat liver allografts and also on the proin-flammatory changes associated with it. Inhibition of oxida-tive stress is one of the possible mechanisms. The possiblerole of CMV in the triggering of acute and chronic allograftrejection makes the iron chelating compounds very interest-ing in the attempts to inhibit and counteract also thesepathophysiological conditions.
Acknowledgments. The authors thank Dr. Roy L. Lobb (BiogenInc., Cambridge, MA), for kindly providing the antibody against ratVCAM-1, and Kaarina Inkinen, Kristina Messina, and StephenVenn for technical assistance and Kari Savelius for animal care.
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ANGIOGENESIS AND ANGIOARCHITECTURE OF TRANSPLANTEDFETAL PORCINE ISLET-LIKE CELL CLUSTERS1
OLLE KORSGREN,2,5 ROLF CHRISTOFFERSON,3,4 AND LEIF JANSSON3
Department of Oncology, Radiology and Clinical Immunology, Department of Medical Cell Biology, andDepartment of Surgery, Uppsala University, Uppsala, Sweden
Normoglycemic, athymic nude mice were implantedwith 3 ml (approximately 250) fetal, porcine islet-likecell clusters under the renal capsule. The angioarchi-tecture of the transplanted islets was studied by mi-crovascular corrosion casts 3 or 52 weeks after im-
plantation. Arterioles were few, and observed mainlyin the older age group. This is likely to be due to thefact that the arterioles were derived from intrarenalblod vessels, i.e., they were not visible on the graftsurface. Within the grafts nests of capillaries, proba-bly supplying a single islet-like cell clusters, could beseen in both groups. Numerous capillary sprouts wereseen within the graft after 3 weeks, and to a slighterextent also after 1 year. Moreover, especially in graftsexamined 3 weeks, but also 52 weeks, after transplan-tation, holes were observed in dilated capillary seg-ments, suggesting that intussusceptive microvasculargrowth occurred in parallel with angiogenesis. A well-developed microvasculature could be observed 52weeks after transplantation, whereas the number ofcapillaries in the implant was less pronounced 3weeks postimplantation. The efferent venules were lo-cated peripherally in the islets and drained immedi-ately into larger veins, derived from capsular veinsclearly seen on the surface of the graft. It is concluded
1 The study was supported by grants from the Swedish MedicalResearch Council (12X-109, 16X-12219, 06P-11813), the SwedishDiabetes Association, the Swedish-American Diabetes Research Pro-gram funded by the Juvenile Diabetes Foundation and the Wallen-berg Foundation, the Åke Wiberg Foundation, the NOVO NordicFund, the Tore Nilsson Foundation, the Magnus Bergvall Founda-tion, the Torsten and Ragnar Soderberg Foundation and the FamilyErnfors Fund.
2 Department of Oncology, Radiology and Clinical Immunology.3 Department of Medical Cell Biology.4 Department of Surgery.5 Address correspondence to: Olle Korsgren, MD, Department of
Department of Oncology, Radiology and Clinical Immunology. Uni-versity Hosptital. S-751 85 Uppsala. Sweden.
