Transplant Immunology 1995; 3: 313-320
Effects of desferrioxamine on human cytomegalovirus replication and expression of HLA antigens and adhesion molecules in human vascular endothelial cells Jindrich Cinatl, Martin Scholz, Bernard Weber, Jaroslav Cinatl, Holger Rabenau, Bernd H Markus, Albrecht Encke and Hans Wilhelm Doerr Institut j?ir Medizinische Virologie, Zentrum der Hygiene, Klinik fiir Allgemeinchirurgie, and Zentrum der Kinderheilkunde, Johann Wolfgang GoetheUniversittit, Frankfurt Received 12 April 1995; accepted for publication 9 May 1995
Abstract: Desferrioxamine (DFO), commonly used in therapy as a chelator of ferric ion in disorders of iron overload, is a potent inhibitor of human cytomegalovirus (HCMV) replication in cultured fibroblast cells. Moreover, DFO has immunomodulatory activity both in vitro and in vivo. We studied DFO effects on HCMV replication in cultured human endothelial cells and on the expression of several cell surface molecules, which mediate interactions of endothelial cells with other cell types in the immune system. The concentrations of DFO required for 50% reduction in the number of endothelial cells expressing HCMV late antigen, ranged for several HCMV strains from 5.2 to 8.8 FM. DFO concentrations ranging from 5 to 40 p.M inhibited cellular DNA synthesis in a dose-dependent manner without any significant effects on the cell viability. DFO at 10 p,M concentration suppressed expression of intercellular adhesion molecule-l (ICAM-1) and endothelial leucocyte adhesion molecule-l (ELAM-l), while it had no significant effect on the expression of vascular cell adhesion molecule-l (VCAM-1). Expression of HLA class I and class II was not influenced by DFO treatment. The results showed that DFO is both effective in inhibition of HCMV replication and expression of ICAM- and ELAM-1 in endothelial cells, a combination that warrants attention to its potential use to prevent HCMV-induced allograft rejection in transplant recipients.
Address for correspondence: Martin Scholz, Transplantations-Immunol. Universitlt, Theodor-Stem-Kai 7, D-60590 Frankfurt a. M., Germany. 0 Edward Arnold 1995
Labor, Klinik fur Allgemeinchirurgie,
Johann Wolfgang Goethe
J Cinatl et al
Human cytomegalovirus (HCMV) was recognized as a major cause of morbidity and mortality among individuals with immunosuppressive disorders, as in bone marrow, cardiac. renal and liver transplant patients and in those with acquired immunodeficiency syndrome.‘-3 In transplant recipients the presence of HCMV infections is observed within the first 6 and it appears to be due to months post-transplantation,4 immunosuppressive regimens used since CMV disease is found only in transplant patients who undergo immunosuppressive treatments.’ For example, inclusion of antithymocyte globulin in immunosuppressive regimen may result in HCMV reactivation, while using cyclosporine A has a minimal effect on reactivation but increases virus replication.h’7 The treatment with FK-506, an immunosuppressant similar in biological action to cyclosporine A, was associated with lower occurrence of HCMV infections.8 However. an optimal immunosuppressive regimen would include a compound with both immunosuppressive and antiviral activity. Desferrioxamine (DFO) is a trihydroxamic acid which can complex with ferric ion to form ferrioxamine,’ and represents the only iron chelator available for clinical studies. Intramuscular and subcutaneous administration of DFO prevents the deleterious effects of iron overload in thalassaemia and sickle cell disease.‘“.” In noniron overload conditions, DFO was used to produce antioxidant effects, antiproliferative effects and antiprotozoal effects and for aluminium chelation.” In addition, several reports describe singular observations in various diseases including immunomodulatory properties and effects on allograft rejection in transplant recipients. A beneficial effect of DFO was reported in two children with graft-versus-host disease (GVHD) after bone marrow transplantation.‘3 The immunomodulatory effects of DFO may be related to its ability to inhibit proliferation of activated lymphocytes.“,” Previously, we reported inhibition of HCMV replication by DFO in cultures of dermal fibroblast cells.lh In the present study we observed effects of DFO on HCMV replication in vascular endothelial cells which are an important site of HCMV infection after transplantation.“.” Moreover, we studied effects of DFO on the expression of several cell surface molecules including intercellular adhesion molecules (ICAM-1). endothelial lymphocyte adhesion molecules (ELAM-l), vascular cell adhesion molecules (VCAM-1) and human leucocyte antigens (HLAs) class I and II which mediate interactions of endothelial cells with other cell types in the immune system.“”
Materials and methods Cells Cultures of human umbilical vein endothelial cells (HUVEC) were established as described previously.” The cells were grown in Iscove’s modified Dulbecco’s medium supplemented with 10% fetal bovine serum (FBS) and 20 ng/ml basic fibroblast growth factor (Boehringer, Mannheim, Germany) in polystyrene culture flasks precoated with human fibron&in (2.5 pg/cm2). The cells were subcultured at 5-day intervals and used at two to six passages in these experiments, Human foreskin fibroblasts (HFF) were grown in Eagle’s Transplant
1995; 3: 313-320
Viruses HCMV laboratory strains including AD169, Towne and Davis were purchased from American Type Culture Collection (Rockville, MA. USA). The viruses were propagated in EMEM supplemented with 4% FBS. Virus titre was determined by examination of immediate early antigen forming units (IEFU) produced in maintenance medium as described previously.” In our hands, this method has a sensitivity comparable to that of plaque forming unit assay. Briefly, medium of infected cultures at fivefold dilutions was incubated with confluent HFF monolayers in 96-well plates. Immunoperoxidase staining of cells using monoclonal antibody directed against 72 kDa immediate early antigen (IEA) of HCMV (DuPont) was performed 24 hours after infection. Stained nuclei were counted microscopically and virus titre was expressed as numbers of IEFU per ml.
Antiviral assay Antiviral activity of DFO was measured by effects on numbers of cells expressing IEA and late antigen (LA). For this purpose HUVEC were infected with different HCMV strains at multiplicity of infection of 10 IEFU per cell. After a 90.minute incubation period HUVEC were washed three times with phosphate-buffered saline and medium without or with different concentrations of DFO was added. Numbers of cells expressing IEA and LA were determined using immunoperoxidase staining 12 hours and 5 days after infection, respectively. To perform immunoperoxidase staining HUVEC were fixed with 1:l mixture of acetone and methanol after trypsinization on cytocentrifuge slides and stained using monoclonal antibody (mAb) directed against 72 kDa IEA and 68 kDa LA both obtained from DuPont (Bad Homburg, Germany). Immunoperoxidase staining was performed as described previously.” The numbers of antigen-positive cells were determined microscopically by examination of at least 500 cells.
Effects on cellular DNA synthesis To measure effects of DFO on DNA synthesis HUVEC were seeded at a density 4 X lo4 cells/cm2 in 96-well plates containing culture medium without DFO. Three days after seeding different concentrations of DFO were added. DNA synthesis was measured in 96.well plates 24 hours after drug addition by quantitative determination of 5-bromo-2’-deoxyuridine (BrdU) incorporated into cellular DNA using ELISA (enzyme-linked immunosorbent assay). A peroxidaselabelled mAb to BrdU and other chemicals were obtained as assay kits from Boehringer (Manheim, Germany). The procedure was performed according to the manufacturer’s instructions. The absorbance of the samples was determined using a multiwell ELISA reader. The results were expressed as percentages of absorbance of untreated control cultures.
Effects on cell viability Cell viability was measured in confluent cultures of HUVEC. For this purpose cells were seeded at a density 4 x IO4 per cm’. Five days after seeding, fresh medium without or with different concentrations of DFO was added. Numbers of viable cells were determined 2 and 5 days after drug addition.
Effects of desferrioxamine
on HCMV replication and expression
Cells were counted using a haemocytometer and cell viability determined by the Trypan blue exclusion method.
of adhesion molecules
(4 AD 169
Effects on expression of cell surface molecules Quantitative analysis of the expression of cell surface molecules was performed by fluorometric measurements as described previously.24X25 Confluent cultures of HUVEC were incubated for 2 days in medium without or with 10 PM DFO. The expression of ICAM-1, ELAM-1 and VCAM-1 was stimulated by treatment of HUVEC for 8-24 hours with 10 U/ml of interleukin-1 (IL-l) (Seromed, Berlin, Germany) or a combination of IL-l/TNF-a (tumour necrosis factor alpha; 500 U/ml), respectively while the expression of HLA class I and HLA class II was stimulated by treatment for 72 hours with 250 U/ml of IFN-y (interferon-gamma; Sigma, Germany). The adhesion molecules ICAM-1, ELAM-1 and VCAM-1 were marked by mouse mAb (clone BBIG-11, BBIG-E6 or BBIG-Vl, respectively; Bierman, Bad Nauheim, Germany). HLA class I and HLA class II (HLA-DR) were marked with mAb W6/32 (Dakopatts) and L243 (Becton Dickinson), respectively (both Dakopatts). HUVEC stained with primary antibodies were treated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Zymed; Ade Laborbedarf, Munich, Germany) for 1 hour at 4°C and fluorescence intensity was evaluated using a Cytofluor (Cytofluor 2300 system; Millipore Eschborn, Germany).
u) = Q) 0
DFO concentration (uM)
5oT (B) m
DFO concentration (V M)
Effects of DFO on HCMV antigen expression To show whether DFO may influence synthesis of HCMV proteins before virus replication occurs, its effects on number of cells expressing IEA were measured. As shown in Figure lA, DFO concentrations ranging from 2.5 to 20 PM had no significant effects on number of cells expressing IEA 24 hours after infection. In contrast, these DFO concentrations inhibited numbers of cells expressing LA in a dose-dependent manner as measured 5 days after infection (Figure 1B). Concentrations of DFO that inhibited 50% of cells expressing LA were 5.2 PM for AD169 strain, 5.9 FM for Towne strain and 8.8 p,M for Davis strain. Effects of DFO on cellular DNA synthesis and cell viability DFO at concentrations ranging from 2.5 to 40 FM inhibited cellular DNA synthesis in a dose-dependent manner (Figure 2). DNA synthesis was completely inhibited after 24 hours of treatment with 40 FM DFO. The DFO concentration required to inhibit 50% of cellular DNA synthesis was 10 r*.M The effects of DFO on cell viability were measured in confluent HUVEC whicn were used for antiviral assays. In the confluent HUVEC cultures cell growth was strongly limited; the cell numbers did not differ significantly between untreated cultures and cultures treated for 2 days with DFO at concentrations ranging from 2.5 to 20 FM (Table 1). In Transplant hmunology
Statistical analysis Data groups were considered to be significantly different when p was lower than 0.05 as determined by the Wilcoxon signed rank assay.
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Figure 1 Effect of DFO on number of HUVEC expressing HCMV IEA (A) and LA (B). DFO at different concentrations was added to a maintenance medium after infection with AD169, Towne and Davis strain. Results are described as the mean 2 SD (bars) of three independent experiments.
I 01 1
DFO concentration (V M) Figure 2 Effect of DFO on cellular DNA synthesis in HUVEC. BrdU incorporation in DNA was measured after 24 hours of treatment.
Each value represents the mean + SD (bars) of triplicate culture of two independent experiments.
J Cinatl et al.
Table 1 Effect of DFO on number of viable cells in confluent HUVEC cultures DFO (PM)
Viable cells X lo-’ per cm’
Cell number X lo-’ per cm’ 48 hours
6.3 + 0.72
7.6 + 0.93”
5.9 k 0.64
7.1 k 0.65 (94) 7.4 + 0.71 (95) 6.3 2 0.53 (92) 5.9 t 0.54 (92) 5.8 5 0.49 (91) 5.8 + 0.39 (93)
6.4 k 0.58
7.8 k 0.61
(93)‘ 5.9 + 0.53
6.2 + 0.64
6.9 + 0.72
(92) 5.8 -t 0.49
6.4 k 0.74
6.3 k 0.48
(93) 5.9 i- 0.66
6.2 2 0.61
6.4 -t 0.61
(92) 5.8 k 0.62
6.3 2 0.69
6.2 k 0.49
(94) 5.7 2 0.54 (91)
a Numbers of viable cells were determined in confluent HUVEC 48 and 120 hours after DFO addition. ’ Each value reuresents the mean ? SD of triplicate cultures from two independent experiments. ’ Values in pare’ntheses are percentage of viable cells. cultures treated for 5 days with 20 FM DFO, up to 20% lower cell numbers were found than in untreated cultures (Table 1). However, cell viability was in all cases similar both in DFOtreated and untreated confluent HUVEC cultures (Table 1). These results showed that DFO exerts its cytostatic effects without any significant cytotoxicity.
Effects of DFO on the surface expression of endothelial HLA Relative fluorescence intensity values for constitutively expressed HLA class I (-A, -B, -C; mAb W6132) was determined in triplicated assays. The mean values of the measured fluorescence units (MFU; SD < 3%) in control cultures (without IFN-7 treatment) ranged between 1379 and 1398 MFU in five independent experiments. Incubation with IFN-y (2.50 LJiml) for 3 days enhanced HLA class I related fluorescence intensity by 3944% (SD ~5%). Resting HUVEC normally do not express HLA class II on their surface membrane. Mean fluorescence intensity for HLA class II (HLA-DR: mAb L243) in untreated controls (SD <5%) ranged between 1394 and 1427 MFU (n = 5). MFU values for IFN-7 induced de novo expression of HLA-DR were enhanced by 26-33% (SD <3%) compared to the control. Before addition of IFN-1/ to the cell cultures HUVEC were preincubated with 10 PM DFO for 2 days. DFO neither affected significantly the constitutive HLA class I expression on untreated control cells nor the IFN-?/ mediated expression (n = 5) of HLA class I and class II. Representative data on the effects of DFO are provided for HLA class I (Figure 3A) and HLA class II (Figure 3B).
Effects of DFO on the surface expression of cell adhesion molecules Resting endothelial cells constitutively express ICAMbut not ELAM-1 and VCAM-1 on their surface membrane. Compared to the MFU values of cells incubated without primary mAbs (FITC background values) the MFU values for the basal expression of ICAMin control cultures (without IL-l treatment) were 23-25% higher (range 2300-2600 MFU; n = 12). MFU values for ELAM-1 (range 1700-2100 MFU; Iz = 12) VCAM-1 and (range Transplant Immunology
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1600-2100 MFU; n = 10) in control cultures did not differ from the FITC background values. Incubation with 10 FM DFO did not induce de nova expression of either adhesion molecule but interestingly reduced basal ICAMexpression by 4650% (Figure 4A). When cells were stimulated with 10 U/ml recombinant IL-1 for 12 hours ICAMspecific MFU values were increased by 6&103% (n = 12). DFO reduced the IL-l-mediated upregulation of ICAMin 10 out of 12 experiments by 4540%. As for ELAM-1 expression (Figure 4B), IL-1 treatment (10 U/ml for 8 hours) induced de novo expression and thus enhanced mAb specific MFU values by 39-70% (n = 12) compared to the control (FITC alone). Furthermore, DFO reduced IL-l-mediated ELAM-1 expression by 27-5 1% in 11 out of 12 independent experiments. The intra-assay SD of triplicates for ICAMand ELAM-1 was always ~5%. In order to induce de novo expression of VCAM-1 HUVEC were stimulated with IL-1 and TNF-m for 8 hours. TNF-a/IL-l enhanced VCAM-1 expression (Figure 4C) by l&21% (n = 10). This upregulated expression was not impaired by preincubation with 10 FM DFO (SD <4%).
Discussion HCMV infection and disease still cause significant morbidity and mortality in transplant recipients.*(’ HCMV infection can be effectively prevented in seronegative graft recipients by providing them with HCMV-seronegative organs and screened blood products.6.27 Unfortunately this approach is limited due to a high HCMV seroprevalence (5&70%) in a general population. Therefore, several approaches to prophylaxis and treatment of HCMV infections have been developed. The treatment strategies include using antiviral substances such as acyclovir, ganciclovir and foscarnet.2G”0 Moreover, HCMV immunoglobulins showed protection from disease when given prophylactically, and synergism with ganciclovir when given as therapy for active disease.2”30 Since HCMV diseases are related to immunosuppression induced by different immunosuppressive regimens,6.7 improvement of immunosuppressive therapy will also reduce the risk of serious disease. For example, using any immunosuppressive
Effects of desferrioxamine
3 E 3
5000 4500 4000
on HCMV replication and expression
of adhesion molecules
Figure 3 Relative mean fluorescence intensity units (MFU) as determined by means of Cytofluor 2300 analysis is depicted for HLA class I (A) and HLA class II (B) expression in HUVEC monolayers. DFO neither modified basal HLA class I (-A, -B, -C) expression (control) nor induced expression of HLA class II (DR). Moreover, EN--y mediated expression was not impaired by DFO. Data are provided as the mean 5 SD of one representative triplicated experiment. FITC-labelled cells without primary mAbs were included to determine the background fluorescence in the
sandwich immunofluorescence staining. agent also exerting antiviral activity may help to manage HCMV disease in transplant recipients. The results of the present study suggest that DFO may fulfil requirements for such a drug. Previously we showed that DFO is a potent inhibitor of HCMV replication in cultured fibroblast cells.16 Inhibitory effects of DFO on HCMV replication were completely prevented by co-incubation with stoichiometric amounts of Fe3+. These findings demonstrated that DFO exerts its primary effect by chelating ferric ion and subsequently inhibiting HCMV replication. However, the mechanism by which DFO exerts its antiviral activity is not clear. HCMV infection is characterized by severalfold increases in the intracellular deoxynucleotides31 which may be required for efficient viral replication. DFO is a potent inhibitor of the de novo synthesis of deoxyribonucleotides due to the inhibition of cellular ribonucleotide reductase which requires iron for its activity.32,33Thus, inhibition of the build-up of the deoxynucleotide pool resulting from inhibition of cellular ribonucleotide reductase may explain inhibition of HCMV replication. On the other hand, mechanisms independent of ribonucleotide reductase could account for antiviral effects. In certain cellular models DFO and other iron chelators have been found to inhibit DNA synthesis by inhibition of ribonucleotide and deoxyribonucleotide incorporation into nucleic acids.34 Such DFO effects may be deleterious only to the rapidly replicating viral DNA and not to the less replicating cellular DNA. The antiviral activity of DFO could also result from its antioxidant effects. Because iron can generate highly reactive radicals, it can initiate or maintain oxidative stress reactions.l* Recently, we have shown that induction of oxidative stress by Transplant Immunology
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buthionine sulfoximine increases HCMV replication in cultured cells and that treatment of infected cells with antioxidants such as ascorbic acid reduces production of infectious virus.3536 Interest in the effects of DFO on immunity stems from both experimental and clinical observations. Bowem et al.37 showed that DFO inhibits the autoimmune neuropathological process in experimental allergic encephalomyelitis in Lewis/ Jc rats. DFO also inhibited chronic pancreatic islet allograft damage in CBA/J recipient mice.3s Weinberg et nLi3 reported a beneficial effect of DFO (SOmg/kg/day for two S-day courses) in two children with GVHD after bone marrow transplantation. The immunomodulatory activity of DFO could stem both from its ability to inhibit free radical production by inflammatory cells and from a direct effect on lymphocyte functions. Several in vitro studies reported inhibitory effects of DFO on the proliferative response of lymphocytes induced by concanavalin A and pokeweek mitogen.39’40 Two inhibitory mechanisms of action are postulated, including an effect mediated by iron chelation and direct inhibition of IL-2 receptors (CD25) on lymphocytes which is not related to iron.14.‘5 Inhibition of CD25 expression was achieved by treatment of T lymphocytes with DFO at a concentration of 300 FM. In the present study we demonstrated that DFO at a pharmacologically attainable concentration of 10 p.M decreases the expression of a member of the superimmunoglobulin family ICAM- and E-selectin ELAM-1 in HUVEC. These findings suggest a novel mechanism which could account for the immunomodulatory activity of the drug observed in vivo. In the process of graft rejection leucocytes emigrate from blood to the site of inflammation resulting in a massive
J Cinatl et al.
infiltration of responders leucocytes into the donor graft.4’.J2 Leucocyte entry into tissue is controlled by the dynamic interaction between adhesion molecules expressed by leucocytes and the endothelium.‘9.20 A number of experimental studies have shown that in vivo treatment with monoclonal antibodies to ICAMand to leucocyte function associated antigen-l (LFA-1; counter-receptor of ICAMon leucocytes) can prevent acute allograft rejection.4346 Zeng et aL4’ demonstrated that pretreatment of pancreatic islet xenografts with mAb to ICAMprolonged graft survival in diabetic B6 mice. Blakely et aL4’ found that downregulation of ELAM-1 induced by treatment with retinoic acid results in prolonged cardiac xenograft survival. Clinical trials are currently under way using anti-ICAMantibodies to inhibit kidney allograft rejection.49 It is conceivable that downmodulation of ICAMand ELAM-1 resulting from DFO treatment could contribute to the prevention of graft rejection after transplantation. It should be also noted that adhesion molecules such as ICAM1 and ELAM-1 may be upregulated in endothelium due to HCMV infection.24.50.s’ Thus, inhibition of virus replication by DFO would additionally account for decreased expression of the adhesion molecules in the endothelium. In summary, the present results demonstrate that nontoxic concentrations of DFO are both effective in inhibition of
HCMV replication and expression of ICAMand ELAM-1 in cultured HUVEC. These in vitro concentrations may be easily achievable in vivo with few toxic adverse effects which are mostly reversible after discontinuation of the drug.i2 For example, subcutaneous infusion of DFO 100 mg/kg over 24 hours demonstrated steady-state DFO levels of 8-20 ~_LM.’ Therefore, DFO deserves further attention as a drug for the prevention of HCMV-induced allograft rejection in transplant recipients. Moreover, it would be of interest to show whether other iron chelators may have antiviral and immunomodulatory activities similar to DFO.
Acknowledgements This research was supported in part by the organization ‘Verein fur krebskranke Kinder, Frankfurt/m. e. V.’ We thank Miss Gesa Meincke, Miss Gabriele Steigmann and Miss Edith Harbich for excellent technical assistance.
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FITC + DFO
FITC + OF0
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Figure 4 The same method as described in Figure 3 was utilized to measure the expression of ICAM(A), ELAM-1 (B) and VCAM-1 (C) in HUVEC monolayers. DFO reduced basal expression of ICAM(p < 0.01) as well as IL-1 induced upregulation of ICAM(p < 0.005) and ELAM-1 (p < 0.05). TNF-(U induced de now expression of VCAM-1 was not impaired. Data are provided as the mean -+ SD of one representative triplicated experiment.
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Effects of cfesferrioxamine
on HCMV replication
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