(p.b.e.) — CYTO — 11/2 — ISSUE — (866054 )—MS 0388

Article No. cyto.1998.0401, available online at http://www.idealibrary.com.on

REDUCTION OF TUMOUR NECROSIS FACTOR aEXPRESSION AND SIGNALLING IN

PERIPHERAL BLOOD MONONUCLEAR CELLSFROM PATIENTS WITH THALASSAEMIA ORSICKLE CELL ANAEMIA UPON TREATMENT

WITH DESFERRIOXAMINE

A. Bellocq,1 D. Israel-Biet,2 J. Cadranel,3 J. Perez,1 B. Fouqueray,1

A. Kanfer,5 R. Girot,4 L. Baud1

Recent evidence indicates that the rate of progression of the HIV-1 disease is significantlyreduced in thalassaemia major patients upon treatment with high doses of desferrioxamine(DFX). The authors have previously demonstrated that in vitro exposure of mononuclear cellsto DFX decreases the bioavailability of tumour necrosis factor a (TNF-a) which has astimulatory effect on HIV-1 replication. In this study, therefore, TNF-a bioavailability frommononuclear cells isolated from 10 patients with thalassaemia or sickle cell anaemia given DFXas compared to 10 untreated subjects has been evaluated. Evidence is presented showing thatDFX treatment reduces TNF-a bioavailability (PQ 0.05) by inhibiting its steady state(PQ 0.05) and by enhancing its inactivation through binding to soluble TNF-a receptor typeII (PQ 0.05). We also show that DFX treatment limits the in vivo activation of NF-kB, atranscription factor involved in both TNF-a gene transcription and TNF-a signalling(PQ 0.005). We conclude that TNF-a bioavailability and signalling are impaired in patientsupon DFX treatment. This mechanism may contribute to delayed progression of the HIV-1infection in vivo.

7 1999 Academic Press

Iron chelation with desferrioxamine mesylate(deferroxamine, DFX) is an important treatment foriron overload from multiple transfusions in patientswith thalassaemia.1 Recent studies demonstrated thatthe rate of progression of the HIV-1 disease inthalassaemia major patients was significantly reducedin those receiving higher doses of this drug.2 However,the mechanisms whereby DFX reduces the progressionof HIV disease were not evaluated in that study. Therisk of the progression of HIV disease is stronglyassociated with the plasma level of HIV RNA.3 Thus,this latency is possibly related to direct inhibition ofHIV-1 replication. In vitro, DFX inhibits HIV-I

replication as evidenced by the reduced expression ofp24 antigen in infected cells,4,5 although this has beendisputed.6 This inhibition is associated with a decreasein the activation of nuclear factor kB (NF-kB) whichparticipates in the induction of HIV-I long terminalrepeat (LTR).7 Alternatively, DFX-induced latencymight be due to indirect inhibition of HIV-I replicationthrough reduction in tumour necrosis factor-alpha(TNF-a) bioavailability. This reasoning is based on theobservation that: (1) TNF-a enhances HIV replicationin infected cells in vitro, as well as in isolated peripheralblood mononuclear cells (PBMC) from HIV-infectedsubjects;7–9 and (2) in vitro exposure of PBMC to DFXboth decreases the release of TNF-a and increases theshedding of soluble TNF-a receptors.10 The latterprocess might further reduce TNF-a bioactivity, asreleased soluble receptors bind to TNF-a and preventits binding to cellular TNF-a receptors.11 However, nodata are available to know whether DFX blood levelstheoretically reached in vivo after subcutaneousinfusion of the drug are sufficient to promote such aresponse.

In the current studies, therefore, we haveevaluated TNF-a bioavailability in culture super-natants of PBMC isolated from patients with

From the 1Service d’Explorations Fonctionnelles and INSERMU489, Hox pital Tenon, 2Laboratoire d’Immunologie Pulmonaire,Hox pital Laennec, 3Service de Pneumologie et de ReanimationRespiratoire, Hox pital Tenon, 4Service d’Hematologie-Immunolo-gie, Hox pital Tenon, 5Hox pital de Jour Pluridisciplinaire, Hox pitalTenon

Correspondence to: Laurent Baud, INSERM U489, Hox pital Tenon,4 rue de la Chine, 75020 Paris, France

Received 15 December 1997; received in revised form 12 June 1998;accepted for publication 1 July 1998

7 1999 Academic Press1043–4666/99/020168+05 $30.00/0

KEY WORDS: HIV-1 replication/ iron chelation/ transcriptionfactor NF-kB/ tumour necrosis factor a

CYTOKINE, Vol. 11, No. 2 (February), 1999: pp 168–172168

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169DFX reduces TNF-a expression and signalling /

Figure 1. Distribution of immunoreactive TNF-a values in culturesupernatants of PBMC from DFX-treated and -untreated subjects.

The median value for each of the two groups is represented by ahorizontal bar.

significantly reduced in culture supernatants of PBMCfrom DFX-treated patients (0.0872 0.015) as com-pared with untreated subjects (0.2512 0.043,PQ 0.005). Additionally, a significant correlation wasobserved between the levels of biologically activeTNF-a and the ratios TNF-a/sTNFRII (P=0.001).

Figure 2. Distribution of immunoreactive sTNFRI values (A),sTNTFRII values (B) and TNF-a/sTNFRII ratios (C) in culturesupernatants of PBMC from DFX-treated and -untreated subjects.

The median value for each of the two groups is represented by ahorizontal bar.

thalassaemia or sickle cell anaemia given DFX ascompared to untreated subjects.

RESULTS

DFX administration reduces TNF-abioavailability

Immunoreactive TNF-a was measurable in super-natants of PBMC after 24 h culture in the absence ofexogenous stimulus (Fig. 1). Its level was significantlyreduced in culture supernatants of PBMC fromDFX-treated patients (351.52 45.9 pg/ml) as com-pared with untreated subjects (791.12 134.1 pg/ml,PQ 0.05). To evaluate further the bioavailability ofTNF-a, the culture supernatants of PBMC wereassayed for TNF-a cytotoxic activity. Its level wasreduced in a similar fashion in DFX-treated patients(28.62 5.5 pg/ml) as compared with untreated subjects(89.92 23.3 pg/ml, PQ 0.05). Reduction in biologi-cally active TNF-a could result from a decrease inTNF-a release and/or an increase in TNF-a inacti-vation by its binding to soluble forms of TNF-areceptors. To address this issue, levels of solubleTNF-a receptor I (sTNFRI) and soluble TNF-areceptor II (sTNFRII) were measured in culturesupernatants of PBMC (Fig. 2). sTNFRII level wassignificantly increased in culture supernatants ofPBMC from DFX-treated patients (47662 662 pg/ml)as compared with untreated subjects (32432 380 pg/ml, PQ 0.05), although sTNFRI level was not altered.The imbalance between TNF-a and sTNFRII wouldexplain the reduced bioactivity of TNF-a upon DFXtreatment. Indeed, the ratio TNF-a/sTNFRII was

Specificcomplexes

Free probe

(I) (II) (III)

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TABLE 1. Serum levels of TNF-a and sTNFR

DFX-treated subjects Untreated subjects

TNF-a ND* NDsTNFRI 1506.32 218.4† 1113.82 222.4sTNFRII 3812.22 404.4 2857.02 164.4

Values are expressed as pg/ml.*ND: not detectable.†Mean2 SE of values obtained from 10 subjects.

DISCUSSION

In this study, we have demonstrated that DFXtreatment for iron overload in patients with thalas-saemia or sickle cell anaemia reduces TNF-abioavailability from circulating mononuclear cells.Whether the differences in the levels of TNF-a that weobserved could be related to factors other than DFXtreatment, (e.g. background pathology) is unlikely.Indeed, identical variability according to DFXtreatment was found among individuals with the samepathology, including two DFX-treated and threeuntreated patients with sickle cell anaemia (data notshown). In addition, these results are consistent withprevious experimental data showing that DFX limitsTNF-a synthesis in rat both in vivo14 and in vitro incultured macrophages15 and mesangial cells.16

The molecular mechanisms underlying the reducedrelease of immunoreactive TNF-a from PBMC ofDFX-treated patients are not yet clear. One possiblemechanism is that DFX decreases TNF-a genetranscription and, hence, TNF-a synthesis. Alterna-tively, DFX might interfere with the cleavage ofmembrane-associated TNF-a (26-kDa molecule), theprecursor of the secretory component of TNF-a(17-kDa molecule). This hypothesis is based on ourprevious finding that DFX treatment inhibits TNF-aprecursor processing in cultured mesangial cells16 aswell as on more recent biochemical characterization ofenzymes involved in TNF-a precursor cleavage.17 Theseenzymes are metalloproteinases, induction of whichrequires oxidative stress.18 Thus, DFX, an iron chelatorpreventing the synthesis of hydroxyl radical (OH

.) via

the Haber–Weiss reaction, would prevent metallo-proteinase-dependent cleavage of TNF-a precursor.

The bioavailability of TNF-a is controlled at thelevel of TNF-a synthesis and TNF-a inactivation bybinding to soluble receptors. In this study, we haveshown that DFX treatment significantly increases therelease of sTNFRII from PBMC, whereas it does notaffect that of sTNFRI. These data are in agreementwith previous studies demonstrating that in vitroexposure of PBMC to DFX increases sTNFRIIrelease.10 Interestingly, the ratio TNF-a/sTNFRII, thatparalleled TNF-a biologic activity, was significantlyreduced in PBMC supernatant from DFX-treatedpatients. Thus, the imbalance between TNF-a andsTNFRII explains the decrease in TNFa bioactivityfollowing DFX treatment.

A second aspect of this study investigated thepossible inhibitory role of DFX treatment on NF-kBactivation in circulating monocytes. We have shownthat DFX treatment significantly decreases NF-kBactivation. In the cytosol, NF-kB is in an inactive formbound to an inhibitory molecule, Ik-Ba, and cannotbind DNA. The major pathway used to activate NF-kB

The circulating levels of TNF-a and sTNFR werealso determined (Table 1). Both immunoreactive andbiologically active TNF-a were undetectable in serum.Again, although not statistically different, thesTNFRII level was increased in serum from DFX-treated patients as compared with untreated subjects.

DFX administration reduces constitutive NF-kBactivity in PBMC

In light of reports demonstrating that TNF-astimulates HIV-1 enhancer by activating NF-kB,12,13 weattempted to determine whether DFX treatment mightaffect NF-kB activation in addition to reducing TNF-aexpression. Nuclei of PBMC isolated from untreatedsubjects contained NF-kB, as shown by retardedmigration of the labelled NF-kB oligonucleotide inelectrophoretic mobility-shift assay (EMSA) exper-iments (Fig. 3). The specificity of bands was assessedin competition experiments. Two main bands disap-peared in the presence of the unlabelled NF-kBoligonucleotide. Scanning densitometry indicated thatthese two bands were significantly reduced in PBMCnuclei from DFX-treated patients (4242 144 arbitraryscanning units) as compared with untreated subjects(20402 181 arbitrary scanning units, PQ 0.005).

Figure 3. EMSA analysis for NF-kB activity in PBMC from sevenuntreated subjects (I) and seven DFX-treated subjects (II).

Nuclear extracts (5 mg) were incubated with 32P-labelled NF-kBoligonucleotide. A 500-fold excess of unlabelled NF-kB oligonucle-otide was added for competitive reaction (III). Arrowheads denotethe specific NF-kB-DNA complexes.

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171DFX reduces TNF-a expression and signalling /

involves the phosphorylation of Ik-Ba at its regulatoryN-terminus on serines 32 and 36. Antioxidant DFXpotentially limits this proteolysis of Ik-Ba bypreventing its phosphorylation with reactive oxygenspecies-driven enzymes.19 The second pathway used toactivate NF-kB involves the phosphorylation of Ik-Baon tyrosine 42.19 This event does not lead todegradation of the molecule, but, rather, targets Ik-Bafor binding to SH2 domain-containing protein thateventually dissociates the NF-kB–IkBa complex. Sincereactive oxygen species induce tyrosine phosphoryl-ation,20 antioxidant DFX potentially blunts thispathway as well.

There was a clear association of low TNF-abioavailability and reduced NF-kB activation inPBMC isolated from DFX-treated patients. We do not,however, know whether the decrease in TNF-aavailability is responsible for the lack of NF-kBactivation or whether, conversely, the lack of NF-kBactivation precludes NF-kB-driven transcription ofTNF-a gene. Whatever the sequence of events, bothlimited TNF-a availability and reduced NF-kBactivation could help in restricting HIV-1 replication.Indeed, in vivo replication of HIV-1 is foundpredominantly in mononuclear cells activated bycytokines in response to HIV-I itself or to opportunisticinfections.7 These cytokines include granulocyte-macrophage colony-stimulating factor (GM-CSF),IL-2, IL-3, IL-6, IL-12 and particularly TNF-a.21

Although TNF-a affects HIV-1 replication throughNF-kB-induced transcription, other cytokines havepredominantly post-transcriptional effects.7

In summary, our data suggest for the first timethat DFX treatment for iron overload blunts the abilityof circulating monocytes to produce bioactive TNF-aand to activate NF-kB. Whether this limitation isresponsible for delaying HIV replication in vivo2

remains to be determined.

MATERIALS AND METHODS

Subjects

Ten HIV-seronegative subjects given DFX (8 patientswith thalassaemia major and 2 patients with sickle cellanaemia: 5 men and 5 women; age 18–33 years) and tenHIV-seronegative untreated subjects (7 healthy laboratoryworkers and 3 patients with sickle cell anaemia: 5 men and5 women; age 22–56 years) entered this study after theirinformed consent had been obtained. DFX was infusedsubcutaneously in an average dose of 39 2 12 mg/kg per dayfor periods longer than 6 years. This administration wasstopped at least 18 h before blood samples were drawn.

Venous blood (5 ml) and heparinized venous blood(25–50 ml) were taken for serum measurements and PBMCisolation, respectively. After centrifugation (100× g for15 min) to remove the platelets, heparinized blood sample

was diluted (1/1) in calcium-free HBSS (Flow Laboratories,Irvine, UK) supplemented with 0.08% EDTA, and PBMCwere isolated by density centrifugation on lymphopaque(Nyegaard, Oslo, Norway).22 They were resuspended inculture medium consisting of RPMI 1640 (Flow Labora-tories) buffered with 20 mM HEPES to pH 7.4 andsupplemented with 10% fetal calf serum (FCS), 100 U/mlpenicillin, 100 mg/ml streptomycin and 2 mM -glutamine,counted after staining with acridine orange, and adjusted toa concentration of 2.5×106 cells/ml.

Measurement of TNF-a and soluble TNF-a receptor levels inPBMC supernatant and serum

Mononuclear cells were cultured for 24 h in humidified95% air, 5% CO2 atmosphere. Levels of TNF-a, sTNFRIand sTNFRII were determined in culture supernatant as wellas in serum by specific immunoassays (R and D Systems,Minnepolis, MN) according to the instructions of thesuppliers. TNF-a biological activity was determined by theassay for cytotoxicity using the murine cell line L929.10

Nuclear extracts and EMSA

Nuclear extracts were prepared from mononuclear cellscultured for 24 h and EMSA was performed using themethod previously described.23

Statistical analysis

A non-parametric test (Mann–Whitney rank sum) wasused to assess differences between DFX-treated and-untreated subjects. Correlations of interest were evaluatedwith Spearman correlation analysis. Statistical significancewas accepted for PQ 0.05. Results are expressed as themean2 SE.

Acknowledgements

This work was supported by the Institut nationalde la Sante et de la Recherche Medicale, the Faculte deMedecine Saint-Antoine, and by grant from Fondationpour la Recherche Medicale. The authors thank MrsN. Knobloch and N. Ourtirane for secretarialassistance.

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