Cellular Microbiology (2004)

6

(9), 839–848 doi:10.1111/j.1462-5822.2004.00406.x

© 2004 Blackwell Publishing Ltd

Blackwell Science, LtdOxford, UKCMICellular Microbiology 1462-5814Blackwell Publishing Ltd, 20046

9839848

Original Article

P. Pino et al.The CD23/NO pathway regulates PRBCs adhesion

Received 13 September, 2003; revised 4 March, 2004; accepted 19March, 2004. *For correspondence. E-mail mazier@ext.jussieu.fr;Tel. (+33) (0)1 40 77 81 08; Fax (+33) (0)1 45 83 88 58. †Both havecontributed equally to the manuscript; ‡B. Dugas is CSO of IsocellNutra SAS, 75015 Paris, France.

Induction of the CD23/nitric oxide pathway in endothelial cells downregulates ICAM-1 expression and decreases cytoadherence of

Plasmodium falciparum

-infected erythrocytes

Paco Pino,

1†

Ioannis Vouldoukis,

1†

Nathalie Dugas,

2

Marc Conti,

1

Josiane Nitcheu,

1

Boubacar Traore,

1

Martin Danis,

1

Bernard Dugas

1‡

and Dominique Mazier

1

*

1

INSERM U511, Immunobiologie Cellulaire et Moléculaire des Infections Parasitaires, CHU Pitié-Salpêtrière Paris VI, 75013 Paris, France.

2

VigiCell sarl, 94240 Kremlin Bicêtre, France.

Summary

Cytoadherence of parasitized red blood cells (PRBCs)to postcapillary venules and cytokine production areclearly involved in the pathogenesis of cerebralmalaria. Nitric oxide and TNF-

aaaa

have been proposedas major effector molecules both in protective andphysiopathological processes during malaria infec-tions. Nitric oxide production has been shown to beinduced by engagement of CD23 antigen. This studyaimed to investigate the potential role of the CD23/nitric oxide pathway in the control of the cytoadher-ence of PRBCs on human endothelial cells. We dem-onstrate that normal human lung endothelial cells(HLECs) are able to express the low affinity receptorfor IgE (Fc

ŒŒŒŒ

RII/CD23), following cell incubation withinterleukin 4 or PRBCs. Ligation of the CD23 antigenby a specific anti-CD23 monoclonal antibody at thecell surface of HLECs was found to induce iNOS mRNAand protein expression, NO release and

P. falciparum

killing. In addition, the specific CD23-engagement onthese cells also induced a significant decrease inICAM-1 expression, an adhesion molecule implicatedin PRBCs cytoadherence. These data not onlydescribed for the first time the expression of a CD23antigen at the cell surface of endothelial cells but alsosuggest a possible new regulatory mechanisms viathe CD23/NO pathway during malaria infection.

Introduction

The CD23 antigen is known to be expressed by a largevariety of haematopoietic cells (Kijimoto-Ochiai, 2002).This antigen has been shown to be implicated in theinitiation and the development of parasitic diseasesincluding malaria (Vouldoukis

et al

., 1995a; Velupillai

et al

., 1997; Perlmann

et al

., 1999; Nacher

et al

., 2002).Pharmacologically, the

engagement

of CD23 antigen atthe surface of immunocompetent cells has been shown toinduce the production of pro-inflammatory cytokines, nitricoxide (NO) and free radicals (Paul-Eugene

et al

., 1995b;Yamaoka and Kolb, 1995; Bayon

et al

., 1998; Kolb

et al

.,2001). In this context, we found that the cell surfaceexpression of the CD23b antigen at the surface of normalhuman endothelial cells was obtained after IL-4+ PRBCstimulation and that the triggering of the CD23 antigen byanti-CD23 monoclonal antibodies stimulated the produc-tion of NO after induction of the inducible form of NOsynthase (iNOS).

Malaria kills an estimated 1.5–2.7 million people everyyear throughout the world (WHO, 1997). The pathogene-sis of cerebral malaria, a leading cause of mortalityresulting from

Plasmodium falciparum

infections, remainspartially understood. However, factors such as anincreased secretion of various pro inflammatory cytok-ines, in particular TNF-

a

, are clearly involved (de Kos-sodo and Grau, 1993; Beeson

et al

., 1999). Furthermore,cytoadherence of parasitized red blood cells (PRBCs), topostcapillary venules, a phenomenon caused by cell–celladhesion via ICAM-1, VCAM-1, E-selectin, PECAM-1/CD31, chondroitin-4-sulphate, CD36, thrombospondinand hyaluronic acid (Craig and Scherf, 2001), is also cen-tral to this pathology. Both mechanisms are inter relatedas the expression of most adhesion molecules is poten-tially modulated by cytokines induced during severemalaria. We recently described that PRBCs adhesioninduce apoptosis in endothelial cells, strengthening thekey function of this phenomenon in cerebral malaria(Pino

et al

., 2003). The proposed critical role for nitricoxide (NO) in the physiopathology of human malaria,remains controversial. In some studies, increased NOderivatives were found associated with coma (Al Yaman

840

P. Pino

et al.

© 2004 Blackwell Publishing Ltd,

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,

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, 839–848

et al

., 1996) and poor outcome (Weiss

et al

., 1998),whereas other reports showed no correlation with dis-ease progression (Agbenyega

et al

., 1997; Taylor

et al

.,1998; Levesque

et al

., 1999). In contrast, other reportssuggested a protective role for NO (Dondorp

et al

., 1998;Perkins

et al

., 1999;Chiwakata

et al

., 2000). Moreover,NO was found to downregulate the expression of ICAM-1and VCAM-1 on endothelial cells (Vidal

et al

., 1992;Takahashi

et al

., 1996), leading to a hypothesis that NOplays a protective role by decreasing the adherence ofinfected erythrocytes (Kremsner

et al

., 1996). The contra-dictory potential effects of NO on the clinical course of

P.falciparum

infection may be explained by the methodsused to quantify or to detect

L

-arginine/NO pathway dur-ing malaria infection. NO is very unstable and reacts rap-idly with various molecular targets, thus measurement ofreactive nitrogen intermediates in biological fluids suchas plasma, cerebrospinal fluid and urine, correlatespoorly with local

in situ

production of NO at the microvascular level.

In this study, we explored the role of nitric oxide fromendothelial cells on

P. falciparum

. In a preliminary analysisof adhesion molecules expressed on activated humanlung endothelial cells (HLECs) (Muanza

et al

., 1996), wedemonstrated the ability of these cells to express surfaceFc

Œ

RII/CD23 following co-culture with infected erythro-cytes, or following activation by IL-4, a well known inducerof CD23 transcription in haematopoietic cells (Paul-Eugene

et al

., 1995a; Vouldoukis

et al

., 1995a). The abil-ity of CD23 to promote, following cross-linking, the func-tional transcription of the inducible NO synthase gene andthe subsequent generation of nitric oxide in most CD23positive cells (Mossalayi

et al

., 1997), is of particular inter-est to physiopathological processes. Using an

ex vivo

model of co-cultures of human PRBCs with HLECs(Muanza

et al

., 1996), we wished to determine: (i) whetherCD23 expression by HLECs is correlated with the abilityof these cells to express NO pathway following CD23stimulation, and (ii) the effect of CD23/NO pathway on thegrowth of

P. falciparum

and the cytoadherence of infectederythrocytes to HLECs.

Results

Induction of CD23b expression by HLECs stimulated by IL-4 and PRBCs

HLECs preparations were tested for their ability to expressCD23 surface antigen. As IL-4 is the major cytokine thatinduces the transcription of CD23 gene, cells were incu-bated with IL-4 during 6 or 24 h before analysis for surfaceCD23 expression. IL-4 was found to induce CD23b mRNA(6 h) and protein expression (24 h) in HLECs (Fig. 1A andB). In addition, IL-4-activated, or PRBC-activated HLECsexpressed CD23, as revealed by fluorescence microscopy(Fig. 2). However, as revealed by flow cytometry andimmunofluorescence microscopy, cell surface expressionof CD23 was obtained when PRBCs were added to IL-4treated HLECs, with 35

±

3.5% of the cells positive com-pared to 17.2

±

1.6% with IL-4 treatment alone (Fig. 3).Non-infected RBC or fixed PRBCs did not induce CD23.In the same way, when PRBCs were co-cultivated withHLECs without physical contact through porous mem-branes as previously described (Pino

et al

., 2003), noinduction of CD23 expression was seen (unpublisheddata).

Induction of the NO pathway in CD23-expressing HLECs

When HLECs were incubated with PRBCs, the inductionof iNOS mRNA (Fig. 4A) and antigen (Fig. 4B) wereobserved, however, no relevant production of nitrites couldbe detected (Fig. 4C). To explain this discrepancy, we canhypothesize that: (i) the produced NO may interact withhaemoglobin or hemozoin and thus be trapped in parasit-ized erythrocytes (ii) NO can interact with superoxideanions to form peroxinitrites and react with endotheliallipids or proteins and thus become undetectable.

In fact, and as already demonstrated in many othersystems (Paul-Eugene

et al

., 1995a; Vouldoukis

et al

.,1995a; Dugas

et al

., 1998), the ligation of the CD23 anti-gen at the cell surface of CD23-bearing and IL-4 activatedHLECs induced the expression of iNOS mRNA expres-sion (controlled to be 99% equivalent to the iNOS expre-

Fig. 1.

mRNA and Western blot analysis for CD23 expression by HLECs. HLECs were cul-tured in the presence or in the absence of IL-4 for 6 h before RNA extraction and CD23 mRNA evaluation (A). Western blot analysis were also performed on HLECs stimulated with IL-4 for 24 h (B). Data correspond to one representative experiment out of four different.

The CD23/NO pathway regulates PRBCs adhesion

841

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ssed in human macrophages) and protein (Fig. 4A and B).The specific engagement of the CD23 molecule at the cellsurface of IL-4 activated HLECs, ultimately induced nitricoxide production as revealed by nitrites production in cell-free supernatants, 18.9

±

1.8

m

M. The addition of 1 mMof L-NMMA completely reversed this phenomenon,4.9

±

0.5

m

M of NO

2

(Fig. 4C).

Regulation of adhesion molecules expression and PRBC cytoadherence to HLECs by the CD23/NO pathway

In addition to its involvement in the control of inflammatoryresponses (Dugas

et al

., 2001), CD23 has been sug-gested to play an important role in the regulation of cellu-lar adhesiveness (Yamaoka and Kolb, 1995). Indeed, the

Fig. 2.

CD23 expression on IL-4, RBC or PRBCs-stimulated HLECs. As described in the

Experimental procedures

, HLECs were cul-tured in the presence or in the absence of IL-4 for 24 h, followed with RBC and PRBCs (3D7 clone) for 24 h. CD23 expression has been evaluated by immunofluorescence on cytospin slides. The immunostaining of CD23 was revealed with an anti-mouse IgG coupled to PE.

Fig. 3.

Synergistic effect between IL-4 and PRBCs in the induction of CD23 expression by HLECs. HLECs were stimulated for 24 h in the presence of IL-4 (A), PRBCs (B) or IL-4 + PRBCs (C). After washing the cells they were stained with the 135 anti-CD23 mAb and specific labelling was revealed with an anti-mouse IgG coupled to FITC for immunoflurescence microscopy (Palo Alto strain) (D) or anti-CD23-PE for flow cytometry to determine the percentage of CD23 positive cells (3D7 clone). *Comparison with controls,

P

<

0.05.

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et al.

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CD23/NO pathway appeared to play an important regula-tory role in the adherence of PRBCs on HLECs (Fig. 5).Activation of HLECs by IL-4 + PRBCs induced the expres-sion of VCAM-1, E-selectin and ICAM-1 expression onHLECs (Table 1). ICAM-1 in particular, plays an importantrole in the adhesion of PRBCs on HLECs (Traore

et al

.,2000). Ligation of the CD23 with the anti-CD23 mAb sig-nificantly decreased the adhesion of PRBCs on HLECs

(both Palo Alto and patient strain). The difference wassignificant between HLECs + IL4 and HLECs + IL-4 + anti-CD23 (

P

<

0.01), and this was completely reversed follow-ing the addition of L-NMMA (Fig. 5). Taken together thesedata suggest that NO produced after the engagement ofthe CD23 antigen at the cell surface of HLECs could playan important regulatory role in the cytoadherence ofPRBCs on HLECs. In addition, following the subsequent

Fig. 4.

iNOS mRNA, protein expression and nitrite production by HLECs. As described in the

Experimental procedures

section, HLECs were cultured in the presence or in the absence of IL-4, anti-CD23, RBC and PRBCs (Palo Alto strain) for 6 h before RNA extraction. iNOS expression was pointed out by RT-PCR (A). iNOS protein expression (B) and nitrites production (C) were evaluated after 24 h of co-culture. Data from iNOS expression represent the mean

±

SD of triplicate experiments whereas iNOS mRNA expression are one representative experiment out of three. *Comparison with controls,

P

<

0.01.

Fig. 5.

Adhesion of PRBCs from different

P. fal-ciparum

strains on HLECs and the role of NO pathway on cytoadherence. As described in the

Experimental procedures

section, the role of NO on the parasite cytoadherence (patient and Palo Alto strains) was evaluated on HLECs pre-activated or not with IL-4 and before and after stimulation with the anti-CD23 mAb. The role of NO in this process was revealed in the pres-ence of 1 mM L-NMMA. Data represent the mean

±

SD of four different experiments. In addition, the adhesion assay was performed using the 3D7 clone of

P. falciparum

and a semiautomatic method of quantification by flu-orescence (B).

The CD23/NO pathway regulates PRBCs adhesion

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CD23-induced NO induction, a marked decrease in para-sitaemia was also observed (2.7%) compared to cellstreated with IL-4 alone (4.9%,

P

<

0.001) strengtheningthe idea that NO produced after ligation of the CD23molecule at the cell surface of HLECs not only regulatescytoadherence of PRBCs but also promotes an importantanti-

P. falciparum

effect.

Discussion

These experiments not only provide the first evidence forCD23 expression by human endothelial cells but alsodemonstrate the interaction between HLECs and infectedRBC as a prerequisite for this phenomenon. These datafit with our previous work demonstrating an enhancementof CD23 expression in human macrophages infected with

L. major

(Vouldoukis

et al

., 1995b). Following cross-link-ing, CD23 surface antigen is known to mediate the induc-tion of iNOS transcription and NO generation in varioushuman blood leucocytes as well as epithelial cells (Dugas

et al

., 1995). However, experimental data indicating anactivation pathway in HLECs cells had yet to be obtained.In the present work, we demonstrated that following treat-ment with IL-4 and ligation of CD23 by specific mAb,endothelial cells co-cultured with PRBCs were able toexpress iNOS and to generate NO. In addition, PRBCsalone can elicit CD23 and iNOS expression in HLECs; itis thus likely that PRBCs adhesion may induce CD23aggregation sufficiently to provide a transducer signalleading to iNOS upregulation. Furthermore, the triggeringof the CD23/NO pathway also affected (i) the expressionof adhesion molecules by the HLECs, and (ii) their abilityto interfere with infected RBCs. Immunostaining analysisindicates that stimulation by IL-4 of HLECs co-culturedwith PRBCs increased the expression of most adhesion

molecules except PECAM-1, whose expression signifi-cantly decreased. In contrast, when IL-4 treatment wasfollowed by an incubation with anti-CD23 mab, a signifi-cant decrease in ICAM-1 expression was observed whilesurface PECAM-1 levels were increased. Modulation ofadhesion molecules following CD23 ligation led us toassay the effect of this phenomenon on interactionbetween infected RBCs and HLECs. Using HLECs andthree different strains of

P. falciparum

parasites, we foundthat, regardless of strain used, IL-4 followed by treatmentwith CD23 mAb significantly decreased adherence ofinfected RBC. This phenomenon was completely reversedfollowing the simultaneous addition of L-NMMA, pointingout the role of NO in mediating the CD23 effect. Subse-quent to NO induction, a marked decrease in parasi-taemia was also observed, confirming previous results onthe antimicrobial effect of NO (Rockett

et al

., 1991).This study provided the critical observation that CD23/

NO pathway is functional in HLECs as: (i)

P. falciparum

-infected RBCs cells induced expression of CD23 onHLECs (ii) there is increased iNOS expression followedby generation of nitrites upon HLECs stimulation by anti-CD23 mAb; and (iii) addition of L-NMMA inhibited CD23-derived effects in HLECs cells. CD23 and its physiologicligand, IgE, have been shown to be expressed in thecourse of numerous infectious diseases including

Plasmo-dium

infections (Vouldoukis

et al

., 1995a). We furthershow that CD23/NO pathway plays a prominent antiplas-modial role either directly through parasite killing, or indi-rectly by inhibiting cytoadherence of parasited RBC,probably caused by a decreased expression of ICAM-1.The anti-

Plasmodium

activity of NO is yet to be charac-terized, but different mechanisms can be hypothesized: (i)the produced NO may interact with haemoglobin andinterfere with parasite hemozoin biosynthesis, and (ii)

Plasmodium

infections induce an important oxidativestress (Pabon

et al

., 2003), and NO can react with super-oxide anions to form peroxinitrites with exhibit high cyto-toxic activities against various cell types includingparasites (Rockett

et al

., 1991; Szabo, 2003).Cytoadherence of PRBCs is clearly multifactorial, and

its extent depends on

P. falciparum

strains and the typeof target cells (Craig and Scherf, 2001). It is interesting tonote that we observed similar results using three geneti-cally different parasite strains: in our model, ICAM-1appeared to be an important receptor for PRBCs adhe-sion to HLECs. The low expression of PECAM-1 followingIL-4 treatment did not reduce cytoadherence of the para-sites which, in this model, appears mostly as a result ofan interaction with ICAM-1, a molecule that increasedfollowing similar IL-4 treatment.

In vivo

, immunohis-tochemical studies showed endothelial expression ofICAM-1 in the brain of fatal cases of cerebral malaria(Turner

et al., 1994). From experimental models, it has

Table 1. ICAM-1, VCAM-1, E-selectin and PECAM-1 expressionafter various stimulation protocols.

Adhesion molecules(% of positive cells)

HLECs+ PRBCs(Control)

HLECs+ IL4+ PRBCs

HLECs+ IL4 + anti-CD23 mAb+ PRBCs

Mabs ICAM-1 9 ± 3 23 ± 4 4 ± 1[P-value] [0.001]* [0.02]*

[< 0.001]**Mabs VCAM-1 14 ± 3 19 ± 3 9 ± 3[P-value] [0.08]* [0.08]*

[0.003]**Mabs E-selectin 10 ± 2 14 ± 3 11 ± 2[P-value] [0.09]* [0.55]*

[0.18]**Mab PECAM-1 18 ± 3 3 ± 1 20 ± 4[P-value] [< 0.001]* [0.55]*

[< 0.001]**

*comparison with controls.** comparison between HLECs + IL-4 andHLECs + IL4 + anti-CD23 mAb.

844 P. Pino et al.

© 2004 Blackwell Publishing Ltd, Cellular Microbiology, 6, 839–848

been proposed that a predominant Th1 type response,with high production of IFN-g and TNF-a, was associatedwith susceptibility to the neurological complications (deKossodo and Grau, 1993). In the immunohistochemicalstudies of brain tissue from patients with fatal cerebralmalaria published to date (Udomsangpetch et al., 1997;Brown et al., 1999), a focal accumulation of Th1 typecytokines was described. However in one patient (Udom-sangpetch et al., 1997; Brown et al., 1999), a differentcytokine profile was described, with no IFN-g nor TNF-a,but where IL-4 was found. With regard to the present studyshowing IL-4 ability to induce expression of ICAM-1 andVCAM-1, together with increased cytoadherence ofPRBC, it may be hypothesized that the observed pathol-ogy is caused by an excessive release of IL-4 in thecerebral micro vasculature presenting no CD23 ligands.

Taken together, our data support a protective role of theNO/CD23 pathway in the pathogenesis of malaria, as itwould lead to a reduction of both parasitaemia andcytoadherence. Previous studies using the HLECs modelshowed that PRBCs from severe cases presentedincreased cytoadherence patterns compared to uncompli-cated cases and that PRBC adherence to endothelial cellsvia ICAM-1 was associated with malaria severity (Traoreet al., 2000). The in vivo relevance of the hypothesis thatthe CD23/NO pathway could diminish complications of P.falciparum malaria is suggested by a study in infectedpatients, where concomittent Ascaris lumbricoides infec-tion was found to be associated with protection from cere-bral malaria (Nacher et al., 2000). As CD23 representsthe major low affinity receptor for IgE, protection againstcerebral malaria observed in Ascaris-infected patientscould have reflected the activation of the CD23/NO path-way in vivo via IgE immune complexes. However, thereport that a significant increase of plasma concentrationof total IgE correlated with cerebral malaria development(Perlmann et al., 1997), implies that excessive CD23 liga-tion might result in subsequent production of high levelsof NO and reactive oxygen species, peroxinitrites gener-ation and cerebral pathology, a phenomenon alreadyobserved in the neuropathogenicity of HIV infection.

In conclusion, circumstances leading to increasedCD23 expression and its ligation via IgE immune com-plexes, such as allergies and helminth infections, may wellinfluence the course of malaria attacks.

Experimental procedures

Reagents

Recombinant human IL-4 (1 ¥ 107 U/mg) was purchased from Rand D Systems. NG-monomethyl-L-arginine (L-NMMA) were pur-chased from Sigma (St Louis, MO). The monoclonal antibodydirected against the CD23 antigen (mAb 135 of IgG1 isotype) hasbeen already shown to upregulate iNOS expression in various

cell types (Dugas et al., 1998); the anti-CD19 mAb (Immunotech,Marseille, France) is an isotype-matched (IgG1) control antibod-ies. The two other anti-CD23 mAbs used in this study wereMHM6 (IgG1) from Dako (Les Ulis France) and IOB8 (clone 25from Immunotech, Marseille, France).

Culture of HLECs

We used primary cultures of the seventh passage of HLECs. Thecells were isolated after enzymatic digestion, then selected usinga continuous gradient and immunomagnetic purification tech-nique (Muanza et al., 1996). The HLECs were seeded at a den-sity of 3 ¥ 103 cells per well in 8-chamber plastic Labteck slides(Chamber slide, Labtek, NUNC) and cultivated at 37∞C with 5%CO2 in M199 medium (Gibco), supplemented with endothelial cellgrowth supplement (ECGS) 10 mg ml-1, Euromedex. After 18–36 h, HLECs were confluent and were ready for use. These cellswere characterized for their expression of Von Willebrand factor,ICAM-1, VCAM-1, CD31, CD36, E/P-selectin and CSA as previ-ously described (Muanza et al., 1996).

P. falciparum parasites

Three types of parasites were used: the strain Palo Alto (FUP),a gift from Arthur Scherf, the clone 3D7, a gift from David Walliker,and parasites isolated from a patient hospitalized in Pitié-Salpêtrière Hospital with cerebral malaria according to WHO. Thepatient was a non-immune Caucasian with cerebral malariareturning form Africa. Parasites were immediately put into cultureas previously described (Trager, 1994). Parasite growth wasmonitored by examination of Giemsa-stained thin blood filmsevery 12 h until isolate reached the pigmented schizont stage.Experiments were performed during the first cycle of in vitroparasite growth.

The 3D7 clone was characterized for adhesion phenotype aspreviously described (Ockenhouse et al., 1991) and adheres atICAM-1 and CD36 at 30 and 45% respectively. The strains weresynchronized and maintained in culture according to Trager andJensen’s technique (Trager, 1994) in a suspension of erythro-cytes in RPMI (Gibco) supplemented with 8.3 g l-1 of Hepes(Sigma, St Louis, MO, USA), 2.1 g l-1 of NaHCO (Merck),0.1 mg ml-1 of gentamycin and 2 g l-1 of dextrose and 0.4% ofAlbumax II (W/V)(Life Technologies).

Expression of CD23b and iNOS on HLECs

Confluent HLECs were treated for 24 h with a suspension of IL-4 (10 ng ml-1, R and D System), then for 36 h with an anti-CD23 mAb (clone 135, IgG1k, F(ab¢)2 (20 mg ml-1) (a kind giftfrom Guy Delespesse, Canada). The interactions between CD23/NO pathway and PRBCs were examined by adding a suspensionof PRBCs (parasitaemia 5%, and 2% haematocrit) to the IL-4and/or CD23-stimulated HLECs for 24 h; blood smears prepara-tions stained with Giemsa were analysed microscopically forparasitemia to determine the effect of the CD23/NO pathway onP. falciparum growth. Parasitaemia were determined on 10 000erythrocytes counted in triplicate (Desjardins et al., 1979).

In addition, supernatants were removed at 24 h and 36 hand frozen at -20∞C before NO derivatives analysis. The slideswere washed three times with PBS then fixed for 30 min with a

The CD23/NO pathway regulates PRBCs adhesion 845

© 2004 Blackwell Publishing Ltd, Cellular Microbiology, 6, 839–848

2% paraformaldehyde/PBS solution and used for CD23 andiNOS immunodetection. Briefly, cells were then incubated for30 min in PBS at 4∞C with 1/100 FITC-conjugated anti-CD23 mAb (IOB8, immunotech, clone 25, Marseille, Lumigny,France) and CD23-PE antibodies (Becton Dickinson). The per-centage of CD23 positive cells was evaluated either by immun-ofluorescence microscopy and flow cytometry analysis(Facscan, Becton Dickinson, Grenoble, France), using one mil-lion of HLECs as previously described (Dugas et al., 2001).Mouse anti-human IgG-FITC or anti-human IgG-PE were usedas controls.

For iNOS analysis, fixed cells were first incubated with nitricoxide synthase 2 (NOS2, C-19) rabbit polyclonal anti-IgG (SantaCruz Biotechnology) at 1 : 50 dilution in PBS. Reactivity with anti-iNOS antibodies was then visualized using biotinylated-rabbit IgGand the avidin-peroxidase method according to the manufac-turer’s instructions (Vector Laboratories, Burlingan, CA). Normalrabbit purified IgG (at 1/1000) was used as control (Immunotech,Marseille Lumigny, France).

CD23 Western blot

For Western blotting, proteins in cell extracts were separated byelectrophoresis on a 15% sodium dodecyl sulphate¯polyacryla-mide gel (SDS¯PAGE), and the proteins transferred onto a nitro-cellulose membrane. The membranes were incubated with 5%BSA in PBS, and then with a monoclonal anti-human CD23antibody (CD23 mAb-135 a kind gift from G. Delepesse) for 1 hat room temperature. After they were washed, the membraneswere then incubated with an alkaline phosphatase-conjugatedgoat anti-mouse IgG antibody (Sigma, diluted 1 : 5000) for 1 h atroom temperature. Immunoreactive bands were visualized withthe alkaline phosphatase conjugate substrate kit (Bio-Rad)according to the manufacturer’s protocol.

Analysis of CD23b mRNA expression

Confluent cells seeded in a 75 mm tissue culture flask wereeither not stimulated or stimulated for 6 h with IL-4 + PBRC. Cellswere lysed with RNABle (Eurobio, Les Ulis, France) and RNAwas extracted by the phenol/chloroform/isopropanol methodaccording to the manufacturer’s instructions. Inducible CD23mRNA expression was investigated by means of reverse tran-scriptase-polymerase chain reaction (RT-PCR) with specificprimer sets, as previously described (Vouldoukis et al., 1995a).The oligonucleotide primer set of sequences in these experi-ments were as follows: CD23b mRNA sense: 5¢-ATGAATCCTCCAAGCCAGGA-3¢, CD23b mRNA antisense: 5¢-TGGCTTTCCAAGTTCTTGGA-3¢, hypoxanthine phosphoribosyl transferase(HPRT) mRNA sense: 5¢-TATGGACAGGACTGAACGTCTTGC-3¢ (HPRT) mRNA antisense: 5¢-GACACAAACATGATTCAAATCCCTGA-3¢. The CD23b message is represented by a 217 bpband, and a 496 bp band represents the HPRT message. Den-sitometry analysis was performed with NIH image software afterscanning of bands. A ratio of CD23b mRNA expression wasdetermined after normalization to HPRT mRNA levels. The CD23gene amplification product was purified after electrophoresis bymeans of a spin column as directed by the manufacturer (Qiagen,Chatsworth, CA, USA) and was sequenced by the ESGS com-pany (Evry, France).

Sequences were further compared to the CD23 mRNAsequence with the BLAST program.

Analysis of iNOS gene expression

Inducible NO-synthase mRNA expression was investigated withreverse transcriptase polymerase chain reaction (RT-PCR). Thesequences of the oligonucleotide primer sets in these experimentswere as follows: iNOS mRNA sense (5¢-ATGCCAGATGGCAGCATCAGA-3¢, exon 8) and iNOS mRNA antisense (5¢-ACTTCCTCCAGGATGTTGTA-3¢, exon 11). The iNOS amplification prod-uct is represented by a 371 bp band, and a 496 bp band indicatedthe HPRT message. iNOS mRNA expression was investigatedwith RT-PCR using a modified single-step guanidinium isothiocy-anate and phenol/chloroform extraction method. Total RNAs wereobtained from 107 stimulated cells/point, washed in PBS (phos-phate-buffered saline, Gibco BRL), lysed in 1 ml of lysis buffer(RNAzole®, Bioprobe, Montreuil sous Bois, France), vortexed andstored at -70∞C until further processing. After thawing, 50 ml ofchloroform isoamyl-alcohol (49 : 1) were added with thoroughvortexing. The samples were then chilled on ice for 15 min andspun at 10 000 g for 20 min. The aqueous phase was recoveredand RNA was precipitated in an equal volume of isopropanol at-20∞C for at least 60 min. The RNA pellet was then washed with75% ethanol, vacuum-dried and resuspended in TE/SDS. Afterincubation at 50∞C for 15 min, the RNA was precipitated overnightat -20∞C and pelleted at 4∞C at 12 000 g for 30 min The RNAwas stored at -70∞C until further processing.

CDNA synthesis. A 5 mg aliquot from each RNA sample wasincubated at 70∞C for 10 min with 5 mg oligo dT (PharmaciaBiotech, Piscataway, NJ) in RNase-free water. After cooling onice, deoxynucleotide triphosphates (10 mM, Pharmacia, St Quen-tin en Yvelines, France), 5¥ cDNA synthesis buffer (Gibco BRL,Cergy-Pontoise, France), 1 ml RNAsin® ribonuclease inhibitor(Promega, Madison, WI), and 200 U of Moloney murine leukaemiavirus reverse transcriptase (MMLV-RT, Gibco BRL) were added.After 1h 30 min at 37∞C, the reverse transcriptase was heat-inactivated at 95∞C for 5 min, and the 20 ml cDNA synthesisreactions were each diluted to 50 ml for use in PCR amplification.

Polymerase chain reaction. The 100 ml PCR reaction consists ofthe following mixture: 5 ml of the above cDNA synthesis reaction,10 ml 10¥ PCR buffer (ATGC, Noisy le Grand, France), 200 mMdeoxynucleotide triphosphates (Pharmacia), oligonucleotideprimers specific for type II NOS (Bioprobe), or the housekeepinggene HPRT (hypoxanthine phosphoribosyl-transferase, Bio-probe) at a concentration of 0.5 mM, 2.5 U of Taq polymerase(ATGC), and sterile water. Samples were run in a Perkin-ElmerDNA thermal cycler (Perkin-Elmer Corporation, Norwalk, CT) for40 cycles (94∞C for 30 s, 60∞C for 30 s, 72∞C for 1 min). Poly-merase chain reaction products were visualized on a 1.5% aga-rose gel stained with ethidium bromide (Sigma). The iNOSmessage is represented by a 371 bp band, and a 496 bp bandindicated the HPRT message.

Sequencing of the iNOS PCR products

Polymerase chain reaction products from endothelial cells stim-ulated by CD23 were cloned into the pCR-Script vector (Strat-

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agene, La Jolla, CA) and transfected into E. Coli competent cells.Clones harbouring recombinant plasmids with inserts of appro-priate size, based on restriction analysis, were sequenced usingthe dideoxy chain termination method with T7 DNA Polymerase(Pharmacia, Uppsala, Sweden). All inserts were sequenced inboth directions and from several independent clones from at leasttwo PCR amplifications.

Determination of nitric oxide derivatives

Culture supernatants of treated HLECs as described above, wereassayed for NO-derivatives by using the Griess reaction modifiedas previously described (Vouldoukis et al., 1995a). L-NMMA wasadded at a concentration of 1 mM on the HLECs in combinationwith IL-4, anti-CD23 and PRBCs to determine the activity andthe specificity of NO-derivatives in the co-culture model.

Induction of adhesion molecules on HLECs

HLECs were stimulated with IL-4 (24 h), anti-CD23 (36 h) andPRBCs (24 h) alone or in combinations as described above. Afterwashings, monolayers of HLEC were first incubated with mousemAb directed against human PECAM-1 (CD31, clone 9G11,isotype IgG1), ICAM-1 (CD54 clone BBIG, I1, isotype IgG1),VCAM-1 (clone BBIG, V1, isotype IgG1) and E-selectin (cloneBBIG-EG, isotype IgG1) (R and D Systems, UK), at 1: 1000dilution in EBSS (Gibco Brl, Paisley Scotland), for 45 min at 4∞C.The cells were washed twice in 500 ml of staining buffer and thenincubated with 50 ml of a biotinylated-mouse IgG following theavidin-peroxidase or alkaline phosphatase method according tothe manufacturer’s instructions (Vector Laboratories, Burlingan,CA). In negative controls, primary or secondary antibodies wereomitted. In addition, mouse anti-human IgG was also used as acontrol. The percentage of HLECs which expressed adhesionmolecules was evaluated by counting means per 500 cells.

Cytoadherence assays

Tests of cytoadherence were carried out as previously described(Udeinya et al., 1981). Suspensions of PRBCs (Palo Alto strain,patient parasites and 3D7 clone) at 10% parasitaemia and 5%haematocrit were washed in cytoadherence medium (RPMI with-out bicarbonate). Three hundred ml of suspension was incubatedon cell monolayers for one hour with gentle continuous shakingat 37∞C. After removal of non-adherent erythrocytes with threewashes using cytoadherence medium, the preparation was fixedfor 30 min with 2% glutaraldehyde, rinsed with PBS and stainedwith Giemsa. Cytoadherence was expressed as the number ofadhered PRBCs per 200 HLECs.

To determine the specific effect of NO on cytoadherence, theL-arginine analogue N-monomethyl-L-arginine (L-NMMA), Sigma(Saint Louis, MO, USA), at concentration of 1 mM, was added atthe time of IL-4 and anti-CD23 treatment for 24 or 36 h respec-tively. After washings, PRBCs from the patient were added for24 h to the HLECs to determine the role of NO derivatives duringcytoadherence.

In order to get a better method to quantify PRBCs adhesion,we developed a new technique where PRBCs were stained witha fluorescent cell tracker (CFDA SE, Molecular Probe, 0.1 mM)as recommended by manufacturer. Only the parasites were

stained and not non-infected erythrocytes. The HLECs werestained with an orange cell tracker (CMTMR, Molecular Probes).Adhesion assays were performed as above, and fluorescence ofadherent PRBCs was quantified using Packard Fusion. Datarepresent a fluorescence ratio, fluorescence of PRBCs/fluores-cence of HLECs.

Statistical analysis

All experiments were performed in triplicate or more. Resultswere expressed as mean and standard deviation. Means werecompared using Students unpaired t-test, or Mann and Withneytest.

Acknowledgements

We thank Dr M. Nacher for helpful suggestions and Dr G.Snounou, Dr P. David, and Dr O. Silvie for helpful and construc-tive discussions.

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