Vol. 57, No. 1INFECTION AND IMMUNITY, Jan. 1989, p. 278-2820019-9567/89/010278-05$02.00/0Copyright ©) 1989, American Society for Microbiology

C-Reactive Protein Protects against PreerythrocyticStages of Malaria

SYLVIANE PIED,1 ANDREAS NUSSLER,' MICHEL PONTET,2 FRANQOIS MILTGEN,3 HUGUES MATILE,4PAUL-HENRI LAMBERT,5 AND DOMINIQUE MAZIERl*

Institut National de la Sante et de la Recherche Medicale U 313 et Departement de Parasitologie, Groupe HospitalierPitie-Salpetriere, 91 Boulevard de l'Hopital, 75013 Paris,' De'partement de Biochimie, Faculte de Medecine des

Saint-P res, Paris,2 and Museum National d'Histoire Naturelle, Paris,3 France, and Hoffman-La Roche, Basel,4 andDepartment of Pathology, World Health Organization, Geneva,5 Switzerland

Received 14 June 1988/Accepted 21 September 1988

We previously reported that low doses of interleukin-1 strongly inhibited in vitro development of the hepaticstages of Plasmodiumfalciparum and P. yoelii. Among several hypotheses, we considered the role of C-reactiveprotein (CRP), a major acute-phase reactant whose concentration increases markedly in infectious disorders.We demonstrated that human hepatocytes cultured in the presence of interleukin-1 released, as early as 30 minafter stimulation, an increased amount of CRP. We then established that CRP bound to the P. falciparum andP. yoelii sporozoite surface membranes, probably via a phosphorylcholine binding site. Experiments in whichCRP was added to rat hepatocyte monolayers during or after inoculation confirmed that the target of theCRP-mediated inhibition was at the very early phase of infection. These in vitro functional activities wereconfirmed in an in vivo model; rats with increased levels of CRP in serum following an injection of turpentineoil were found to be largely protected against an inoculation of P. yoelii sporozoites. The same results wereobserved in animals inoculated with sporozoites previously incubated in purified CRP or in sera of ratspretreated with turpentine oil. The latter effect was inhibited after incubation of serum from turpentine-injected rats with anti-CRP serum.

The establishment of protection against preerythrocyticstages of malaria parasites depends on both humoral andcell-mediated immune responses (20a, 22, 30, 33). Particularattention has recently been given to the possible role ofcytokines in protective mechanisms at the preerythrocyticstage of the infection (8, 18, 21, 29).Gamma interferon and interleukin-1 (IL-1) were shown to

strongly inhibit Plasmodium falciparum sporozoite develop-ment in primary cultures of functional hepatocytes. Neitherof these two cytokines directly affects free sporozoites, buttheir points of action appear to differ. Time experiments withinterferon indicate a postpenetration (intrahepatocyte) cellu-lar mechanism, in contrast to IL-1, which exerts its effect atthe very early phase of infection (21).One of the hypotheses that could explain IL-1 activity is

related to the secretion of inflammatory proteins by IL-1-stimulated hepatocytes, in particular C-reactive protein(CRP) (1, 6, 13). CRP, a major acute-phase reactant found intrace amounts in normal human serum, shows markedlyincreased levels in sera of individuals with many conditions,including infectious disorders (17, 26). CRP participation inhost defense systems against infection is in part related to itscapacity to bind to the phosphorylcholine-containing sub-stances in microorganism membranes (10, 32).We investigated the release of CRP in primary cultures of

human hepatocytes after stimulation with human recombi-nant IL-1. We then analyzed the possible deleterious effectsof CRP on sporozoites and on hepatic stages of Plasmodiumspp. Finally, using inflammatory substances (e.g., turpentineoil), we investigated the possible influence of the nonspecificinduction of CRP in vivo on the infection of rats by P. yoeliisporozoites.

* Corresponding author.

MATERIALS AND METHODSCRP. CRP was purified from human ascitic fluid or from

normal rat serum by a three-step chromatography procedureinvolving passage through columns of phosphoethanol-amine-Sepharose 4B, protein A-Sepharose, and SephacrylS-300 (27). The purity of CRP isolated by this procedure hasbeen documented elsewhere (7). Preparations were checkedby sodium dodecyl sulfate-polyacrylamide gel electrophore-sis (4 to 30% acrylamide), which indicated a single band witha molecular weight of 24,000. CRP was radiolabeled by theiodogen method with carrier-free Na1251 (CEA, Saclay,France) and lodobeads (Pierce Chemical Co., Rockford, Ill.)(9). Iodine-labeled protein was separated from free iodine ona Sephadex G-25 column. The specific activity of variouspreparations was 1 + 0.25 ,uCi/,ug. 125I-CRP retained itsbinding capacity, since more than 95% of the iodinatedprotein did bind to a gel substituted by phosphorylcholine orphosphoethanolamine. This binding was specific, since inhi-bition occurred in the presence of a calcium chelator agentand/or phosphorylcholine.

Antisera. Rabbit polyclonal anti-human CRP serum waspurchased from Biosys, Compiegne, France. Polyclonalanti-rat CRP was raised in a rabbit immunized with purifiedrat CRP in the presence of complete Freund adjuvant.

IL-1. Human recombinant IL-1 was purchased from Hoff-man-LaRoche, Basel, Switzerland.

Sporozoites. Sporozoites were obtained from the salivaryglands of Anopheles stephensi infected with P. falciparumNF54 or with P. yoelii 17X or 265BY.

Culture of hepatic stages of malaria parasites. Human androdent hepatocytes were isolated by collagenase perfusion ofliver biopsies as previously described (19). The cells werecultured in eight-chamber plastic Lab-Tek slides (MilesLaboratories, Inc., Elkhart, Ind.) at a concentration of100,000 cells per well and were maintained for 24 h before P.

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yoelii sporozoite inoculation and/or addition of humanrecombinant IL-1 at a concentration of 5 IU/mi.The parasites were obtained by dissection of the salivary

glands of A. stephensi. The infestation procedure of therodent hepatocyte cultures was carried out as describedpreviously (20). Parasites were detected by immunofluores-cence assay (IFA) with a murine monoclonal antibodyrecognizing hepatic stages of P. yoelii.CRP secretion in liver cell cultures. (i) Qualitative assay.

Samples (50 VI) of supernatants of cultures incubated fordifferent lengths of time with human recombinant IL-1 (5 IU/ml) or control medium were mixed with an equal volume of2 x Laemmli sample buffer (15) and boiled for 5 min. Themixture was electrophoresed under reducing conditions in12.5% acrylamide slab gels as described by Laemmli (15).Resolved proteins were either stained with Coomassie blueor transferred to nitrocellulose paper by electroblotting (31).In the second instance, saturation with 5% nonfat milk inphosphate-buffered saline (PBS) was performed for 2 h atroom temperature. CRP was revealed by incubating thenitrocellulose strips with a 1/100 dilution of rabbit anti-human CRP serum in blocking buffer for 12 h and then withI251-labeled Staphylococcus aureus protein A (Amersham,France). Dots were exposed for 3 days at -70°C on X-OmatAR film (Eastman Kodak Co., Rochester, N.Y.).

(ii) Quantitative assay. An enzyme immunoassay was usedto measure the amount of human CRP present in liver cellculture supernatants incubated for 18 h with human recom-binant IL-1 (5 IU/ml). In brief, 10 ,ug of rabbit anti-humanCRP immunoglobulin was attached, by passive adsorptionovernight at 4°C, to polystyrene microplates (Nunc, Becton-Dickinson, Grenoble France). Purified human CRP, dilutedin culture medium, was used to generate the reference curve,which was developed by using alkaline phosphatase-conju-gated rabbit anti-human CRP.

In vitro inhibition of the Plasmodium hepatic stage by CRP.Inhibition assays were performed by adding various concen-trations of human or rat CRP to culture medium at the timeof sporozoite inoculation. The cultures were inoculated byadding 40,000 P. yoelii sporozoites to culture medium con-taining or not containing CRP. At 3 h later, the medium waschanged and cultures were maintained in the absence ofCRP. In other experiments, CRP was added 3 and 24 h afterinoculation. All experiments were performed in duplicate.The percentage of inhibition of parasite development wasassessed by comparing the number of schizonts in CRP-treated cultures with the number in the corresponding con-trol cultures.

In vitro effect of CRP on the erythrocyte stages of P.falciparum. The effect of CRP on the P. falciparum erythro-cyte stages was evaluated by using an in vitro microtest;parasite growth was estimated by [3H]hypoxanthine incor-poration (3). Nine different dilutions of CRP in RPMI 1640medium were prepared (10 to 0.039 ,ug/ml) and added towells of 96-well flat-bottom plates at the beginning of theculture. The cultures were grown for 48 h.

CRP-parasite interactions. (i) Binding of '251-CRP to spo-rozoites. Binding studies were performed at 37°C for 3 h in1.5-ml Eppendorf microtest tubes coated with human serumalbumin. Suspensions of infected or noninfected salivaryglands in calcium buffer (5 mmol of CaCl2 per liter, 150 mmolof NaCl per liter, 5 mmol of NaN3 per liter, 20 mmol of Trishydrochloride [pH 7.4] per liter) containing 0.05 ,ug of1251-CRP were incubated with gentle agitation in a finalvolume of 120 i1,l. Incubations were terminated by theaddition of 1 ml of calcium buffer, followed by centrifugation

at 10.000 x g for 7 min at 4°C. The activity present in thesupernatant was determined in a gamma counter (CorningGlass Works, Corning, N.Y.), and all results are means offour determinations.

(ii) IFA. The fixation of CRP was investigated by incubat-ing P. yoelii or P. falciparium sporozoites for 1 h at 37°C withpurified human or rat CRP at concentrations of 5, 25, and 50,ug/ml in calcium buffer. After three washings, the sporozo-ites were transferred to poly-L-lysine-treated slides as pre-viously described (28a). CRP binding was determined byIFA with anti-human CRP reagent as the primary antibodyand a fluorescein-tagged rabbit anti-human immunoglobulinas the secondary antibody. The same experiment was per-formed with free P. falciparum merozoites isolated by themethod of Heidrich et al. (11).

In vivo experiments. Five groups of 2-month-old femaleWistar rats (IFFA CREDO, Lyon, France) were used. Eachrat in group 1 (total of seven rats) was injected subcutane-ously with 1.0 ml of turpentine oil. At 24 h later, each rat wasinoculated intravenously with 7,000 P. yoelii sporozoites in0.5 ml of PBS. Each rat in group 2 (control group for group1, also containing seven rats) received a subcutaneousinjection of 1.0 ml of PBS. Subsequent treatment was thesame as that for group 1 rats. Each rat in group 3 (total of fiverats) was inoculated intravenously with 7,000 sporozoiteswhich had been previously incubated for 30 min at 37°C in0.5 ml of rat serum collected 24 h after turpentine oilinjection and diluted 1/5 in PBS. The rats in group 4 (total offive rats) were inoculated under the same conditions as thosein group 3, but the rat serum was previously incubated withanti-CRP for 30 min at 37°C before the sporozoites wereadded. Each rat in group 5 (total of five rats) was inoculatedintravenously with 7,000 sporozoites which had been mixedfor 30 min at 37°C with 100 ,ug of purified rat CRP in 0.5 mlof PBS.Blood smears, taken daily from days 3 to 15 after sporo-

zoite inoculation, were stained with Giemsa stain and exam-ined for P. yoelii erythrocyte stages.

RESULTS

CRP secretion by human hepatocytes in response to IL-1stimulation. Cultures of human hepatocytes incubated for 18h with 5 IU of human recombinant IL-1 per ml secreted CRPin larger amounts than did untreated cultures. As measuredby enzyme-linked immunosorbent assay, IL-1 induced afivefold increase in the level of CRP detected in culturesupernatants: 24 ,ug/ml versus 4.8 j±g/ml for untreated cul-tures. Time course analysis of CRP release, as assessed by asemiquantitative method, revealed a progressive increase inthe CRP level detected in culture supernatants; this increasewas detected as early as 30 min after IL-1 stimulation (Fig.1).

Effect of CRP on hepatic-stage Plasmodium cultures. Whenpurified CRP was added to rat hepatocyte cultures at the

CCRP CC:I 1 2 3 4 5 6

FIG. 1. Western immunoblot analysis of secreted CRP. CRP isvisualized by using rabbit anti-human CRP antiserum followed by1251-labeled protein A. Lanes 1 to 6 show the amount of CRP in thesupernatant of cells incubated for 30 min and 1, 5, 10, 15, and 20 h,respectively, with IL-1. Abbreviations: Ccrp, Control purified CRP;Ccm, control culture medium.

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*" . iii11..ll2.5 5 25 5 1 2 3 4 5

CRP (CJg/sl )

FIG. 2. Effect of dose of rat CRP on P. yoelii schizont develop-ment in cultured rat hepatocytes. Bars with standards errors repre-sent the average inhibitory effect of CRP compared with theuntreated controls.

time of sporozoite inoculation, parasite schizogony was

inhibited in a dose-dependent manner (Fig. 2). Comparativestudies of the effect of human and rat CRP on P. yoeliidevelopment in rat hepatocytes demonstrated the speciesspecificity of CRP activity, since 50% inhibition was ob-tained with 25 ,ug of rodent CRP per ml, whereas 300 ,ug ofhuman CRP per ml was necessary to achieve a similarinhibition (Fig. 3). Neutralization experiments in whichanti-CRP was added to culture medium at 37°C 30 min beforecell treatment demonstrated the specificity of the CRPactivity. When CRP was added 3 h after sporozoite inocu-lation, no significant inhibition was found (data not shown).

Effect of CRP on erythrocyte-stage Plasmodium cultures.No inhibition was observed when purified CRP was added toerythrocyte-stage P. falciparum cultures.

CRP-parasite interaction. (i) Binding of 1251-CRP to in-fected and noninfected salivary glands. To investigate thepossible binding of '25I-CRP to sporozoites, we comparedthe binding of 125I-CRP to P. yoelii-infected salivary glandswith the binding of '25I-CRP to noninfected salivary glands(Fig. 4). It appears that '25I-CRP bound to the sporozoites,since the binding to salivary glands was increased threefoldwhen the glands were infected. The complete inhibition ofthe binding observed in the presence of phosphorylcholinesuggests that the CRP binding is mediated by phosphoryl-choline-binding sites.

V. Inhibition

14000

12000

100

6000

2000-

A

B

ISO ISG+PC NISO NISG+PC

FIG. 4. Binding of CRP to P. yoelii sporozoites. The amount ofCRP bound to infected salivary glands (ISG) is compared with thatbound to an equal number of noninfected salivary glands (NISG). Inexperiment A, 240,000 sporozoites were used; in experiment B,24,000 were used. The effect of phosphorylcholine (PC) is shown foreach binding assay. The results are expressed as the mean andstandard deviation of four determinations. Symbols: 1, 100-,ulsuspension of infected or noninfected salivary glands; E, 10-pAlsuspension of infected or noninfected salivary glands.

(ii) IFA analysis. The binding of CRP to the parasite wasfurther studied by IFA. Significant binding to unfixed live P.yoelii sporozoites (Fig. 5) and P. falciparum merozoites(data not shown) was observed. No fluorescence appearedwhen the experiment was performed with calcium-free me-dium, confirming the importance of this divalent ion in theestablishment of the interaction.

In vivo effect of CRP on P. yoelii sporozoite development inrats. In vivo experiments were carried out to find the effectof CRP on protection against sporozoite challenge. In group1 rats, which were pretreated with turpentine oil, only onerat exhibited parasitemia, which was weak and was imme-diately resolved. In group 2 (the PBS-treated control group),five of the seven rats were infected. In group 3 and 5 rats,which were given sporozoites that were previously incu-bated in either rat serum or purified rat CRP, no parasitemiawas observed. In contrast, group 4 rats, which were givenCRP that was neutralized before sporozoite incubation, wereall found to be infected.

DISCUSSION

In a previous report, we showed that low doses of naturalor recombinant IL-1 strongly inhibit the in vitro hepaticdevelopment of P. falciparum and P. yoelii, since 98%

2.5 5 25

CRP (CYf.l)

FIG. 3. Comparative study of human (O) and rat (0) CRP on

development of P. yoelii sporozoites in rat hepatocyte primaryculture.

FIG. 5. IFA of P. yoelii sporozoites. The sporozoites weretreated with human purified CRP followed by rabbit anti-humanCRP serum.

v. Inhihition

89-

709-

69-

59-

49-

39-

209-

19-

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CPI1of,CRPsund

.

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C-REACTIVE PROTEIN PROTECTS AGAINST MALARIA 281

inhibition was achieved by treating hepatocyte primarycultures with 5 IU of IL-1 per ml. Analysis of time courseexperiments indicated that IL-1 activity did not affect theparasite maturation or the sporozoite itself but was probablydue to an indirect effect on the process of liver cell invasionby the sporozoite (21).Among several hypotheses that could explain the mode of

action of the IL-1, we considered the role of the acute-phaseproteins which are synthesized at a higher rate when hepa-toma cell lines (6) or murine (28) and rabbit (16) hepatocytesare stimulated. One of the acute-phase proteins, the CRP,has been shown to play a significant role in nonspecific hostdefense mechanisms against bacteria (23, 24). In this study,we demonstrated that human hepatocytes cultured in thepresence of human recombinant IL-1 released, as early as 30min after stimulation, an increased amount of CRP. SinceCRP bound to the P. falciparum and P. yoelii sporozoitesurface membranes in a calcium-dependent manner andprobably via a phosphorylcholine-binding site, this interac-tion is similar to that of CRP with bacterial membranes.Experiments in which CRP was added to rat hepatocytemonolayers during or after inoculation with P. yoelii sporo-zoites confirmed that the CRP-mediated inhibition occurredat the very early phase of infection. Lastly, the antiplasmo-dial activity displayed by CRP was substantiated in an invivo model. Rats injected with turpentine oil, which causesa substantial increase in the CRP level in serum (14), werefound to be largely protected against P. yoelii sporozoiteinoculation. More specifically, sporozoites preincubated inacute-phase serum were no longer infectious when adminis-tered to untreated rats. This antiinfectious activity wasspecific, since it was abolished by anti-CRP antibodies andsince it could be similarly achieved by preincubation ofsporozoites with purified rat CRP. In contrast, despite astrong fixation of CRP on P. falciparum merozoites, noinhibition of the erythrocytic stages was observed in vitro.These findings indicate that CRP, by binding to sporozoite

surface components, can interfere with plasmodial infection.CRP can prevent sporozoite penetration into the hepatocyteby masking recognition sites involved in the sporozoite-hostcell interaction. Complexed CRP may also activate thecomplement system (5, 12), which, in turn, can damage ordestroy the sporozoite. Bound CRP also appears to influencethe early stages of parasite maturation after penetration intohepatocytes. Besides these mechanisms, additional CRP-triggered events may occur in vivo: CRP capacity to modu-late neutrophil functions (4) or platelet (2) or macrophage(34, 35) activity or to enhance parasite opsonisation (25) canalso modify the host-parasite interaction in favor of theformer.Whether the CRP level in the sera of patients is increased

during the acute phase of malaria infection is presently underinvestigation. If it is, this might be a mechanism of resistanceto sporozoite-induced superinfection. Similarly, it would beof interest to determine the extent to which acute-phasereactants, other than CRP, affect the nonspecific resistanceto plasmodial infection during acute inflammatory states inindividuals exposed to natural malaria infection.

ACKNOWLEDGMENTS

This research was supported in part by the United NationsDevelopment Program/World Bank/World Health Organization Spe-cial Program on Training and Research in Tropical Diseases and bythe Commission des Communautds Europeennes. S. P. is a recipientfellow of the Association des Universites Partiellement ou Totale-ment de Langue Francaise.

We are grateful to D. Frommel for a critical review of themanuscript; to Y. Charoenvit, Naval Medical Research Institute,Bethesda, Md., for the gift of monoclonal antibodies; and to J. R. L.Pinck, Hoffman-La Roche, Basel, Switzerland, for having providedthe IL-1.

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