Increased expression and secretion of ICAM-1 during experimental

infection with Trypanosoma cruzi

SUSANA LAUCELLA 1, ROSALBA SALCEDO2, ESMERALDA CASTANOS-VELEZ3, ADELINA RIARTE 1,ERNESTO H. DE TITTO1, MANUEL PATARROYO2, ANDERS ORN2 & MARTI N E. ROTTENBERG2

1 Instituto Nacional de Chagas Dr M. Fatala Chaben, Buenos Aires, Agentina2 Microbiology and Tumor Biology Center, Doktorsringen 13, Karolinska Institute, S-171 77 Stockholm, Sweden3 Immunopathology Laboratory, Karolinska Hospital/Institute, Stockholm, Sweden

SUMMARY

In the present study we demonstrate that spleens and heartsfrom BALB/c mice infected with the virulent Tulahue´n or thelow virulent CA-I strains ofTrypanosoma cruzi, containsubstantially higher ICAM-1 transcripts than uninfectedcontrols. ICAM-1 expression in heart cells was alsoincreased at the protein level, as measured by flow cyto-metry, ELISA and immunohistochemistry. The adhesivereceptor was observed not only on inflammatory cells butalso on sarcolemma of cardiac myocytes fromT. cruziinfected mice. ICAM-1 expression was higher during theacute phase than in the chronic phase of infection, andparalleled the density of inflammatory leukocytes. Elevatedtitres of soluble ICAM-1 (s-ICAM-1) were detected in serafrom mice during the acute phase of infection with CA-I orTulahuen parasites. Cytokines, including IFN-°, IL-1®, IL-6and TNF-® have been shown to modulate expression ofICAM-1. Spleens and hearts from mice infected with CA-I orTulahuen strains showed increased accumulation of mRNAsspecific for these cytokines, which peaked during the acutephase of infection. However, IFN-° activity was not neces-sary for ICAM-1 induction, as its levels were also increasedduring infection in IFN-° receptor knock-out (IFN-°Rÿ)mice. Upregulation of ICAM-1 expression might be a directconsequence of parasite infection, since its density on celllines of different lineages was enhanced after 24 or 48 h ofinfection withT. cruzi.

Keywords T. cruzi, Chagas’ disease, ICAM-1, s-ICAM-1,cytokines, interferon-°

INTRODUCTION

Trypanosoma cruzi, the causal agent of Chagas’ disease, isan obligate intracellular parasite that infects 16–20 millionpeople in Central and South America (Takle & Snary 1993).Following the initial infection, the acute phase ensues. Thisphase is characterized by the presence of numerous circulat-ing parasites and it is terminated when the humoral andcellular immune responses are potent enough to control theparasite load. Patients or experimentally infected animalsthen enter a chronic phase of infection, characterized bylow-level parasitaemia. Among millions of chronicallyinfected individuals, a fraction will develop one of severallong term sequelae of Chagas’ disease. The most serious ofthese is a persistent inflammatory cardiomyopathy thatmay lead to congestive heart failure and death. The abilityof T-cells to transfer some features of heart disease intonaive syngeneic animals (Hontebeyrie-Joskowiczet al.1987, dos Santoset al. 1992), and the presence of crossreactivity between parasite and host antigens (Petry & vanVoorhis 1991, Hernandez-Munainet al. 1992, Bonfaet al.1993), have prompted many to propose that immunemechanisms may be involved in pathogenesis of Chagas’heart disease.

Recognition of parasite antigens by the immune systemresults in the release of cytokines which play a pivotal rolein the activation of trypanocidal effector mechanisms.However, cytokines might also be required to create theappropriate milieu for immunopathology. For example,cytokines might modulate cellular recruitment and thusinfluence the intensity of inflammatory foci. They mayalso affect resident cells by inducing inappropriate expres-sion of cell-surface receptors. Expression of these moleculesin a local or systemic fashion, might convert myocardialand/ or skeletal muscle cells from immunologically inertcells into targets for cytotoxic T-cells.

The intercellular adhesion molecule-1 (ICAM-1, CD54)is a cytokine-induced glycoprotein that contains five

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Correspondence: M. E. RottenbergReceived: 22 September 1995Accepted for publication: 15 January 1996

immunoglobulin-like domains, a transmembrane region anda cytoplasmic domain. ICAM-1 is expressed on a multitudeof cell lineages and performs several vital roles in thenormal functioning of the immune system. For example,lymphocytes and other leucocytes interact with ICAM-1expressed on endothelial cells allowing the recruitment intotissues undergoing inflammatory responses. Also, T-cellrecognition of ICAM-1 on antigen presenting cells stabilizesthe cell-cell interaction and reduces the threshold forantigen-specific activation (Springeret al. 1987, 1990).Thus, ICAM-1 is involved in directing cells to sites ofinflammation, as well as in activating them once theyarrive. Moreover, ICAM-1 expression renders target cellssusceptible to lymphocyte mediated cytotoxicity (Dohlstenet al. 1991). A soluble form of ICAM-1 (s-ICAM-1) lackingthe transmembrane and intracytoplasmic domains has beenrecently described (Rothleinet al. 1991). Although thepossibility that s-ICAM-1 inhibits T-cell-target cell inter-action has been suggested by Beckeret al. (1993), thephysiological function of s-ICAM-1 is still not clear.Interestingly, its release from cells is affected by the samestimuli that upregulate cellular ICAM-1 (Rothleinet al.1991).

Whether ICAM-1-LFA-1 interaction plays a role inresistance and pathology during infection withT. cruzihasnot been elucidated.

The purpose of this study was to measure perturbations ofICAM-1 levels in myocardial cells and inflammatory leu-kocytes during infection with strains ofT. cruzipossessingdifferent virulence and tropism.

MATERIALS AND METHODS

Mice and infections

Eight-ten week old BALB/c mice were used unless other-wise indicated. C57Bl/6 mice homozygous for disruptedIFN- receptor (R) gene or for null mutation, generatedusing embryonic stem cell technology as described byHuang (1993), were used in some experiments. Groups ofmice were infected with 50 trypomastigotes of the virulentand reticulotropic Tulahue´n (Taliaferro & Pizzi 1955) or 104

low virulent and miotropic CA-I (Gonzalez Cappaet al.1980) parasites isolated from peripheral blood of infectedmice. Parasitaemia was periodically measured and mortalitywas registered.

Cell cultures

L-929 fibroblasts (ATCC CRL 6319), monocyte-like U-937(ATCC CRL 1593), myelomonocytic WEHI-3 (ATCC TIB68) and macrophage-like P388.D1 cells (ATCC CCL 46)

were maintained in DMEM medium withL-glutamine(2 mM) and supplemented with 5% fetal calf serum(GIBCO Laboratories, Paisley, Scotland), 100 U/ml penicil-lin and 100�g/ml streptomycin. When indicated, cells werecocultured with either lipopolysaccharide 500 ng/ml (LPS,Calbiochem, La Jolla, CA, USA) orT. cruzi parasites.T.cruzi trypomastigotes (Tulahue´n), collected from the super-natant of L-929 cell monolayers infected seven days before,were used forin vitro experiments.

Competitive PCR assay

The accumulation of cytokines and ICAM-1 mRNA inspleen and hearts from infected mice orin vitro infectedcell lines was measured in a qualitative and a competitivePCR assay (Gillilandet al. 1990). RNA from freshlyextracted organs or cultured cells was obtained using gua-nidinium acid thiocyanate and phenol-chloroform extraction(Chomczynsky & Sacchi 1987). 2�g of RNA were dena-tured and reverse transcribed using random hexanucleotides(N6 Pharmacia, Uppsala, Sweden). Thereafter, 10–50 ng ofcDNA were amplified in a 20�l PCR reaction mixturecontaining 2�l 10� PCR buffer (Boehringer, Mannheim,Germany), 3�l dNTP (0.125 mM), 0.1�l Taq polymerase(5 U/ml) (Boehringer), 7.5�l cDNA, and 2�l of competitorfragments with a different length but using the same primersas the target DNA. The IFN- competitor was built byligating an unrelated DNA fragment (188 bp) into a Pst siteof the subcloned PCR product. The�-actin competitor wasbuilt by deleting a MseI fragment from a plasmid containingthe whole cDNA sequence. TNF-�, IL-1-� and IL-6 com-petitors were constructed using composite primers and anexogenous DNA fragment as described (Siebert & Larrick1992). Competitors were amplified by PCR, purified(Qiagen, Studio City, Ca, USA) and quantified in a spectro-photometer.

The primer sequences for the amplification of the cDNAwere:

Sense IFN: 50 AAC GCT ACA CAC TGC ATC TTG G 30

Antisense IFN- : 50 GAC TTC AAA GAG TCT GAG G 30

Sense TNF-�. 50 ATG AGC ACA GAA AGC ATG ATCCGC 30

Antisense TNF-� 50 CC AAA GTA GAC CTG CCC GGACTC 30

Sense IL-1-� 50 ATG GCC AAA GTT CCT GAC TTGTTT 30

Antisense IL-1-� 50 C CTT CAG CAA CAC GGG CTGGTC 30

Sense IL-6 50 ATG AAG TTC CTC TCT GCA AGA GACT 30

Antisense IL-6 50 CA CTA GGT TTG CCG AGT AGATCT C 30

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Sense ICAM-1 50 GAT GCT CAG GTA TCC ATC CAT C 30

Antisense ICAM-1 50 CAC CGT GAA TGT GAT CTCCTT G 30

Sense�-actin: 50 GTG GGC CGC TCT AGG CAC CAA 30

Antisense�-actin: 50 CTC TTT GAT GTC ACG CACGAT TTC 30.

Ten or three-fold serial dilutions of the competitor wereamplified in the presence of a constant amount of cDNA.Reactions were carried out for 28 to 45 cycles in a thermalcycler (Perkin-Elmer Cetus, Ct, USA) using an annealingstep at 55oC (for IFN- ) or 60oC (IL-1�, IL-6, TNF-� or�-actin). The PCR products were then resolved by 1.8%agarose gel electrophoresis and photographed. The nega-tive of the photograph was analysed by laser densitometry,and the absolute values of the PCR products were deter-mined. The ratios of the absorbance of the relevant PCRproduct pairs were plotted against the concentration of thecompetitor DNA used. The intersection curve where theamounts of target and competitor are equal was extra-polated to the x-axis (concentration of the competitoradded).

Determination of soluble and cellular ICAM-1

A capture ELISA was used to measure levels of s-ICAM-1in the serum from individual mice. Anti murine ICAM-13E2 MoAb (4�g/ml) (Pharmigen, San Diego, CA, USA)was used as capture antibody , and biotinylated anti mouseICAM-1 (MALA-2) (5 �g/ml) (Prietoet al. 1989) was usedfor detection. The subsequent addition of peroxidase-conjugated streptavidin (Dakopatts, Glostrup, Denmark),followed by the substrate (o-phenylen-diamine, Sigma, StLouis, MO, USA), yielded a colorimetric result which wasmeasured at 492 nm. Quantification of s-ICAM-1 was per-formed by comparing to a standard curve using mouserecombinant soluble ICAM-1 (Welder, Lee & Takei 1993),a gift of Dr F. Takei, Terry Fox Laboratory, Vancouver,Canada. Results are expressed as nanograms of s-ICAM-1per ml of serum.

A similar assay was used to determine the concentration ofcellular ICAM-1 in tissues after Triton-X100 solubilization.In this case results are expressed as nanograms of ICAM-1per mg of protein as measured at 280 nm.

Immunohistochemistry assay

Freshly dissected hearts were frozen in liquid nitrogen.Cryostat sections (8�M thick) were prepared, air driedand fixed in acetone for 10 min. They were blockedfor endogenous peroxidase activity with 0.3% H2O2 inTris saline buffer pH: 7.5 containing 1% bovine serum

albumin. After washing with Tris saline buffer, sectionswere incubated with rat anti-murine ICAM-1 (MALA-2) ornormal rat serum as negative control for 60 min at roomtemperature. Thereafter, slides were stained withbiotinylated rabbit anti rat-IgG antibody, washed andincubated with avidin-biotinylated peroxidase complex(ABComplex-HRP, Dakopatts). Specific staining wasvisualized using diaminobenzidine as chromogen. Slideswere counterstained with haematoxylin and mounted withresin.

Quantification of tissular parasitism and lesions

Hearts were fixed in 10% neutral buffered formalin andprocessed for conventional paraffin embedding. The wholeheart was cut sagitally into two parts, each containing thefour cavities. Sections were cut at 2�m, deparaffinized andstained with haematoxylin and eosin. A single blind micro-scopic evaluation of the two sets of serial sections from eachorgan was performed in precoded slides. Histologicalanalysis was performed by histocytometry with the aid ofan ‘integrationsplatte’ eyepiece (Zeiss) with 100 hits. Pre-sence or absence of inflammatory cells for every hit wasscored. Presence of amastigote nests and number of nuceliof cardiocytes per field were also recorded. Thirty-fields/histological section were counted in each section using anOlympus microscope with an objective lens of�40, cover-ing an area of 5 mm2.

Flow cytometric analysis

Hearts from infected mice or controls were removed andminced. Cell suspensions were obtained after treatment with1 mg/ml collagenase (Sigma) for 15 min at 37oC in agyratory shaker. Residual multicellular aggregates wereremoved by sedimentation thereafter. Heart cells werewashed twice with 0.5% BSA in PBS containing NaN3and then stained for ICAM-1 using biotinylated anti-ICAM-1 antibody (MALA-2), followed by streptavidin-phycoerythrin (Dakopatts). Control samples without thefirst antibody were also analysed by flow cytometry.ICAM-1 expression on cell lines was measured in a similarway. LB-2 mouse anti human MoAb was used for stainingof ICAM-1 on U-937 cells.

RESULTS

Expression and secretion of ICAM-1 during infectionwith T. cruzi

All mice infected with 104 CA-I parasites were

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Figure 1 Kinetics of parasitaemia duringmurine infection withT. cruzi.The meanparasitaemia�SE of the mean from BALB/cmice (seven animals per group) infected witheither 50 Tulahue´n or 104 CA-I bloodstreamform trypomastigotes are depicted.gTulahuen; [ CA-I.

Figure 2 Immunofluorescence analysis of ICAM-1 expression on cardiocytes from mice after 0 or 20 days of infection withT. cruzi, Tulahuenstrain. Hearts were minced and treated with collagenase for 15 min, 378C. Cell suspensions from Tulahue´n-infected (20 days) or non-infected micewere stained with biotinylated anti-ICAM-1 MoAb (MALA-2) followed by streptavidin-FITC before analysis in a flow cytometer. A controlwithout addition of the first antibody is also depicted.

alive at the end of the experimental period (150 daysof infection). Parasitaemia peaked one month afterinfection and slowly declined thereafter (Figure 1). Onthe contrary, 20–40% of mice died after infectionwith 50 Tulahue´n parasites. Parasitaemia, which washigher as compared to CA-I infection, peaked threeweeks after infection (Figure 1). The number of parasitenests in the heart was also higher in Tulahue´n (0.60�0.16 amastigote nests per 1000 cardiocytes at the peakof parasitaemia,n�8) than in CA-I infected mice(0.06� 0.04, n�8) (P < 0.05 Mann-Whitney-WilcoxonU-test).

Heart lysates from mice during the acute phase ofinfection with the two different strains ofT. cruzi showedincreased ICAM-1 expression, as measured by a sandwichELISA (Figure 2; Table 1). During the chronic phase ofinfection ICAM-1 levels were only slightly elevated, ifaltered at all (Table 1). A fraction of cardyocytes in a heartcell suspension obtained during the acute phase ofT. cruziinfection showed an intense staining of ICAM-1 as deter-mined by flow cytometry (Figure 2). Mean fluorescenceintensity was 350�48 (four individual mice) at 0 days,675�118 (4) at 20 days and 437�8 (3) 150 days afterinfection with Tulahue´n.

ICAM-1 immunostaining was low or undetectable onsarcolemma of cardiac myocytes and fibroblasts from unin-fected mice, while endocardium and capillary endotheliawere stained with the specific antibody (Figure 3). Incontrast, ICAM-1 was expressed on the sarcolemma ofcardiac myocytes obtained 20 days after infection withCA-I or Tulahuen, in parallel with the presence of extensivecell infiltrations (Figure 3, Table 1). Presence of ICAM-1 atthis time after infection was observed non-uniformly overthe myocardium, and was stronger around the areas ofinflammation (Figure 3). Mice chronically infected withTulahuen or CA-I showed weaker or no immunostainingfor ICAM-1, and reduced inflammatory foci as compared toanimals during the acute phase of infection (data notshown).

The presence of ICAM-1 transcripts in hearts andspleens from mice infected withT. cruzi was assessed byqualitative PCR. The amount of mRNA present in thereactions was standardized to equal amounts of RNAencoding �-actin. Organs from mice during the acutephase of infection with CA-I or Tulahue´n showed higheramounts of ICAM-1 mRNA than those from non infectedmice (Figure 4).

Sera from mice during the acute phase of infection withCA-I or Tulahuen contained elevated levels of s-ICAM-1(Figure 5). High s-ICAM-1 levels were also measured insera from mice chronically infected with CA-I. On thecontrary, s-ICAM-1 levels in sera obtained after 60 days

of infection with Tulahue´n were similar to those from non-infected controls (Figure 5).

Levels of cytokine transcripts in hearts and spleensfrom T. cruzi infected mice

Cytokines have been shown to modulate expression ofdifferent cell adhesion molecules, including ICAM-1.Increased accumulation of IFN- , IL-1�, IL-6 and TNF-�mRNAs was detected in hearts and spleens from miceduring the acute phase of infection with CA-I or Tulahue´n,as measured by qualitative and competitive PCR (Figure 6,Table 2). Levels of cytokine transcripts decreased thereafterif infected with the Tulahue´n strain ofT. cruzi,but remainedelevated in spleens, and to a lower extent in hearts, frommice infected with CA-I (Figure 6, Table 2).

Role of IFN- in expression of ICAM-1 duringinfection with T. cruzi

We analysed whether expression of ICAM-1 was dependenton the presence of IFN- , a cytokine that plays a pivotal rolein the outcome of infection withT. cruzi. This appeared notto be the case, since IFN- R-mice, displaying a dramati-cally enhanced susceptibility to infection withT. cruzi asmeasured by parasitaemia and cumulative mortality,showed enhanced expression of the adhesive receptorduring infection as compared to non infected controls(Figure 7). ICAM-1 was expressed by inflammatory cells

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Table 1 ICAM-1 levels and density of inflammatory leucocytes inhearts from mice duringT. cruzi infection

Week of ICAM-1 ng/mg Lesion areainfection Strain protein (%)

0 – 4.7�0.8 0.6�0.43 CA-I 9.2�0.9* 7.0�3.2*3 Tulahuen 7.8�0.8* 3.1�1.1*

21 CA-I 5.8�1.1 1.5�0.3*21 Tulahue´n 4.5�1.5 1.0�0.3

A sandwich ELISA was used to measure the concentration ofICAM-1 in detergent lysates of hearts fromT. cruzi-infected or non-infected mice. Data shows the mean nanogram of ICAM-1 per mg oftotal protein�SEM in heart tissue from individual mice (4–5animals per group), at 0, 20 or 150 days after infection with Tulahue´nor CA-I parasites. The lesion area was measured in formaline fixed,paraffin embedded sections. Results are expressed as mean ofinflammatory cells per area (%)�SEM from 6–8 mice per group.* Differences with non-infected controls are significant (P < 0.05,Mann-Whitney-Wilcoxon,U-test).

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Figure 3 Immunohistochemical study of ICAM-1 expression. Ventricular myocardium from non infected mice was stained with anti-ICAM-1MoAb (a). Only undetectable levels were observed on sarcolemma of cardiac myocytes. On the contrary, vascular endothelial cells (a) andendocardium (not shown) expressed the receptor. Ventricular myocardium from mice infected (20 days) with Tulahue´n (b) or CA-I (c) stainedwith ICAM-1. Note the clear reactions of sarcolemma of cardiac myocytes (b, c) and infiltrating cells (c).

Figure 4 Reverse transcriptase-PCR analysis of ICAM-1 mRNA expression in spleens and hearts from mice 0 or 33 days after infection with CA-Ior Tulahuen parasites. Each lane represent a single mouse. The amount of mRNA present in the reactions was previously standardized to equalamounts of RNA encoding�-actin as previously measured in a competitive PCR assay.

Figure 5 Levels of serum s-ICAM-1 in mice infected withT. cruzi.A sandwich ELISA was used to measure s-ICAM-1 levels in mice at differenttime points after infection with (a) CA-I or (b) Tulahue´n. Each point represents one mouse and the bars represent mean�SEM. Levels ofs-ICAM-1 from mice infected with CA-I are higher than those from the non-infected group (P < 0:05 Mann-Whitney-WilcoxonU-test). s-ICAM-1levels were also increased 12, 24 and 30 days after infection with Tulahue´n as compared to the non-infected group (P<0.05 Mann-Whitney-Wilcoxon U-test).

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Figure 6 Qualitative PCR analysis of IFN- , IL-1-�, IL-6 and TNF-� mRNA accumulation in spleens (a) and hearts (b) from mice duringinfection with CA-I or Tulahue´n parasites. The amount of mRNA present in the reactions was standardized to equal amounts of RNA encoding�-actin previously measured in a competitive PCR assay.

as well as cardiac myocytes in hearts obtained from IFN- R– mice (Figure 7). The density of inflammatory cells inhearts obtained from IFN- Rÿ mice (16.1� 4.0 % n�4)was similar to that in wild type infected controls (15.8� 2.2% n� 4).

Expression of ICAM-1 after in vitro infection withT. cruzi

We then addressed whether enhanced ICAM-1 expressionin vivo was a direct consequence of parasite infection.We showed that human (U-937) or murine (WEHI andP-388.D1) cell lines had increased surface expression ofICAM-1 after 24h (U-937) or 48 h (WEHI-3 and P388.D1)of infection with T. cruzi trypomastigotes (Table 3).Coincubation with non infective epimastigotes fromT. cruzi, was on the contrary not able to induce ICAM-1expression (data not shown). ICAM-1 was constitutivelyexpressed by all cell lines and coculture with LPS, aknown stimuli of ICAM-1, elicited an enhanced expres-sion of the receptor (Table 3). P.388.D1 cells alsoshowed enhanced accumulation of ICAM-1 mRNA at 24and 48 h but not at 4 h after infection withT. cruzi(Figure 8). The amount of mRNA present in the reactions

was standardized to equal amounts of RNA encoding�-actin (Figure 8).

DISCUSSION

In this study we demonstrate that heart cells from miceinfected with different strains ofT. cruziexhibited enhancedexpression of ICAM-1, as visualized by immunohisto-chemistry and measured by flow cytometry and captureELISA assays, or by the steady-state levels of ICAM-1mRNA. We observed no correlation of ICAM-1 expressionwith number of circulating parasites, tissue parasitism in theheart and virulence of the infecting strain. However, presenceof ICAM-1 paralleled the intensity of inflammatory foci inthe same tissue. Both number of inflammatory cells andexpression of ICAM-1 peaked early after infection anddecreased thereafter. During the acute phase of infectionICAM-1 was not only detected on infiltrating leucocytesbut also on sarcolemma of cardiocytes. Mice chronicallyinfected withT. cruzishowed weaker or no immunostainingfor ICAM-1. Accordingly, ICAM-1 expression was detectedon inflammatory leucocytes, but not on myocardiocytes frompatients with chronic Chagas’ disease (d’Avila-Reiset al.1993). Thus, our data does not suggest a role for ICAM-1 in

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Table 2 Cytokine mRNA levels in the spleen or heart during infection with Tulahue´n or CA-I.

A: Spleen Moles of cytokine mRNA per mole of�-actin mRNA

IFN- TNF-� IL-1-� IL-6Weeks afterinfection Tulahue´n CA-I Tulahuen CA-I Tulahuen CA-I Tulahuen CA-I

0 <0.42 0.64 <0.44 0.22 0.36 0.58 <0.02 nd1 0.37 nd 0.27 nd. 0.18 nd 0.03 nd3 1.93 0.64 0.51 0.38 2.31 1.00 0.14 nd5 1.63 5.86 1.17 1.14 0.11 1.00 0.73 nd

>9 0.20 10.16 0.73 3.47 0.59 1.73 0.01 nd

B: Heart Moles of cytokine mRNA per mole of�-actin mRNA

IFN- TNF-� IL-1-� IL-6Weeks afterinfection Tulahue´n CA-I Tulahuen CA-I Tulahuen CA-I Tulahuen CA-I

0 <0.62 <1.16 <0.58 <0.66 0.33 <0.34 <0.006 0.031 <1.14 nd 0.98 nd 0.33 nd 0.03 nd3 0.62 5.85 1.22 1.95 0.41 1.00 0.41 0.905 3.33 16.43 nd 10.42 2.82 0.99 0.42 1.55

>9 <1.14 3.52 0.46 3.47 <0.33 0.56 0.06 0.52

Total RNA was obtained from spleens (A) or hearts (B) pooled from three mice and transcribed into cDNA. Two�g of cDNA were amplified withIFN- , TNF-�, IL-1�, IL-6 or �-actin primers in the presence of three-fold serial dilutions of the respective competitors. The moles of cytokinemRNA per mole of�-actin mRNA, of one of two independent experiments are depicted.

chronic Chagas’ disease, but rather in the acute phase ofinfection. Whether events during the acute phase of infectionmodulate the outcome of the chronic stage is still unclear.Myocardial cells expressing ICAM-1 duringT. cruzi infec-tion could become potential targets for NK- and T-cellmediated cytotoxicity. Increased ICAM-1 expression inheart cells has been previously described in associationwith inflammatory conditions. For example, ICAM-1 iselevated in murine and human myocarditis (Sekoet al.1993, Toyozakiet al. 1993) and levels in endobiopsies arecorrelated with cardiac allograft rejection (Sekoet al. 1993,Toyozaki et al. 1993). Moreover, the severity of allograftrejection and cell infiltration during viral myocarditis werereduced when ICAM-1 was blocked by administration ofspecific antibodies, indicating the involvement of the adhe-sion molecule in these events (Isobeet al. 1992, Sadahiro,McDonald & Allen 1993). However, induction of ICAM-1expression might be also involved in adhesive interactions

resulting in activation of specific lymphocytes that participatein the control ofT. cruzigrowth.

Sera from mice during the acute phase of infectionwith CA-I or Tulahuen contained elevated amounts ofs-ICAM-1 as compared with those from non-infected con-trols. We found no correlation between levels of s-ICAM-1with the virulence of the infecting parasite and the kineticsof parasitaemia. However, while levels of s-ICAM-1returned to control values after 60 days of infection withTulahuen, titres were still elevated after 150 days ofinfection with CA-I. In accordance, preliminary resultsindicate that s-ICAM-1 levels are increased in sera frompatients with acute Chagas’ disease (Laucellaet al. unpub-lished results). If s-ICAM-1 is a hallmark for inflammatoryprocess, parasite strain dependent differences might berelated to the presence of infiltrating leucocytes in tissuesother than heart.

ICAM-1 is induced on target cells by pro-inflammatory

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Figure 7 Modulation of ICAM-1 expression during infection withT. cruziof IFN- R-mice. (a) Kinetics of parasitemia in IFN- Rÿ (g) and wildtype mice (IFN- R+,l) (seven per group) infected intraperitoneally with 104 CA-I bloodstream forms ofT. cruzi.In parenthesis cumulativemortality at 50 days after infection. One of two representative experiments is depicted. * Differencesvswild type controls were significant(P < 0.05, Mann-Whitney-U-Wilcoxon test). Immunohistochemical study of ICAM-1 expression in hearts obtained IFN- Rÿ (b, d) or wild typemice (c) at 0 (b) or 30 (c, d) days after infection with 104 CA-I parasites.

cytokines such as IFN- , TNF-�, IL-1-� and IL-6 (Dustinet al. 1986, Rothleinet al. 1988, Prietoet al. 1992). Serafrom mice infected withT. cruzishow higher IFN- , TNF-�and IL-6 levels (Nabors & Tarleton 1991, Truyenset al.1994, Riveraet al. 1995), in parallel with higher amounts ofIFN- mRNA in spleens (Nabors & Tarleton 1991). How-ever, cytokine expression duringT. cruziinfection has beenproven to be organ-specific (Lafailleet al. 1990, Rottenberget al. 1993). We now show that accumulation of IFN- ,TNF-�, IL-6 and IL-1� mRNAs is enhanced in heart andspleen from mice during acute infection with CA-I orTulahuen. Accumulation of these cytokine transcriptsduring the chronic phase exhibits a differential organ loca-lization, somewhat related with the biological characteris-tics of the infecting strain (Gonzalez-Cappaet al. 1980).Lymphocytes infiltrating the heart might be locally releasingcytokines, which, in turn, through ICAM-1 induction, might

facilitate their continuous recruitment and activation, result-ing in either pathology or parasite control. Of such cyto-kines, IFN- has been demonstrated to play a relevant rolein the control of infection withT. cruzi(Reed 1988, Torricoet al. 1991, Petrayet al. 1993). However, IFN- was notnecessary for ICAM-1 enhanced expression, neither oninfiltrating cells nor on cardiocytes, as mice geneticallydeleted of IFN- R also exhibited a similar pattern ofICAM-1 staining as compared to wild type controls afterT. cruzi infection.

In vitro infection withT. cruzienhanced the expression ofICAM-1 in human and murine cell lines in a time dependentfashion. TNF-�, IL-1 and IL-6 which are secreted bymonocytes after infection withT. cruzi in vitro mightaccount for the increased expression of ICAM-1 at themRNA and protein levels inin vitro infected cells. Thelack of correlation between parasitaemia levels and ICAM-1expressionin vivo, and above all the small percentage ofinfected cardyocytes, suggest that cytokines produced byinflammatory cells rather than a direct effect of parasiteinfection are of importance in induction of ICAM-1. How-ever, parasite-induced ICAM-1 might play a relevant role inrecruitment and triggering of inflammatory cells early afterinfection withT. cruzi.

In summary, we demonstrate that ICAM-1 expression isinduced on myocardial cells and high s-ICAM-1 levels arepresent in serum during infection with different strains ofT. cruzi. Parasite infection of host cells is a direct stimulusfor increased expression of the adhesion molecule. ICAM-1expression is paralleled by the accumulation of infiltratingleucocytes and inflammatory cytokines. It remains to beclarified whether increased cellular and s-ICAM-1 levelshave a causative relation to resistance and/or pathology ofT. cruzi infection.

ACKNOWLEDGEMENTS

This work was supported by the Swedish Agency forResearch Cooperation with Developing Countries (SAREC).

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Table 3 ICAM-1 expression after infection withT. cruzi in vitro

Cell lines (mean fluorescenceintensity)

Treatment Anti-ICAM-1 WEHI-3 P388.D1 U-937

None No 26 104 19T. cruzi No 28 173 27None Yes 215 561 108LPS 24 h Yes 389 1099 167T. cruzi24 h Yes 255 648 199T. cruzi48 h Yes 411 1233 nd

Immunofluorescence analysis of ICAM-1 expression on monocyticcell lines after 0, 1 or 2 days of infection withT. cruzi, Tulahuenstrain. Cells (106 per ml) were infected with culture derivedtripomastigotes (3� 106 per ml) or cocultured with 0.5�g/ml LPS.Cells were stained with biotinylated anti-murine ICAM-1 MoAb(MALA-2) (WEHI-3 and P388.D1) or anti-human ICAM-1 (MoAb)(LB2) followed by streptavidin-phycoerythrin before analysis in aflow cytometer. A control without addition of the first antibody isalso shown. One of two representative experiments for each cell lineis depicted.

Figure 8. Reverse transcriptase-PCR analysisof ICAM-1 mRNA expression in P388.D1 cellsat 0, 4, 24 or 48 h after infection withT. cruzi(Tulahuen). P388.D1 cells (106 per ml) wereinfected with culture derived trypomastigotes(3�106 per ml). At the indicated time pointscells were washed and RNA was extracted asdescribed in materials and methods. Theamount of mRNA present in the reactions waspreviously standardized to equal amounts ofRNA encoding�-actin as previously measuredin a competitive PCR assay.

The IFN- R– mutant mice were kindly provided by Dr MichelAguet (Institute of Molecular Biology, University of Zu¨rich,Switzerland). We would also like to thank Dr Fumio Takei(Terry Fox Labs, British Columbia Cancer Agency) for the giftof recombinant soluble ICAM-1 and Dr Robert Harris forcritical reading of the manuscript.

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