rickettsia conorii infection enhances vascular cell adhesion

1142

Rickettsia conorii Infection Enhances Vascular Cell Adhesion Molecule-1– andIntercellular Adhesion Molecule-1–Dependent Mononuclear Cell Adherence toEndothelial Cells

Francoise Dignat-George, Nadine Teysseire, Laboratoire d’Hematologie et d’Immuno-Hematologie, Unite deFormation et de Recherche (UFR) de Pharmacie-27, and Unite desMurielle Mutin, Nathalie Bardin, Ghislaine Lesaule,

Rickettsies, Unite Propre de Recherche et d’Enseignement SuperieurDidier Raoult, and Jose SampolAssocies–0054, UFR de Medecine-27, Marseille, France

Leukocyte adherence to the endothelium is an essential component of the inflammatory responseduring rickettsial infection. In vitro, Rickettsia conorii infection of endothelial cells enhances theexpression of adhesive molecules E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascu-lar cell adhesion molecule-1 (VCAM-1) in a time- and dose-dependent manner. Rickettsial lipopoly-saccharide does not seem to be involved, because polymyxin B does not reduce their expression.The intracellular presence of the organism and de novo host protein synthesis are required forexpression of cell adhesive molecules, since rickettsial inactivation by formol and pretreatment ofcells with cycloheximide inhibits an increase in expression. The contribution of interleukin-1a (IL-1a) to this endothelial adhesive phenotype was shown by inhibitory experiments 8 and 24 h afterinfection with IL-1 receptor antagonist and IL-1a blocking antibodies. Enhanced adherence ofmononuclear cells to infected endothelial cells involved VCAM-1– and ICAM-1–dependent mecha-nisms at the late phase of the inflammatory response. This endothelial adhesive phenotype mayconstitute a key pathophysiologic mechanism in R. conorii– induced vascular injury.

Mediterranean spotted fever is caused by Rickettsia conorii, highly regulated and that it controls the nature of leukocyterecruitment in response to cytokines, such as interleukin-1which is usually transmitted by the tick Rhipicephalus san-

guineus [1, 2]. At the site of inoculation, the microorganisms (IL-1) or tumor necrosis factor [8, 9]. Early expression ofE-selectin mediates the adherence of polymorphonuclear cells,disseminate in the circulating blood to enter endothelial cells

(EC), causing generalized vasculitis and inflammatory response whereas late expression of ICAM-1 and VCAM-1 lasts §24h [12–14], resulting in increased adherence of mononuclear[2–5], with increased vascular permeability and local accumu-

lation of blood leukocytes in the surrounding tissues. Inflam- cells (MNC), but not of neutrophils [15–18].The ability of Rickettsia rickettsii to induce E-selectin ex-matory infiltrates in skin biopsy samples of typical tache noire

have been characterized for human R. conorii infection [6] and pression and early polymorphonuclear cell adherence on cul-tured EC has been described [19]. In the present study, wefor experimental guinea pig skin lesions [7]. These studies

showed that early perivascular neutrophilic infiltration was fol- used a similar in vitro model of endothelial infection to investi-gate both the early and late events during infection withlowed 24 h later by dense accumulation of lymphocytes and

monocytes, which were often associated with focal vascular R. conorii. We studied E-selectin, VCAM-1, and ICAM-1 mod-ulation by R. conorii and its consequences with regard to theinjury.

EC play an active role in the local initiation and amplification adhesiveness of infected EC for MNC.of the inflammatory process [8, 9] by expressing leukocyteadhesion molecules that appear on the luminal surface in re-

Materials and Methodssponse to inflammatory stimuli. The best known molecules arereceptors belonging to the selectin family (E-selectin, Monoclonal antibodies (MAbs) and reagents. E-selectinP-selectin) and the immunoglobulin superfamily (intercellular (CD62E), VCAM-1 (CD106), and ICAM-1 (CD54) expressionadhesion molecule [ICAM]-1 and -2, and vascular cell adhesion was measured by using MAbs 1–2 B6 (IgG1), 1G11 (IgG1; Immu-

notech, Marseille), and F2b 1-8 (IgG1; Biocytex, Marseille) asmolecule [VCAM]-1) [10, 11]. In vitro studies have shownfirst-layer MAbs and fluorescein isothiocyanate F(ab�)2 sheep anti-that the expression of E-selectin, VCAM-1, and ICAM-1 ismouse IgG (Silenus, Hawthorn, Australia) as second-layer MAb.The 6D1 MAb that binds to bladder cancer cells but not to ECwas used as an isotype-matched MAb of irrelevant specificity (6D1

Received 8 January 1996; revised 25 November 1996. MAb) [20]. Recombinant human IL-1 receptor antagonist (IL-1Reprints or correspondence: Prof. Jose Sampol, Laboratoire d’Hematologie Ra) and polyclonal antibodies neutralizing anti-human IL-1a and

et d’Immuno-Hematologie, U.F.R. de Pharmacie, 27, Bd. Jean Moulin, 13385IL-1b were obtained from British Biotechnology (Abingdon,Marseille Cedex 5, France.Oxon, UK), cycloheximide and polymyxin B sulfate from Sigma

The Journal of Infectious Diseases 1997;175:1142–52(Grenoble, France), and Ficoll-Paque from Pharmacia Fine Chemi-� 1997 by The University of Chicago. All rights reserved.

0022–1899/97/7505–0016$01.00 cals (Uppsala, Sweden).

/ 9d27$$my19 04-07-97 09:13:17 jinfa UC: J Infect

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1143JID 1997;175 (May) R. conorii Infection and Endothelium Adherence

Figure 1. Flow cytometric analysis of control(A) and R. conorii–infected endothelial cells (EC;B), after staining with anti–E-selectin monoclonalantibody (MAb; 1-2B6, 10 mg/mL), anti–VCAM-1 (MAb 1G11, 10 mg/mL), anti–ICAM-1 (MAbF2b-1-8, 10 mg/mL), and irrelevant (IR) MAb(6D1, 10 mg/mL). In this representative experi-ment, 70% of 8-h infected EC in positively stainedpeak induced E-selectin expression. After infectionfor 24 h, ú80% of infected EC had increasedVCAM-1 and ICAM-1 expression.

EC culture. Human umbilical vein EC (HUVEC) were har- R. conorii. R. conorii (ATCC ‘‘7’’ strain VR617) was grownin Vero cells. When the Vero cells were highly infected, they werevested from fresh human umbilical cord veins as described by

Jaffe et al. [21]. The cells were maintained and subcultured in harvested by use of glass beads and disrupted by passage througha needle. Cell debris was eliminated by low-speed centrifugation.RPMI 1640 (Sigma) containing 20% fetal calf serum (FCS;

Dutscher, Brumath, France) and EC growth supplement (1.5 Purified rickettsiae were collected in the supernatant and placedin vials, which were incubated with 1 mL of serial dilutions ofmg/mL; Sigma) without antibiotics. All culture reagents were endo-

toxin-free. We used only 100% pure cultures as assessed by mor- the inoculum, and shown by indirect immunofluorescence with ananti–R. conorii rabbit immunoglobulin (1/200 in PBS) and goatphologic and immunologic criteria (von Willebrand’s factor ex-

pression). When confluent, HUVEC were washed with PBS and anti-rabbit immunoglobulin conjugated to fluorescein isothiocya-nate (FITC 1/200 in PBS; Immunotech). We determined the titerinfected with R. conorii. In some experiments, HUVEC were cul-

tured with cycloheximide (20 mg/mL) or polymyxin B sulfate of the inoculum infectivity by counting the plaques per vial, whichwere expressed as plaque-forming units per milliliter [22]. Titers(7 mg/mL) added 1 h before infection and throughout postinfection

incubations or with IL-1 Ra (100 ng/mL) and anti-human IL-1a were 107–108 pfu/mL in the following experiments.In inoculum dilution assays, negative controls were HUVEC-and IL-1b neutralizing antibodies (10 mg/mL) added to the culture

during rickettsial infection. For each experiment, viability of EC cultured with uninfected Vero cells harvested by the same proce-dures and materials as used for the inoculum. In some experiments,was determined by trypan blue dye exclusion test (for EC count)

and by iodide propidium staining (for flow cytometry analysis). infection was performed with formalin-fixed R. conorii that were



1144 Dignat-George et al. JID 1997;175 (May)

Figure 2. Time course of E-selectin, VCAM-1, and ICAM-1 expression on R. conorii (RC)–infected endothelial cells (EC) vs. uninfectedcells (C). EC were stained by indirect immuno-fluorescence to determine E-selectin expression(A) and VCAM-1 and ICAM-1 expression (B).Antigenic densities were measured by flow cy-tometry (quantitative indirect immunofluores-cence assay) 4, 8, 12, and 24 h after infection.Results are antigenic sites/cell (mean { SD) of5 determinations.




Figure 3. Effect of inoculum dilution on E-selectin (A) andVCAM-1 and ICAM-1 (B) expression. Endothelial monolayers wereincubated with pure infectious inoculum (107 –108 pfu/mL) and after1/10 and 1/100 dilutions were stained to determine E-selectin, VCAM-1, and ICAM-1 expression as in figure 1. After infection times of 8h for E-selectin and 24 h for VCAM-1 and ICAM-1, no. of antigenicsites/cell was measured. Results are mean { SD of 4 experiments. Põ * .05, ** .005, *** .001, significant difference from control (C).RC, R. conorii; NS, not significant.

fixed by incubating purified rickettsiae with 0.1% formaldehydeovernight. The fixative was removed by three successive centrifu-gations and rickettsiae were washed in PBS. In formalin assays,HUVEC were exposed to the same number of fixed rickettsialparticles as the control.

Immunofluorescence and quantitative flow cytometry analysis.Figure 4. Effect of uninfected Vero cells or polymyxin B treat-Infected or uninfected HUVEC were rapidly detached by treatmentment on E-selectin, VCAM-1, and ICAM-1 expression. Humanwith 1 mL of a solution of 0.05% trypsin and 0.53 mM EDTA forumbilical vein endothelial cells were treated with culture medium30 s at 37�C. After trypsin neutralization, indirect immunofluores-alone (C), uninfected lysed Vero cells (V), or purified R. conoriicence labeling was done as previously described, with first-layer(RC) rickettsiae in absence (0) or presence (/) of polymyxin B,

MAbs under saturating concentrations (10 mg/mL for each MAb) then stained at 8 h for E-selectin (A) and at 24 h for VCAM-1and fluorescein-labeled MAbs at 1/100 dilution as the second (B) and ICAM-1 (C) as described in figure 1. Results are no. oflayer [22]. antigenic sites/cell (mean { SD of 4 different experiments). Only

For flow cytometry, we used an Epics Profile II equipped with statistical analysis of experiments done in absence of polymyxinfour decade logarithmic amplifiers (Coulter, Margency, France) as with C vs. uninfected V or vs. purified RC are shown. P õ * .05,

significant difference from control; NS, not significant.described [22]. Mean fluorescence intensity of the positive popula-




nate–conjugated goat anti-mouse IgG (1/200). Coverslips werethen mounted in 50% glycerol–PBS. Cell preparations were as-sessed with a Leica confocal microscope.

MNC adhesion assay. MNC were isolated from freshly drawnheparinized blood by Ficoll-Paque gradient centrifugation as de-scribed [24]. The resulting cell suspension contained ú99% mono-cytes and lymphocytes (by Coulter counter; Coultronics,Margency, France). MNC adherence to endothelial monolayerswas quantitated by a fluorescent plate reader. In brief, MNC werestained with 2�,7�-bis-(2-carboxyethyl)-5 and 6-carbofluoresceinacetoxymethyl (BCECF; molecular weight, 821 g/mol; MolecularProbes, Eugene, OR) for 30 min at 37�C and then washed twiceat 4�C with PBS supplemented with 0.02% bovine serum albumin.Cell suspension was adjusted to 5 1 105/mL in RPMI containing10% FCS and maintained at 4�C. Viability was ú95% after MNCwere stained. Aliquots (100 mL) of stained MNC were layeredon uninfected or infected HUVEC plated in 96-well flat-bottomfibronectin-coated plates and incubated for 30 min at 37�C under5% CO2. Nonadherent MNC were removed by two gentle wash-ings.

The remaining fluorescence was determined by plate reader(Fluoroscan; Labsystems, Helsinki). The excitation and emissionfilters, respectively, had band widths of 20 and 25 nm and werecentered at 485 and 530 nm, which corresponds well with BCECFexcitation and emission maxima. Results were expressed in arbi-trary units of fluorescence intensity (mean of 10 wells measuredin 4 experiments). In the same experiments, MNC adherence wasinhibited by preincubating HUVEC for 30 min at 37�C with acombination of anti–E-selectin (1-2 B6), anti–VCAM-1 (1G11),or anti–ICAM-1 (F2b 1-8) MAbs or with individual MAbs. TheseMAbs, which have been characterized for their blocking properties[25–27], were used at 10 times the saturating dilution as deter-Figure 5. Effect of formaldehyde-fixed rickettsiae on E-selectin,mined by flow cytometry and were present during the adherenceVCAM-1, and ICAM-1. Endothelial cell (EC) cultures were infectedassay and during the preincubation step. Inhibition experimentsfor 8 (A) or 24 h (B) with live R. conorii (RC) or formaldehyde-with irrelevant MAb 6D1 were performed concurrently under thefixed RC (RC / formaldehyde) as described in Methods. For each

condition, E-selectin, VCAM-1, and ICAM-1 expression was mea- same conditions.sured. Results are mean { SD of 3 different experiments. P õ * .05, Statistical analysis. Results are shown as the mean { SD of*** .005, **** .0005 significant difference from control (C); NS, not three to five experiments. Statistical analysis was done by Student’ssignificant. t test and Mann-Whitney test. Differences were considered signifi-

cant at P õ .05.

tion was measured. Antigen expression was quantified by the QIFItechnique [23], which is based on the linear relationship between Resultsmean fluorescence intensity and the number of antigenic sites per

E-selectin, VCAM-1, and ICAM-1 expression were increasedcell (antigen density). Fluorescence intensity is converted into anti-on R. conorii– infected HUVEC. Cell adhesive moleculegen density by using standards represented by microbeads (Qifikit;

Dako, Trappes, France) labeled in conjunction with the cells. Fluo- (CAM) expression on infected and uninfected HUVEC wasrescence intensity measured by different standards was used to studied by flow cytometry after indirect immunofluorescencecalculate the standard regression line, thus allowing conversion of staining with MAbs against E-selectin (1-2B6), VCAM-1the fluorescence intensity of the positive population into antigen (1G11), and ICAM-1 (F2B1-8). Compared with controls (figuredensity (expressed in sites per cell). 1A), fluorescence histograms obtained at 8 (E-selectin) and

Immunofluorescence and confocal microscopic analysis. Cells 24 h (VCAM-1, ICAM-1) of infection showed a shift in meanto be examined by immunofluorescence microscopy were grown

fluorescence intensity (figure 1B). In this experiment, 70% ofto confluence on glass coverslips, fixed with 3% paraformaldehyde

the infected cells showed induction of E-selectin and ú80%in PBS (30-min incubation), and stained with a mixture of rabbitof 24-h infected cells exhibited increases in VCAM-1 andanti-human von Willebrand’s factor polyclonal antibody (1/200)ICAM-1.and mouse anti–R. conorii MAb (1/200). After extensive washing,

E-selectin, VCAM-1, and ICAM-1 were modulated in a time-cells were incubated with a second mixture of FITC-conjugatedanti-rabbit IgG (1/200) and tetramethylrhodamine B isothiocya- and dose-dependent manner. Standards of fluorescence




Figure 6. Immunofluorescence and confocal micro-scopic analysis of von Willebrand’s factor (vWF) andrickettsiae distribution in endothelial cells. A, Cells in-fected with live rickettsiae single-stained with anti-vWFantibody revealed by fluorescein isothiocyanate (FITC)–conjugated anti-rabbit IgG. C, Cells infected with liverickettsiae double-stained with mixture of rabbit anti-vWF antibody and mouse anti–R. conorii monoclonalantibody (MAb) revealed by FITC-conjugated anti-rabbitIgG and tetramethylrhodamine B isothiocyanate–conju-gated goat anti-mouse IgG. B, D, Cells infected, respec-tively, by live and fixed rickettsiae stained with anti–R.conorii MAb alone. Coverslips were mounted in 50%glycerol-PBS.

allowing transformation of fluorescence intensity into mean higher than controls and fell to values near that of control with1/100 dilution. Since R. conorii is grown in Vero cells, HUVECabsolute number of antigenic sites per cell (antigenic density)

were used to quantitate E-selectin, VCAM-1, and ICAM-1 incubated with lysed uninfected Vero cells were studied con-currently under the same conditions. Their effect on CAMmodulation. Increased expression of E-selectin, VCAM-1, and

ICAM-1 differed in terms of intensity and kinetics. Nondetect- expression was compared with those obtained with infectedVero cells at 8 h (E-selectin) and 24 h (ICAM-1, VCAM-1)able on uninfected HUVEC, E-selectin was induced after 4 h

of infection, peaked at Ç240 1 103 sites per cell at 8 h, and after infection. No effect was detectable on E-selectin (figure4A), whereas a nonsignificant effect 2-fold higher than controldeclined quickly to near baseline levels (õ50 1 103 sites/cell)

within 24 h (figure 2A). The up-regulation of VCAM-1 and values was obtained for VCAM-1 and ICAM-1 (figure 4B, C).We next analyzed the contribution of rickettsial lipopolysac-ICAM-1 was also time-dependent, but it was slower and

reached higher antigenic densities (figure 2B). Between 0 and charide (LPS) as a potential inducer of CAM expression. Tothis end, we infected HUVEC in the presence of polymyxin8 h of infection, VCAM-1 mean density increased from 182

to 845 1 103 sites per cell, whereas its expression at 8, 12, B. No significant decrease in E-selectin, VCAM-1, or ICAM-1 expression was detected (figure 4).and 24 h after infection appeared statistically similar. From 4

to 24 h after infection, ICAM-1 expression regularly increased E-selectin, VCAM-1, and ICAM-1 expression requires denovo protein synthesis and intracellular rickettsiae. To deter-with time from 712 to 3511 1 103 sites per cell. Throughout

the infection period, ICAM-1 and VCAM-1 expressions, re- mine if increased endothelial adhesiveness was conditioned byintracellular presence of rickettsiae, we treated HUVEC withspectively, were õ100 and 200 1 103 sites per cell on unin-

fected HUVEC. formaldehyde-fixed R. conorii before infection. In contrast tolive rickettsiae, fixed rickettsiae failed to induce increases inThe effect of inoculum dilution on E-selectin, VCAM-1, and

ICAM-1 expression was studied after infection times corre- E-selectin, VCAM-1, or ICAM-1 expression on EC (figure 5).We showed that formaldehyde blocks R. conorii entry into thesponding to optimal stimulation, as indicated by antigenic den-

sities (i.e., 8 h for E-selectin and 24 h for VCAM-1 and ICAM- cell by confocal microscopy experiments. First, the intracellularpresence of unfixed rickettsiae was confirmed by double-labeling1; figure 3). Pure inoculum titers (107 –108 pfu/mL) induced a

significant increase in the expression of the three adhesive immunofluorescence, which showed that the microorganism iscolocalized within the cytoplasm with von Willebrand’s factor,receptors. In experiments with 1/10 dilution of pure inoculum,

their expressions were significantly reduced but remained an intracellular endothelial antigen (figure 6A, C). In contrast,




infected cells and numbers of R. conorii organisms per cell(not shown).

IL-1 Ra and anti–IL-1a antibodies inhibited E-selectin,VCAM-1, and ICAM-1 expression. Having established a linkbetween the intracellular presence of rickettsiae and the in-creased expression of CAM, we investigated if this increasewas a direct effect or an indirect process involving autocrineproduction of soluble mediators. Since the expression pattern ofE-selectin, ICAM-1, and VCAM-1 observed during rickettsialinfection was similar to that previously described in responseto inflammatory cytokines, we performed experiments to estab-lish the potential involvement of IL-1, which was previouslyshown to increase E-selectin expression on cytomegalovirus-infected EC [27]. When R. conorii– infected HUVEC werecultured in the presence of IL-1 Ra, E-selectin expression wastotally inhibited (figure 8A), whereas increases in ICAM-1 andVCAM-1 expression were significantly reduced (63% and 60%,respectively; figure 8B). CAM expression by R. conorii–in-fected HUVEC was inhibited similarly by anti–IL-1a neu-tralizing antibodies (figure 8), whereas IL-1b antibodies hadno significant effect on E-selectin, VCAM-1, or ICAM-1 (datanot shown).

Enhanced MNC adherence to 24 h–infected EC. We inves-tigated whether E-selectin, VCAM-1, and ICAM-1 up-regula-tion was correlated with increased endothelial adhesiveness forMNC. Whereas relatively few MNC adhered to uninfected EC,an infection period of 8 or 24 h (figure 9) led to a significantincrease in MNC adherence. This effect was dependent onCAM expression, since a combination of MAbs blocking E-selectin, VCAM-1, and ICAM-1 reduced MNC adherence by

Figure 7. Effect of cycloheximide (C) on E-selectin, VCAM-1, and 76% at 8 h and by 72% at 24 h, whereas the presence of theICAM-1 expression. Control and infected endothelial cells (EC) were irrelevant 6D1 MAb had no significant effect. In experimentstreated with cycloheximide (CHX 10 mg/mL) for 1 h before infection

with each MAb alone, significant inhibition was demonstratedand during 8 h of infection by R. conorii (RC). E-selectin (A) and

for E-selectin and ICAM-1 at 8 h (61% and 66%, respectively).VCAM-1 and ICAM-1 (B) expression was measured as described inIn contrast, at 24 h after infection, the blocking effect of E-Methods in control cells in presence (C / CHX) or absence of C

and in RC-infected EC in presence (RC / CHX) or absence (RC) of selectin was comparable to that of the irrelevant 6D1 MAb,C. Results are mean { SD of 4 different experiments. P õ * .05, whereas significant inhibition was observed for VCAM-1 and** .005, significant difference from control; NS, not significant.

ICAM-1 (64% and 69%, respectively).

Discussionwhen formalin-fixed rickettsiae were used, no specific labeling ofrickettsial component was detectable compared with live organ- The model presented demonstrates that R. conorii infection

modulates endothelial adhesiveness for MNC by enhancingisms (figure 6B, D).Finally, to assess if the increase observed in CAM expression ICAM-1 and VCAM-1 expression. A pathway involving auto-

crine production of IL-1a may account for a significant partwas dependent on EC protein synthesis, we treated cultureswith 10 mg/mL cycloheximide for 1 h before and during the of this endothelial adhesive phenotype. Activation of EC in

response to rickettsial infection is of critical importance incourse of rickettsial infection. The effect of cycloheximide wasstudied after 8 h of treatment, since longer incubation periods the interaction of circulating leukocytes with the vessel wall,

because changes in the expression of adhesive receptors regu-are not compatible with cell viability. Cycloheximide com-pletely blocked the increase in E-selectin (figure 7A) and inhib- late the number and type of leukocytes emigrating from blood

to inflammatory tissues. Indeed, we show that R. conorii canited 93% and 94% of VCAM-1 and ICAM-1 expression, re-spectively (figure 7B). Such treatment, however, did not affect induce the synthesis and surface expression of the adhesive

molecules E-selectin, VCAM-1, and ICAM-1 in a tightly regu-the infectious process, since we verified that treated and un-treated cultures were comparable in terms of percentages of lated process. Whereas E-selectin peaked at 6–8 h of infection,




Figure 8. Effect of interleukin-1 receptorantagonist (IL-1 Ra) and anti–IL-1a mono-clonal antibody (MAb) on E-selectin,VCAM-1, and ICAM-1 expression. Endo-thelial cells were infected with R. conorii(RC) or cultured without organisms (con-trol: C). Then cells (uninfected or infected)were cultured in medium alone (C, RC) orin presence of IL-1 Ra (C / IL-1 Ra, RC/ IL-1 Ra) or in presence of anti–IL-1aMAb (C or RC / anti–IL-1a MAb). E-selectin (A) and ICAM-1 and VCAM-1 (B)expression were measured 8 and 24 h afterinfection, respectively. Results (antigenicsites/cell) are mean { SD of 3 experiments.P õ * .05, ** .005, *** .0005, significantdifference from infected cells; NS, not sig-nificant.

VCAM-1 and ICAM-1 densities were elevated for 24 h. The explanation is that the Mabs used in these experiments do notcompletely inhibit binding of MNC.kinetics described parallel responses to inflammatory cytokines

[8, 9]. Our in vitro model showed that the expression pattern ofadhesive molecules may influence the type of leukocyte infil-Our findings confirm those found during the early phase of

rickettsial infection (R. rickettsii was shown to cause maximal tration and supports observations made in vivo concerning thecellular composition of the inflammatory infiltrate during rick-induction of E-selectin 6–8 h after the onset of the infection

[19]). We have extended the earlier study by exploring the late ettsial infection [6, 7, 28]. This cellular response is not specificto rickettsiae, since enterovirus, poliovirus, herpesvirus, andphase of the inflammatory rickettsial response. We found that

increased expression of VCAM-1 and ICAM-1 was maintained cytomegalovirus are also responsible for increased adhesion ofleukocytes to infected EC [29–35].for §24 h and was associated with greater adherence of MNC

to EC. This enhanced adhesiveness was dependent on endothe- Of interest, in biopsies from tache noire, the number ofmicroorganisms identified has correlated with the degree oflial adhesive molecule induction, since it was significantly re-

duced by MAbs blocking VCAM-1 and ICAM-1. In contrast, inflammatory infiltration and subsequent tissue injury [5]. Simi-larly, our experiments on inoculum dilution showed that theE-selectin was not involved, as shown by very low cell surface

expression of E-selectin at 24 h. The combination of antibodies level of expression of E-selectin, VCAM-1, and ICAM-1 de-creased proportionally with the number of rickettsiae inocu-used to block adhesion never resulted in complete inhibition,

suggesting that other CAMs are involved in the residual adhe- lated. Thus, the resultant leukocyte accumulation might play apathogenic role in the formation of rickettsial lesions, becausesion observed, most likely ICAM-2 [11]. An equally plausible




Figure 9. Adherence of mononuclear cells(MNC) to uninfected cells (C), cells infected withR. conorii (RC), and infected cells after additionof irrelevant 6D1 monoclonal antibody (MAb),anti–E-selectin MAb, anti–VCAM-1 MAb, anti–ICAM-1 MAb, or combination of 3 MAbs. AllMAbs were used at 101 saturating dilution. MNCadherence to endothelial cell monolayers was de-termined at 8 (A) and 24 (B) h after infection byfluorescence plate reader as described in Methods.Results (mean fluorescence intensity { SD of 10wells) are representative of 4 experiments. * P õ.05; NS, not significant.

they release oxygen radicals and proteases involved in the de- vitro was unequivocally demonstrated by the confocal micro-scopic study, which directly visualized the colocalization of thevelopment of necrotic vasculitis [36, 37].

Several experiments were done to study the mechanisms unfixed rickettsiae and von Willebrand’s factor within the cellbut no fixed rickettsiae. In agreement with other studies, thesecausing increased CAM expression. The addition of cyclohexi-

mide (an inhibitor of eukaryotic protein synthesis) to HUVEC observations confirm that formalin-fixed R. conorii have losttheir ability to enter HUVEC [41, 42]. Although fixation mightduring the course of rickettsial infection completely blocked

E-selectin induction and inhibited VCAM-1 and ICAM-1 ex- have changed the conformation of some components of the mi-croorganism surface, fixed rickettsiae are still able to adhere topression. Since 8 h of cycloheximide treatment did not affect

the infectious process, this inhibitory effect stresses the neces- HUVEC [41]. Thus, it is also possible that a microorganism cellwall component is recognized at the HUVEC plasma membrane,sity for de novo protein synthesis, in agreement with the molec-

ular mechanisms regulating CAM expression [10, 11]. leading to EC activation. This was shown for gram-negativebacteria by recognition of LPS in the outer membrane of HU-In our experiments, rickettsiae inactivated with formaldehyde

before inoculation were unable to enhance CAM expression, VEC [43]. In our experiments, the contribution of rickettsiae-derived LPS can be excluded, since polymyxin B did not affectsuggesting that penetration of the microorganism and its pres-

ence in the cytoplasm are necessary for EC stimulation. The CAM expression. These observations together with the fact thatformalin-fixed rickettsiae did not induce CAM expressionhypothesis was supported by complementary experiments. Rick-

ettsiae are obligate intracellular bacteria with a major tropism strongly suggest that adherence of R. conorii to the EC mem-brane is not sufficient to signal these events.for EC in vivo [38–40]. Rickettsiae infection of HUVEC in




Numerous reports have documented that other endothelial tools to monitor endothelial activation or damage in Mediter-ranean spotted fever.responses induced in vitro by rickettsial infection are abolished

when microorganisms are fixed [41, 42]. Similarly, guinea pigsgiven intradermal injections of live rickettsiae, but not formal-dehyde-killed ones, developed scabs at the sites of inoculation, Acknowledgmentsshowing that the lesion required rickettsial infectivity to form

We thank Bussotti Brigitte and Lo Presti Martine for secretarialand is not a host reaction to R. conorii components [7]. Inhibi-support, P. Menut for graphic illustrations, Laurence Pascal andtion of rickettsial protein synthesis by substances such as chlor-Herve Tissot-Dupond for statistical analysis, and Immunotech andamphenicol should be investigated to determine whether CAMBiocytex companies for providing antibodies.

induction is simply due to the presence of intracytoplasmicrickettsiae or whether actively metabolizing and spreading mi-croorganisms are required.

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2. Walker DH. Pathology and pathogenesis of the vasculotropic rickettsioses.Several studies have shown that EC stimulation results in pro- In: Biology of rickettsial diseases. Vol 1. Boca Raton, FL: CRC Press,duction of IL-1 in two forms—associated with the cell mem- 1988:115–32.

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rickettsia conorii infection enhances vascular cell adhesion

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