in vitro and in vivo interactions of haemophilus ducreyi with host

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INFECTION AND IMMUNITY, Feb. 2002, p. 899–908 Vol. 70, No. 2 0019-9567/02/$04.000 DOI: 10.1128/IAI.70.2.899–908.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved. In Vitro and In Vivo Interactions of Haemophilus ducreyi with Host Phagocytes Hinda J. Ahmed, 1 Catharina Johansson, 1 Liselott A. Svensson, 1 Karin Ahlman, 1 Margareta Verdrengh, 2 and Teresa Lagergård 1 * Department of Medical Microbiology and Immunology 1 and Department of Rheumatology, 2 University of Göteborg, S-413 46 Göteborg, Sweden Received 18 June 2001/Returned for modification 22 August 2001/Accepted 1 November 2001 We investigated the phagocytosis of Haemophilus ducreyi both in vitro and in vivo. Human granulocyte and monocyte phagocytosis of opsonized and nonopsonized, fluorescence-labeled H. ducreyi was assessed by flow cytometry. Both Escherichia coli and noncapsulated H. influenzae were included as controls. The maximal percentage of granulocytes taken up by H. ducreyi was 35% after 90 min. In contrast, 95% of H. influenzae bacteria were phagocytosed by granulocytes after 30 min. These results indicated that H. ducreyi phagocytosis was slow and inefficient. Bacterial opsonization by using specific antibodies increased the percentage of granulocytes phagocytosing H. ducreyi from 24 to 49%. The nonphagocytosed bacteria were completely resistant to phagocytosis even when reexposed to granulocytes, indicating that the H. ducreyi culture comprised a mixture of phenotypes. The intracellular survival of H. ducreyi in granulocytes, in monocytes/macrophages, and in a monocyte cell line (THP-1) was quantified after application of gentamicin treatment to kill extracellular bacteria. H. ducreyi survival within phagocytes was poor; approximately 11 and <0.1% of the added bacteria survived intracellularly after 2 and 20 h of incubation, respectively, while no intracellular H. influenzae bacteria were recovered after 2 h of incubation with phagocytes. The role of phagocytes in the development of skin lesions due to H. ducreyi was also studied in vivo. Mice that were depleted of granulocytes and/or monocytes and SCID mice, which lacked T and B cells, were injected intradermally with approximately 10 6 CFU of H. ducreyi. Within 4 days of inoculation, the granulocyte-depleted mice developed lesions that persisted through- out the experimental period. This result reinforces the importance of granulocytes in the early innate defense against H. ducreyi infection. In conclusion, H. ducreyi is insufficiently phagocytosed to achieve complete eradication of the bacteria. Indeed, H. ducreyi has the ability to survive intracellularly for short periods within phagocytic cells in vitro. Since granulocytes play a major role in the innate defense against H. ducreyi infection in vivo, bacterial resistance to phagocytosis probably plays a crucial role in the pathogenesis of chancroid. Haemophilus ducreyi is the causative agent of chancroid, a sexually transmitted disease that is characterized by mucocu- taneous ulcerative lesions on the external genitals and occa- sionally painful swollen regional lymph nodes. The disease is common in developing countries, particularly in Africa and Southeast Asia, but localized outbreaks have been reported in Canada, the United States, and Europe (10, 25). The disease has received renewed attention following reports that genital ulcers facilitate the transmission of human immunodeficiency virus (HIV) in populations in which genital ulcers are endemic (17, 36, 40). The pathogenesis of chancroid is not well understood, and information concerning essential H. ducreyi virulence compo- nents is scarce. The known H. ducreyi virulence factors include lipooligosaccharide (LOS), which may contribute to ulcer for- mation by inducing the migration of inflammatory cells (7, 18). H. ducreyi also expresses cell surface components that are similar to human cell receptors and that may prevent host immune recognition of the bacteria (22). In addition, the bac- teria have the ability to bind to extracellular matrix protein (5). Other identified virulence factors include pilus-like structures; heat shock proteins (8, 11, 27); and components that enable the bacteria to survive within the host, including outer mem- brane proteins, hemoglobin-binding proteins, and periplasmic superoxide dismutase (6, 30, 37). Two H. ducreyi toxins, the cytolethal distending toxin (CDT) and hemolysin, have been shown to destroy various human cells (9, 20, 28, 34, 38, 39). Previous results showed that the bactericidal activities of specific antibodies, including antibodies to LOS, are insuffi- cient for the killing of H. ducreyi, although many other gram- negative bacteria can be killed in this manner (12). It has also been suggested that virulent, in contrast to avirulent, H. ducreyi strains are relatively resistant to killing by human neutrophils in vitro (12, 19). Several animal models, including rabbits, pigs, and mice, have been used to study the pathogenesis of and host immune responses involved in H. ducreyi infection (15, 16, 18, 31). All these models of infection result in intraepidermal lesions con- taining infiltrates of polymorphonuclear leukocytes (PMNL), monocytes, macrophages, and T cells. Similar cell populations were described for early-stage disease in humans; in these studies, bacteria were injected intradermally into the forearm skin of volunteers (26, 33, 37). In human infection studies such as these, although H. ducreyi was found to be associated with fibrin and collagen in the dermis of the pustule, no intracellular bacteria were observed, suggesting that H. ducreyi remains extracellular in the early stages of infection (3, 4). Examination * Corresponding author. Mailing address: Department of Medical Microbiology and Immunology, University of Göteborg, Guldhedsga- tan 10, S-413 46 Göteborg, Sweden. Phone: 46 31 3424758. Fax: 46 31 820160. E-mail: [email protected]. 899 on February 11, 2018 by guest http://iai.asm.org/ Downloaded from

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Page 1: In Vitro and In Vivo Interactions of Haemophilus ducreyi with Host

INFECTION AND IMMUNITY, Feb. 2002, p. 899–908 Vol. 70, No. 20019-9567/02/$04.00�0 DOI: 10.1128/IAI.70.2.899–908.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.

In Vitro and In Vivo Interactions of Haemophilus ducreyiwith Host Phagocytes

Hinda J. Ahmed,1 Catharina Johansson,1 Liselott A. Svensson,1 Karin Ahlman,1Margareta Verdrengh,2 and Teresa Lagergård1*

Department of Medical Microbiology and Immunology1 and Department of Rheumatology,2

University of Göteborg, S-413 46 Göteborg, Sweden

Received 18 June 2001/Returned for modification 22 August 2001/Accepted 1 November 2001

We investigated the phagocytosis of Haemophilus ducreyi both in vitro and in vivo. Human granulocyte andmonocyte phagocytosis of opsonized and nonopsonized, fluorescence-labeled H. ducreyi was assessed by flowcytometry. Both Escherichia coli and noncapsulated H. influenzae were included as controls. The maximalpercentage of granulocytes taken up by H. ducreyi was 35% after 90 min. In contrast, 95% of H. influenzaebacteria were phagocytosed by granulocytes after 30 min. These results indicated that H. ducreyi phagocytosiswas slow and inefficient. Bacterial opsonization by using specific antibodies increased the percentage ofgranulocytes phagocytosing H. ducreyi from 24 to 49%. The nonphagocytosed bacteria were completely resistantto phagocytosis even when reexposed to granulocytes, indicating that the H. ducreyi culture comprised amixture of phenotypes. The intracellular survival of H. ducreyi in granulocytes, in monocytes/macrophages, andin a monocyte cell line (THP-1) was quantified after application of gentamicin treatment to kill extracellularbacteria. H. ducreyi survival within phagocytes was poor; approximately 11 and <0.1% of the added bacteriasurvived intracellularly after 2 and 20 h of incubation, respectively, while no intracellular H. influenzae bacteriawere recovered after 2 h of incubation with phagocytes. The role of phagocytes in the development of skinlesions due to H. ducreyi was also studied in vivo. Mice that were depleted of granulocytes and/or monocytesand SCID mice, which lacked T and B cells, were injected intradermally with approximately 106 CFU of H.ducreyi. Within 4 days of inoculation, the granulocyte-depleted mice developed lesions that persisted through-out the experimental period. This result reinforces the importance of granulocytes in the early innate defenseagainst H. ducreyi infection. In conclusion, H. ducreyi is insufficiently phagocytosed to achieve completeeradication of the bacteria. Indeed, H. ducreyi has the ability to survive intracellularly for short periods withinphagocytic cells in vitro. Since granulocytes play a major role in the innate defense against H. ducreyi infectionin vivo, bacterial resistance to phagocytosis probably plays a crucial role in the pathogenesis of chancroid.

Haemophilus ducreyi is the causative agent of chancroid, asexually transmitted disease that is characterized by mucocu-taneous ulcerative lesions on the external genitals and occa-sionally painful swollen regional lymph nodes. The disease iscommon in developing countries, particularly in Africa andSoutheast Asia, but localized outbreaks have been reported inCanada, the United States, and Europe (10, 25). The diseasehas received renewed attention following reports that genitalulcers facilitate the transmission of human immunodeficiencyvirus (HIV) in populations in which genital ulcers are endemic(17, 36, 40).

The pathogenesis of chancroid is not well understood, andinformation concerning essential H. ducreyi virulence compo-nents is scarce. The known H. ducreyi virulence factors includelipooligosaccharide (LOS), which may contribute to ulcer for-mation by inducing the migration of inflammatory cells (7, 18).H. ducreyi also expresses cell surface components that aresimilar to human cell receptors and that may prevent hostimmune recognition of the bacteria (22). In addition, the bac-teria have the ability to bind to extracellular matrix protein (5).Other identified virulence factors include pilus-like structures;

heat shock proteins (8, 11, 27); and components that enablethe bacteria to survive within the host, including outer mem-brane proteins, hemoglobin-binding proteins, and periplasmicsuperoxide dismutase (6, 30, 37). Two H. ducreyi toxins, thecytolethal distending toxin (CDT) and hemolysin, have beenshown to destroy various human cells (9, 20, 28, 34, 38, 39).

Previous results showed that the bactericidal activities ofspecific antibodies, including antibodies to LOS, are insuffi-cient for the killing of H. ducreyi, although many other gram-negative bacteria can be killed in this manner (12). It has alsobeen suggested that virulent, in contrast to avirulent, H. ducreyistrains are relatively resistant to killing by human neutrophilsin vitro (12, 19).

Several animal models, including rabbits, pigs, and mice,have been used to study the pathogenesis of and host immuneresponses involved in H. ducreyi infection (15, 16, 18, 31). Allthese models of infection result in intraepidermal lesions con-taining infiltrates of polymorphonuclear leukocytes (PMNL),monocytes, macrophages, and T cells. Similar cell populationswere described for early-stage disease in humans; in thesestudies, bacteria were injected intradermally into the forearmskin of volunteers (26, 33, 37). In human infection studies suchas these, although H. ducreyi was found to be associated withfibrin and collagen in the dermis of the pustule, no intracellularbacteria were observed, suggesting that H. ducreyi remainsextracellular in the early stages of infection (3, 4). Examination

* Corresponding author. Mailing address: Department of MedicalMicrobiology and Immunology, University of Göteborg, Guldhedsga-tan 10, S-413 46 Göteborg, Sweden. Phone: 46 31 3424758. Fax: 46 31820160. E-mail: [email protected].

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of Wright-stained smears of a lesion from a laboratory-ac-quired H. ducreyi infection showed the presence of PMNL andboth intracellular and extracellular rod-shaped bacteria (41).In clinical cases of chancroid, where smears of the lesion exu-date were examined by using fluorescence microscopy and aspecific monoclonal antibody (MAb) against H. ducreyi LOS,both phagocyte-engulfed and extracellular H. ducreyi bacteriawere observed (2).

Neutrophils and monocytes/macrophages are the earliesttypes of leukocytes entering the tissue in response to invadingpathogens. The major role of neutrophils in inflammatory andimmune responses is thought to be bacterial phagocytosis, fol-lowed by the killing of bacteria via the generation of reactiveoxygen intermediates and the release of lytic enzymes. Neu-trophils play a crucial protective role in the early phases ofmany infectious diseases, such as Staphylococcus aureus skininfection, and are important for a beneficial outcome (24, 43).The monocytes/macrophages phagocytose bacteria, act as an-tigen-presenting cells, and secrete large numbers of regulatoryproducts. A common detrimental outcome of infection is thatof macrophage-mediated tissue destruction (44). However, therole of phagocytic cells in host-parasite interactions in chan-croid is poorly understood and requires further investigation.

The aims of this study were to elucidate the kinetics andefficiency in vitro of the phagocytosis of opsonized and non-opsonized H. ducreyi strains by human phagocytes and thecapacity of the bacteria to survive within phagocytic cells. Inaddition, we investigated the role of granulocytes and mono-cytes in the early defense against intradermally injected H.ducreyi in a mouse model.

MATERIALS AND METHODS

Mice. Female BALB/c mice 5 to 6 weeks old and SCID mice were obtainedfrom B&K Universal AB (Stockholm, Sweden). They were housed in the Ex-perimental Biomedicine Animal Facility of the University of Göteborg. Micewere maintained under standard conditions of light and temperature and fedstandard laboratory chow and water ad libitum. All animal experiments wereapproved by the Animal Ethics Committee of the University of Göteborg.

Bacterial strains and cultivation. Four H. ducreyi strains obtained from theCulture Collection, University of Göteborg (CCUG), and one strain obtainedfrom the American Type Culture Collection (ATCC) were included in this study.Strains CCUG 7470, CCUG 27022, and ATCC 35000 have a nonasaccharideLOS structure (long LOS) and produce CDT, except for strain CCUG 27022,which is not a toxin producer (1, 23, 28). Strain CCUG 4438 has a hexasaccharideLOS structure (short LOS) and does not produce toxin (1, 28). The noncapsu-lated H. influenzae strain, CCUG 7566, which has been shown to be 100%sensitive to phagocytic killing by PMNL, was used as a control (12).

H. ducreyi was cultivated on chocolate agar plates as previously described (28).Since H. ducreyi aggregates on solid medium, bacteria were further cultured inliquid medium and incubated in an anaerobic jar with rotation at 100 rpm for 15to 16 h at 33°C as previously described (28). The liquid medium used for thecultivation of H. ducreyi was brain heart infusion broth supplemented with 1%hemin-histidine (Sigma Chemical Company, St. Louis, Mo.), 0.04% L-histidine(Fluka Chemie AG, Buchs, Switzerland), 10% fetal calf serum (FCS), 1% Iso-VitaleX, and 3 �g of vancomycin (Department of Bacteriology, SahlgrenskaHospital, Göteborg, Sweden)/ml. H. influenzae was cultivated on chocolate agarplates for 24 h at 37°C in a CO2-enriched atmosphere.

Animal experiments. Six groups of mice, with 10 to 15 mice/group, were usedin this study. One group of BALB/c mice was depleted of granulocytes byintraperitoneal injections of partially purified MAb RB6-8C5 (DNAX, ResearchInstitute of Molecular and Cellular Microbiology, Palo Alto, Calif.). The MAbwas administered at 1 mg of protein/ml 1 day before challenge with bacteria andon days 2 and 4 postchallenge (24, 43). A control group of 15 mice was injectedwith an unrelated MAb, CT17/13 (specific for cholera toxin), in the same man-ner. In order to deplete peripheral monocytes, a group of BALB/c mice was

treated with etoposide (VePesid; Bristol-Mayers SQUIBB, Bromma, Sweden)(44). This cytostatic drug leads to a selective decrease in monocyte numbers inperipheral blood (44). Etoposide was diluted to 2 mg/ml in phosphate-bufferedsaline (PBS), and 200 �l was injected subcutaneously once a day throughout theexperimental period. Another group of mice was depleted of both granulocytesand monocytes. The efficacies of those treatments were investigated by flowcytometric analysis of peripheral blood cells. Treated mice showed 95 and 50%fewer granulocytes and monocytes, respectively, than control mice. The extent ofleukocytopenia was similar to that described in previous studies (24, 43, 44).Groups of T- and B-cell-deficient mice (SCID) and naive BALB/c mice (control)were also included in this study. All mice were injected intradermally with 106

CFU of H. ducreyi strain CCUG 7470 that had been cultured in liquid mediumfor 15 h. Lesion development was examined over a period of 10 days (18).Bacteria were cultivated from lesions at days 2, 5, and 10.

In addition, four groups of mice (five mice per group) that were (i) granulocytedepleted, (ii) monocyte depleted, (iii) SCID, or (iv) untreated and immunocom-petent (naive) were injected intradermally with two doses, 150 and 15 �g, ofpurified LOS from H. ducreyi strain CCUG 7470 (1, 18). Lesion development wasmonitored as described above.

ELISA. An enzyme-linked immunosorbent assay (ELISA) was performed asdescribed previously (12). Briefly, the H. ducreyi bacteria were suspended in PBSto an optical density at 600 nm of 0.3. Heat-killed (65°C for 80 min) or non-heat-killed bacteria were used to coat microtitration plates (Greiner, Labortech-nik GmbH, Frichenhausen, Germany) overnight at room temperature (23°C).Plates were blocked with 1% (wt/vol) bovine serum albumin (Sigma) in PBS. A100-�l quantity of rabbit immune serum against surface antigens (24-kDa pro-tein, 60-kDa heat shock protein, and LOS) was added per well (starting with adilution of 1:100 and then diluted 10-fold in dilution buffer [0.1% bovine serumalbumin–PBS–Tween 20]) and incubated overnight at room temperature. Non-immunized rabbit serum and antiserum to H. ducreyi LOS were used as referencesera. The conjugate used was alkaline phosphatase-labeled anti-rabbit immuno-globulin G (Jackson ImmunoResearch Laboratories); for reaction development,1 mg of disodium p-nitrophenyl phosphate (Sigma) per ml in 1 M diethanol-amine–0.5 mM MgCl2 (pH 9.8) was used as a substrate. The dilution giving anabsorbance value approximately 0.2 �log10 unit greater than the backgroundvalue at A405 was determined as the endpoint titer. Anti-LOS serum was used asa positive control because the LOS structure is not affected by heat.

Fluorescence labeling and opsonization of bacteria. Fluorescein isothiocya-nate (FITC; Sigma) was used to label bacterial strains as described previously byGentry et al. (14). Briefly, bacteria grown in liquid medium for 15 to 16 h werewashed twice in PBS and heat killed for 80 min at 65°C in a water bath. In certainexperiments, live H. ducreyi was used. Bacteria were pelleted, suspended in 2 mlof PBS containing 1 mg of FITC/ml, and incubated for 1 h at 4°C. After beingwashed in PBS, the labeled H. ducreyi and H. influenzae bacteria were blockedwith 0.1% gelatin suspended in Hank’s balanced salt solution (GHBSS) (14).

For the opsonization of H. ducreyi strain CCUG 7470, homologous rabbitantisera were raised against (i) the oligosaccharide part of LOS conjugated totetanus toxoid as described earlier (1), (ii) the 24-kDa surface protein, (iii) the60-kDa heat shock protein (11), and (iv) whole-cell sonicate. The immunizationprocedure was described previously (12). The antibody titers are summarized inTable 1.

Bacteria suspended in PBS were incubated for 30 min at 37°C with heat-inactivated (56°C for 30 min) immune serum (12). As controls, inactivated babyrabbit serum and PBS were included. To reduce the cross-reactivity of naturalrabbit antibodies with H. ducreyi antigens, all sera were diluted 1:400. The

TABLE 1. Reciprocal antibody titers of sera raised against purifiedhomologous and surface antigens of H. ducreyi

strain CCUG 7470 in an ELISA

Antigen against which rabbitantiserum was raised

ELISA antibody titer (�log10) to:

Homologousantigen

Whole cells(surface antigen)

H. ducreyi bacteria NAa 7.224-kDa surface protein 4.0 4.560-kDa heat shock protein 4.0 6.8Oligosaccharide component

of LOS3.5 3.8

None (control: baby rabbit) NAa 2.4

a NA, not applicable.

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heat-inactivated serum was previously shown to lack complement activity but topossess functional antibody (12). Following opsonization, the bacteria werewashed and resuspended in GBHSS to a final concentration of 108 CFU/ml anddivided into aliquots. Labeled bacteria were examined by using fluorescencemicroscopy to confirm uniform staining.

Isolation of human phagocytic cells. Granulocytes and monocytes were iso-lated from heparinized blood of healthy blood donors as described previously(34). Briefly, granulocytes were prepared by dextran sedimentation followed byFicoll-Hypaque density centrifugation (Amersham Pharmacia Biotech AB, Upp-sala, Sweden) and hypotonic lysis to remove erythrocytes as described previously(34). The granulocytes were washed twice with Krebs-Ringer phosphate buffercontaining 1 mM Ca2� (pH 7.2), resuspended in RPMI 1640 (Life Technologies,Täby, Sweden) to a concentration of 106 cells/ml, and kept on ice until used lateron the same day.

Monocytes were selected from a fraction of peripheral blood mononuclearcells situated between the plasma and Ficoll-Hypaque and were suspended inRPMI 1640 containing 5% active human AB serum. Cells were seeded at aconcentration of 106 per well in 96-well microtiter tissue culture plates to allowmonocytes to adhere to the surface (34).

Monocytes were maintained in cell cultures in RPMI 1640 supplemented with10% inactivated FCS and 1% L-glutamine for 5 to 6 days to allow differentiationinto macrophages (13).

In addition, the human monocyte cell line THP-1 (ATCC T1B 202), derivedfrom the peripheral blood of a 1-year-old boy with acute monocytic leukemia,was maintained in RPMI 1640 with 10% FCS (42). Cell viability, determined atthe start of each experiment by the trypan blue exclusion test, was greater than95%.

Phagocytosis assays. The phagocytosis of opsonized and nonopsonized H.ducreyi was investigated by two methods in vitro.

(i) FACS assay. The phagocytosis of opsonized and nonopsonized H. Thephagocytosis of FITC-labeled H. ducreyi was measured by flow cytometry (fluo-rescence-activated cell sorting [FACS]; Becton Dickinson, San Jose, Calif.) (14).A commercial test kit (Phagotest; Orpegen Pharma, Heidelberg, Germany) wasused, and test procedures were performed according to the manufacturer’sinstructions. Heparinized whole blood (100 �l; containing approximately 8,000white cells/�l) was mixed with 20 �l of bacterial suspension (108 CFU/ml); theratio of phagocytes to bacteria was approximately 1:25. Samples were mixed welland incubated for 10, 30, 60, and 90 min at 37°C in a water bath. Negative controlsamples were kept on ice. FITC-labeled opsonized Escherichia coli bacteria thatwere highly phagocytosed within a 10-min period were included in each assay asa test control. A noncapsulated strain of H. influenzae was used for comparison.Placing the samples on ice stopped phagocytosis. The quenching solution allowedthe discrimination of attached and internalized bacteria by suppressing the flu-orescent green color of surface-attached bacteria but not that of internalizedremaining bacteria. DNA staining was used to distinguish live cells from bacterialaggregates. Samples were analyzed by flow cytometry with blue-green excitationat 488 nm by use of an argon ion laser and FACSCalibur CellQuest software. Thecell populations were analyzed by means of forward light scatter and side lightscatter. A live gate (gating for live cells) was set (red fluorescence), and a controlmarker containing a sample maintained at 0°C was used to exclude backgroundcell autofluorescence (fluorescence intensity) in each of the histograms. Thepercentages of granulocytes and monocytes that ingested bacteria as well as theirfluorescence intensity channel means were recorded. The mean and standarddeviation (SD) for three independent experiments were calculated.

In separate FACS assays, 100 �l of bacteria (heat killed or live H. ducreyi orE. coli) at 6 � 107 CFU/ml was inoculated with 100 �l of heparinized wholeblood and assayed as described above. Supernatants containing nonphagocy-tosed bacteria were subsequently reincubated with fresh phagocytes for 30 min at37°C and analyzed by FACS.

(ii) Fluorescence microscopy. To examine the locations of intracellular bacte-ria, isolated granulocytes (106 cells/ml) in RPMI 1640 were incubated with H.ducreyi (107 CFU/ml) for 60 min at 37°C with gentle shaking. The reaction wasstopped by placing the samples on ice. Cytocentrifugation was used to transferthe samples to cytoslides (Shandon, Inc.). The cells were washed twice with PBSafter every step. The cells were fixed in 2% paraformaldehyde in PBS for 30 minat 4°C and then pretreated with a 1:500 dilution of MAb MAHD7 (1 mg/ml),which is specific for H. ducreyi LOS, for 30 min at room temperature in a humidchamber. The cells were permeabilized with 0.5% Triton X-100 for 3 min atroom temperature and incubated for 30 min at room temperature with a 1:100dilution of a goat anti-mouse antibody conjugated to Rhodamine Red-X (Jack-son ImmunoResearch Laboratories). The cells were again incubated with MAbMAHD7 for 60 min at 37°C. Finally, the cells were incubated for 30 min at 37°Cwith a 1:100 dilution of a goat anti-mouse antibody conjugated to FITC (Sigma).

The glass slides were mounted, examined at a magnification of �100 by usingfluorescence microscopy (Zeiss, Oberkochen, Germany), and photographed.

Bacterial survival assays. The survival in human phagocytic cells of H. ducreyistrains CCUG 7470, CCUG 27022, and CCUG 4438 and the noncapsulated H.influenzae strain was examined over a 20-h incubation period. The assay wascarried out as previously described (29), with some modifications. Briefly, H.ducreyi strains grown in liquid medium (4 � 107 to 8 � 107 CFU/ml) were addedto sterile plastic tubes containing approximately 1 � 106 to 3 � 106 granulocytesor THP-1 monocytes per ml or to 96-well plates containing adherent macro-phages. The samples were incubated at 37°C in the presence of 5% CO2 for 1 hto allow phagocytosis. The samples were then washed twice with PBS to removenonadherent bacteria; resuspended in fresh medium containing 200 �g of gen-tamicin (Gibco/BRL)/ml, i.e., a concentration of antibiotic sufficient to kill ex-tracellular bacteria (35); and further incubated for 1 h. Subsequently, the sam-ples were washed twice with PBS (for the 2-h survival assay), or the medium wasreplaced with RPMI 1640 containing 50 �g of gentamicin/ml to prevent theextracellular growth of any released bacteria (for the 20-h survival assay). At the

FIG. 1. Phagocytic activity of gated granulocyte populations ana-lyzed by using histograms of cell number versus fluorescence intensity(FL1-H). Cells exposed to FITC-labeled, opsonized bacteria at 0°C(broken line) were used as a control marker (M1) to exclude thebackground. The percentage of granulocytes phagocytosing at 37°C(solid line) over 30 min (M1 area) was determined by FACS analysis.(a) E. coli (test control). (b) H. ducreyi. All values are expressed as themean and SD for at least three independent experiments. Note thatmore than half of the granulocytes do not ingest H. ducreyi.

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end of the 2- and 20-h incubation periods, the samples were processed for viablecount determinations.

The cell-bacterium mixtures were washed three times with PBS. The cells werelysed by adding 1 ml of ice-cold distilled water to each sample, followed byvigorous shaking, and were allowed to stand for 10 min. The cell lysates wereserially diluted 10-fold in PBS, and aliquots were plated on chocolate agar toassess the numbers of viable intracellular bacteria. Two controls for each strainwere included in the assay. The first control served to estimate the total numbersof viable bacteria per tube or well at the start of the experiment (time zero), andin the second control, gentamicin was added to the bacterial culture in order toestimate the efficacy of antibiotic killing. Results are expressed as the number ofbacteria recovered from the bacterium-cell mixture exposed to gentamicin (via-ble intracellular bacteria) divided by the number of bacteria in the inoculum attime zero (initial inoculum).

Statistics. The two-tailed Student t test was used to assess differences betweenpaired means. Results showing P values of �0.05 were considered statisticallysignificant.

RESULTS

H. ducreyi interactions with human phagocytic cells. Thekinetics of granulocyte and monocyte phagocytosis of H. du-creyi were studied at different times by using flow cytometry.Examples of flow cytometric histograms obtained following

phagocytosis of either opsonized E. coli (test control) or op-sonized H. ducreyi for 30 min are shown in Fig. 1a and b,respectively. After 10 min of incubation, 85% � 4.2% of thegranulocytes ingested E. coli, whereas 14% � 2.3% to 18% �2.3% of the granulocytes ingested different H. ducreyi strains.After 1 h of incubation, the corresponding numbers were 97%� 7.0% and 30% � 4.7%, respectively.

The kinetics of phagocytosis of the four nonopsonized H.ducreyi strains and the noncapsulated H. influenzae strain (con-trol) are shown in Fig. 2a. A striking difference was observedbetween the H. influenzae and H. ducreyi strains with regard tothe outcome of phagocytosis. After 30 min, 95% � 1.5% ofgranulocytes had taken up H. influenzae; in comparison, 19%� 1.4% to 22% � 1.4% of granulocytes had ingested thedifferent H. ducreyi strains (P � 0.0002 ). These findings indi-cate that all four H. ducreyi strains were phagocytosed to someextent by granulocytes; however, the H. ducreyi phagocytosiswas slow, requiring longer incubation times than E. coli and H.influenzae. There was no significant difference in the process ofphagocytosis among the different H. ducreyi strains.

FIG. 2. (a) Kinetics of phagocytosis by human granulocytes of four nonopsonized H. ducreyi strains and one noncapsulated H. influenzae strain.FACS was used to determine the percentage of phagocytosing granulocytes. Hd, H. ducreyi; Hi., H. influenzae. (b) Kinetics of phagocytosis bymonocytes of four nonopsonized H. ducreyi strains and one noncapsulated H. influenzae strain. The percentage of phagocytosing monocytes wascalculated from the experiments shown in panel a. (c) Phagocytosis by human granulocytes of H. ducreyi strain CCUG 7470 opsonized with seraspecific for different antigens. The percentage of phagocytosing granulocytes was determined by FACS. WCbact, whole-cell sonicate. All valuesare expressed as the mean and SD for at least three independent experiments.

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The mean fluorescence intensity of bacterium/cell mixturesin FACS analyses was used as a measure of the relative numberof bacteria per cell. The mean mean fluorescence intensitiesfor granulocytes that had phagocytosed H. ducreyi and H. in-fluenzae were 781.5 � 236.8 and 1,339 � 196.5, respectively.After 60 min, granulocytes containing ingested H. ducreyi ex-hibited significantly lower mean mean fluorescence intensitiesthan granulocytes containing ingested H. influenzae (P �0.032). These results indicate less efficient granulocyte phago-cytosis of H. ducreyi than of H. influenzae.

The kinetics of phagocytosis of the four H. ducreyi strainsand the noncapsulated H. influenzae strain by monocytes weresimilar to those seen with granulocytes and showed that ahigher percentage of monocytes ingested H. influenzae than H.ducreyi (Fig. 2b). Twice as many granulocytes as monocytesphagocytosed H. ducreyi.

Furthermore, we used fluorescence microscopy to confirmthe FACS results. The features of H. influenzae and H. ducreyiphagocytosis differed with regard to the number of cells havingingested bacteria and the number of bacteria in each cell. After30 min of phagocytosis, few phagocytes contained H. ducreyi,and there were many clumped bacteria, whereas at this timealmost 80% of the cells were filled with H. influenzae (data notshown). Bacterial aggregation occurred even though the H.ducreyi strains were grown in liquid medium and the cultureswere vortexed vigorously with glass beads in order to disruptclumps and guarantee single-bacterium suspensions. We can-not exclude the possibility that bacterial clumping might havehad some influence on the phagocytosis of H. ducreyi.

In addition, we used a double-immunofluorescence methodto determine the intracellular location of H. ducreyi in granu-locytes. The extracellular bacteria stained red, whereas theintracellular bacteria stained green or yellow-green (Fig. 3).

In order to study the antigen specificity of opsonizing anti-bodies, antisera directed against different bacterial surfacecomponents were used (Table 1). The results obtained forbacterial phagocytosis by granulocytes are summarized in Fig.2c. Phagocytosis was enhanced by the opsonization of bacteriawith specific antibodies. The highest level of opsonization wasnoted for antiserum to the O-side component of LOS (P �0.003). For example, after 60 min of incubation, 49% � 5.6%of granulocytes had ingested bacteria opsonized with antibodyto LOS; in contrast, 28% � 2.8% of granulocytes had taken upbacteria coated with antibody against the 24-kDa protein. Thesurface antibody titers in sera tested against heat-killed andlive bacteria showed no significant statistical difference whenanalyzed by an ELISA. The results indicate that specific anti-bodies promote phagocytosis and that anti-LOS antibodieshave the highest opsonizing capacity compared with antibodiesto other surface antigens. By FACS analysis, when the phago-cytosis of opsonized heat-killed H. ducreyi was compared withthat of opsonized live H. ducreyi, a slightly but not statisticallysignificantly higher percentage (approximately 5%) of granu-locytes phagocytosed the live bacteria.

Further experiments were performed to investigate whethercertain subpopulations of heat-killed or live H. ducreyi hadpronounced antiphagocytic activity. Nonphagocytosed bacteriawere reexposed to phagocytes, and the efficacy of phagocytosis

FIG. 3. Localization of H. ducreyi in isolated granulocytes. Cells were processed for double-immunofluorescence staining after 30 min ofexposure to H. ducreyi and examined by fluorescence microscopy. The extracellular bacteria stained red, whereas the intracellular bacteria stainedgreen or yellow-green.

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was examined by FACS. The results showed that previouslynonphagocytosed bacteria were not phagocytosed during asubsequent exposure to phagocytes (Table 2). It is noteworthythat even when high concentrations of H. ducreyi and E. coliwere used, the resistance to phagocytosis of previously non-phagocytosed H. ducreyi bacteria was significantly higher thanthat of previously nonphagocytosed E. coli bacteria. Theseresults indicated that a subpopulation of H. ducreyi was resis-tant to ingestion by granulocytes and that bacterial compo-nents other than LOS might mediate this resistance.

Survival in human phagocytes. The survival of H. ducreyistrains in granulocytes, monocytes/macrophages, and mono-cytic cell line THP-1 was assessed. The H. influenzae strain wasused as a control. H. ducreyi strains survived in phagocytic cellswhile noncapsulated H. influenzae was completely killed within2 h (Table 3). H. ducreyi survival in granulocytes and macro-phages was low, 11 and 2.7% of the inoculum, respectively,after 2 h of incubation. Similarly, up to 6.6% of the H. ducreyiinoculum was found to have survived in THP-1 monocytesafter 2 h of incubation (Table 3). After 20 h of incubation, H.ducreyi survival in all the phagocytic cells was extremely low,i.e., �0.1%. Interstrain differences in the ability of H. ducreyi tosurvive in phagocytes were slight; however, strain CCUG 4438was recovered in small numbers, possibly reflecting its slowergrowth compared with that of the other strains (data notshown).

Role of phagocytic cells in ulcer development following in-tradermal injection of H. ducreyi in the mouse model. We alsoevaluated the importance of phagocytic cells in early host de-fense against H. ducreyi in vivo. Mice that were selectivelydepleted of granulocytes and/or monocytes or that lacked Tand B cells were injected intradermally with about 106 CFU of

live bacteria, and the development of skin lesions was moni-tored. Mice that were granulocyte depleted or granulocyte plusmonocyte depleted developed hemorrhagic nodules 2 days af-ter bacterial inoculation; in almost all cases, these nodulesruptured on day 4 (Table 4). In the monocyte-depleted group,2 of 15 mice developed slight skin disruptions at day 4. Thecontrol groups (untreated mice and mice injected with an un-related MAb) as well as SCID mice developed a nodule within2 days; in about 50% of cases, the nodule healed completelywithin 10 days. Bacteria were recovered from the skin lesionsof granulocyte- and/or monocyte-depleted mice up to day 5postinfection. Examples of skin lesions that developed in dif-ferent groups of mice are presented in Fig. 4. These findingsdemonstrate the crucial role of granulocytes in inhibiting ulcerdevelopment early in H. ducreyi infection.

In a previous study, LOS was implicated in lesion develop-ment in mice; however, high doses of intradermally injectedpurified LOS were used (18). In the present study, injections ofhigh doses of an LOS preparation resulted in the developmentof lesions in two of five naive, control mice after 4 days. Onlyone of five SCID mice showed slight skin disruptions. Thegranulocyte- and/or monocyte-depleted mice did not show anyskin lesions. The results indicate that bacterial LOS, even inhigh doses, is not responsible for the development of skindisruptions in leukopenic mice.

DISCUSSION

Human phagocytic cells, such as granulocytes, monocytes,and macrophages, defend against different pathogens by in-gesting and killing the invaders. The effectiveness of phagocytic

TABLE 2. Resistance of the nonphagocytosed H. ducreyi population to repeated phagocytosis by granulocytes, as measured by FACS

Bacterial population and incubationa% Phagocytosisb after the following min:

10 30 60

H. ducreyiFirst 28 � 0.7 (26.8 � 4.9) 36 � 1.4 (40 � 5.6) 44 � 0.7 (53 � 1.4)Second (supernatant of the first incubation) 4 � 5.0 (5 � 1.4) 3 � 2.1 (7 � 2.8) 2 � 1.4 (3 � 2.2)

E. coli (control)First 90 � 1.3 91 � 3.0 93 � 2.1Second (supernatant of the first incubation) 67 � 0.8 41 � 7.0 38 � 1.5

a See Materials and Methods for bacterial concentration and procedures for the phagocytosis of bacteria.b Results are reported as the mean and SD for three experiments. For H. ducreyi, values are for heat-killed (live) bacteria tested with antiserum to LOS.

TABLE 3. Survival of H. ducreyi in phagocytic cells and in the monocyte cell line THP-1

Straina

% of the initial inoculum recovered from the following cellsb after the indicated h:

Granulocytes (macrophages) THP-1 cells

2 20 2 20

H. ducreyiCCUG 7470 (CDT�, long LOS) 11.00 � 1.4 (2.71 � 1.1) 0.03 � 0.2 (0.09 � 0.01) 6.46 � 0.28 0.02 � 0.00CCUG 27022 (CDT�, long LOS) 7.50 � 0.1 (1.66 � 0.6) 0.03 � 0.0 (0.08 � 0.001) 5.13 � 0.48 0.02 � 0.001CCUG 4438 (CDT�, short LOS) 2.00 � 0.2 (1.10 � 0.2) 0.001 � 0.0 (0.004 � 0.00) 1.51 � 0.18 0.001 � 0.002

H. influenzae 0 0 0 0

a CDT� and CDT�, presence or absence of CDT production.b Results are reported as the mean and SD for three independent experiments.

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cells in eliminating H. ducreyi bacteria was addressed in thisstudy.

Previously reported in vitro studies showed that H. ducreyiwas localized both inside and outside of PMNL (19). Similarly,in patients with chancroid, bacteria were seen to be eitherengulfed by phagocytic cells or localized extracellularly (2). Ina recent study with a human model of early-stage chancroid, nointracellular H. ducreyi bacteria were found, although the bac-teria were associated with professional phagocytes (3).

In the present study, phagocytosis and intracellular killing ofdifferent H. ducreyi strains by human phagocytes were deter-mined in vitro by using FACS analysis, microscopy, and a

survival assay with gentamicin treatment. We found that H.ducreyi bacteria were, in general, poorly phagocytosed by bothhuman granulocytes and human monocytes compared to H.influenzae and E. coli. There were no significant differences inthe ingestion of different H. ducreyi strains, despite interstraindifferences in LOS structures and the production of CDT.Opsonization with antibodies to surface components increasedthe uptake of H. ducreyi; however, phagocytosis was not com-plete, since less than half of the granulocyte population in-gested bacteria. It is noteworthy that serum with moderatelevels of antibodies specific for the O-side component of LOSwas far more effective in bacterial opsonization than sera spe-cific for other surface-localized antigens with high homologousantibody titers. However, no marked differences in phagocyto-sis were observed when live bacteria or heat-killed bacteriawere analyzed. It is also possible that the bacteria express otherstructures that influence their resistance to phagocytosis. Wealso discovered that a subpopulation of the H. ducreyi cultureused in the phagocytosis assay was completely resistant tophagocytosis by granulocytes. This was true for both heat-killed and live bacteria. The reason for the insufficient phago-cytosis of H. ducreyi observed here and in other studies (19, 45)is so far unclear. One possible explanation is that bacterialaggregation impairs the ingestive capacity of granulocytes. Al-

FIG. 4. Lesion formation at 4 days postinoculation in mice injected with 106 CFU of H. ducreyi. (A) Granulocyte-depleted mouse showing adeveloped skin lesion. (B) Monocyte-depleted mouse showing slight inflammation. (C and D) SCID (C) and control (untreated) (D) mice showingnodule development.

TABLE 4. Skin lesions in mice after intradermal injectionof 106 CFU of H. ducreyi

Mouse groupNo. of lesions/no. ofmice tested at day 4

postinfection

Granulocyte depleted............................................................. 14/15Monocyte depleted................................................................. 2/15Granulocyte plus monocyte depleted .................................. 12/15SCID (T- and B-cell deficient) ............................................. 0/10Control (treated with an unrelated MAb) .......................... 0/15Control (untreated)................................................................ 0/10

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ternatively, H. ducreyi may express different surface structures,such as those deployed by gonococci, e.g., pili, that promoteattachment to but impede engulfment by professional phago-cytes (32).

Another bacterial strategy for avoiding host defense mech-anisms is the capacity to survive the microbicidal milieu in thedeveloping phagolysosome (21). We observed that only a smallbut reproducible number of H. ducreyi bacteria survived inhuman phagocytic cells, while noncapsulated H. influenzae wasrapidly and completely killed. Our results indicate that a frac-tion of the bacteria can survive for only a short period of timeafter phagocytosis in human phagocytic cells, in the absence ofany apparent intracellular multiplication. Similar findings werereported by Wood et al., who showed that H. ducreyi did notsurvive in the human macrophage-like cell line U-937 after24 h of incubation (45). Since neither the bacterial populationnor the phagocytosis conditions resemble those in vivo, theseresults need to be assessed with caution. Therefore, H. ducreyiprobably can be considered a transiently intracellular pathogenbut not a typical intracellular pathogen, such as Mycobacteriumtuberculosis (21).

The present in vitro results indicate the ability of H. ducreyito avoid phagocytosis by both granulocytes and monocytes andthe capacity to survive for a limited time in phagocytic cells. Tostudy the role of phagocytic cells in lesion development in vivo,infection studies with H. ducreyi were carried out with micedepleted of granulocytes and/or monocytes and with mice thatlacked T and B cells. Mice were previously used to study H.ducreyi pathogenesis. However, in the earlier study, large bac-terial numbers (more than 107 CFU) were needed to elicitdermonecrotic lesions, and intradermal injections of highdoses of LOS resulted in the development of similar lesions(18). In this study, in order to study the role of neutrophilleukocytes, mice were depleted of granulocytes by injections ofMAb RB6-8C5, which is directed against differentiation anti-gens on myeloid cells (24). This treatment has been shown tocause pronounced early neutropenia, with depletion of at least90% of peripheral blood granulocytes (24). All granulocyte-depleted mice injected with 105 CFU of H. ducreyi developedskin lesions within 4 days, and the lesions persisted for 10 daysafter infection. In contrast, naive and SCID mice developedtransient nodules only. Importantly, lesion development ingranulocyte-depleted mice was not due to the presence ofLOS, since even a high dose of LOS (150 �g) did not causelesions in these mice. These results emphasize the crucial andindependent role of neutrophils in the early defense against H.ducreyi bacteria. As shown in the present study, monocytes playa minor role, since monocyte-depleted mice with an intactgranulocyte population only occasionally developed skin le-sions. The recruitment of neutrophils during the initial stagesof H. ducreyi infection is likely to be critical for the develop-ment of ulcers in chancroid.

In conclusion, the experiments described in this study dem-onstrate that (i) H. ducreyi is weakly phagocytosed by granu-locytes and monocytes, as a subpopulation of the bacteria resistphagocytic ingestion; (ii) H. ducreyi is capable of short-termsurvival inside human phagocytic cells; (iii) granulocytes play avery important role in the defense against H. ducreyi infection;and (iv) variations in the chemical structure of LOS and in

CDT production by H. ducreyi may not play major roles in theprocesses of phagocytosis and survival.

Phagocytic killing seems to be an important host defensemechanism against H. ducreyi, and bacterial resistance tophagocytosis may represent a pathogenic strategy for success-fully establishing disease. This characteristic of H. ducreyi maybe relevant in vivo for the maintenance of chancroid and bac-terial persistence in tissues or ulcers. Further studies areneeded to define the components and mechanisms by which H.ducreyi inhibits effective phagocytosis.

ACKNOWLEDGMENTS

This work was supported by the Swedish Agency for Research Co-operation with Developing Countries (SIDA/SAREC) and the Swed-ish Medical Research Council (grant 12630).

We thank Vincent Collins for revising the English text of the manu-script.

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