INFECTION AND IMMUNITY,0019-9567/99/$04.0010

Sept. 1999, p. 4963–4967 Vol. 67, No. 9

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Immune Cells Are Required for Cutaneous Ulceration in aSwine Model of Chancroid

LANI R. SAN MATEO,1 KRISTEN L. TOFFER,1 PAUL E. ORNDORFF,2

AND THOMAS H. KAWULA1*

Department of Microbiology and Immunology, University of North Carolina School of Medicine,Chapel Hill, North Carolina 27599,1 and Department of Microbiology, Pathology and Parasitology,

College of Veterinary Medicine, North Carolina State University,Raleigh, North Carolina 276062

Received 29 April 1999/Returned for modification 10 June 1999/Accepted 22 June 1999

Cutaneous lesions of the human sexually transmitted genital ulcer disease chancroid are characterized bythe presence of intraepidermal pustules, keratinocyte cytopathology, and epidermal and dermal erosion. Theselesions are replete with neutrophils, macrophages, and CD41 T cells and contain very low numbers of cells ofHaemophilus ducreyi, the bacterial agent of chancroid. We examined lesion formation by H. ducreyi in a pigmodel by using cyclophosphamide (CPA)-induced immune cell deficiency to distinguish between host andbacterial contributions to chancroid ulcer formation. Histologic presentation of H. ducreyi-induced lesions inCPA-treated pigs differed from ulcers that developed in immune-competent animals in that pustules did notform and surface epithelia remained intact. However, these lesions had significant suprabasal keratinocytecytotoxicity. These results demonstrate that the host immune response was required for chancroid ulceration,while bacterial products were at least partially responsible for the keratinocyte cytopathology associated withchancroid lesions in the pig. The low numbers of H. ducreyi present in lesions in humans and immune-competent pigs have prevented localization of these organisms within skin. However, H. ducreyi organisms werereadily visualized in lesion biopsies from infected CPA-treated pigs by immunoelectron microscopy. Thesebacteria were extracellular and associated with necrotic host cells in the epidermis and dermis. The relativeabundance of H. ducreyi in inoculated CPA-treated pig skin suggests control of bacterial replication by hostimmune cells during natural human infection.

Chancroid is a sexually transmitted cutaneous genital ulcerdisease caused by Haemophilus ducreyi (reviewed in references6, 10, and 17). This disease is a serious public health concern inmany tropical developing nations, where genital ulcerating dis-eases contribute significantly to the spread of human immuno-deficiency virus (HIV) (18).

Chancroid ulcers are characterized by surface pustules com-posed of neutrophils and necrotic debris, keratinocyte cytopa-thology, and dense dermal inflammatory infiltrates (2, 7, 14).Neutrophils, macrophages, and CD41 T cells migrate to thesite of H. ducreyi infection in a delayed-type hypersensitivityresponse involving predominantly Th1-type cytokines (7, 12,13). Bacterial products that might play a role in chancroidulceration include factors that cause cell damage in vitro, suchas cytotoxin (1) and hemolysin (11), and factors that enablebacterial survival in chancroid animal models, including a he-moglobin-binding protein (15) and a periplasmic superoxidedismutase (our unpublished data). However, the unique inter-actions of bacterial molecules and elements of the host im-mune response that result in the characteristic histopathologyof chancroid ulceration are not known.

Our laboratory has developed a juvenile pig model of chan-croid that mimics human infection. H. ducreyi infection of pigsresults in disease that resembles human chancroid from thedevelopment of ulcers histologically similar to chancroid inhumans, with an immune cell infiltrate of neutrophils, macro-phages, and T cells, to the lack of protective immunity (4). To

determine the role of the host immune response in the histo-pathology and progression of chancroid, we examined the ef-fect of immune cell deficiency on ulceration induced by H.ducreyi in the pig model of chancroid.

Histopathology of lesions induced by H. ducreyi in immune-competent and immune cell-deficient pigs. To define the roleof the host immune cells in chancroid pathology, we deter-mined the effect of induced immune cell deficiency on ulcerformation caused by H. ducreyi in the pig model. Pigs wererendered immune cell deficient by intravenous administrationof cyclophosphamide (CPA) (Mead Johnson, Princeton, N.J.)at 50 mg per kg 4 days preinoculation and 20 mg per kg everyother day thereafter until the day of biopsy (8). Pigs treatedwith CPA showed a 76% reduction in total leukocyte numbers

* Corresponding author. Mailing address: Department of Microbi-ology and Immunology, Campus Box 7290, University of North Caro-lina School of Medicine, Chapel Hill, NC 27599. Phone: (919) 966-9699. Fax: (919) 962-8103. E-mail: kawula@med.unc.edu.

TABLE 1. Effect of CPA on peripheral blood leukocyte numbersin pigs

Circulatingleukocyte type

No. of cells/mm3a

Mean %reductionb P value

Baseline 1CPA

Neutrophils 8,079 6 1,518 1,101 6 1,013 87 0.009Monocytes 678 6 179 6 6 6 99 0.029Lymphocytes 6,498 6 941 3,360 6 607 49 0.031

Total 15,425 6 1,587 3,650 6 777 76 ,0.001

a Leukocyte numbers are presented as the mean 6 standard error for fourpigs. Baseline values were determined before treatment with CPA and 4 daysprior to infection with H. ducreyi. Values for post-CPA administration weredetermined on the day of infection with H. ducreyi and 4 days after beginningCPA treatment.

b % reduction 5 [(baseline 2 CPA)/baseline] 3 100.

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FIG. 1. Hematoxylin-eosin-stained cross-sections of H. ducreyi-infected skin from immune-competent pigs. (A) Uninfected skin (actual magnification, 3105). (B)Day 2 after infection with H. ducreyi (353). Cells in the ulcer with darkly staining nuclei are neutrophils, macrophages, and lymphocytes.

FIG. 2. Hematoxylin-eosin (A and B)- and toluidine blue (C)-stained cross-sections of ear skin from cyclophosphamide-treated pigs. (A) Uninfected skin (353).(B) Day 2 after infection with H. ducreyi (353). m, epidermal microbreak; n, degenerating neutrophils with pycnotic nuclei; b, bacterial colonies in the dermis. (C) Day7 after infection with H. ducreyi (3105). r, regenerated surface layers of the epidermis; x, area wherein cell debris and edema replace the orderly strata of epidermalcells.

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by the day of infection. Numbers of circulating polymorpho-nuclear leukocytes (neutrophils), lymphocytes, and monocytesall significantly decreased after administration of CPA (Table1) [all P , 0.05 by t test]).

Pigs were inoculated on the ears with an estimated delivereddose of 104 CFU of H. ducreyi 35000 by using an allergendelivery device as previously described (4). H. ducreyi-infectedsites and control sites inoculated with phosphate-buffered sa-line (PBS) were biopsied on days 2 and 7 by using 6-mm skinpunches (Acuderm, Ft. Lauderdale, Fla.). Samples for bright-field microscopy were fixed in 4% paraformaldehyde in PBS at4°C, embedded in paraffin, sectioned, and stained with hema-

toxylin and eosin (Histopathology Reference Laboratory,Richmond, Calif.).

In response to H. ducreyi infection, immune-competent pigsdeveloped skin lesions with the characteristic features of chan-croid disease. In immune-competent pigs 2 and 7 days afterinoculation, most H. ducreyi-infected sites showed surface mi-cropustules consisting of neutrophils and tissue debris, com-plete erosion of the epidermis, ballooning and cytopathologyof keratinocytes surrounding the micropustule, and near con-fluence of inflammatory cells in the dermis below the micro-pustule (Fig. 1).

In contrast, a typical day 2 lesion from a CPA-treated pig

FIG. 3. Electron micrographs of pig skin showing H. ducreyi labeled with gold particles. (A and B) H. ducreyi in the middle of necrotic pig skin cells at lowmagnification (A) and high magnification (B). (C and D) H. ducreyi microcolony surrounding a necrotic pig cell in a section from an infected neutropenic pig seen atlow magnification (C) and at high magnification (D). Each bar 5 1 mM.

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had a mildly acanthotic but intact epidermis with a microbreakat the site of inoculation bordered by regenerative keratino-cytes beginning to repair the microbreak. The dermis had largeclumps of bacteria and degenerating neutrophils with pycnoticnuclei (Fig. 2B). Day 7 lesions were similar to day 2 lesions inthat surface epithelia were fully intact (Fig. 2C). Epidermalerosion, a requirement for the transmission of HIV and H.ducreyi itself (5, 9, 18), did not develop in immune cell-defi-cient animals, suggesting that the host immune response isrequired for ulceration and, consequently, that host immuneresponses may play a role in the spread of chancroid, as well asHIV. The participation of the immune response in tissue injuryhas been observed in many other disease models. In one study,the elimination of circulating neutrophils in a guinea pig modelof Pseudomonas-induced acute respiratory distress syndromesignificantly diminished lung injury while stimulation with re-combinant granulocyte colony-stimulating factor significantlydecreased survival (16).

Death of epidermal keratinocytes in the spinous layer wasevident in H. ducreyi-infected skin of CPA-treated pigs 7 daysafter inoculation (Fig. 2C), suggesting that some tissue damagemay occur in the absence of a normal host immune responseand as a direct result of the action of specific bacterial factors.H. ducreyi has been shown to cause cell death in vitro (3),through the actions of a secreted cytolethal distending toxin (1)and a surface-associated hemolysin (11).

Localization of H. ducreyi in infected pig skin. Although weconsistently recovered viable H. ducreyi from infected immune-competent pigs, our attempts to localize bacteria in skin biop-sies by using various stains and bright-field microscopy wereunsuccessful. Other groups have experienced the same diffi-culty (12–14). Consequently, to localize H. ducreyi within in-fected pig skin, we used transmission electron microscopy(TEM). We surveyed ultrathin sections embedded in LR whiteon pioloform-treated nickel grids (Ted Pella, Inc., Redding,Calif.) probed with rabbit polyclonal anti-H. ducreyi antiserumand goat anti-rabbit immunoglobulin G conjugated to 15-nm-diameter gold particles (3).

Visible H. ducreyi organisms were rare in immune-compe-tent pigs, appearing as single cells within necrotic macrophagesand granulocytes, amidst collagen fibers in the dermis, or be-tween necrotic keratinocytes (Fig. 3A and B). In contrast, H.ducreyi cells were readily visible by TEM in skin of infectedimmune cell-deficient pigs. Clumps of bacteria were visiblepredominantly in the dermis amidst host cell debris and extra-cellular matrix components and often surrounding or adjacentto host cell ghosts (Fig. 3C and D).

We have determined that H. ducreyi numbers at an inocu-lated site 2 days postinoculation of a CPA-treated pig can be asmuch as 1 order of magnitude greater than the number in asimilarly-infected site on an immune-competent pig (our un-published data). The abundance of H. ducreyi cells in infectedCPA-treated pigs and their relative rarity in immune-compe-tent pigs provide evidence that neutrophils, lymphocytes, andmacrophages may control H. ducreyi growth and that the hostimmune response may play a role in the clearance of chan-croid.

The immune cell-deficient pig model promises to be a valu-able tool with which to study bacterial and host factors in-volved in chancroid pathogenesis. Engineered H. ducreyi vari-ants with mutations in genes putatively involved in immuneevasion and survival of host bactericidal mechanisms can becompared in immune-competent and CPA-treated pigs. Theincreased ability to visualize the infected site in CPA-treatedpigs due to the absence of overwhelming numbers of immunecells could also be exploited for the analysis of cytopathic or

cytotoxic effects of specific H. ducreyi gene products. The in-creasing availability of monoclonal antibodies to pig immunecells will also allow future studies involving the selective de-pletion of entire classes of immune cells or molecules to definethe contributions of these factors to chancroid pathology andH. ducreyi clearance.

In summary, we have provided evidence that the host im-mune response to H. ducreyi infection is required for the de-velopment of chancroid ulcers and the associated loss of epi-dermal integrity necessary for H. ducreyi transmission. Wehave also observed keratinocyte cytopathology occurringlargely in the absence of immune cells, suggesting that H.ducreyi may contribute to the tissue damage associated withchancroid. The immune cell-deficient pig model can be furtherused to dissect bacterial and host contributions to disease pro-gression in chancroid.

This work was supported by grants NIAID AI42824 (T.H.K.) andNRSA 1 F31 AI09565-01 (L.R.S.M.).

We gratefully acknowledge our collaborator, Glen Almond, for valu-able advice on pigs; Patty Routh, John Horton, and Re Bai for tech-nical assistance with the pigs; Stephen Knight for help with light mi-crography; Victoria Madden and Robert Bagnell for help with electronmicroscopy; John Woosley for help with dermatohistopathology; JanneCannon for helpful discussions; and past and present members of theKawula laboratory, particularly Marcia Hobbs, Franca Zaretzky, andGina Donato, for practical and moral support. We are especially grate-ful to Myron Cohen for his idea to use induced neutropenia.

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Editor: D. L. Burns

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