Haemophilus Ducreyi Infection and Its Relevance to HIV-1 Acquisition This Information Is Current As of September 25, 2021

Evolution of the Cutaneous Immune Response to Experimental Haemophilus ducreyi Infection and Its Relevance to HIV-1 Acquisition This information is current as of September 25, 2021. Tricia L. Humphreys, Carol T. Schnizlein-Bick, Barry P. Katz, Lee Ann Baldridge, Antoinette F. Hood, Robert A. Hromas and Stanley M. Spinola J Immunol 2002; 169:6316-6323; ; doi: 10.4049/jimmunol.169.11.6316 Downloaded from http://www.jimmunol.org/content/169/11/6316

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Evolution of the Cutaneous Immune Response to Experimental Haemophilus ducreyi Infection and Its Relevance to HIV-1 Acquisition1,2

Tricia L. Humphreys,3* Carol T. Schnizlein-Bick,* Barry P. Katz,* Lee Ann Baldridge,† Antoinette F. Hood,‡ Robert A. Hromas,*§ and Stanley M. Spinola*¶ͦͦ

Haemophilus ducreyi causes the sexually transmitted disease chancroid, which facilitates HIV-1 transmission. Skin biopsies were obtained from subjects experimentally infected with H. ducreyi to study the evolution of the immune response and immunophe- notypes relevant to transmission of HIV-1. Compared with peripheral blood, there was an enrichment of T cells and macrophages after 48 h of infection in the skin. Neutrophils became the predominant cell type by 7–9 days. By immunohistochemistry, macrophage-inflammatory protein-1␣ was not present early in infection, but was abundant at later stages. RANTES was present throughout the papular and pustular stages of experimental infection, but not present in uninfected control skin. Stromal cell- Downloaded from derived factor-1 was present at low levels in all samples examined. Macrophages in lesions had significantly increased expression of CCR5 and CXCR4 compared with peripheral blood cells, and CD4 T cells had significant up-regulation of CCR5. The magnitude of increased expression of these receptors was not replicated when PBMCs were incubated with H. ducreyi or H. ducreyi lipooligosaccharide in vitro. Together with the disruption of mucosal and skin barriers, the presence of cells with up-regulated HIV-1 coreceptors in H. ducreyi-infected lesions may provide an environment that facilitates the acquisition of R5 (CCR5), X4 http://www.jimmunol.org/ (CXCR4), and dual-tropic HIV-1 strains. The Journal of Immunology, 2002, 169: 6316–6323.

aemophilus ducreyi is the etiologic agent of chancroid, a at which time patients generally seek medical attention. Because genital ulcer disease (GUD).4 Like other GUDs, chan- patients wait until the ulcerative stage to seek treatment, little is H croid increases acquisition of HIV-1 (1, 2). Chancroid is known about the initial stages of disease. endemic in certain areas of Africa and Asia, accounting for up to To characterize the immune response to H. ducreyi infection and 56% of GUD (3–8), and many chancroid endemic countries have study the pathogenesis of chancroid, we developed a human model HIV-1 seroprevalence rates exceeding 8% (9). GUDs may enhance of experimental infection that closely mimics the papular and pus-

HIV-1 transmission by up to 100-fold per sexual contact, although tular stages of natural chancroid (13–15). In the model, the bacteria by guest on September 25, 2021 the exact magnitude of this cofactor effect is difficult to estimate are delivered to the epidermis and dermis on the upper arm with an (10–12). Simulation models predict that GUD had a major effect allergy testing device. One of the strengths of the model is infec- on the spread of HIV-1 in Africa in the 1990s, accounting for the tion of a relevant site of infection, human skin. The histopathology acquisition of up to 40–80% of HIV-1 infections (10). of experimental infection closely resembles that of naturally oc- In naturally occurring chancroid, H. ducreyi most likely enters curring disease (16–18). In the human model, an estimated deliv- the skin through breaks in the epithelium during intercourse. H. ered dose as low as 1–2 CFU may result in papule formation. In ducreyi infects both genital and nongenital skin, as well as mucosal contrast, animal models require inoculation with 104-107 CFU to surfaces and regional lymph nodes. Papules appear first, followed in 2–3 days by pustules. The pustules ulcerate after several weeks, establish infection (19–21). In the human model, sites may progress into pustules or resolve spontaneously. The infection is terminated after 14 days or when a painful pustule develops, gen- Departments of *Medicine, †Anatomic Pathology, ‡Dermatology, §Biochemistry, erally 7–9 days after inoculation. For subject safety, infection is ¶ ͉͉ Microbiology and Immunology, and Pathology and Laboratory Medicine, Indiana not allowed to progress to ulcers. The model is useful in that it University, Indianapolis, IN 46202 mimics the first 2 wk of an infection that in nature is probably Received for publication July 19, 2002. Accepted for publication October 1, 2002. present for 3–6 wk before patients with ulcers seek treatment (22). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance The local immune response to experimental infection consists of with 18 U.S.C. Section 1734 solely to indicate this fact. epidermal pustules filled with polymorphonuclear leukocytes and a 1 This work was supported by Grants AI31494 and AI27863 from the National In- perivascular infiltrate of T cells, macrophages, and a few B cells in stitutes of Allergy and Infectious Diseases. T.L.H. was supported by T32AI07367 from the National Institutes of Allergy and Infectious Diseases. The human challenge the dermis (14, 18). By immunohistopathologic analysis, the ma- trials were also supported by Grant MO1RR00750 to the General Clinical Research jority of T cells are CD4, CD45RO cells. In experimental infec- Center at Indiana University. tion, H. ducreyi associates with the polymorphonuclear leukocytes 2 Portions of this work were presented in abstract form at the International Congress in the pustule and with macrophages at the base of the pustule, but of Sexually Transmitted Disease in Berlin, Germany, 2001, and at the American Society for Microbiology meeting in Salt Lake City, UT, 2002. is not taken up by the phagocytes (23). By escaping phagocytic 3 Address correspondence and reprint requests to Dr. Tricia L. Humphreys, Indiana killing, the bacteria sustain the immune response that most likely University School of Medicine, 435 Emerson Hall, 545 Barnhill Drive, Indianapolis, damages the skin (22). Although the immunohistopathology of IN 46202. E-mail address: [email protected] experimental and natural infection is well described, no quantita- 4 Abbreviations used in this paper: GUD, genital ulcer disease; FSC, forward scatter; LOS, lipooligosaccharide; MIP-1␣, macrophage-inflammatory protein 1␣; SDF-1, tive studies that address kinetics of cell recruitment have been stromal cell-derived factor 1; SSC, side scatter. performed.

HIV-1 is primarily sexually transmitted. HIV-1 uses CD4 as a populations of macrophages and T cells and later evolves into receptor for entry into cells, along with the coreceptors CCR5 and polymorphonuclear leukocyte predominance. We also show that CXCR4 (24, 25). HIV-1 has been classified according to corecep- HIV-1 coreceptors CCR5 and CXCR4 are enriched on macro- tor use. R5 viruses use CCR5 as a coreceptor for entry, while X4 phages, and CCR5 is enriched on T cells throughout experimental viruses use CXCR4 as a coreceptor (26). Some primary isolates of infection. HIV-1 can use both coreceptors, while many laboratory-adapted strains use CXCR4 only. Although the type of virus that is trans- Materials and Methods mitted has not been directly studied, the majority of virus in semen Human subjects are R5 (27). Shortly after seroconversion, most individuals have Twenty-two healthy adult volunteers donated 37 tissue specimens for this R5 virus circulating in their peripheral blood, although some in- study. Biopsies designated CS were obtained from seven subjects who dividuals have X4 virus or both types (28). Individuals with a participated in a previous human challenge trial (15, 18). Tissue specimens homozygous ⌬32 mutation in CCR5 are resistant to infection with for FACS analysis were obtained from 10 volunteers (189 and 195–204) HIV-1, confirming the importance of R5 viruses in transmission who were infected for the purposes of this study, 2 subjects who partici- (29–32). To our knowledge, no data exist on the type of virus pated in a mutant parent trial (186 and 190) (33), and 2 subjects (164RR and 172RR) who participated in a reinfection trial (Table I). One unin- acquired by persons who are infected with other sexually transfected volunteer donated normal skin (18). Informed consent was obtained mitted diseases. from the subjects in accordance with the guidelines for human experimen- GUDs such as chancroid are thought to facilitate HIV-1 acqui- tation of the U.S. Department of Health and Human Services and the In- sition by disrupting the mucosal integrity and by recruiting acti- stitutional Review Board of Indiana University-Purdue University (Indianapolis, IN). Downloaded from vated macrophages and CD4 T cells to the skin. Although recruit- The enrollment procedures, exclusion criteria, preparation of the bacte- ment of CD4-expressing cells has been demonstrated in both ria, and inoculation procedures for the human challenge experiments are natural and experimental H. ducreyi infection, the hypothesis that described in detail elsewhere (13–15). Each volunteer was inoculated at these cells have up-regulated chemokine receptors that serve as two or three sites on the upper arm with live H. ducreyi 35000HP (a human HIV-1 coreceptors has not been tested. passaged isolate of 35000) and at one site with heat-killed 35000HP. Up to three biopsy specimens were obtained from each subject (Table I). Some In this study, we describe the evolution of recruitment of neu- subjects were assigned to biopsy 1, 2, or 4 days after inoculation. Other trophils, macrophages, T cells, and B cells to sites experimentally subjects were biopsied when they developed painful pustules, which http://www.jimmunol.org/ infected with H. ducreyi. Given the relationship between chancroid ranged from 6 to 10 days after inoculation. All 14 subjects who donated and HIV-1 acquisition, we also examined experimental lesions for tissue for FACS analysis also donated peripheral blood for FACS analysis at the time of biopsy. After biopsy, infected subjects were treated with two the T cell- and monocyte-attractant chemokines RANTES (CCL5), doses of ciprofloxacin. macrophage-inflammatory protein (MIP)-1␣ (CCL3), and stromal cell-derived factor 1 (SDF-1; CXCL12), and examined macro- Immunohistochemical analysis phages and CD4 T cells for up-regulation of their respective re- Nine banked specimens (designated CS or control) of Formalin-fixed, par- ceptors, CCR5 and CXCR4. Our data demonstrate that the cuta- affin-embedded tissue were deparaffinized and hydrated. Ag retrieval was neous immune response to H. ducreyi initially consists of enriched performed with citrate buffer (DAKO Target Retrieval, pH 6.0; DAKO, by guest on September 25, 2021

Table I. Sources of tissue samples

No. of Biopsies Contributed Day of Reference or Subject No. Inoculuma Biopsy Live Heat Killed Analysesb Source

Control 0 NAc NA NA I 18 CS5 44 7 1 I 15, 18 CS7 44 9 1 I 15, 18 CS8 44 8 1 I 15, 18 CS10 27 4 1 I 15, 18 CS11 30 4 1 I 15, 18 CS12 30 1 2 I 15, 18 CS16 18 1 1 1 15, 18 186 60 6 1 L 33 189 57 7 1 1 L,Cd This study 190 54 10 1 L 33 195 51 7 2 L This study 196 71 2 3 L This study 197 61 7 2 1 L,C This study 199 61 2 2 1 L This study 200 74 7 1 L This study 201 74 2 1 1 L,Ce This study 202 90 2 2 1 C This study 203 38 7 1 C This study 204 56 2 1 1 C This study 164RR 56 9 1 1 C Unpublished 172RR 56 7 1 1 C Unpublished

a Estimated delivered dose of bacteria in CFU. All sites were inoculated with strain 35000HP or heat-killed 35000HP. b Analyses for which the sample was used. I, immunohistochemistry; L, major cell lineages; C, coreceptor; L, C, both lineage and coreceptor. c NA, not applicable; uninfected subject donated normal skin. d Infected site used for both studies. Control biopsy used only for lineage study. e Infected site used for coreceptor study. Control biopsy used for lineage study. 6318 H. ducreyi INFECTION INCREASES CORECEPTOR EXPRESSION

Carpenteria, CA) at 95°C for 20 min. Samples were cooled and rinsed with Comparisons of the levels (Abs bound per cell) of chemokine receptors

TBS (DAKO), then treated with 3% H2O2 (Fisher Scientific, Pittsburgh, in 2-day vs endpoint lesions were made using a Student’s t test after a log PA) for 15 min. Nonspecific binding was blocked with normal donkey transformation was applied to the data. Comparisons among site types serum for 30 min. Polyclonal primary Abs directed against MIP-1␣, (control, infected, and blood) were performed using a mixed model RANTES, and SDF-1 (R&D Systems, Minneapolis, MN) were diluted ANOVA. The model included a fixed site-type effect and a random effect 1/25, and samples were incubated overnight at 4°C. Biotinylated sec- for subject to account for correlation of sites within the same subject. If a ondary Ab (donkey anti-goat IgG; Jackson ImmunoResearch Labora- significant site-type effect was detected, pairwise comparisons among the tories, West Grove, PA) was diluted 1/100 and incubated 30 min. Signal three types were performed using a Tukey-Kramer adjustment for multiple was detected with streptavidin-HRP (DAKO; 30 min), followed by dia- comparisons. minobenzidine (DAKO; 3 min). Tissues were counterstained with May- er’s hematoxylin. As controls, normal skin and tonsil were probed with Preparation of lipooligosaccharide (LOS) each Ab, or the primary Ab was omitted. Samples were analyzed by a dermatopathologist and scored positive if at least three separate sections LOS was purified from H. ducreyi, as previously described (34). Briefly, exhibited cytoplasmic staining. Samples were only included in the anal- cells were harvested from overnight growth on chocolate agar plates and ysis if both positive and negative controls for that set of samples stained lysed with a French press. The lysate was treated with lysozyme, DNase, appropriately. RNase, and proteinase K. LOS was extracted with hot phenol, then sub- jected to two rounds of low and high speed centrifugation. The LOS prep- Immunophenotyping using four-color flow cytometry aration was examined for protein and nucleic acid contamination. There were Ͻ2 ␮g/ml protein (Pierce, Rockford, IL; bicinchoninic acid assay), Fourteen subjects contributed 28 biopsies for flow cytometry. A total of 13 and no protein bands were visible by silver staining. After agarose gel biopsies were used to determine major cell lineages, 11 were used to de- electrophoresis, there were no nucleic acids visible by ethidium bromide termine chemokine receptor density, and 4 were used for both analyses. We staining.

analyzed 17 biopsies (12 live, 5 heat killed) for major lineages and 15 Downloaded from biopsies (9 live, 6 heat killed) for chemokine receptor density. Entire 5-mm In vitro stimulation of PBMCs punch biopsies were minced in RPMI 1640 (Life Technologies, Rockville, For these studies, PBMCs were used from three infected subjects and two MD) to release leukocytes from the tissue. To maximize cell yield, some additional uninfected healthy volunteers. PBMCs were isolated from pe- samples were digested with collagenase and DNase (Sigma-Aldrich, St. ripheral blood by Ficoll-Hypaque density gradient centrifugation. PBMCs Louis, MO), and others were digested with EDTA (Sigma-Aldrich). En- were incubated with 1 ␮g/ml LOS or 106–107 live H. ducreyi for6hat zyme and EDTA treatments did not affect major cell lineage markers. En- 37°C with 5% CO (35). PBMCs were washed, stained for CCR5 and zyme treatment can affect chemokine receptors, so only EDTA treatment 2

CXCR4, and examined by ﬂow cytometry, as above. Comparisons of the http://www.jimmunol.org/ was used in those experiments. For the coreceptor expression experiments, levels of chemokine receptors on PBMCs treated with LOS or H. ducreyi skin biopsy specimens were obtained from H. ducreyi-naive subjects and vs untreated controls were made using a mixed model ANOVA after a log two volunteers who were participating in a reinfection trial. The level of transformation. If a signiﬁcant site-type effect was detected, pairwise com- coreceptor expression from reinfected subjects was similar to that of naive parisons were performed using a Tukey-Kramer adjustment for multiple subjects. Therefore, we pooled data from both naive and reinfected subjects comparisons. for this analysis.

Abs used Results Evolution of the cellular immune response to H. ducreyi To determine the phenotype of the leukocytes recruited to infected sites (n ϭ 18) and heat-killed control sites (n ϭ 10), cells isolated from biopsy We analyzed cell populations present at 17 sites inoculated with specimens were stained with ﬂuorescent Abs (BD Biosciences, San Jose, live or heat-killed H. ducreyi after 2 days of infection or at clinical by guest on September 25, 2021 CA). The following Abs were used, conjugated to FITC, allophycocyanin, endpoint (7.3 Ϯ 1.3 days) (Table I). Using CD45 vs SSC gating PE, or PerCP: CD3 (clone SK7), CD4 (SK3), CD8 (SK1), CD14 (M␾P9), and lineage-speciﬁc markers, we determined numbers of neutro- CD19 (4G7), and CD45 (2D1). ϩ ϩ Quantitative analysis of chemokine receptors was performed using the phils, tissue macrophages (CD14 ), T cells (CD3 ), and B cells QuantiBRITE system (BD Biosciences), in which Abs are labeled in a 1:1 ratio with PE. Calibrated beads were used to construct a standard curve from which Abs bound per cell were calculated from the geometric mean in the PE channel. Abs bound per cell for macrophages/monocytes and CD4 T cells isolated from biopsy specimens and peripheral blood were determined after subtracting out the values for the isotype controls. The Abs included CCR5 (2D7, PE 1:1), CXCR4 (12G5, PE 1:1), and mouse IgG2a as an isotype control.

Staining and analysis Cells isolated from biopsies were pelleted, suspended in Ab solutions, and incubated 30 min on ice. RBCs were lysed with FACS lysing solution (BD Biosciences), washed once in PBS, fixed in PBS with 2% paraformalde- hyde, and analyzed on a FACSCalibur flow cytometer (BD Biosciences). A threshold was set to eliminate debris, and the percentages and absolute numbers of lymphocytes, monocytes/macrophages, and neutrophils were determined utilizing a gating strategy using CD45 (pan-leukocyte marker) vs 90°C side scatter (SSC). To further analyze lymphocyte subpopulations, the lymphocyte population was gated using forward scatter (FSC) vs SSC, and then the percentages and absolute numbers of T and B lymphocytes were determined using quadrant statistics from dot plots of CD3 vs CD19. FIGURE 1. FACS analysis of major cell lineages from skin biopsy To assess chemokine receptor density, cell types were isolated for anal- specimens. Neutrophils were defined by positive CD45 staining as well as ysis by gating on FSC vs SSC for either lymphocytes or monocytes/mac- FSC and SSC. Macrophages were defined as CD45ϩ/CD14ϩ cells. T cells rophages. These populations were further purified for analysis by using a were obtained by first gating on the lymphocyte population using FSC vs second gating strategy whereby lymphocytes were anchor gated based on SSC, anchor gated on CD45, then defined as CD3ϩ/CD19Ϫ. B cells were bright CD4 fluorescence and monocytes/macrophages were anchor gated ϩ ϩ Ϫ CD45 /CD19 /CD3 cells inside the same lymphocyte gate. Populations based on bright CD14 fluorescence. These double-gated populations were were expressed as cell number Ϯ SD, and all possible events were col- then analyzed using a single-parameter histogram for the mean fluorescence intensity of either CCR5 or CXCR4. To account for the size differ- lected from each sample. n, The number of biopsy specimens obtained. ence between blood monocytes and tissue macrophages, the FSC vs SSC Control biopsies were obtained from sites inoculated with heat-killed H. gate was sized to include populations with a broad FSC, but to exclude ducreyi at day 2 (n ϭ 2) and at endpoint (day 7, n ϭ 3). The data from populations with high SSC characteristics such as neutrophils. control sites were similar and were pooled. The Journal of Immunology 6319

FIGURE 2. FACS analysis of T cell subsets. T cells were identified by first gating on the lymphocyte population using FSC vs SSC, then by CD45ϩ/CD3ϩ anchor gating. The CD3 population was then examined for CD4 and CD8 subsets. Populations were expressed as cell number Ϯ SD, and all possible events were collected from each sample. n, The number of biopsy specimens obtained. Control biopsies were obtained from sites in- Downloaded from oculated with heat-killed H. ducreyi 2(n ϭ 2) and 7 days (n ϭ 2) after inoculation. The data from control sites were similar and were pooled. FIGURE 3. Immunohistochemical staining of skin biopsy sections. A, CS8 probed with anti-MIP-1␣. B, CS5 probed with anti-RANTES. C, CS10 probed with anti-SDF-1. D, Normal skin probed with anti-SDF-1. ϩ (CD19 ). We further characterized T cells as Th (CD4) or T cy- Arrows point to brown-staining positive cells. Images are representative totoxic (CD8). The skin biopsies were all 5 mm in diameter, and sections. Original magnification, ϫ400 each sample was analyzed in its entirety by flow cytometry, with http://www.jimmunol.org/ the number of events collected corresponding to the number of cells present in each sample. There were ϳ20,000 total leukocytes present in control biopsy Immunohistochemical analysis of MIP-1␣, RANTES, and SDF-1 specimens obtained 2–7 days after inoculation with heat-killed Because macrophages and T cells were the predominant mononu- bacteria (Fig. 1). After 2 days of infection, there were ϳ50,000 clear cells present in lesions, we analyzed banked Formalin-fixed, leukocytes per biopsy. Infected endpoint specimens contained paraffin-embedded samples from eight additional lesions and one ϳ200,000 leukocytes. There was an initial influx of T cells and biopsy specimen of uninfected skin for representative chemokines macrophages into the lesions, with T cells accounting for nearly 50% and macrophages accounting for 20% of the total leukocytes associated with T cell and macrophage recruitment (Table II). by guest on September 25, 2021 ␣ present at day 2. Approximately 30% of cells were neutrophils MIP-1 was expressed by the mononuclear cells in the upper der- after 2 days. By endpoint, ϳ75% of the cells in the lesion were mis, but not in cells deeper in the dermis (Fig. 3A, and data not ␣ neutrophils (Fig. 1). In contrast, the numbers of accumulated mac- shown). In addition, MIP-1 was not expressed in the early, pap- rophages and T cells did not change greatly from 2 days to end- ular stage of infection nor in the uninfected control, but was abun- point. B cells made up a minority of the cells at all time points dant at the pustular stage (Table II). Biopsies probed with anti- investigated. The T cell populations in all samples were predom- RANTES Ab showed positive staining in mononuclear cells (Fig. inantly CD4 cells, with a ratio of ϳ2:1 (CD4:CD8) in both pe- 3B). RANTES expression was evident at the earliest stages of in- ripheral blood and the 2-day biopsy specimens (Fig. 2). By end- fection examined, but not present in the uninfected control (Table point, the ratio of CD4 to CD8 cells increased to ϳ3:1. The FACS II). Probing biopsy tissue with anti-SDF-1 Ab resulted in low lev- analysis of the cellular infiltrate reported in this study closely re- els of positive staining in mononuclear cells and occasionally ker- sembles published immunohistologic data, although our earlier re- atinocytes, endothelial cells, or neutrophils (Fig. 3, C and D). Bi- ports underestimated the number of macrophages present in the opsies from different clinical stages (papule, pustule, and no lesions (18). infection) exhibited similar levels of SDF-1 expression.

Table II. Expression of T cell- and monocyte-attractant chemokines in infected skin

Homeostatic Inﬂammatory Days of Clinical Subject No. Infection Appearance SDF-1 RANTES MIP-1␣

Control 0 Uninfected ϩa ϪϪ CS16 1 Papule NDb ϩϪ CS12ac 1 Papule ϩϩϪ CS12b 1 Papule ϩϩϪ CS10 4 Papule ϩϩϩ CS11 4 Pustule ϩϩϩ CS5 7 Pustule ϩϩϩ CS8 8 Pustule ϩϩϩ CS7 9 Pustule ϩϩϩ

a Results are based on staining of at least three separate sections from each specimen. b ND due to lack of specimen. c Biopsy specimens CS12a and CS12b were obtained from different sites from the same subject. 6320 H. ducreyi INFECTION INCREASES CORECEPTOR EXPRESSION

FIGURE 4. FACS analysis of leukocytes isolated from biopsy specimens. Representative histogram of CCR5 and CXCR4 expression on mac- Downloaded from rophages/monocytes and CD4 T cells. Gray line represents peripheral blood cells. Black line represents cells from biopsy.

In vivo expression of CCR5 and CXCR4 ␣ The chemokines RANTES and MIP-1 bind to the HIV-1 core- http://www.jimmunol.org/ ceptor CCR5, and SDF-1 binds to CXCR4, another HIV-1 coreceptor. We examined T cells and macrophages from 15 additional biopsy specimens for cell surface expression of CCR5 and CXCR4. We compared the receptor densities on monocytes/macrophages and CD4 T cells from peripheral blood to control biopsy specimens, 2-day infected sites, and endpoint infected samples. Representative histograms are depicted in Fig. 4, and the absolute receptor density is shown in Fig. 5. Macrophages isolated from sites infected with heat-killed or live H. ducreyi had significantly by guest on September 25, 2021 increased levels of both CCR5 and CXCR4. CCR5 levels in tissue macrophages were increased (range, 16- to 36-fold) relative to FIGURE 5. Density of CCR5 (A) and CXCR4 (B) on macrophages/ levels of peripheral blood monocytes ( p Ͻ 0.0001), and CXCR4 monocytes and CD4 T cells. Cells were analyzed using QuantiBRITE such levels were increased (range, 7- to 10-fold) relative to that of blood that Abs bound per cell are directly related to the number of receptors per monocytes ( p Ͻ 0.0001). CD4 T cells from biopsy specimens had cell. Values for isotype controls were subtracted out to compensate for a more modest (range, 2.1- to 2.6-fold), but statistically significant background staining. Data are expressed as mean Ϯ SD. Asterisks denote increase in surface expression of CCR5 in the skin ( p ϭ 0.003). statistical differences from peripheral blood (p Յ 0.003). CXCR4 levels on CD4 T cells were not statistically different from peripheral blood ( p ϭ 0.43). Thus, macrophages present during experimental infection expressed increased levels of CXCR4 and with LOS ( p ϭ 0.03), and increased CCR5 levels 2.2-fold in re- CCR5, and CD4 T cells expressed increased levels of CCR5. sponse to H. ducreyi ( p ϭ 0.01). Exposure of monocytes to LOS There was no statistical difference in coreceptor density between or H. ducreyi had no significant effect on CXCR4 levels relative to macrophages and T cells isolated from heat-killed control sites and controls. Thus, the pattern and magnitude of the up-regulation of infected sites. CCR5 and CXCR4 on macrophages and CCR5 on T cells in vivo were not replicated in vitro. In vitro expression of CCR5 and CXCR4 Previous reports indicated that bacterial products such as LOS af- Discussion fect expression of CCR5 and CXCR4 in vitro. To determine In this study, we examined the kinetics of leukocyte infiltration in whether H. ducreyi and/or LOS were responsible for the changes skin biopsy specimens from experimental H. ducreyi infection. in coreceptor expression noted in vivo, we measured CCR5 and After 2 days of infection, about one-half of the infiltrating leuko- CXCR4 expression on PBMCs stimulated for 6 h with purified cytes were T cells, followed in abundance by neutrophils and mac- LOS or 106-107 CFU of live H. ducreyi. LOS was used at a con- rophages. Between day 2 and endpoint (7.3 Ϯ 1.4 days), there was centration (1 ␮g/ml) that is equivalent to ϳ107 CFU of H. ducreyi an influx of neutrophils into the infected site, and little change in (36). CD4 T cells stimulated with LOS in vitro did not exhibit any the number of accumulated T cells and macrophages. We detected change in coreceptor expression relative to untreated controls (data expression of the chemokines MIP-1␣, RANTES, and SDF-1 in not shown). CD4 T cells up-regulated CXCR4 1.3-fold in response experimental lesions, as well as up-regulation of their respective to live H. ducreyi, a small, but statistically significant increase receptors, CCR5 and CXCR4. In addition to the physical disrup- ( p ϭ 0.008). Levels of CCR5 on CD4 T cells incubated with live tion of mucosal and skin barriers that occurs during chancroid, our H. ducreyi were unchanged relative to controls. Monocytes exhib- data suggest that H. ducreyi infection provides an environment that ited significantly decreased levels of CCR5 (1.9-fold) when treated may enhance the acquisition of X4, R5, or dual-tropic HIV-1. The Journal of Immunology 6321

MIP-1␣ and RANTES are inflammatory chemokines that serve up-regulation of receptor density on T cells, although acquisition as T cell and monocyte attractants (37). SDF-1 is a homeostatic of the phenotype at the site of infection cannot be excluded. chemokine responsible for normal lymphocyte and monocyte traf- Although H. ducreyi infection leads to accumulation of tissue ficking through tissues (38). As might be expected for inflamma- macrophages that had significant increases in receptor density for tory chemokines, MIP-1␣ and RANTES were highly expressed in both CCR5 and CXCR4 relative to peripheral blood monocytes, in the infected lesions, but not expressed in uninfected control tissue. vitro stimulation of monocytes with H. ducreyi LOS decreased SDF-1, in contrast, was expressed in all tissues examined. The expression of CCR5, while stimulation with live H. ducreyi re- receptors for these chemokines, CCR5 and CXCR4, serve as sulted in a small (2.2-fold), but statistically significant increase in HIV-1 coreceptors. In vitro, the presence of SDF-1 prevents in- CCR5 expression. Incubation of monocytes with LOS or H. du- fection of transfected HeLa cells and PHA-stimulated PBMCs by creyi had no significant effect on levels of CXCR4. Thus, the up- HIV-1 (39, 40), but RANTES and MIP-1␣ do not suppress HIV-1 regulation of coreceptors on monocytes by H. ducreyi in vivo is at replication in monocytes/macrophages (41, 42). Thus, the up-reg- least an order of magnitude greater than in vitro. In contrast to our ulation of MIP-1␣ and RANTES in chancroidal lesions may not findings during infection, the effects of purified bacterial products block HIV-1 entry via CCR5. on CCR5 and CXCR4 expression on monocytes and macrophages We found a highly statistically significant increase in density of have been variable (35, 45, 50–54). In general, an increase in one CCR5 and CXCR4 on tissue macrophages relative to peripheral coreceptor is accompanied by a decrease or no change in expres- blood monocytes in response to experimental infection with H. sion of the other coreceptor. Changes in the levels of chemokine ducreyi. A statistically significant increase in density of CCR5 was receptors caused by treatment of blood monocytes with bacterial also present on infiltrating T cells relative to peripheral blood T products in vitro may not reflect changes that occur in vivo due to Downloaded from cells. Sites infected with heat-killed bacteria contained fewer leu- the complex microenvironment that promotes recruitment and ac- kocytes than sites infected with live bacteria, but these leukocytes cumulation of macrophages at sites of infection. also had increased surface expression of CCR5 and CXCR4 that To our knowledge, this is the first demonstration that a bacterial was similar to levels present on leukocytes recruited to infected infection leads to accumulation of macrophages with up-regulation sites. Wounding of the skin and injection of heat-treated bacteria of both CCR5 and CXCR4 and CD4 T cells that express CCR5 at

are probably sufficient to create a microenvironment that recruits T the site of disease. We did not examine other skin infections and http://www.jimmunol.org/ cells and macrophages with up-regulated receptors to the skin. cannot exclude the possibility that the changes observed are not H. Bacterial replication that occurs in the model (43) most likely in- ducreyi specific. Nonetheless, the up-regulation of both CCR5 and creases the number of inflammatory cells that accumulate at in- CXCR4 by experimental H. ducreyi infection may have major im- fected sites compared with mock-infected sites. plications for HIV-1 acquisition. First, R5 strains of HIV-1 are the We did not examine coreceptor expression levels in normal skin. most common sexually transmitted strains (55–57). The striking A 5-mm punch biopsy of an experimental chancroid lesion is 0.19 up-regulation of CCR5 on macrophages and the influx of macro- cm2, and contains approximately the same number of T cells as is phages and CCR5-expressing T cells into the lesion provide a pop- found in 10 cm2 of human facial skin (J. Campbell, personal com- ulation of R5-susceptible cells. The increased expression of munication). Thus, we would have needed to obtain 50 punch bi- CXCR4 also offers an environment that could allow for acquisition by guest on September 25, 2021 opsies of normal skin to obtain sufficient cells to perform a com- of X4 viruses. Finally, the infiltrating leukocytes could readily pro- parison between inoculated and uninfected skin, which was mote acquisition of dual-tropic strains. Thus, H. ducreyi infection impractical. To our knowledge, the level of CCR5 and CXCR4 not only disrupts epithelial barriers, but provides a microenviron- expression on macrophages in normal skin has not been deter- ment that could readily lead to acquisition of R5, X4, and dual- mined. However, the majority of CD4 cells found in normal skin tropic HIV-1. express CCR5 (44). Thus, the CD4 cells recruited to experimental In the human infection model, volunteers are infected until they chancroid lesions differ from those that traffic to normal skin in develop a painful pustule or for 2 wk. The average duration of numbers, but not in ability to express CCR5. This is consistent infection for naturally occurring chancroid is estimated to be 5 wk with the concept that CCR5 expression may be a common pheno- (58). Due to a lack of readily available specimens, the expression type of tissue infiltrating T cells (44). levels of CCR5 and CXCR4 on macrophages and T cells in lesions We detected a 2.1- to 2.6-fold increase in CCR5 expression on of naturally occurring chancroid have not been determined. How- T cells recruited to infected sites vs those found in peripheral ever, the histopathology of experimental and natural disease is blood. The percentage of CD4 T cells that were CCR5ϩ in lesions nearly identical (16–18). We speculate that increased coreceptor was ϳ2.4-fold higher than those in peripheral blood. Similarly, expression may occur in natural disease. Together with disruption there are no changes in the level of CCR5 expression on T cells of mucosal barriers, recruitment of cells with increased coreceptor obtained from the skin of volunteers injected with synthetic Trepo- expression may be responsible for the relationship between chan- nema pallidum lipopeptides, although the percentage of T cells croid and HIV-1 acquisition. In future studies, we plan to isolate T expressing CCR5 recruited to these sites is increased (45). Com- cells and macrophages from experimental lesions and test their pared with peripheral blood, sites of inflammation are enriched for susceptibility to primary isolates of R5, X4, and dual-tropic HIV-1. CCR5ϩ T cells (46, 47). In peripheral blood, CCR5 is expressed on a subset of memory/effector T cells, while CXCR4 is present on Acknowledgments naive T cells (48, 49). The T cells present in experimental H. We thank Margaret Bauer, Jim Campbell, Ray Johnson, and Justin Radolf ducreyi lesions are predominantly memory/effector cells in that ϩ for their thoughtful criticism of the manuscript; Cliffton Bong and Kate they are CD45RO (18). Similarly, T cells that infiltrate the skin Fortney for their assistance with the human challenge trials; and the vol- following injection of T. pallidum lipopeptides are also predomi- unteers who participated in the study. nantly memory/effector cells (45). In vitro stimulation of PBMCs with LOS or live H. ducreyi did not affect CCR5 levels on T cells. Therefore, for CCR5, the increased receptor expression reported References for T cells most likely reflects selective migration of CCR5ϩ/ 1. Wasserheit, J. N. 1992. Epidemiological synergy. interrelationships between hu- ϩ man immunodeficiency virus infection and other sexually transmitted diseases. CD45RO memory/effector T cells into the skin rather than an Sex. Transm. Dis. 19:61. 6322 H. ducreyi INFECTION INCREASES CORECEPTOR EXPRESSION

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