The chemokine receptors (CCRs) CCR4 and CCR10, and the cutaneous lymphocyte antigen (CLA), have each been proposed as critical mediators of skin-specific T H lymphocyte homing in mice and humans. CLA initiates skin homing by mediating E-selectin-dependent tethering and rolling within cutaneous venules, but the specific roles of CCR4 and CCR10 are unclear. We have generated an antihuman CCR10 monoclonal antibody (mAb; 1B5) to illuminate the individual contributions of these molecules. This mAb allows us to compare CCR10, CCR4, and CLA expression within human T H populations. The mAb 1B5 recognizes functional CCR10 expression, as chemotactic responsiveness to cutaneous T-cell-attracting chemokine (CTACK)/CCL27 (a CCR10 ligand) parallels the staining of T H subsets. We find CCR10 expressed by only a minority (approximately 30%) of bloodborne, skin-homing (CLA ؉ /CCR4 ؉ ) T H cells. However, essentially all members of the relatively small "effector" (CLA ؉ / CCR4 ؉ /CD27 ؊ /CCR7 ؊ ) skin-homing T H population express CCR10. Most skininfiltrating lymphocytes in allergic delayed-type hypersensitivity (DTH) and bacterial chancroid skin lesions express both CCR4 and CLA, but only about 10% express CCR10. This suggests for the 2 models of T H skin homing studied here that CCR10 ؉ T H cells have no advantage over other CLA ؉ /CCR4 ؉ T H cells in homing to cutaneous sites. We conclude that the skin-homing T H compartment is itself divided into distinct subpopulations, the smaller of which expresses both CCR4 and CCR10, and the larger of which expresses only CCR4. Thus, CCR10 is unlikely to be necessary for cutaneous homing of T H cells in the models studied here. CCR10 may instead play a role in the movement of specialized "effector" cutaneous T H cells to and/or within epidermal

Human subjects were experimentally infected with Haemophilus ducreyi for up to 2 weeks. Bacterial suspensions were delivered into the epidermis and dermis through puncture wounds made by an allergy-testing device. Subjects developed papular lesions that evolved into pustules resembling natural disease. Some papular lesions resolved spontaneously, indicating that host responses may clear infection. Bacteria were shed intermittently from lesions, suggesting that H. ducreyi may be transmissible before ulceration. Host responses to infection consisted primarily of cutaneous infiltrate of polymorphonuclear leukocytes, Langerhans cells, macrophages, and CD4 T cells of alpha beta lineage. Expression of HLA-DR by keratinocytes was associated with the presence of interferon-gamma mRNA in the skin. There was little evidence for humoral or peripheral blood mononuclear cell responses to bacterial antigens. The cutaneous infiltrate of CD4 cells and macrophages provides a mechanism that facilitates transmission of human immunodeficiency virus by H. ducreyi.

Four subjects were experimentally infected with Haemophilus ducreyi. Lesions developed only at sites where live bacteria were inoculated on abraded skin. No subject developed fever, lymphadenopathy, or disseminated infection during a 3-day observation period. Two subjects who were rechallenged 2 months after initial infection also developed lesions. The amount of H. ducreyi recovered from 10 of 12 biopsies that were semiquantitatively cultured varied widely. Similar histologic features were present in initial and second infections. The epidermis contained pustules; the dermis contained an infiltrate of T cells and macrophages and reactive endothelial cells. Keratinocytes and T cells expressed HLA-DR, consistent with a delayed-type hypersensitivity response. The subjects did not mount humoral responses to bacterial proteins and to lipooligosaccharides after primary and secondary challenges. Thus, human experimental infection with H. ducreyi is well tolerated and safe. Recruitment of T cells and macrophages into chancroid lesions may partially explain the association between chancroid and human immunodeficiency virus transmission.

In a previous study, Haemophilus ducreyi was found in the pustule and dermis of samples obtained at the clinical end point in the human model of infection. To understand the kinetics of localization, we examined infected sites at 0, 24, and 48 h after inoculation and at the clinical end point. Immediately after inoculation, bacteria were found predominantly in the dermis but also in the epidermis. Few bacteria were detectable at 24 h; however, by 48 h, bacteria were readily seen in the pustule and dermis. H. ducreyi was associated with polymorphonuclear leukocytes and macrophages in the pustule and at its base, but was not associated with T cells, Langerhans' cells, or fibroblasts. H. ducreyi colocalized with collagen and fibrin but not laminin or fibronectin. Association with phagocytes, collagen, and fibrin was seen as early as 48 h and persisted at the pustular stage of disease. Optical sectioning by confocal microscopy and transmission electron microscopy both failed to demonstrate intracellular H. ducreyi. These data identify collagen and fibrin as potentially important targets of adherence in vivo and strongly suggest that H. ducreyi remains extracellular throughout infection and survives by resisting phagocytic killing in vivo.

Human volunteers were challenged with Haemophilus ducreyi. Twenty subjects were inoculated with 2 doses (approximately 30 cfu) of live and 1 dose of heat-killed bacteria at 3 sites on the arm. Eight subjects were assigned to biopsy 1 or 4 days after inoculation, and 12 were biopsied after they developed a painful pustular lesion or were followed until disease resolved. Papules developed at 95% of 40 sites infected with live bacteria (95% confidence interval [CI], 83. 1%-99.4%). In 24 sites followed to end point, 27% of the papules resolved, 69% (95% CI, 47.1%-86.6%) evolved into pustules, and 4% remained at the papular stage. Recovery rates of H. ducreyi from surface cultures ranged from 13% to 41%. H. ducreyi was recovered from biopsies of 12 of 15 pustules and 1 of 7 papules, suggesting that H. ducreyi replicates between the papular and pustular stages of disease.

Haemophilus ducreyi must adapt to the environment of the human host to establish and maintain infection in the skin. Bacteria generally utilize stress response systems, such as the CpxRA two-component system, to adapt to hostile environments. CpxRA is the only obvious two-component system contained in the H. ducreyi genome and negatively regulates the lspB-lspA2 operon, which encodes proteins that enable the organism to resist phagocytosis. We constructed an unmarked, in-frame H. ducreyi cpxA deletion mutant, 35000HP⌬cpxA. In human inoculation experiments, 35000HP⌬cpxA formed papules at a rate and size that were significantly less than its parent and was unable to form pustules compared to the parent. CpxA usually has kinase and phosphatase activities for CpxR, and the deletion of CpxA leads to the accumulation of activated CpxR due to the loss of phosphatase activity and the ability of CpxR to accept phosphate groups from other donors. Using a reporter construct, the lspB-lspA2 promoter was downregulated in 35000HP⌬cpxA, confirming that CpxR was activated. Deletion of cpxA downregulated DsrA, the major determinant of serum resistance in the organism, causing the mutant to become serum susceptible. Complementation in trans restored parental phenotypes. 35000HP⌬cpxA is the first H. ducreyi mutant that is impaired in its ability to form both papules and pustules in humans. Since a major function of CpxRA is to control the flow of protein traffic across the periplasm, uncontrolled activation of this system likely causes dysregulated expression of multiple virulence determinants and cripples the ability of the organism to adapt to the host.

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The lipooligosaccharides (LOS) of strains of Haemophilus ducreyi, Neisseria gonorrhoeae, Neisseria meningitidis, and Neisseria lactamica contain epitopes that are antigenically and structurally similar to carbohydrates present in human glycosphingolipids. LOS from strains of Haemophilus influenzae and H. influenzae biogroup aegyptius were tested for the binding of monoclonal antibodies (MAbs) that bind to human glycosphingolipids possessing Gal beta 1-4GlcNAc (MAb 3F11) and Gal alpha 1-4Gal beta 1-4Glc (MAb anti-Pk). In solid-phase radioimmunoassays, the LOS of 18 of 19 H. influenzae type b (Hib), 8 of 19 nontypeable H. influenzae, and 10 of 20 H. influenzae biogroup aegyptius strains bound MAb anti-Pk. The LOS of 13 of 19 Hib, 10 of 16 nontypeable H. influenzae, and 2 of 18 H. influenzae biogroup aegyptius strains bound MAb 3F11. Neuraminidase treatment of the strains increased the binding of MAb 3F11 by more than twofold in 47% of the H. influenzae strains, suggesting that sialic acid occluded the LOS structure recognized by MAb 3F11. The material released from neuraminidase-treated Hib LOS was confirmed to be sialic acid by high-performance anion-exchange chromatography. A recombinant plasmid containing genes involved in Hib LOS biosynthesis directed the expression (assembly) of the 3F11 epitope in Escherichia coli. These studies demonstrate that H. influenzae and H. influenzae biogroup aegyptius express at least two LOS epitopes that are similar to those present in human glycosphingolipids. Sialic acid was present on the LOS of some H. influenzae strains and prevented the binding of MAb 3F11 to its epitope. The oligosaccharide portion of sialylated LOS may also resemble sialylated oligosaccharides present in human glycosphingolipids (gangliosides).