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Keywords:

  • T-cell;
  • homing receptor;
  • infection;
  • skin

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We investigated the regulation of T-cell homing receptors in infectious disease by evaluating the cutaneous lymphocyte antigen (CLA) in human leprosy. We found that CLA-positive cells were enriched in the infectious lesions associated with restricting the growth of the pathogen Mycobacterium leprae, as assessed by the clinical course of infection. Moreover, CLA expression on T cells isolated from the peripheral blood of antigen-responsive tuberculoid leprosy patients increased in the presence of M. leprae (2·4-fold median increase; range 0·8–6·1, n = 17), but not in unresponsive lepromatous leprosy patients (1·0-fold median increase; range 0·1–2·2, n = 10; P < 0·005). Mycobacterium leprae specifically up-regulated the skin homing receptor, CLA, but not α47, the intestinal homing receptor, which decreased on T cells of patients with tuberculoid leprosy after antigen stimulation (2·2-fold median decrease; range 1·6–3·4, n = 3). Our data indicate that CLA expression is regulated during the course of leprosy infection and suggest that T-cell responsiveness to a microbial antigen directs antigen-specific T cells to the site of infection.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

To combat infection, T cells migrate from their site of activation, a lymph node, to the site of disease, usually peripheral tissues. Regulation of cell surface receptors that mediate tissue-specific homing in the context of bacterial infection has not been thoroughly investigated. Activated T cells express homing receptors, which direct their migration from lymphoid tissue, home to discrete locations and extravasate into the tissue. Cutaneous lymphocyte antigen (CLA) is a homing receptor expressed on T cells that infiltrate the skin.1–3 The CLA molecule is a post-translational modification of P-selectin glycoprotein ligand 1 (PSGL-1 or CD162) a protein that binds to P-selectin and mediates tethering and rolling on endothelium for extravasation into tissue.4 Whereas CD162 is expressed ubiquitously on circulating T cells, CLA is expressed on T cells that escape specifically into inflammatory sites in the skin as opposed to other organs.5

We sought to evaluate the expression of CLA in bacterial infection using leprosy as a model. T cells infiltrate the skin lesions of leprosy patients across the spectrum of immunological responses to the pathogen, Mycobacterium leprae. However, the T cells of patients with tuberculoid leprosy, whose disease is self-limited, produce interferon-γ (IFN-γ),6,7 a cytokine that is considered protective against mycobacterial infection.8 In contrast, patients with lepromatous leprosy, those with disseminated disease, do not mount an effective T-cell response to the pathogen in their lesions. The T cells of lepromatous patients do not proliferate or produce IFN-γ in response to M. leprae; instead, the T cells derived from lepromatous patients produce interleukin-4 (IL-4),6,7 a cytokine that promotes antibody production but that is not protective against mycobacterial disease. We reasoned that CLA might play a role in the migration of T-cell subsets into leprosy lesions; therefore we investigated the regulation of CLA expression in the cutaneous immune response to leprosy infection.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Patient samples and bacterial extracts

Leprosy patients (Table 1) were recruited on a volunteer basis from the ambulatory population seen at the Hansen's Disease Clinic at the University of Southern California Los Angeles County Medical Center. Clinical classification of patients with symptomatic M. leprae infection was performed according to the criteria of Ridley and Jopling.9 Skin biopsy specimens (6 mm diameter) containing both epidermis and dermis were obtained by standard punch technique following informed consent. Biopsy specimens were embedded in optimal cutting temperature (OCT) medium (Ames Co., Elkhart, IN) and snap-frozen in liquid nitrogen. Blood samples for isolation of peripheral blood mononuclear cells (PBMC) were obtained by venepuncture from leprosy patients.

Table 1.   Patient characterization
Patient classificationTuberculoid leprosyLepromatous leprosy
Number2314
Gender (male/female)16/714/2
Age in years  (mean/median)57·1/5546·3/46
Age range (years)30–7428–70
Time since treatment began  (mean/median in years)13·9/814·8/7
Time since treatment  began (range in years) 0·17–34 0·02–32

Extracts of M. leprae (obtained from Dr Patrick Brennan, Colorado State University, through National Institutes of Health contract N01 AI25469) were prepared by probe sonication as described previously.10

Measurement of CLA expression in vivousing immunohistochemistry

CLA and CD162 expression were detected in leprosy lesions using immunohistochemistry. Sections (3–5 μm thick) were acetone-fixed and blocked with either normal horse serum or normal mouse serum before incubation with monoclonal antibodies (Table 2) for 60 min, followed by either biotinylated horse anti-mouse immunoglobulin G (IgG) or biotinylated mouse anti-rat IgM for 30 min. Primary antibody was visualized with the ABC Elite system (Vector Laboratories, Burlingame, CA), which uses avidin and a biotin–peroxidase conjugate for signal amplification. The ABC reagent was incubated for 1 hr followed by the addition of substrate (3-amino-9-ethylcarbazole) for 10 min. Slides were counterstained with haematoxylin and mounted in Crystal Mount (Biomeda Corp., Foster City, CA). To preserve the signal that was detected on slides, the samples were mounted the following day with Permount (Fisher Scientific; Fair Lawn, NJ). The level of CLA-positive and CD162-positive cells in dermal granulomas was quantified by calculating the percentage of positive cells based on the total number of cells (i.e. haematoxylin-stained nuclei) within the granuloma.

Table 2.   Antibodies used to evaluate CLA and CD162 in leprosy lesions
Antibody specificityCloneIsotype Source1
  • 1

    BD Pharmingen, San Diego, CA; Biomeda Corp., Foster City, CA; Sigma, St Louis, MO.

CLAHECA-452Rat IgMBD Pharmingen
CD3B355·1Mouse IgG3Biomeda Corp.
CD68Y1/82 AMouse IgG2bBD Pharmingen
CD162KPL-1Mouse IgG1BD Pharmingen
Isotype controlMOPC-21Mouse IgG1Sigma
Isotype controlMOPC-141Mouse IgG2bSigma
Isotype controlR4-22Rat IgMBD Pharmingen

Double immunofluorescence was performed by serially incubating cryostat tissue sections with mouse anti-human monoclonal antibodies of different isotypes [e.g. B355.1 (anti-CD3, IgG3)], and either HECA-452 (anti-CLA, rat IgM; Pharmingen, San Diego, CA) or KPL-1 (anti-CD162; IgG1, Pharmingen) followed by incubation with isotype-specific, fluorochrome (fluorescein isothiocyanate; Caltag Laboratories; Burlingame, CA) or Alexa Fluor 568 (Molecular Probes; Eugene, OR) -labelled goat anti-mouse immunoglobulin antibodies. HECA-452 (anti-CLA), however, was amplified with a biotinylated mouse anti-rat followed by incubation with streptavidin tetramethylrhodamine isothiocyanate (Southern Biotechnology Associates, Inc., Birmingham, AL). Slides were observed on a Nikon Labophot-2 microscope (Nikon Instruments, Inc., Melville, NY) and images were digitally acquired and manipulated using a Spot camera and version 3.2.4 software (Diagnostic Instruments, Inc., Sterling Heights, MI).

Double immunofluorescence of sections and cells was also examined with a Leica-TCS-SP inverted confocal laser scanning microscope fitted with krypton and argon lasers at the Carol Moss Spivak Cell Imaging Facility in the UCLA Brain Research Institute. Sections and cells were illuminated with 488 and 568 nm of light after filtering through an acoustic optical device. Images decorated with fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, Alexa 488, or Alexa 568 (Molecular Probes) were recorded simultaneously through separate optical detectors with a 530-nm band-pass filter and a 590-nm long-pass filter, respectively. Pairs of images were superimposed for colocalization analysis.

Measurement of T-cell homing receptor expression in vitrousing flow cytometry

To measure CLA expression on T cells after in vitro activation with M. leprae, PBMCs were isolated by density gradient centrifugation (Ficoll-Paque, Amersham-Pharmacia, Piscataway, NJ). Freshly isolated PBMCs (1 × 106/ml) were cultured (X-VIVO 15 medium, Cambrex, Bio-Whittaker Inc, Walkersville, MD) in a 24-well plate with M. leprae (3 μg/ml, prepared by probe sonication11), or anti-CD3 (10 μg/ml) for 12 days to induce CLA expression.12 Cells were maintained in culture medium supplemented with recombinant IL-2 (1 nm, Chiron Diagnostics, Norwood, MA) on day 5 and day 8 of culture. Time–course studies demonstrated that maximal levels of CLA expression in antigen-stimulated cultures occurred at day 12. Cell surface expression of CLA was determined by flow cytometry using a CLA-specific monoclonal antibody and a CD3-specific monoclonal antibody on PBMCs from leprosy patients. Cells evaluated for CLA were first gated on lymphocytes and then CD3 expression. Results are therefore reported as CLA-positive T cells. Expression of the integrin α47 was examined under the same conditions, using antibodies for integrin α4 (anti-CD49d, Serotec, Raleigh, NC) and integrin β7 (anti-β7, Pharmingen). To determine the role of superantigens in the up-regulation of CLA on T cells in leprosy patients, T cells were isolated from the blood of leprosy patients using RosetteSep (Stem Cell Technologies, Vancouver, BC, Canada), cells were cultured with M. leprae as described above, and CLA expression was evaluated by flow cytometry.

Measurements of T-cell responsiveness

To assess T-cell responsiveness, PBMCs (1 × 106/ml) were stimulated with M. leprae (3 μg/ml, 5 days) and proliferation was determined by [3H]thymidine incorporation assays.11 Cytokine levels in culture supernatants (48 hr) were measured by enzyme-linked immunosorbent assay using commercially available antibodies (IFN-γ, Pharmingen; IL-4, IL-10, IL-12, Biosource, Camarillo, CA). Expression of CD86 was determined by flow cytometry (clone 2331, Pharmingen).

Statistical analysis

For statistical comparisons between tuberculoid and lepromatous patients, the Mann–Whitney U-test was applied. Non-parametric methods were used because the data were not normally distributed. P < 0·05 was considered significant.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

CLA expression in leprosy lesions correlates with clinical form of the disease

To examine the regulation of T-cell homing receptors in the context of bacterial infection, we compared the levels of CLA in tuberculoid and lepromatous leprosy lesions. We found CLA to be expressed in the granulomas of tuberculoid leprosy patients, at much higher levels than in the lesions of patients with lepromatous leprosy (Fig. 1a, one representative patient each) in contrast to CD162 expression, which was equivalent across the leprosy spectrum. When CLA expression was enumerated in five patients, it was found to be significantly higher in tuberculoid patients (median = 5·4, range 3·3–8·6, n = 5) compared to lepromatous patients (median = 1·5, range 0·7–2·1, n = 5; P < 0·05) (Fig. 1b). In contrast, CD162 was expressed at equal levels in the cutaneous lesions of patients with tuberculoid (median = 9·5, range 8·2–21·8, n = 5) and lepromatous (median = 11·7, range 3·9–19·1, n = 6; P = not significant) leprosy. The data indicate that CLA expression in leprosy lesions correlates with the ability to limit growth of the pathogen in vivo. The increase in CLA is probably not the result of a simple increase in T cells infiltrating the lesions of tuberculoid patients because T-cell numbers are comparable in the cutaneous infiltrate of tuberculoid and lepromatous patients.13,14

image

Figure 1.  Expression of T-cell homing receptors in leprosy lesions. (a) Immunohistochemical analysis of CLA and CD162 expression in one representative tuberculoid leprosy patient (T-lep) and a lepromatous patient (L-lep). Original magnification 200 ×. (b) Expression of CLA (left panel) and CD162 (right panel) in several leprosy patients quantified as % positive cells in a granuloma. Horizontal bars indicate means. Mean values of CLA and CD162 levels were compared between T-lep and L-lep patients using non-parametric methods and P-values are indicated. n.s. = not significant.

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In addition to T cells, CLA is expressed on monocytes and granulocytes15 and although granulocytes are not common in leprosy lesions, monocytes are prominent in the granulomas of leprosy lesions. To determine the phenotype of CLA-positive cells in tuberculoid lesions, we labelled cells with CLA and CD3, a T-cell marker. We found that the majority of, though not all, CLA-positive cells were CD3-positive (Fig. 2a), indicating that the primary CLA-positive cells in leprosy lesions were T cells. To examine the expression of CLA on myeloid cells in the skin lesions of leprosy patients, we labelled cells with CLA and CD68 because it has been demonstrated that CD68-positive cells express CLA in skin.16 We found that CD68-positive cells were effectively devoid of CLA expression (Fig. 2b), confirming that virtually all of the CLA-positive cells in the skin lesions of tuberculoid leprosy patients were CD3-positive T cells (Fig. 2b).

image

Figure 2.  CLA is expressed on T lymphocytes in tuberculoid leprosy lesions. (a) Fluorescence micrographic image of a tuberculoid leprosy lesion. Colocalization of T cells (CD3-green, left panel) with CLA (red, central panel) is visualized in merged image (right panel). (b) Confocal laser micrographic images of a tuberculoid leprosy lesion. Macrophages (CD68-red, upper left panel) and CLA (green, upper central panel) do not colocalize (merge, upper right panel), whereas T cells (CD3-red, lower left panel) and CLA (green, lower central panel) do colocalize (merge, lower right panel). Inset in merged image emphasizes the region of colocalization. Original magnification 200 ×.

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Cutaneous homing receptor expression correlates with T-cell responses to M. leprae in vitro

The data from Fig. 1 suggested that CLA expression in leprosy lesions was correlated with T-cell responsiveness in vivo. To investigate whether CLA expression is increased upon T-cell activation in the context of infection, we cultured PBMCs from leprosy patients with M. leprae and measured CLA expression on T cells. PBMCs from tuberculoid patients cultured with M. leprae showed an increase (3·6-fold) in T-cell expression of CLA (Fig. 3a, one representative experiment). In contrast, T cells of a lepromatous patient did not up-regulate CLA (Fig. 3a, one representative experiment). However, CLA expression was induced in the lepromatous patient by anti-CD3 antibodies (Fig. 3b, right panel), indicating that lepromatous patients had the capacity to up-regulate CLA.

image

Figure 3.  Antigen-dependent up-regulation of CLA expression on T cells from leprosy patients. (a) PBMCs of one representative tuberculoid patient (upper three histograms) and lepromatous patient (lower three histograms) were cultured with media, M. leprae, or anti-CD3 antibody. Cells were collected 12 days after antigen stimulation and CLA and CD3 expression were analysed by flow cytometry. The histograms shown were first gated for T cells using CD3 expression. The thin line represents labelling with isotype control antibody and the dark line represents labelling with antibody to CLA. (b) Fold-change in CLA expression (left panel, % CLA-positive T cells after culture with M. leprae / % CLA-positive T cells after culture with media) and T-cell proliferation (right panel) for the same group of tuberculoid leprosy (T-lep) and lepromatous leprosy (L-lep) patients. Δ c.p.m. = c.p.m. M. leprae − c.p.m. media. Horizontal bars indicate means. Mean values of CLA expression on T cells and T-cell proliferation were compared between T-lep and L-lep patients using non-parametric methods and P-values are indicated. (c) Comparisons of CLA expression (left panel) and IFN-γ production (right panel). Fold-change in CLA expression is determined as in (b). IFN-γ values are expressed as the means of triplicate values. ΔIFN-γ = IFN-γM. leprae − IFN-γ media.

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We further explored our hypothesis that CLA expression correlates with antigen responsiveness by examining several donors. CLA expression was reproducibly up-regulated by M. leprae in tuberculoid patients (2·4-fold median increase; range 0·8–6·1, n = 17) in comparison to lepromatous patients, in whom, on average, no increase in CLA was observed (0·9-fold median increase; range 0·1–2·2, n = 10; P < 0·001; Fig. 3b). The up-regulation of CLA was reproducible; in two tuberculoid donors, CLA up-regulation was observed in two independent experiments, i.e. in cells obtained on two distinct dates. The fold CLA increases for donor 1 (1·4 and 1·5) and donor 2 (2·4 and 3·8) demonstrated a consistency with regard to the ability to up-regulate CLA in vitro after M. leprae stimulation.

To determine the M. leprae antigen responsiveness of the patients examined in this study, T-cell proliferation assays were performed. The T cells of tuberculoid patients exhibited vigorous responses to M. leprae, in contrast to lepromatous patients of which the T-cell responses to M. leprae were significantly weaker [median values of Δ counts per minute (c.p.m) (i.e. c.p.m. M. leprae − c.p.m. media) were 15 631 (range 0–91 925; n = 17) for tuberculoid leprosy and 2789 (range 0–6080; n = 10) for lepromatous leprosy; P < 0·05; Fig. 3b]. The data indicate that CLA expression is induced on T cells by activation with M. leprae, and suggest that expression of CLA correlates with T-cell responsiveness to the pathogen in vitro.

Several mechanisms for the regulation of CLA on T cells have been described, including superantigen stimulation and cytokine production.17 To determine whether superantigens in M. leprae are responsible for the up-regulation of CLA on T cells in leprosy patients, T cells were isolated from patient blood using RosetteSep and cultured with M. leprae. In the presence of M. leprae extract, CLA expression did not increase on purified T cells (fold increases 0·7 and 1·1, n = 2), in contrast to PBMCs where M. leprae induced an increase in CLA expression on T cells (fold increases 2·0 and 1·8). We interpret these data to indicate that M. leprae-stimulated expression of CLA is not superantigen mediated, but is mediated through major histocompatibility complex (MHC)–peptide–T-cell receptor stimulation.

Cytokines have been shown to regulate the expression of CLA on T cells. T cells derived from patients with tuberculoid leprosy produce IFN-γ in response to M. leprae stimulation, whereas antigen-unresponsive T cells from patients with lepromatous leprosy only weakly produce IFN-γ. A distinct set of patients was evaluated for CLA expression relative to their levels of T helper type 1 (Th1) and Th2 cytokines. To evaluate the effect of cytokines and antigen-presenting functions of cells stimulated with M. leprae, we measured T-cell cytokines (IFN-γ and IL-4) and monocyte cytokines (IL-12 and IL-10) on PBMCs stimulated with M. leprae. We found that in response to M. leprae, PBMCs from tuberculoid leprosy patients (n = 6) produced higher levels of IFN-γ (Fig. 3c) than those from lepromatous patients (n = 6, P < 0·01). Levels of M. leprae-induced IL-4, IL-10 and IL-12 were comparable in the two patient populations.

The difference in cytokine production between patients with tuberculoid and lepromatous leprosy in vitro, together with earlier studies by van Wely et al.18 indicating that Th1 T cells expressed higher levels of fucosyltransferase VII (FucTVII), led us to hypothesize that blocking expression of IFN-γ could prevent the up-regulation of CLA on tuberculoid T cells exposed to M. leprae in vitro. Neutralization of IFN-γ did not show an appreciable effect (mean antibody-mediated inhibition = 0%, n = 5) on the M. leprae-mediated increase in CLA expression on T cells, indicating that IFN-γ is not required for up-regulation of CLA in vitro in response to M. leprae.

T cells require two signals from an antigen-presenting cell: antigen coupled to an MHC molecule and costimulation. To examine a role for antigen presentation in CLA expression, levels of the costimulatory protein CD86 on monocytes were examined after M. leprae stimulation in vitro. The levels of CD86 variably increased or decreased in both patient groups. The change (antigen-stimulated minus unstimulated cultures) in median fluorescence intensity in the tuberculoid patient group (n = 5) ranged from − 118·0 to + 67·5 (mean − 27·9) whereas in the lepromatous group (n = 5) the change in median fluorescence intensity ranged from − 57·0 to + 55·0 (mean + 5·1). These data suggested that changes in levels of costimulatory proteins did not modify expression of CLA.

Specificity of regulation of skin homing receptors in leprosy

Distinct homing receptors are expressed on T cells that localize to the skin in comparison to those that localize to the intestinal tract, which express integrin α47.19 To determine the specificity of homing receptor expression on T cells of leprosy patients, we therefore examined α47 expression on T cells of tuberculoid patients after stimulation with M. leprae. In contrast to CLA, expression of α47 decreased in response to M. leprae(Fig. 4). Moreover, we found in lepromatous patients that α47 expression decreased in response to M. leprae stimulation (Fig. 4b), although not to the extent that was detected for tuberculoid patients. We speculate that the comparative lack of α47 down-regulation in lepromatous patients is a function of their relative T-cell unresponsiveness. A polyclonal T-cell stimulus, anti-CD3 antibody, elicited no change in the expression of α47 in comparison to cells cultured in media alone. The data demonstrate specificity to the regulation of homing receptor expression in leprosy, with M. leprae stimulation leading to increased skin homing receptors and decreased intestinal homing receptors.

image

Figure 4.  Reciprocal regulation of homing receptor expression on T cells of tuberculoid leprosy patients. (a) Histograms showing CLA up-regulation (left panels) and dot plots showing integrin α47 down-regulation (right panels) in response to M. leprae stimulation for one representative tuberculoid patient. (b) Fold change in CLA (left panel, % CLA-positive T cells after culture with M. leprae / % CLA-positive T cells after culture with media) and integrin α47[right panel, (% integrin α47 + T cells after culture with M. leprae)/(% integrin α4/β7 + T cells after culture with media)] expression in leprosy patients. Closed symbols, T-lep patients; open symbols, L-lep patients; each symbol represents an individual patient.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In the course of an immune response to infection, T lymphocytes migrate from their site of activation, namely lymphoid tissue, to the site of infection. Trafficking of T cells from lymphoid tissue to non-lymphoid tissue is directed by specific receptors that induce the migration of lymphocytes out of the circulation and into an inflammatory site. Homing of T cells to the skin is mediated by cutaneous lymphocyte antigen, CLA and E-selectin, two related, though not identical, homing receptors.20 We evaluated CLA expression in the cutaneous lesions of leprosy patients, the primary site of infection by M. leprae. We found that CLA expression was readily detected in lesions of patients with limited versus progressive infection and that CLA expression on T cells in vitro corresponded with antigen responsiveness to M. leprae. Our data suggest that CLA expression is a useful marker of T-cell antigen responsiveness to bacterial infection in the skin.

We provide evidence that CLA expression correlates with the ability to restrict the growth of a cutaneous bacterial pathogen in vivo in humans. In the context of viral infection, CLA is preferentially expressed on circulating CD8 T cells specific for a skin-tropic herpesvirus in contrast to T cells that recognize a herpesvirus with no skin tropism.21 Together with our data, these studies suggest that CLA-positive cells mediate inflammation in the skin and may contribute to protection against cutaneous infection. T cells directed to the skin via CLA may also mediate unwanted inflammatory responses. CLA-positive T cells have previously been shown to be enriched in the inflammatory lesions of psoriasis where Th1 cytokine-producing cells are thought to have a pathological role.22 So although CLA expression correlates with immune protection in the context of bacterial and viral infection, in the absence of infection, it is possible that some of the CLA-positive T cells can mediate tissue injury.

One mechanism to explain divergent CLA expression in leprosy lesions may be T-cell responsiveness to M. leprae. We found that M. leprae increased CLA expression in vitro in the T cells of responsive tuberculoid patients, but not in unresponsive lepromatous patients. In contrast, expression of an intestinal T-cell homing receptor was decreased in response to antigen. CLA expression is also induced via superantigens,17,23 which activate the Vβ chains of T-cell receptors outside the CDR3 region, suggesting that the up-regulation of CLA in vitro by M. leprae could be the result of superantigens stimulating T-cell receptors.24 In the present study, up-regulation of CLA in leprosy was not through superantigen stimulation and was therefore more probably the result of MHC–peptide–T-cell receptor recognition. Since CLA up-regulation correlated with antigen responsiveness in tuberculoid leprosy patients, our data suggest that T-cell responsiveness to a cutaneous pathogen programmes them to migrate to the site of infection.

In addition to T-cell responsiveness, cytokine patterns in leprosy may be a mechanism of CLA induction and maintenance. T cells from lesions of tuberculoid patients produce Th1 or type 1 cytokines.6,7 We found that antigen-stimulated cultures from tuberculoid patients produced higher levels of IFN-γ than cultures from lepromatous patients. Type 1 cytokine-producing cells express high levels of fucosyltransferase VII (FucTVII),18 the enzyme that generates the CLA carbohydrate25 and maintains this high level of expression after Th1 differentiation and migration into inflammatory sites.26 Although tuberculoid leprosy patients produced IFN-γ, we showed that CLA expression was not directly regulated by IFN-γ. IL-12, a cytokine that drives Th1 responses27 and is abundantly present in tuberculoid lesions,28 also increases CLA expression17 through activation of FucTVII.29 Moreover, IL-12 was recently shown to enhance CLA expression on herpes simplex virus type 2-specific CD4 T cells.30

Similarly, the relative lack of CLA expression in lepromatous leprosy might be the result of cytokine patterns in the lesions. T cells from lepromatous patients produce Th2 or type 2 cytokines, including IL-4.7 IL-4 inhibits CLA expression by reducing FucTVII expression on T cells.31 IL-10, also overexpressed in lepromatous lesions,32 is a potent inhibitor of IL-12 production,33 indicating that IL-10 may inhibit CLA expression indirectly via inhibition of IL-12. Expression of a type 2 cytokine pattern on T cells in lepromatous leprosy therefore, may prevent an antigen-reactive T-cell population from entering the site of infection and eliminating the pathogen. Therefore, the cytokine pattern in leprosy lesions is likely to modulate T-cell migration into skin by regulating adhesion receptors on T cells. The increase in CLA expression is probably not simply the result of an influx of T cells into the lesions of tuberculoid patients because the numbers of T cells in inflammatory infiltrates of both tuberculoid and lepromatous patients were equivalent.13,14 Rather, T-cell expression of CLA may be down-regulated in the Th2 context of lepromatous lesions;6,7 alternatively, T cells of lepromatous patients may localize to skin directed by CCR4, a chemokine receptor expressed on Th2 cells.34

We propose that T-cell responsiveness and cytokine patterns in leprosy are likely determinants of CLA expression in leprosy. Moreover, our data suggest that T-cell responsiveness to a microbial antigen directs antigen-specific T cells to the site of infection where they contribute to the outcome of disease. Although not a ligand for E-selectin, we speculate that CLA may be of use as a diagnostic marker for cutaneous infectious diseases requiring T-cell reactivity for effective immunity21,35 and as an indicator of the efficacy of vaccines for cutaneous infectious diseases, including leprosy.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors thank Dr Joy Frank and Tony Mottino for use of the fluorescence microscope, Dr Matthew Schibler and the Carol Moss Spivak Cell Imaging Facility in the UCLA Brain Research Institute for the use of the confocal laser microscope, and the UCLA Flow Cytometry Core Laboratory for the use of their facilities. This investigation received financial support from the National Institutes of Health (AI22553 R. L. M) and the United Nations Development Programme/World Bank/World Health Organization Special Programme for Research and Training in Tropical Diseases.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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    Yamamura M, Uyemura K, Deans RJ, Weinberg K, Rea TH, Bloom BR, Modlin RL. Defining protective responses to pathogens: cytokine profiles in leprosy lesions. Science 1991; 254:2779.
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    Cooper AM, Dalton DK, Stewart TA, Griffin JP, Russell DG, Orme IM. Disseminated tuberculosis in interferon-gamma gene-disrupted mice. J Exp Med 1993; 178:22437.
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    Ridley DS, Jopling WH. Classification of leprosy according to immunity. A five-group system. Int J Lepr 1966; 34:25573.
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    Beckman EM, Melian A, Behar SM, et al. CD1c restricts responses of mycobacteria-specific T cells. Evidence for antigen presentation by a second member of the human CD1 family. J Immunol 1996; 157:2795803.
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