Immunohistochemical assessment of cell populations in leprosy-spectrum lesions and reactional forms.

In situ immunophenotyping of leprosy lesions can improve our understanding of the biology of inflammatory cells during the immune response to Mycobacterium leprae antigens. In the present study, biopsies from 10 healthy controls and 70 leprosy patients were selected, 10 for each of the following conditions: clinical tuberculoid (TT), borderline tuberculoid (BT), borderline borderline (BB), borderline lepromatous (BL), lepromatous (LL), reversal reaction (R1), and erythema nodosum leprosum (R2). Qualitative and quantitative immunohistochemical analyses were performed to detect CD3, CD4, CD8, FoxP3, CD20, CD138, CD1a, CD57, CD15, CD117, CD68, and CD163. In addition, histochemistry was employed to identify eosinophils. The amount of CD3+ and CD4+ T cells was higher in TT than in LL patients. CD8+ T cells were predominant in T lymphocyte infiltrations in the basal layer of the epidermis. The number of FoxP3+ cells was similar among different forms of the disease, but was higher in BL and LL than in R2 individuals. CD20+ lymphocytes were most abundant in TT samples, while CD138+ plasma cells displayed no detectable differences. Epithelioid macrophages from the center of TT and R1 granulomas exhibited the M1 phenotype (CD68+CD163-), whereas those in LL granulomas showed the M2 phenotype (CD68+CD163+). There was a gradual decrease in the amount of CD1a+ cells from the TT towards the LL form of the disease. A significant increase in the number of neutrophils was observed only in R2 samples. All the cells investigated, except eosinophils, participated in the immunopathogenesis of leprosy.

Immunohistochemical assessment of cell populations in leprosy-spectrum lesions and reactional forms

In situ immunophenotyping of leprosy lesions can improve our understanding of the biology of inflammatory cells during the immune response to Mycobacterium leprae antigens. In the present study, biopsies from 10 healthy controls and 70 leprosy patients were selected, 10 for each of the following conditions clinical tuberculoid (TT), borderline tuberculoid (BT), borderline borderline (BB), borderline lepromatous (BL), lepromatous (LL), reversal reaction (R1), and erythema nodosum leprosum (R2). Qualitative and quantitative immunohistochemical analyses were performed to detect CD3, CD4, CD8, FoxP3, CD20, CD138, CD1a, CD57, CD15, CD117, CD68, and CD163. In addition, histochemistry was employed to identify eosinophils. The amount of CD3+ and CD4+ T cells was higher in TT than in LL patients. CD8+ T cells were predominant in T lymphocyte infiltrations in the basal layer of the epidermis. The number of FoxP3+ cells was similar among different forms of the disease, but was higher in BL and LL than in R2 individuals. CD20+ lymphocytes were most abundant in TT samples, while CD138+ plasma cells displayed no detectable differences. Epithelioid macrophages from the center of TT and R1 granulomas exhibited the M1 phenotype (CD68+CD163-), whereas those in LL granulomas showed the M2 phenotype (CD68+CD163+). There was a gradual decrease in the amount of CD1a+ cells from the TT towards the LL form of the disease. A significant increase in the number of neutrophils was observed only in R2 samples. All the cells investigated, except eosinophils, participated in the immunopathogenesis of leprosy.

Expression of costimulatory molecules (CD80, CD86, CD28, CD152), accessory molecules (TCR alphabeta, TCR gammadelta) and T cell lineage molecules (CD4+, CD8+) in PBMC of leprosy patients using Mycobacterium leprae antigen (MLCWA) with murabutide and T cell peptide of Trat protein.

In leprosy, cell-mediated immunity (CMI) is more significant than humoral response to eliminate intracellular pathogen. T cell defect is a common feature in lepromatous leprosy (LL) patients as compared to tuberculoid type (TT) patients. For efficient initiation of CD4+, T cell response requires T cell receptor (TCR) activation and costimulation provided by molecules on antigen-presenting cells (APC) and their counter receptors on T cells. In our previous study, the defective T cell function in LL patients was restored to a proliferating state with the release of TH1 type cytokines using mycobacterial antigen(s) with two immunomodulators (Murabutide (MDP-BE) and T cell epitope of Trat protein of Escherichia coli) by presenting the antigen in particulate form in vitro to PBMC derived from leprosy patients. This observation prompted us to study the expression of the costimulatory molecules (CD80, CD86, CD28, CD152), other accessory molecules (TCR alphabeta/gammadelta) and T cell lineage molecules (CD4+ and CD8+) during constitutive and activated state of peripheral blood mononuclear cells (PBMC) derived from normal and leprosy individuals using different formulations of Mycobacterium leprae total cell wall antigen (MLCWA), Trat and MDP-BE using flow cytometric analysis. An increased surface expression of CD80, CD86 and CD28 but decreased CD152 expression was observed when PBMC of normal, BT/TT (tuberculoid) and BL/LL (lepromatous) patients were stimulated in vitro with MLCWA+MDP-BE+Trat peptide using liposomal mode of antigen delivery, while opposite results were obtained with the antigen alone. Antibody inhibition study using antihuman CD80 or CD86 completely abolished the T cell lymphoproliferation, thereby reconfirming the importance of these costimulatory molecules during T cell activation/differentiation. Though the liposome-entrapped antigen formulation has no effect on expression of alphabeta/gammadelta T cell receptor, the constitutive levels of TCR gammadelta were high in lepromatous patients. Thus, TCR bearing gammadelta appears to have a negligible regulatory role in peripheral blood of leprosy patients. The percentage of cells positive for CD4+ are increased in inducible state in all the three groups, while CD8+-positive cells were decreased in LL patients, thereby reconfirming the fact that priming of CD4+ cells are necessary for producing final effector functions. Lastly, intracellular cytokine staining experiment indicated that CD4+ cells are the major producers of IFN-gamma but not NK cells. The study highlights the reversal of T cell anergy especially in lepromatous patients through the modulation of costimulatory molecule expression under the influence of Th1 cytokines, i.e., IL-2 and IFNgamma.

B7-1, but not CD28, is crucial for the maintenance of the CD4+ T cell responses in human leprosy.

We used human leprosy as a model to compare patterns of costimulatory molecule expression in respect to the clinical/immunologic spectrum of disease. We found that B7-1, B7-2, and CD28 transcripts dominated in tuberculoid leprosy patients, who have potent T cell responses to Mycobacterium leprae. In contrast, CTLA-4 was more strongly expressed in lesions from lepromatous patients, who manifest specific T cell anergy to the leprosy bacterium. T cell clones from tuberculoid lesions were CD4+CD28+ or CD4+CD28-, and T cell clones from lepromatous lesions were predominantly CD8+CD28-. The M. leprae-specific recall response of CD4+ T cell clones from tuberculoid lesions was blocked by anti-B7-1 mAb, but not by anti-B7-2 mAb or CTLA-Ig. However, anti-CD28 and anti-CTLA-4 mAbs did not block activation of clones from tuberculoid lesions, suggesting that B7-1 may utilize another costimulatory pathway. Peripheral blood T cell responses in the lepromatous form were strongly regulated by CD28 during T cell activation, in contrast to the tuberculoid form. Thus, B7-1 costimulation could play a role in maintaining a strong immune response to the pathogen.

Lack of expression of intercellular adhesion molecule ICAM-1 in lepromatous leprosy patients.

It is conceivable that an abnormal expression of cell-adhesion molecules can contribute to the poor inflammatory response seen in some inflammatory skin diseases. Adhesins are cell-surface molecules that are expressed by many cell types. The main function of adhesins appears to be the promotion of cellular interactions, such as those occurring between immune cells. The epidermis of patients with inflammatory skin diseases exhibits an increased expression of ICAM-1, and it has been postulated that such increased expression can be important in the genesis of cutaneous inflammation. The expression of cell-adhesion molecules (LFA-1, LFA-2, LFA-3 and ICAM-1) in skin lesions of leprosy patients was studied, as well as the in vitro expression of these molecules induced with gamma interferon (IFN-gamma). A lack of expression of ICAM-1 in the epidermis of lepromatous patients was noted; in addition, no expression of ICAM-1 was seen in the nearly normal skin from these patients incubated with IFN-gamma. A similar expression of the four molecules studies was noted in the dermis of both the lepromatous and tuberculoid types of leprosy. The epidermis of the lepromatous leprosy patients appears to have a defective expression of ICAM-1.