T-cell release of granulysin contributes to host defense in leprosy.

A novel mechanism by which T cells contribute to host defense against microbial pathogens is release of the antimicrobial protein granulysin. We investigated the role of granulysin in human infectious disease using leprosy as a model. Granulysin-expressing T cells were detected in cutaneous leprosy lesions at a six-fold greater frequency in patients with the localized tuberculoid as compared with the disseminated lepromatous form of the disease. In contrast, perforin, a cytolytic molecule that colocalizes with granulysin in cytotoxic granules, was expressed at similar levels across the spectrum of disease. Within leprosy lesions, granulysin colocalized in CD4+ T cells and was expressed in CD4+ T-cell lines derived from skin lesions. These CD4+ T-cell lines lysed targets by the granule exocytosis pathway and reduced the viability of mycobacteria in infected targets. Given the broad antimicrobial spectrum of granulysin, these data provide evidence that T-cell release of granulysin contributes to host defense in human infectious disease.

Evidence for clonal selection of gamma/delta T cells in response to a human pathogen.

T cells bearing gamma/delta antigen receptors comprise a resident population of intraepithelial lymphocytes in organs such as skin, gut, and lungs, where they are strategically located to contribute to the initial defense against infection. An important unsolved question about antigen-driven gamma/delta T cell responses regards the breadth of their T cell receptor (TCR) repertoire, since many specific epithelial compartments in mice display limited diversity. We have examined the diversity of TCR delta gene expression among human gamma/delta T cells from skin lesions induced by intradermal challenge with Mycobacterium leprae. We show that the vast majority of gamma/delta cells from M. leprae lesions use either V delta 1-J delta 1 or V delta 2-J delta 1 gene rearrangements and, within a given region of the lesion, display limited junctional diversity. This contrasts markedly with the extensive diversity of gamma/delta T cells from peripheral blood of these same individuals, as well as skin from normal donors. These results indicate that the gamma/delta response to M. leprae involves the selection of a limited number of clones from among a diverse repertoire, probably in response to specific mycobacterial and/or host antigens.

Analysis of naturally occurring delayed-type hypersensitivity reactions in leprosy by in situ hybridization.

Analysis of tissue lesions of the major reactional states of leprosy was undertaken to study the immune mechanisms underlying regulation of cell-mediated immunity and delayed-type hypersensitivity (DTH) in man. In situ hybridization hybridization of reversal reaction biopsy specimens for INF-gamma mRNA expression revealed a 10-fold increase in specific mRNA-containing cells over that observed in unresponsive lepromatous patients. Expression of huHF serine esterase, a marker for T cytotoxic cells, were fourfold increased in reversal reaction and tuberculoid lesions above that detected in unresponsive lepromatous individuals. Immunohistology of reversal reactions confirmed a selective increase of Th and T cytotoxic cells in the cellular immune response. Of interest, the microanatomic location of these serine esterase mRNA-containing cells was identical to the distribution of CD4+ cells. Analysis of erythema nodosum leprosum (ENL) lesions revealed differences in the underlying immune processes in comparison with reversal reaction lesions. Although phenotypic Th cells predominated in ENL lesions, IFN-gamma and serine esterase gene expression were markedly reduced. We suggest that reversal reactions represent a hyperimmune DTH response characterized by a selective increase of CD4+ IFN-gamma producing cells and T cytotoxic cells, which result in the clearing of bacilli and concomitant tissue damage. In contrast, ENL reactions may be viewed as a transient diminution of Ts cells and activity leading to a partial and transient augmentation in cell-mediated immunity, perhaps sufficient to result in antibody and immune complex formation, but insufficient to clear bacilli from lesions.

Defining protective responses to pathogens: cytokine profiles in leprosy lesions.

The immunological mechanisms required to engender resistance have been defined in few infectious diseases of man, and the role of specific cytokines is unclear. Leprosy presents clinically as a spectrum in which resistance correlates with cell-mediated immunity to the pathogen. To assess in situ cytokine patterns, messenger RNA extracted from leprosy skin biopsy specimens was amplified by the polymerase chain reaction with 14 cytokine-specific primers. In lesions of the resistant form of the disease, messenger RNAs coding for interleukin-2 and interferon-gamma were most evident. In contrast, messenger RNAs for interleukin-4, interleukin-5, and interleukin-10 predominated in the multibacillary form. Thus, resistance and susceptibility were correlated with distinct patterns of cytokine production.

Interferon-gamma differentially regulates interleukin-12 and interleukin-10 production in leprosy.

The ability of monocytes to influence the nature of the T cell response to microbial pathogens is mediated in part by the release of cytokines. Of particular importance is the release of IL-12 and IL-10 by cells of the monocyte/macrophage lineage upon encountering the infectious agent. IL-12 promotes cell mediated immunity (CMI) to intracellular pathogens by augmenting T-helper type 1 responses, whereas IL-10 downregulates these responses. The ability of IFN-gamma to modulate the balance between IL-12 and IL-10 production was examined by studying leprosy as a model. In response to Mycobacterium leprae stimulation, IFN-gamma differentially regulated IL-12 and IL-10 production resulting in upregulation of IL-12 release and downregulation of IL-10 release. Furthermore, we determined that the mechanism by which IFN-gamma downregulates IL-10 was through the induction of IL-12. The data suggest a model of lymphocyte-monocyte interaction whereby the relative presence or absence of IFN-gamma in the local microenvironment is a key determinant of the type of monocyte cytokine response, and hence the degree of CMI in the host response to infection.

Serum macrophage migration inhibition activity in patients with leprosy.

We have found that 26 of 54 (48%) untreated patients with leprosy had serum migration inhibitory activity, and that this was present in tuberculoid, borderline, and lepromatous forms of the disease. Patients with active recreational states; i.e., reversal reactions, Lucio's reaction, or erythema nodosum leprosum, were particularly apt to have this inhibitory activity. The prevalence of inhibitory activity did not vary significantly with treatment, dinitrochlorobenzene responsiveness, tuberculin responsiveness, or serum lysozyme levels.

Dinitrochlorobenzene responsivity in disseminated coccidioidomycosis: an inverse correlation with complement-fixing antibody titers.

The immunologic status of 25 patients with disseminated coccidiodomycosis was evaluated by serum anticoccidioidin complement-fixing antibody levels, coccidiodin skin tests, and dinitrochlorobenzene (DNCB) sensitization. In the 10 patients who had disseminated disease and a complement-fixing titer of 1:32 or less, responses to DNCB were similar to those of 20 controls. In the 15 patients with disseminated disease and a complement-fixing titer of 1:64 or more, responses to DNCB were statistically significantly diminished compared to controls (p = 0.002). Since the complement-fixing titer is associated with extent of dissemination, these results signify a relationship between diminished DNCB responses and extensive dissemination. Of the several hypotheses which might explain this relationship, we find the most attractive is that of a nonspecific deficiency of cell-mediated immunity developing secondarily to extensive disseminated disease.

Comparison of S-100 and OKT6 antisera in human skin.

The monoclonal antibody OKT6 and antisera against S-100 protein have both been advocated as immunologic markers of Langerhans cells in the skin. S-100 antiserum has an advantage in its ability to stain Langerhans cells in paraffin tissues. In order to evaluate whether these antibodies stain equivalent numbers of Langerhans cells in skin, we compared the staining patterns of S-100 antiserum and OKT6 antibody on biopsy specimens from 40 patients with leprosy using immunoperoxidase techniques. Utilizing OKT6 antibody, greater numbers of positive Langerhans cells were found in the epidermis in tuberculoid leprosy, reversal reaction, and erythema nodosum leprosum than in lepromatous leprosy. However, these differences were not observed with the S-100 antiserum and, overall, fewer cells were found as compared with the OKT6 antibody. In the dermis both antibodies stained "dendritic cells" that were found encircling granulomas in tuberculoid leprosy and reversal reaction. Staining in lepromatous leprosy granulomas, in contrast to the epidermal staining pattern, revealed rare OKT6-positive cells, while S-100 cells were numerous and were more diffusely distributed throughout the granuloma. Our results indicate that antiserum to S-100 protein and OKT6 antibody stain morphologically similar cells (dendritic cells), but do not provide comparable results concerning distribution and frequency of these cells.

Selective expansion of V delta 1 + T cells from leprosy skin lesions.

T cells bearing gamma delta T-cell receptors (TCRs) are prominent residents of murine epidermis and appear to be important participants in the immune response to infection in human skin. The Mitsuda reaction in leprosy, induced by intradermal challenge with Mycobacterium leprae, provides an opportunity to study the cellular events that mediate a form of delayed-type hypersensitivity (DTH) in skin. T cells bearing gamma delta TCRs comprise a significant proportion of the T-cell population in these DTH reactions. Presently we have generated T-cell lines from Mitsuda reactions in vitro and compared their TCR repertoire to that found in situ. gamma delta T cells comprised 20-40% of lines derived from these skin lesions, but < 10% of lines derived from the peripheral blood of the same individuals. Flow-cytometric analysis of variable (V) chain usage in T-cell lines derived from skin lesions indicated that V delta 1 was predominant. Evaluation of the TCR repertoire using PCR indicated that V delta 1-J delta 1 and V gamma 2-J gamma P gene rearrangements were prevalent. In comparison, V delta 2-J delta 1 gene rearrangements predominated in situ. Furthermore, nucleotide sequence analysis of the V-J junction of one T-cell line revealed limited genetic diversity of the gamma delta TCR. These findings suggest that the V delta 1 subpopulation of gamma delta T cells in Mitsuda skin reactions selectively outgrows from leprosy skin lesions in vitro. Such V delta 1 + T-cell lines should be useful for determining the relevant antigens and restriction elements in this response to a pathogen in skin.

Hypercalcemia and abnormal 1,25-dihydroxyvitamin D concentrations in leprosy.

Two patients with lepromatous leprosy and hypercalcemia are presented. Serum immunoreactive parathyroid hormone and urinary cyclic adenosine monophosphate concentrations were suppressed. Serum 1,25-dihydroxyvitamin D [1,25-(OH)2D] concentrations were elevated in one patient and normal in the other. Urinary hydroxyproline excretion was slightly high in both patients. Hypercalcemia resolved excretion was slightly high in both patients. Hypercalcemia resolved with prednisone therapy. Abnormal 1,25-(OH)2D production and/or metabolism may play a role in the pathogenesis of hypercalcemia in some patients with leprosy.

Erythema nodosum leprosum in a general hospital.

We report 32 patients with erythema nodosum leprosum (ENL). Twenty-two patients developed ENL before beginning chemotherapy. The recognition that ENL in the absence of chemotherapy is not an uncommon event is of importance, not simply as a fact in and of itself, but because this observation raises provocative questions when placed in the context of current ideas concerning ENL. ENL should be regarded as a manifestation of leprosy, not necessarily as a complication of its therapy.

Lucio's phenomenon and diffuse nonnodular lepromatous leprosy.

The records of ten patients with Lucio's phenomenon showed clinical and histopathological changes similar to those described by others. Lucio's phenomenon is a syndrome distinct from erythema nodosum leprosum as indicated by an absence of fever, leukocytosis and tenderness, a failure to respond to thalidomide, and a restriction to patients with diffuse nonnodular lepromatous leprosy. Lymphopenia associated with splenomegaly in three patients and glomerulonephritis in one patient were unexpected findings of unknown relevance.

Apparently normal skin in lepromatous leprosy: histopathological findings.

Biopsy specimens of apparently uninvolved skin from 34 patients with lepromatous leprosy were studied histologically. Bacilli were found in 30 of 31 specimens from clinically polar or near-polar lepromatous patients but not in the three from nonpolar patients. A predominantly perivascular distribution of infiltrate and bacilli is consistent with a hematogenous spread of infection. Subclinical, diffuse lepromatous leprosy is found in patients with nodular lesions and may precede the development of nodules. Study of apparently uninvolved skin may be helpful in classifying patients, in interpreting immunologic responses, and in elucidating the natural history of the illness.

Immunoglobulin deposits in lepromatous leprosy skin. Presence of deposits in apparently uninvolved skin and occurrence of serum antiepithelial antibodies.

Immunoglobulin deposits were detected in ten of 13 biopsy specimens from apparently uninvolved skin of patients with lepromatous leprosy. There were deposits of IgM at the dermoepidermal junction in the skin of five patients, and deposits of IgM along the dermal collagen and elastic fibers in the skin of the other five. The deposits were eluted with acid buffers and high molarity salt solution. Circulating IgG antibodies to intercellular substance of epithelial cells, similar to those present in pemphigus vulgaris, were found in 25% of patients with lepromatous leprosy who were studied. These antibodies appeared to be different from the skin-bound immunoglobulin deposits.

Primary dapsone-resistant Hansen's disease in California. Experience with over 100 Mycobacterium leprae isolates.

We found that in the years 1978 through 1981 only one of 54 previously untreated patients with Hansen's disease was found to harbor dapsone-resistant Mycobacterium leprae. That single strain was only partially resistant, ie, it was resistant to 0.0001% dapsone in a mouse diet but not to higher concentrations. During the years 1983 through 1988, M leprae from 47 previously untreated patients presenting to clinics in San Francisco, Calif, and Los Angeles, Calif, grew in mice. None of these strains was found to be dapsone resistant. Thus, from 1978 through 1988 only one of 101 M leprae isolates obtained from skin biopsy specimens from patients with leprosy was found to be resistant to dapsone. We have concluded that primary dapsone resistance still does not appear to be a significant problem in California. Owing to the fact that our single resistant case and those reported from international sources are, in general, partially resistant, the potential importance of partial dapsone resistance is discussed.

Immunologic responses in patients with lepromatous leprosy.

Immunologic responses were measured in 46 patients with lepromatous leprosy. These patients were not distinguishable from controls on the basis of responses to soluble intradermal antigens, sensitization to contactants, peripheral blood T- and B-cell percentages, in vitro lymphocyte responses to a mitogen, or the prevalence of autoantibodies. Generalized immunologic abnormalities in patients with lepromatous leprosy are neither predisposing causes nor necessary accompaniments of lepromatous leprosy, but are probably remote sequellae of the illness. By implication, the generalized immunologic abnormalities reported in other diseases are likely to be remote sequellae of the particular illness.

Tissue and blood T-lymphocyte subpopulations in erythema nodosum leprosum.

To study T lymphocytes in erythema nodosum leprosum (ENL), monoclonal antibodies were used to identify T-lymphocyte subpopulations in the blood and skin lesions of patients with ENL and patients with nonreactional lepromatous leprosy. The blood of nonreactional lepromatous patients had a lymphopenia and a proportionate reduction in pan T cells, helper-inducer, and suppressor-cytotoxic subsets, but a normal helper-suppressor ratio, as compared with controls. Patients with ENL did not differ significantly from the controls. In skin lesions, an admixture of helper and suppressor phenotypes among foamy histiocytes was found. The ENL tissue had more numerous cells of the helper-inducer phenotype and fewer of the suppressor-cytotoxic phenotype, as compared with nonreaction lepromatous tissues. In 22 patients with simultaneous examination of tissue and blood T-cell subsets, there was no correlation between tissue and blood helper-suppressor ratios, indicating that some sort of selection process brings lymphocytes into tissues from peripheral blood.

Lepromin skin testing in the classification of Hansen's disease in the United States.

Hansen's disease, or leprosy, although a relatively uncommon disease in the United States, continues to be important because of its implications--physical, psychological, and social--for the patient. Prognosis and treatment of the disease are based largely on clinical classification, which ranges from the multibacillary "lepromatous" to the paucibacillary "tuberculoid" forms, depending on the patient's specific immune capabilities. Traditionally, skin testing with lepromins--suspensions of the etiologic agent of Hansen's disease, Mycobacterium leprae--have been used as adjuncts to clinical parameters for classification in endemic areas. However, these have not been systematically studied in the United States. This report describes the results obtained from skin testing 38 volunteers (22 patients and 16 uninfected persons) with standard lepromin preparations. These results support the adjunctive value of lepromins for clinically classifying Hansen's disease in our "hypoendemic" population.

Dermal ultrastructure in leprosy.

We studied the ultrastructure of the dermal inflammatory response in 18 patients with leprosy. Biopsy specimens from 14 lepromatous patients, including four with Lucio's phenomenon and four with erythema nodosum leprosum, were compared with biopsy specimens from one borderline lepromatous and three borderline tuberculoid patients. In all, the dermal infiltrate consisted of macrophages, lymphocytes, and mast cells. This infiltrate was predominantly perivascular, and chronic reactive changes were found in the small dermal vessels. The macrophages contained phagocytized organisms within membrane-bound vacuoles and a wide variety of lysosomal residual dense bodies. Intraendothelial organisms were occasionally seen, especially in biopsy specimens from the patients with Lucio's phenomenon. The greatest number of mast cells were also seen in the infiltrate in those cases. The frequent close association of macrophages with lymphocytes and mast cells suggests an interrelationship between these cells that appears typical of the host response to leprosy.

Granuloma annulare. Identification of cells in the cutaneous infiltrate by immunoperoxidase techniques.

To characterize the immunopathologic process of granuloma annulare, frozen sections of eight specimens were evaluated with monoclonal antibodies directed against T lymphocytes and the monocyte-macrophage series, in conjunction with immunoperoxidase techniques and with histochemical staining. The predominant lymphocyte was an activated T lymphocyte (Leu 1+, HLA-Dr+) with an excess of helper-inducer phenotype (Leu 3a+), as compared with suppressor-cytotoxic phenotype (Leu 2a+). OKT-6+ Langerhans' cells were observed in the epidermis, and numerous OKT-6+ cells were observed in the perivascular and granulomatous infiltrate. The use of four monoclonal antibodies, having specificity against peripheral blood monocyte antigens, revealed three different staining patterns in the granulomas. Finally, mast cells were present in perivascular and granulomatous infiltrates. Our results demonstrate that the cutaneous infiltrate of granuloma annulare contains all of the principal cell types that characterize cell-mediated immune responses.