Characterization of the cellular immune response in American cutaneous leishmaniasis

The in vitro and in vivo cellular immune reactivity of 49 patients with American cutaneous leishmaniasis (ACL) was evaluated using mitogens and parasite antigens. Patients were examined before treatment and were classified on the basis of clinical and histopathological criteria as suffering localized cutaneous leishmaniasis (LCL, 32 patients) or mucocutaneous leishmaniasis (MCL, 11 patients). A small group (6 patients) of treated diffuse cutaneous leishmaniasis (DCL) patients was also examined. The lymphocyte proliferative responses to PHA were significantly lower than those of controls (87 individuals, from either endemic or nonendemic zones) in LCL, and particularly MCL. Con A responses were, however, effectively normal in these patients. Both in vivo and in vitro cellular immune responses to leishmanial antigens were significantly greater in MCL and LCL patients than in the controls, the intensity of the reactions being by far the greatest in MCL. DCL patients demonstrated a complete absence of specific immune responsiveness both in vivo and in vitro. The significance of these results in the mechanisms leading to the resolution of the infection or production of pathologic lesions is discussed.     

In Situ Characterization of the Cutaneous Immune Response in Ethiopian Cutaneous Leishmaniasis

Cryostat sections from 10 patients with localized cutaneous leishmaniasis (LCL) and eight patients with diffuse cutaneous leishmaniasis (DCL) from Ethiopia were studied with immunofluorescence methods for the phenotypic characterization of cells in the lesions. Higher numbers of Leu 2+ and Leu 3+ cells (P less than 0.005) were found in LCL than in DCL, while the Leu 3a + b/Leu 2a ratios were the same. No differences were found in the numbers of transferrin receptor, HLA-DR, and HLA-DQ expressing cells in the granulomas. Significantly (P less than 0.0001) lower numbers of IL-2 receptors expressing (Tac+) cells were found in DCL than LCL lesions, suggesting interference in the activation of the T cells. IL-2-containing cells were absent in DCL and were found in LCL lesions. Epidermal keratinocytes above the LCL but not the DCL lesions expressed HLA-DR (but not HLA-DQ) antigen, suggesting a lower gamma-interferon production in the DCL granulomas. The number of Langerhans' cells (Leu 6+) was higher in the epidermis of DCL (P less than 0.005) than in LCL, while a lower number of Leu 6+ cells were seen in the dermal lesions (P less than 0.001). These observations could account for some of the mechanisms responsible for the disturbed immunostimulation and immunoregulation observed in the lesions of DCL.     

T-cell subpopulations, expression of interleukin-2 receptor, and production of interleukin-2 and gamma interferon in human American cutaneous leishmaniasis

Leukocyte subpopulations, the expression of the interleukin-2 (IL-2) receptor, and the production of IL-2 and gamma interferon (IFN-gamma) were studied in the peripheral blood mononuclear cells of American cutaneous leishmaniasis patients that had been stimulated in vitro with either leishmanial antigen or mitogen (phytohemagglutinin M). The 75 patients examined were classified as having either the localized (LCL; 66 patients), mucocutaneous (MCL; 5 patients), or the rare diffuse (DCL; 4 patients) form of the disease. Patients with DCL, who are characterized by their defective cell-mediated immune response to leishmanial antigen, failed to express the IL-2 receptor and did not produce IFN-gamma when exposed to the antigen but did so when stimulated by phytohemagglutinin M. Both LCL and MCL patients showed strong proliferative responses to leishmanial antigen; these were by far the greatest in MCL patients. Both groups had significantly increased IL-2 receptor expression and IFN-gamma production after exposure to either antigen or mitogen, and these were highest in the MCL patients. Concerning the leukocyte subpopulations evaluated (CD2, CD4, CD8, CD20, MO2), the most significant findings were a decrease of both CD4+ cells and the CD4/CD8 ratio in MCL patients compared with the other groups. Considering IL-2 production, in response to phytohemagglutinin M both MCL and LCL patients showed amounts of IL-2 comparable to those of the controls. Our results help explain the anergy of T cells from DCL patients to leishmanial antigen, which could lead to a defective production of IFN-gamma and possibly contribute to their incapacity to kill the Leishmania parasite. Concerning MCL patients, the significantly increased expression of IL-2 receptor, decreased expression of the CD4 (helper-inducer of suppression) phenotype, and elevated IFV-gamma production might partially explains the state of hypersensitivity and mucosal damage exhibited by these patients.     

Mechanisms associated with immunoregulation in human American cutaneous leishmaniasis

Mechanisms possibly involved in the regulation of the immune response were evaluated in 49 patients with American cutaneous leishmaniasis (ACL). The patients were classified on the basis of clinical and histopathological criteria as suffering localized (LCL), mucocutaneous (MCL) or diffuse (DCL) forms of the disease. A significant leishmanial antigen-induced suppression of in vitro mitogen responsiveness was demonstrated in the DCL group, but not in the other two diseases states. Lack of suppressive activity was particularly evident in MCL, this being the group that presented the highest in vivo and in vitro reactivity to the parasite antigens. In fact, a significant inverse correlation was found between the degree of suppression and the antigen-induced lymphocyte proliferative response. In contrast, a mixture of mononuclear cells from MCL patients and normal subjects showed higher that expected responses to mitogen, while this increase was not observed in co-cultures of DCL and normal mononuclear cells. Due to their possible modulatory influences, circulating immune complexes were also evaluated in these patient groups, higher levels being found in MCL and DCL patients than in either LCL or controls. The possible mechanisms involved in the regulation of the immune response to the protozoan in the complex disease spectrum of ACL are discussed in relation to anergy in DCL and hyperresponsiveness in MCL.     

Monocyte chemotactic protein-1 stimulates the killing of leishmania major by human monocytes, acts synergistically with IFN-gamma and is antagonized by IL-4

We recently demonstrated that monocyte chemotactic protein-1 (MCP-1) is strongly expressed in lesions of patients with self-healing localized cutaneous leishmaniasis (LCL) whereas it is scarce in those of chronic diffuse cutaneous leishmaniasis (DCL). This finding indicated that MCP-1 may contribute to the healing process. In the present study, we analyzed the capacity of MCP-1 to trigger leishmanicidal activities. The results show that MCP-1 directly stimulates the elimination of intracellular Leishmania parasites by human monocytes, a potential that correlates with the induction of reactive oxygen intermediates. Release of NO was not detected. To understand the cross-talk between the chemokine and T cell-associated cytokines, we studied the influence of the Th1 cytokine IFN-gamma and the Th2 cytokine IL-4 on MCP-1-mediated activation of human monocytes. The data demonstrate that IFN-gamma and MCP-1 synergistically activate monocytes to clear intracellular parasites, whereas IL-4 abrogates the effect of MCP-1. Furthermore, IL-4 inhibits MCP-1 expression by infected monocytes, a finding that may explain the lack of MCP-1 in chronic lesions. The data suggest a novel model for macrophage activation in cutaneous leishmaniasis. In lesions of LCL, the synergistic action of MCP-1 and IFN-gamma may stimulate the killing of parasites by macrophages and promote healing, whereas the presence of IL-4 in DCL lesions may favor the suppression of MCP-1 and, together with the lack of IFN-gamma, the progression of disease.     

Demonstration of an indomethacin-sensitive mechanism regulating immune reactivity in American cutaneous leishmaniasis patients

We investigated some aspects of the regulation of the immune response that were sensitive to the effect of indomethacin (INDO), an inhibitor of prostaglandin synthesis, in 84 patients with American cutaneous leishmaniasis (ACL), and in normal controls. The patients were classified on the basis of clinical and histopathological criteria as suffering localized (LCL), mucocutaneous (MCL) or diffuse (DCL) forms of the disease. The responses in vitro to mitogens (PHA and Con A) and leishmanial antigens were evaluated in the presence or absence of INDO. It was found that the drug significantly increased in vitro the mitogenic stimulation by PHA of peripheral blood mononuclear cells from LCL, MCL and DCL patients, but the effect was less evident in the controls. Considering specific responses to leishmanial antigens, we showed that in the presence of INDO, these were significantly increased in LCL patients, but not in MCL or DCL. Also, only in LCL was an inverse correlation found between the initial response to leishmanial antigen and the increase caused by INDO. Significant correlations were found between the INDO-induced enhancement of PHA and Con A responses in the patient groups, but not in the controls. In LCL patients there was a significant correlation between the increases caused by INDO of the mitogen and antigen responses. It can be suggested that an indomethacin-sensitive (prostaglandin dependent) suppressor mechanism operates in LCL patients, that is possibly responsible for the modulation of the immune response against the parasite. In MCL, where this suppressive mechanism is apparently not functional, the response to the parasite is intense, and a possible consequence of this could be tissue damage. Our results indicate, however, that the anergy observed in DCL patients is not due to an involvement of prostaglandins in the suppression of the specific immune response.     

Expression of inducible nitric oxide synthase in skin lesions of patients with american cutaneous leishmaniasis

Cytokine-inducible (or type 2) nitric oxide synthase (iNOS) is indispensable for the resolution of Leishmania major or Leishmania donovani infections in mice. In contrast, little is known about the expression and function of iNOS in human leishmaniasis. Here, we show by immunohistological analysis of skin biopsies from Mexican patients with local (LCL) or diffuse (DCL) cutaneous leishmaniasis that the expression of iNOS was most prominent in LCL lesions with small numbers of parasites whereas lesions with a high parasite burden (LCL or DCL) contained considerably fewer iNOS-positive cells. This is the first study to suggest an antileishmanial function of iNOS in human Leishmania infections in vivo.     

In situ characterization of the cellular immune response in American cutaneous leishmaniasis.

American cutaneous leishmaniasis is a spectrum of granulomatous disease caused by related species of an intracellular parasite. The host response in localized cutaneous leishmaniasis (LCL) is effective in that few organisms can be found in tissue lesions. In contrast, diffuse cutaneous leishmaniasis (DCL) patients mount a poor response with numerous parasites present in multiple skin lesions. Immunopathological correlates were sought in LCL and DCL with immunoperoxidase techniques using monoclonal antibodies directed against T lymphocyte subpopulations and interleukin-2 in tissue lesions. Both LCL and DCL granulomas showed a mixture of T lymphocyte subpopulations with the ratio of helper:suppressor phenotypes less than one. This ratio and localization of cells is more similar to the ineffective lepromatous leprosy granuloma than the effective tuberculoid leprosy granuloma. In contrast, interleukin-2 was identified in equivalent numbers of cells in LCL and tuberculoid leprosy, an order of magnitude greater than DCL and lepromatous leprosy lesions. Cells expressing Tac, the receptor for interleukin-2, were present in approximately equal numbers in all disorders. The immunological effectiveness of granulomas appear to related less to the numbers and location of T cell phenotypes than to the functional aspects of these cells, particularly the ability to generate lymphokines.     

Cytokine expression in the liver during the early phase of murine tularemia.

Cytokine expression was determined in the livers of mice inoculated subcutaneously with Francisella tularensis LVS. During the first 48 h of infection, there was a logarithmic increase of bacteria in the liver, with a doubling time of 2.5 h. Within 48 h, tumor necrosis factor alpha (TNF-alpha), interleukin 10 (IL-10), IL-12, and gamma interferon (IFN-gamma) mRNAs were expressed, and production of TNF-alpha and IFN-gamma was demonstrated. There was no expression within 96 h of mRNA from IL-2, IL-3, or IL-4. After subcutaneous inoculation of heat-killed LVS, no expression of any of the cytokine mRNAs and no increase in the levels of TNF-alpha or IFN-gamma occurred. The expression of TNF-alpha, IL-12, and IFN-gamma is held to be important to evoke an early T-cell-independent host defense against F. tularensis as well as to drive the expansion of a protective Th1 cell response.     

Enhancement of a TH1 immune response in amphotericin B-treated mucocutaneous leishmaniasis

In an attempt to investigate the effects of treatment of human leishmaniasis, the cytokines produced by peripheral blood mononuclear cells (PBMCs) of patients with cutaneous leishmaniasis (CL) and mucocutaneous leishmaniasis (MCL) under treatment with amphotericin B were determined during the active disease from cocultures of cells and Leishmania (Viannia) braziliensis antigens. PBMC of these patients exhibited a nonsignificant marginal increased production of TNF-alpha upon antigen stimulation. However, under the same antigenic stimulus, patients with active MCL presented higher IFN-gamma production compared to patients with CL. This increased IFN-gamma production was accompanied by a drastically augmented IL-12 synthesis from cells of MCL patients. The highlighted T cell responses could be relevant for sound control measures of protozoan infections with emphasis on the combined usage of immunoenhancing agents and antiprotozoal drugs.     

Cytokine profiles of BAL T cells and T-cell clones obtained from human asthmatic airways after local allergen challenge.

BACKGROUND: This study assessed the heterogeneity of cytokine expression in asthma before and after local allergen challenge. METHODS: BAL T cells were obtained 10 min or 24 h after local endobronchial allergen challenge in atopic asthmatic subjects. T cells were cloned by direct limiting dilution. mRNA expression was assessed by RT-PCR, and cytokine protein production by ELISA. RESULTS: Unstimulated baseline BAL T cells expressed mRNA for IFN-gamma, IL-13, and TNF-alpha. A minority of samples expressed IL-4 and IL-5, but no IL-3 mRNA was detected. PHA stimulation increased expression of IL-3, IL-4, and IL-5 mRNA in 4/6 samples. IL-13 and GM-CSF mRNA were found in BAL cells after allergen challenge, but expression of IFN-gamma was reduced. Both IL-4 and IL-3 were strongly upregulated after PHA stimulation, while the expression of TNF-alpha and IFN-gamma was reduced, compared to equivalent baseline samples. Seventeen panels of BAL T-cell clones were derived (average cloning efficiency 1/40 T cells). Seven panels survived to 8 weeks for analysis. Clones derived 4 h after saline challenge showed strong mRNA signals for IL-13, IL-4, and IFN-gamma, whereas clones derived 24 h after allergen challenge expressed IL-13, GM-CSF, IL-3, IL-4, and often IL-5 (i.e., closer to the Th2 profile). There was considerable heterogeneity in the patterns of cytokine mRNA and protein production by different clones. CONCLUSIONS: T cells from asthmatic airways produce IL-13, IFN-gamma, and TNF-alpha, but after allergen challenge, type 2 cytokines are upregulated. mRNA and protein analysis provide complementary information on airways T-cell cytokine profiles.     

High Serum-Soluble Interleukin-2 Receptor is not Associated with the Immunosuppression in Diffuse Cutaneous Leishmaniasis

Diffuse Cutaneous Leishmaniasis (DCL) is a rare complication of Leishmania aethiopica -induced cutaneous leishmaniasis which is associated with non-self healing and in vivo and in vitro antigen-specific non-responsiveness. Such antigen-specific unresponsiveness is also observed in visceral leishmaniasis (VL). The non-responsiveness seen in VL disease is believed to be due, in part, to serum-derived factors, including raised serum soluble IL-2 receptor (sIL-2R). Raised sIL-2R in serum was not a consistent feature of DCL in our study (range: 787–4546 U/ml) but was frequently observed in sera of patients with other dermatological disorders (range: 474–3313 U/ml) and some patients with the simple local cutaneous leishmaniasis (LCL; range: 556–4247 U/ml). The level of sIL-2R in the sera of DCL patients was not indicative of the disease state. Sera from DCL patients did not reduce proliferation of the IL-2-dependent CTLL cell line nor reduce PHA-driven mononuclear cell proliferation, although sera from VL patients could. Both DCL and VL sera could reduce the L. aethiopica -driven proliferation. Furthermore addition of serial dilutions of recombinant IL-2 to CTLL cultured in VL or DCL sera containing high sIL-2R levels did not alter the effect of such sera on proliferation. We conclude therefore, that raised sIL-2R in serum is not associated with the immunosuppression in DCL.     

In situ production of gamma interferon, interleukin-4, and tumor necrosis factor alpha mRNA in human lung tuberculous granulomas

Human tuberculous granulomas from five adults undergoing surgery for hemoptysis were analyzed by nonradioactive in situ hybridization for tumor necrosis factor alpha (TNF-alpha), gamma interferon (IFN-gamma), and interleukin-4 (IL-4) gene expression. All of the patients produced TNF-alpha mRNA. Three patients stained positive for both IFN-gamma and IL-4 mRNA; the other two stained positive for IFN-gamma but not IL-4 mRNA. Heterogeneity between the granulomas was observed in those patients staining positive for both IFN-gamma and IL-4 mRNA; these patients exhibited granulomas having IFN-gamma and not IL-4 mRNA as well as granulomas positive for both cytokine mRNAs. There was no evidence of caseation in these granulomas, and the cytokine patterns may represent events in the evolution of the granuloma. However, in those granulomas exhibiting caseous necrosis, very little IFN-gamma or IL-4 mRNA was observed, implying that progression of the granuloma is accompanied by a down regulation of T-cell responses. TNF-alpha mRNA expression was highest in patients with both IFN-gamma and IL-4 mRNA. Populations of CD68 positive macrophage-like cells within the granulomas produce mRNA for TNF-alpha, IFN-gamma, and IL-4. This implies that macrophages within the tuberculous granuloma may not be dependent on T-cell cytokines for modulation of their function but may be able to regulate their own activation state and that of the surrounding T cells. These findings have implications on the delivery of immunotherapies to patients with tuberculosis.     

A Th1-associated increase in tumor necrosis factor alpha expression in the spleen correlates with resistance to blood-stage malaria in mice.

We investigated the kinetics of tissue-specific mRNA expression and systemic production of tumor necrosis factor alpha (TNF-alpha) and the kinetics of splenic expression of mRNAs of gamma interferon (INF-gamma) and interleukin-4 (IL-4), cytokines that may regulate TNF-alpha production, during the early phase of blood-stage infection with Plasmodium chabaudi AS. Northern blot analysis revealed that resistant C57BL/6 mice, which clear the infection by 4 weeks, had higher levels of TNF-alpha mRNA in the spleen and liver early during infection that did susceptible A/J mice, which succumb to the disease 10 days after initiation of infection. Treatment of resistant mice with a polyclonal anti-TNF-alpha antibody confirmed the protective role of TNF-alpha early during the course of infection. Furthermore, resistant C57BL/6 mice also expressed high levels of mRNA of IFN-gamma (a Th1 marker) and low levels of mRNA of IL-4 (a Th2 marker) in the spleen, whereas susceptible A/J mice had low levels of IFN-gamma mRNA but high levels of TNF-alpha mRNA in the liver and had high levels of TNF-alpha protein in serum, as measured by enzyme-linked immunosorbent assay, later during infection just before death occurred. These results demonstrate that a Th1-associated increase in TNF-alpha mRNA expression in the spleen early during infection correlates with resistance to P. chabaudi AS, whereas increased TNF-alpha mRNA levels in the liver and excessive levels of the TNF-alpha protein in serum later during infection correlate with susceptibility. Thus, the role of the TNF-alpha during malaria appears to depend on the timing and site of its expression and the presence of cytokines regulating its production.     

Differential cytokine and chemokine production characterizes experimental autoimmune meningitis and experimental autoimmune encephalomyelitis

After primary immunization with myelin/oligodendrocyte glycoprotein, CD28(-/-) mice developed experimental autoimmune meningitis (EAM) rather than experimental autoimmune encephalomyelitis (EAE). Cytokine and chemokine production in EAE and EAM were compared to understand the differences in disease phenotype. T cells from the central nervous system lesions of mice with either EAE or EAM expressed intracellular TNF-alpha. Splenic T cells from mice with EAM produced TNF-alpha and IL-6 but no IL-2. Conversely, EAE-derived splenic T cells produced TNF-alpha and IL-2 but no IL-6. Altered T cell differentiation in EAM was not due to a Th1 to Th2 shift, because equivalent amounts of T cell IFN-gamma mRNA were produced in both diseases. Neutrophils also produced inflammatory mediators such as TNF-alpha and IL-6 in EAM. Autocrine production of MIP-2 mRNA was observed in neutrophils from mice with EAM but not EAE. Therefore, distinct patterns of cytokines and chemokines distinguish EAE and EAM.     

Elevated levels of interferon-gamma, interleukin-10, and interleukin-6 during active disease in Indian kala azar

We have evaluated levels of 6 cytokines in sera of 35 patients of kala azar (KA), 29 post kala azar dermal leishmaniasis (PKDL), and 18 healthy controls using cytometric bead array technology. Results indicated significantly high levels of interferon gamma (IFN-gamma), interleukin (IL)-10, and IL-6 during active KA, while tumor necrosis factor alpha (TNF-alpha), IL-2, and IL-4 were minimal. Serum level of cytokines in PKDL was comparable to the controls while TNF-alpha was significantly elevated compared to KA or control. At post-treatment stage, KA patients showed a significant decrement in the levels of IFN-gamma, IL-10, and IL-6; however, IL-6 remained significantly elevated above control levels. Further, comparison of cytokine levels in children and adults revealed elevated level of IL-10 in pediatric cases. SAG unresponsive cases showed significantly elevated levels of IFN-gamma in comparison with the responsive cases. The results depict that type1 response is not depressed during active KA and suggest the possibility that unresponsiveness to type1 stimuli may prevail.     

Synthesis and regulation of accessory/proinflammatory cytokines by intestinal epithelial cells.

Intestinal epithelial cells (IEC) have been shown to act as antigen-presenting cells (APC) in vitro and may have this capacity in vivo. In order to determine whether IEC, like other APC, are able to produce accessory cytokines which may play a role in T cell activation, we assessed the accessory cytokine profile of IEC constitutively or after stimulation. We measured expression, production and regulation of accessory cytokines (IL-1 beta, IL-6, tumour necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta) by the presence of mRNA as well as secreted protein. Freshly isolated IEC from surgical specimens were cultured in the presence or absence of lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), IL-1 beta or TNF-alpha. mRNA was assessed by a specific RNAse protection assay which controlled for contaminating cell populations while protein secretion was measured by ELISA (IL-1) or bioassay (TNF and IL-6). Neither IL-1 beta nor TNF-alpha were detectable in cultured IEC supernatants, supporting the lack of macrophage contamination. All IEC spontaneously secreted IL-6 at levels comparable to those of macrophages. IEC IL-6 mRNA also increased approximately 200-fold during the first 24 h of culture. LPS, IFN-gamma or TNF-alpha had no effect on spontaneous IL-6 production, and neither resulted in the secretion of IL-1 beta or TNF-alpha. However, IL-1 beta up-regulated IL-6 synthesis by 6-7-fold. IEC express a profile of cytokine mRNAs distinct from conventional APC (low level constitutive IL-6 expression but no detectable IL-1 beta, TGF-beta or TNF-alpha), adding to their uniqueness as APC.