CD4(+) CD25(high) forkhead box protein 3(+) regulatory T lymphocytes suppress interferon-γ and CD107 expression in CD4(+) and CD8(+) T cells from tuberculous pleural effusions.

Tuberculous pleural effusion is characterized by a T helper type 1 (Th1) profile, but an excessive Th1 response may also cause tissue damage that might be controlled by regulatory mechanisms. In the current study we investigated the role of regulatory T cells (Treg ) in the modulation of Th1 responses in patients with tuberculous (TB) pleurisy. Using flow cytometry we evaluated the proportion of Treg (CD4(+) CD25(high) forkhead box protein 3(+) ), interferon (IFN)-γ and interleukin (IL)-10 expression and CD107 degranulation in peripheral blood (PB) and pleural fluid (PF) from patients with TB pleurisy. We demonstrated that the proportion of CD4(+) CD25(+) , CD4(+) CD25(high) FoxP3(+) and CD8(+) CD25(+) cells were increased in PF compared to PB samples. Mycobacterium tuberculosis stimulation increased the proportion of CD4(+) CD25(low/neg) IL-10(+) in PB and CD4(+) CD25(low/neg) IFN-γ(+) in PF; meanwhile, CD25(high) mainly expressed IL-10 in both compartments. A high proportion of CD4(+) CD107(+) and CD8(+) CD107(+) cells was observed in PF. Treg depletion enhanced the in-vitro M. tuberculosis-induced IFN-γ and CD4(+) and CD8(+) degranulation responses and decreased CD4(+) IL-10(+) cells in PF. Our results demonstrated that in TB pleurisy Treg cells effectively inhibit not only IFN-γ expression but also the ability of CD4(+) and CD8(+) cells to degranulate in response to M. tuberculosis.

CD3 expression distinguishes two gammadeltaT cell receptor subsets with different phenotype and effector function in tuberculous pleurisy.

Tuberculous pleurisy is a naturally occurring site of Mycobacterium tuberculosis (Mtb) infection. Herein, we describe the expression of activation, natural killer (NK) and cell migration markers, as well as effector functions from gammadeltaT cells in peripheral blood (PB) and pleural effusion (PE) from tuberculosis patients (TB). We observed a decreased percentage of circulating gammadeltaT from TB patients and differential expression of NK as well as of chemokine receptors on PB and PE. Two subsets of gammadeltaT cells were differentiated by the CD3/gammadeltaT cell receptor (gammadeltaTCR) complex. The gammadeltaTCR(low) subset had a higher CD3 to TCR ratio and was enriched in Vdelta2(+) cells, whereas most Vdelta1(+) cells belonged to the gammadeltaTCR(high) subset. In PB from TB, most gammadeltaTCR(high) were CD45RA(+)CCR7(-) and gammadeltaTCR(low) were CD45RA(+/-)CCR7(+)CXCR3(+). In the pleural space the proportion of CD45RA(-)CCR7(+)CXCR3(+) cells was higher. Neither spontaneous nor Mtb-induced interferon (IFN)-gamma production was observed in PB-gammadeltaT cells from TB; however, PE-gammadeltaT cells showed a strong response. Both PB- and PE-gammadelta T cells expressed surface CD107a upon stimulation with Mtb. Notably, PE-gammadeltaTCR(low) cells were the most potent effector cells. Thus, gammadeltaT cells from PB would acquire a further activated phenotype within the site of Mtb infection and exert full effector functions. As gammadeltaT cells produce IFN-gamma within the pleural space, they would be expected to play a beneficial role in tuberculous pleurisy by helping to maintain a T helper type 1 profile.

Activation of peripheral blood neutrophils from patients with active advanced tuberculosis.

Activation of peripheral blood neutrophils (PMN) was investigated in order to determine whether they might contribute to the inflammatory process during active advanced tuberculosis. Receptors for the Fc portion of IgG (FcgammaR) (FcgammaRI, FcgammaRII, and FcgammaRIIIB), CD66 (degranulation marker), and receptors for tumor necrosis factor-alpha (TNF-R55 and TNF-R75) were analyzed on PMN obtained from normal controls and tuberculosis patients (TB-PMN). Functional parameters such as cytotoxicity, superoxide anion generation triggered by N-formyl-methionyl-leucyl-phenyl-alanine (FMLP), and TNF-alpha and IL-1beta production were evaluated. A high expression of TNF-R55, CD66, and FcgammaRIIIB and the appearance of FcgammaRI were detected in TB-PMN. In addition, cytotoxicity, superoxide anion release, and TNF-alpha and IL-1beta production were enhanced in TB-PMN. Thus, in tuberculosis, the activation of PMN outside the focus of infection strongly suggests the possibility of a systemic inflammation that could modulate the inflammatory response.

Interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) are necessary in the early stages of induction of CD4 and CD8 cytotoxic T cells by Mycobacterium leprae heat shock protein (hsp) 65 kD.

Cytotoxic T cells (CTL) may play an important role in host defence against mycobacterial infections. CD4 CTL are preferentially induced by mycobacteria, but both CD4 and CD8 CTL may be necessary components of a protective immune response. The 65-kD mycobacterium heat shock protein (hsp65) is a poor inducer of CTL in multibacillary leprosy (MB) patients. In this study we evaluate the possible role of cytokines in modulating the cytotoxic activity of CTL from leprosy patients and normal individuals (N) against autologous macrophages presenting Mycobacterium leprae hsp65. Our results show that hsp65-specific CTL were generated from both CD4 and CD8 lymphocytes. In N, individual cytokines as well as the combination of them were able to modify the hsp65-induced cytotoxic activity. The effect of cytokines on leprosy patients' lymphocytes was different in MB and paucibacillary (PB) patients. Thus, IL-6, IL-2, IFN-gamma or TNF-alpha did not modify the generation of hsp65-CTL from either MB (with or without an erythema nodosum episode (ENL)) or PB. In all the patients the simultaneous addition of two cytokines was required in order to increase CTL generation. In MB, IL-6 plus IFN-gamma or IL-2 increased both CD4 and CD8 CTL, while TNF-alpha plus IFN-gamma up-regulated only CD4 CTL. In PB, CD8 CTL were prominent with IL-6 plus IFN-gamma, while the increase was significant in CD4 CTL with IL-6 plus IL-2. Down-regulation of CTL was observed by addition of IL-4, IL-10, anti-IFN-gamma or anti-TNF-alpha in N controls. Our data demonstrate that IFN-gamma and TNF-alpha must be present for at least the first 60 h of the induction stage in order to generate full hsp65 CTL. Hence, IFN-gamma and TNF-alpha would be key factors in the generation of hsp65 CTL.

Differential development of CD4 and CD8 cytotoxic T cells (CTL) in PBMC across the leprosy spectrum; IL-6 with IFN-gamma or IL-2 generate CTL in multibacillary patients.

In the present study we evaluated the contribution of CD4 and CD8 T cells on the antigen-specific cytotoxic activity induced by whole Mycobacterium leprae in leprosy patients and normal controls (N) as well as the modulation of this activity by some cytokines. Peripheral blood mononuclear cells (PBMC) from N or from leprosy patients were stimulated with antigen in the presence or absence of cytokines for 7 days. M. leprae-stimulated PBMC were depleted of CD4 or CD8 antigen-bearing cells and employed as effector cells in a 4-hr [31Cr]-release assay against autologous M. leprae-pulsed macrophages. Our results demonstrate that both CD4 and CD8 T cells contribute to M. leprae-induced cytotoxic activity, with differences observed in paucibacillary (PB) and multibacillary (MB) patients. CD8-mediated cytotoxic activity is higher than that of CD4 cells in PB patients, while in MB patients CD4 cytotoxicity is predominant. Our data also demonstrate that the generation of CD4 and CD8 cytotoxic T lymphocytes (CTL) can be modulated differentially by interleukin-4 (IL-4), IL-6, gamma interferon (IFN-gamma), or IL-2. Although MB patients developed the lowest CTL response, cytokines such as IL-6 plus IL-2 or IFN-gamma were able to generate both CD4 and CD8 cytotoxic T cells from MB patients. In PB patients, IL-6 plus IFN-gamma displayed the highest stimulation on CD8 effector cells. Thus, an important role may be assigned to IL-6, together with IL-2 or IFN-gamma, in the differentiation of M. leprae-specific CTL effector cells.

[Evaluation of cytokine production in leprosy patients]. / Evaluación de la producción de citoquinas en enfermos de lepra.

The aim of the present study was to evaluate the cytokine production by peripheral blood mononuclear cells (PBMC) of leprosy patients when the cells were stimulated in culture by ConA, PPD or M.leprae. We measured IL-2, IL-4, IFN-gamma and IL-6 in cell-free supernatants by enzyme linked immunoassays. Our results do not suggest a clear association of a clinical form of leprosy with either Th1 or Th2 cytokine secretion profile in PBMC of leprosy patients.

Lack of cytotoxic activity against Mycobacterium leprae 65-kD heat shock protein (hsp) in multibacillary leprosy patients.

Cytotoxic T cells play an important role in host defence mechanisms, as well as in the immunopathology of leprosy. In this study, we evaluated whether Mycobacterium leprae hsp18, hsp65 and Myco. tuberculosis hsp71 could induce cytotoxic T cell activity against autologous macrophages pulsed with these hsp. Paucibacillary (PB) patients and normal controls generated more effector cells than multibacillary (MB) patients with all three hsp tested. There was no cross-reactivity between any of the hsp tested. Mycobacterium leprae hsp65 induced cytotoxic responses only in those MB patients undergoing an erythema nodosum leprosum (ENL) episode. Although hsp65 and hsp18 induced similar proliferation in MB patients, a high proportion of these patients did not generate cytotoxic effector cells in response to hsp65. Hence, those T cells reacting to hsp65 may play an important role in the control of Myco. leprae infection.

IFN-gamma, IL-6 and IL-4 modulate M. leprae- or PPD-specific cytotoxic T cells in leprosy patients.

Specific cytotoxic T cells against intracellular pathogens may be generated in vitro. On the other hand it is well known that cytokines can regulate almost every aspect of immune function. The aim of this study was to evaluate the effect of some cytokines on the generation of cytotoxic T cells with specificity for Mycobacterium leprae- or PPD-pulsed autologous macrophages from leprosy patients and normal controls. Peripheral blood mononuclear cells from M. bovis BCG-immunized controls or from leprosy patients were stimulated with antigen, in the presence or absence of cytokines, for 7 days. These were used as effector cells in a 4-h [51Cr]-release assay. Our results show that development of cytotoxic T cells may be enhanced by gamma-IFN, IL-6 or the combination of IL-6 and IL-2. Addition of IL-2 or TNF-alpha alone did not modify the generation of cytotoxic activity. IL-4 down-regulated the cytotoxic response and gamma-IFN was able to counteract this effect. Hence, the generation of specific cytotoxic T cells can be modulated by cytokines. Whether this cytotoxic mechanism contributes to protection or tissue damage in M. leprae infection remains to be determined.

T-cell-mediated cytotoxicity against Mycobacterium antigen-pulsed autologous macrophages in leprosy patients.

The involvement of CD4+ T lymphocytes in the defense mechanisms against intracellular pathogens is widely recognized. Little information is available on the generation and specificity of the cytotoxic cells that eliminate human monocytes/macrophages infected with mycobacteria. In this work, we tested whether mononuclear cells from leprosy patients could generate cytotoxic T-cell activity against autologous macrophages pulsed with Mycobacterium leprae or purified protein derivative (PPD) in a 4-h 51Cr release assay. Peripheral blood mononuclear cells from normal Mycobacterium bovis BCG-immunized controls or from leprosy patients stimulated with antigen for 7 days were used as effector cells. Paucibacillary (PB) patients and normal controls yielded more active effector cells in this system than multibacillary (MB) patients. MB patients were able to develop cytotoxicity against M. leprae, BCG, or PPD, in contrast with the immunological anergy widely described. We did not find cytotoxicity against unpulsed macrophages. Cross-reactivity was observed between PPD, BCG, and M. leprae. Only antigen-pulsed autologous macrophages were suitable as target cells. M. leprae-induced cytotoxic cells were found in both CD4+ CD8- and CD4- CD8+ T-cell subsets, whereas CD4+ cells were the main component of PPD-induced cytotoxicity. In MB patients, BCG-induced cytotoxic cells were better killers of M. leprae-pulsed macrophages than cells induced by M. leprae. This is an interesting finding in view of the ongoing vaccination trials. The involvement of CD4- or CD8-mediated cytotoxicity may be important in the balance between protection and tissue or nerve damage.

Suppression of T cell proliferation induced by concanavalin A in hemophilia patients.

Concanavalin A (Con-A)-induced suppression of T cell proliferation was studied in 48 patients with severe hemophilia. Two groups of patients were defined according to the proliferative response when increasing numbers of Con A-induced cells were added to a constant number of phytohemagglutinin (PHA)-stimulated autologous T cells: In group A (60%) and in normal controls, higher suppression was achieved when more Con A-induced cells were added; in Group B, increasing numbers of Con A-induced cells produced no suppression of stimulated PHA-triggered proliferation. This effect could be corrected in Group B by inducing suppression in the presence of inhibitors of the oxidative metabolism of arachidonic acid. No correlation was found between the suppression profile and HIV-1 or HBV serology. Clinical evolution, as judged by signs and symptoms of AIDS related complex tended to be better in Group B than in Group A patients. It is suggested that decreased Con A-induced suppression in Group B may represent part of a normal regulatory process that involves products of arachidonic acid oxidative metabolism.

Suppression of T cell proliferation induced by concanavalin A in hemophilia patients.

Concanavalin A (Con-A)-induced suppression of T cell proliferation was studied in 48 patients with severe hemophilia. Two groups of patients were defined according to the proliferative response when increasing numbers of Con A-induced cells were added to a constant number of phytohemagglutinin (PHA)-stimulated autologous T cells: In group A (60

) and in normal controls, higher suppression was achieved when more Con A-induced cells were added; in Group B, increasing numbers of Con A-induced cells produced no suppression of stimulated PHA-triggered proliferation. This effect could be corrected in Group B by inducing suppression in the presence of inhibitors of the oxidative metabolism of arachidonic acid. No correlation was found between the suppression profile and HIV-1 or HBV serology. Clinical evolution, as judged by signs and symptoms of AIDS related complex tended to be better in Group B than in Group A patients. It is suggested that decreased Con A-induced suppression in Group B may represent part of a normal regulatory process that involves products of arachidonic acid oxidative metabolism.

[Immunosuppression in leprosy]. / Immunosupresión en lepra.

Many authors tried to show that in lepromatous leprosy (LL), suppressor mechanisms are involved in the immune response. We have previously shown that non-specific suppression (Con A induced) was impaired in LL patients and tends to normalize during the erythema nodosum leprosum episode (ENL). In this system we have shown that CD8+ cells (Leu 2a+) can interfere with the generation of Con A-induced suppression. We also observed that a high percentage of LL patients had an increased spontaneous suppression. In these patients, the number of Leu 2a+ cells added in the assay did not correlate with the suppression values. On the other hand, we had demonstrated that the monocyte suppressor system may have an important role, due to the release of soluble factors (PGE2). We evaluated M. leprae-induced suppressor cell function using a two step assay, on T cell proliferation. The results of this study indicate that the ability of M. leprae to induce suppressor activity was lower in LL patients than in tuberculoid (TT), intermediate clinical forms (BB, BL, BT) and BCG-immunized controls. On the other hand, we determined that the proportion of peripheral blood mononuclear cells (PBMC) bearing the Leu 8+ antigen (associated to suppressor inducer cells) was low in LL and tends to normalize during the ENL episode. Suppression of proliferation could not be overcome with exogenous IL-2 and was not related to the induction of Tac antigen. The ability of LL, TT, ENL and normal cells to proliferate upon PHA or Con A stimulus was similar.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunosupresión en lepra. / [Immunosuppression in leprosy]

Many authors tried to show that in lepromatous leprosy (LL), suppressor mechanisms are involved in the immune response. We have previously shown that non-specific suppression (Con A induced) was impaired in LL patients and tends to normalize during the erythema nodosum leprosum episode (ENL). In this system we have shown that CD8+ cells (Leu 2a+) can interfere with the generation of Con A-induced suppression. We also observed that a high percentage of LL patients had an increased spontaneous suppression. In these patients, the number of Leu 2a+ cells added in the assay did not correlate with the suppression values. On the other hand, we had demonstrated that the monocyte suppressor system may have an important role, due to the release of soluble factors (PGE2). We evaluated M. leprae-induced suppressor cell function using a two step assay, on T cell proliferation. The results of this study indicate that the ability of M. leprae to induce suppressor activity was lower in LL patients than in tuberculoid (TT), intermediate clinical forms (BB, BL, BT) and BCG-immunized controls. On the other hand, we determined that the proportion of peripheral blood mononuclear cells (PBMC) bearing the Leu 8+ antigen (associated to suppressor inducer cells) was low in LL and tends to normalize during the ENL episode. Suppression of proliferation could not be overcome with exogenous IL-2 and was not related to the induction of Tac antigen. The ability of LL, TT, ENL and normal cells to proliferate upon PHA or Con A stimulus was similar.(ABSTRACT TRUNCATED AT 250 WORDS)