Roberta Placido

Apoptosis of human monocytes/macrophages in Mycobacterium tuberculosis infection.

Tuberculosis (TB) is still a major health problem, both as a single disease entity and as a cofactor in AIDS. The interaction between macrophage and Mycobacterium tuberculosis (MTB) is a critical step in the establishment of an early chronic infection. This study analyses the capacity of MTB to induce apoptosis in cells obtained by broncho‐alveolar lavage (BAL) from patients with reactive pulmonary tuberculosis and from AIDS patients with disseminated pulmonary tuberculosis. Apoptosis was increased three‐fold in BAL cells obtained from patients with pulmonary tuberculosis and even more markedly in alveolar macrophages of MTB‐infected AIDS patients, compared with controls. Apoptosis was analysed and characterized by propidium iodide (PI) incorporation, terminal deoxy transferase (TDT)‐mediated dUTP‐biotin nick end labelling (TUNEL), and tissue transglutaminase (tTG) expression. The MTB–macrophage interaction was also investigated in vitro by infecting monocyte‐derived macrophages (MDM) with MTB (virulent strain H37Rv). The induction of apoptosis by MTB required viable bacteria, was dose‐dependent, and was restricted to H37Rv. Infection with either Mycobacterium avium complex (MAC) or HIV‐1 and treatment with heat‐killed MTB failed to induce apoptosis.

Heat-shock protein expression on the membrane of T cells undergoing apoptosis.

Heat‐shock proteins (hsp) represent a highly conserved family of proteins, normally localized in the cytoplasm and nucleus, whose expression is induced in situations involving cell stress. This paper reports the unusual translocation of hsp to the cell membrane of T cells undergoing apoptosis. We observed that glucocorticosteroid‐induced thymocyte death is associated to the surface expression of hsp  60 and hsp  70 in a discrete fraction of apoptotic cells. hsp surface expression is closely related to a thymic subset of immature CD3low/− T cells. The expression of surface hsp  60 appears early after treatment with dexamethasone (3 hr) whereas the membrane expression of hsp  70 follows different kinetics and peaks later. Morphological analysis of the hsp+ apoptotic cells suggest that this subset represents late‐stage apoptotic cells at their minimal volume before fragmentation into apoptotic bodies. Membrane expression of hsp is also associated with apoptosis in peripheral blood mononuclear cells from AIDS patients cultured in vitro. Altogether, we show that a discrete fraction of cells undergoing apoptosis expresses membrane hsp  60 and hsp  70, supporting the hypothesis that apoptosis causes a radical alteration in the expression of cell surface molecules. Surface hsp expressed during apoptosis may constitute a novel immune‐context able to generate packages of self‐ and exogenous antigens, originating from degradation of altered cells.

Enhanced Production of Tumor Necrosis Factor-α and Interleukin-6 Due to Prolonged Response to Lipopolysaccharide in Human Macrophages Infected In Vitro with Human Immunodeficiency Virus Type 1

Elevated levels of circulating tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 have been detected in human immunodeficiency virus (HIV) type 1 infection. The overproduction of these cytokines could contribute to AIDS pathogenesis. Thus, the expression of TNF-alpha and IL-6 in human macrophages infected with HIV-1 was investigated. HIV-1 infection, per se, did not induce any TNF-alpha or IL-6 production or cytokine-specific mRNA expression. In contrast, HIV-1 primed macrophages to a prolonged TNF-alpha and IL-6 response to lipopolysaccharide (LPS) stimulation with respect to uninfected cells. Time-course analysis and flow cytometry demonstrated that cytokine production stopped at 6 h in uninfected macrophages but continued up to 24 h in HIV-1-infected cells. RNA studies suggested that HIV-1 interfered with late steps of cytokine synthesis. No modulation of membrane CD14 was found to account for the enhanced response to LPS. Finally, the effect of HIV-1 on cytokine response could not be abolished by the antiviral compound U75875.

Infection of Human Monocytes with Mycobacterium tuberculosis Enhances Human Immunodeficiency Virus Type 1 Replication and Transmission to T Cells

Mycobacterium tuberculosis and human immunodeficiency virus type 1 (HIV-1) are virulent intracellular pathogens that invade and multiply within macrophages. The effect of M. tuberculosis on HIV-1 infection and replication was analyzed in vitro using human monocyte-derived macrophages (MDM) isolated from peripheral blood mononuclear cells by countercurrent centrifugal elutriation. Preinfection of MDM with M. tuberculosis followed by HIV-1 infection resulted in an increase in p24 release, reverse transcriptase activity, and infective virus production. In contrast, no increase in HIV-1 production was observed when MDM were infected with Mycobacterium avium complex or heat-killed M. tuberculosis. Coinfected MDM were potent stimulators of T cell proliferation, while HIV-1-infected MDM failed to present exogenous tuberculin to T cells. Furthermore, coinfected MDM showed an increased capacity to transmit HIV-1 to activated T cells. These results suggest that M. tuberculosis infection can both up-regulate HIV-1 infection and replication within MDM and increase the efficiency of virus transmission from infected MDM to T cells.

Evidence for a role of γδ T cells in demyelinating diseases as determined by activation states and responses to lipid antigens

Abstract In this report we review current information on the phenotypic and functional properties of γδ T cells in demyelinating disorders. The results support the conclusion that although γδ T cells show evidence of activation in patients with either multiple sclerosis (MS) or Guillain Barre syndrome (GBS), differences exist in the phenotypic and functional properties of these cells between the two diseases. In particular, our data indicate that in patients with MS the Vδ2 subset is activated and that these cells can be induced to secrete high levels of proinflammatory cytokines. In contrast, in patients with GBS, the Vδ1 subset is expanded and can be induced to secrete cytokines more associated with a humoral response.

Phenotypic and functional properties of γδ T Cells from patients with Guillain Barré syndrome

Abstract In this study we have examined the phenotypic and functional properties of circulating γδ T cells in patients with Guillain Barre syndrome (GBS), in normal healthy controls, and in patients with active multiple sclerosis (MS). Cells expressing the Vδ2 T cell receptor showed elevated expression of the C-lectin receptor NKRP1A in both GBS and MS, suggestive of an activated state. However, in patients with GBS these cells failed to respond to pyrenil-pyrophosphate derivatives and Vδ2+ T cell clones derived from these patients released lower levels of IFNγ than Vδ2+ clones derived from controls and MS patients. In contrast, in patients with GBS the Vδ1+ subset was expanded, showed elevated expression of NKRP1A and Vδ1+ clones derived from these patients secreted high levels of IL-4. Our findings of expanded NKRP-1A+, IL-4-producing Vδ1 T cells in the GBS patients suggests the possibility that these cells are activated by the recognition of non-protein antigens in an MHC-unrestricted manner and contribute to the humoral response to glycolipids that is a hallmark of this disease.

HIV-1 does not alter in vitro and in vivo IL-10 production by human monocytes and macrophages

The present study analyses the ability of HIV‐1 to modulate IL‐10 production in cells of monocyte‐macrophage lineage cultured in the presence of macrophage colony‐stimulating factor (M‐CSF). Both monocytes and macrophages spontaneously produced low amount of IL‐10. Lipopolysaccharide (LPS) induced a strong IL‐10 response in fresh monocytes and in M‐CSF‐treated macrophages. In contrast, macrophages cultured in the absence of M‐CSF exhibited a marked decrease in their susceptibility to LPS stimulation. M‐CSF increased the IL‐10 response of macrophages to LPS by enhancing both the expression of membrane‐bound CD14, the protein that serves as LPS receptor, and the sensibility of CD14‐expressing cells to LPS stimulation. Neither spontaneous nor LPS‐induced expression of IL‐10 was modulated in monocytes and macrophages by infection with eight monocytotropic strains, as demonstrated by ELISA and cytofluorimetric analysis. In contrast, all the HIV‐1 strains primed macrophages for an increased IL‐6 response to LPS stimulation. To determine whether IL‐10 production was associated with in vivo infection, monocytes from AIDS individuals were analysed for IL‐10 production. We found that neither spontaneous nor LPS‐induced IL‐10 production were different between healthy controls and HIV‐infected patients. Taken together, these data strongly suggest that HIV‐1 infection of monocytes‐macrophages does not play a significant role in the regulation of IL‐10 in infected patients. This study also emphasizes the role of M‐CSF activation in the regulation of the cytokine response in macrophages.

Characterization of the Immune Response of Human Cord-Blood Derived γδ T Cells to Stimulation with Aminobisphosphonate Compounds

Vγ9Vδ2 T lymphocytes have been shown to respond to a variety of non-peptide antigens including alkylamines and phosphoantigens. Recently, aminobisphosphonates have also been shown to stimulate this subset of γδ+ T cells. In this study we analyzed the proliferative responses of freshly isolated γδ T lymphocytes obtained from human cord blood when challenged with pyrophosphomonoesters or aminobisphosphonates. Nitrogen-containing aminobisphopsphonates, in contrast to phoshoantigens, readily stimulated expansion of Vδ2Vγ9 cells in human cord blood. Expanded cells displayed an activated mature phenotype, and were capable of producing TNFα and IFNγ but not perforin following secondary stimulation, consistent with the development of a regulatory, as opposed to cytotoxic, phenotype. This approach may provide a useful strategy for a new approach to the treatment of neonatal pathologies.

Mycobacterium avium infection in BALB/c and SCID mice.

BALB/c and severe combined immunodeficient (SCID) mice were inoculated intraperitoneally with Mycobacterium avium and the numbers of cfu were monitored for 70 days in spleen, liver, lung, kidney, brain and peritoneum. While BALB/c mice formed typical granulomas and controlled bacterial growth in organs, a delay in development of lesions and a modest containment of infection were observed in SCID mice. In the spleen of BALB/c mice, in which bacterial growth was contained, macrophages (Mo) and natural killer (NK) cell numbers increased > or = 4.2 times and T- and B-cell numbers increased > or = 1.8 times after 42 days of infection; conversely, a low recruitment of mononuclear cells was observed in the spleen of SCID mice, where M. avium proliferated efficiently. Unlike visceral organs, a pronounced decrease in the number of cfu was observed in the peritoneum of BALB/c mice, concomitantly with a > or = 31.7-fold increase in Mo and NK cells and a > or = 9.1-fold increase in T and B cells. In the peritoneum of SCID mice only a bacteriostatic effect was observed despite a > or = 56.7-fold increase in Mo and NK cells and a > or = 22.3-fold increase in T and B cells. These results suggest that while an intact immune response can efficiently control M. avium infection in the spleen and peritoneum of BALB/c mice, cells of the innate immune system such as Mo and NK cells play a role in the containment of bacterial growth in the peritoneum, but not spleen, of SCID mice.