Biomedical subjects

Ramakrishna Vankayalapati

Publications and source records attributed to Ramakrishna Vankayalapati.

10 recordsLinked to original sources

PubMed2006

Vimentin expressed on Mycobacterium tuberculosis-infected human monocytes is involved in binding to the NKp46 receptor.

We previously showed that human NK cells used the NKp46 receptor to lyse Mycobacterium tuberculosis H37Ra-infected monocytes. To identify ligands on H37Ra-infected human mononuclear phagocytes, we used anti-NKp46 to immunoprecipitate NKp46 from NK cells bound to its ligand(s) on H37Ra-infected monocytes. Mass spectrometry analysis identified a 57-kDa molecule, vimentin, as a putative ligand for NKp46. Vimentin expression was significantly up-regulated on the surface of infected monocytes, compared with uninfected cells, and this was confirmed by fluorescence microscopy. Anti-vimentin antiserum inhibited NK cell lysis of infected monocytes, whereas antiserum to actin, another filamentous protein, did not. CHO-K1 cells transfected with a vimentin construct were lysed much more efficiently by NK cells than cells transfected with a control plasmid. This lysis was inhibited by mAb-mediated masking of NKp46 (on NK cells) or vimentin (on infected monocytes). ELISA and Far Western blotting showed that recombinant vimentin bound to a NKp46 fusion protein. These results indicate that vimentin is involved in binding of NKp46 to M. tuberculosis H37Ra-infected mononuclear phagocytes.

Animals↗

PubMed2005

Role of NK cell-activating receptors and their ligands in the lysis of mononuclear phagocytes infected with an intracellular bacterium.

We studied the role of NK cell-activating receptors and their ligands in the lysis of mononuclear phagocytes infected with the intracellular pathogen Mycobacterium tuberculosis. Expression of the activating receptors NKp30, NKp46, and NKG2D were enhanced on NK cells by exposure to M. tuberculosis-infected monocytes, whereas expression of DNAX accessory molecule-1 and 2B4 was not. Anti-NKG2D and anti-NKp46 inhibited NK cell lysis of M. tuberculosis-infected monocytes, but Abs to NKp30, DNAX accessory molecule-1, and 2B4 had no effect. Infection of monocytes up-regulated expression of the NKG2D ligand, UL-16 binding protein (ULBP)1, but not expression of ULBP2, ULBP3, or MHC class I-related chain A or chain B. Up-regulation of ULBP1 on infected monocytes was dependent on TLR2, and anti-ULBP1 abrogated NK cell lysis of infected monocytes. The dominant roles of NKp46, NKG2D, and ULBP1 were confirmed for NK cell lysis of M. tuberculosis-infected alveolar macrophages. We conclude that NKp46 and NKG2D are the principal receptors involved in lysis of M. tuberculosis-infected mononuclear phagocytes, and that ULBP1 on infected cells is the major ligand for NKG2D. Furthermore, TLR2 contributes to up-regulation of ULBP1 expression.

Carrier Proteins↗

PubMed2004

NK cells regulate CD8+ T cell effector function in response to an intracellular pathogen.

We studied the role of NK cells in regulating human CD8+ T cell effector function against mononuclear phagocytes infected with the intracellular pathogen Mycobacterium tuberculosis. Depletion of NK cells from PBMC of healthy tuberculin reactors reduced the frequency of M. tuberculosis-responsive CD8+IFN-gamma+ cells and decreased their capacity to lyse M. tuberculosis-infected monocytes. The frequency of CD8+ IFN-gamma+ cells was restored by soluble factors produced by activated NK cells and was dependent on IFN-gamma, IL-15, and IL-18. M. tuberculosis-activated NK cells produced IFN-gamma, activated NK cells stimulated infected monocytes to produce IL-15 and IL-18, and production of IL-15 and IL-18 were inhibited by anti-IFN-gamma. These findings suggest that NK cells maintain the frequency of M. tuberculosis-responsive CD8+IFN-gamma+ T cells by producing IFN-gamma, which elicits secretion of IL-15 and IL-18 by monocytes. These monokines in turn favor expansion of Tc1 CD8+ T cells. The capacity of NK cells to prime CD8+ T cells to lyse M. tuberculosis-infected target cells required cell-cell contact between NK cells and infected monocytes and depended on interactions between the CD40 ligand on NK cells and CD40 on infected monocytes. NK cells link the innate and the adaptive immune responses by optimizing the capacity of CD8+ T cells to produce IFN-gamma and to lyse infected cells, functions that are critical for protective immunity against M. tuberculosis and other intracellular pathogens.

Antibodies, Blocking↗

PubMed2004

IS6110 functions as a mobile, monocyte-activated promoter in Mycobacterium tuberculosis.

The mobile insertion sequence, IS6110, is an important marker in tracking of Mycobacterium tuberculosis strains. Here, we demonstrate that IS6110 can upregulate downstream genes through an outward-directed promoter in its 3' end, thus adding to the significance of this element. Promoter activity was orientation dependent and was localized within a 110 bp fragment adjacent to the right terminal inverted repeat. Transcripts from this promoter, named OP6110, begin approximately 85 bp upstream of the 3' end of IS6110. Use of green fluorescent protein (GFP) expression constructs showed that OP6110 was upregulated in M. tuberculosis during growth in human monocytes and in late growth phases in broth. Analysis of natural insertion sites in M. tuberculosis showed that IS6110 upregulated expression of several downstream genes during growth in human monocytes, including Rv2280 in H37Rv and the PE-PGRS gene, Rv1468c, in the clinical strain 210, which is a member of the Beijing family. Transcription between IS6110 and downstream genes was confirmed by reverse transcription polymerase chain reaction. The ability to activate genes during infection suggests that IS6110 has the potential to influence growth characteristics of different strains, and indicates another mechanism by which IS6110 can impact M. tuberculosis evolution.

Artificial Gene Fusion↗

PubMed2003

The CD14 receptor does not mediate entry of Mycobacterium tuberculosis into human mononuclear phagocytes.

Prior reports have suggested that CD14 mediates uptake of Mycobacterium tuberculosis into porcine alveolar macrophages and human fetal microglia, but the contribution of CD14 to cell entry in human macrophages has not been studied. To address this question, we used flow cytometry to quantify uptake by human monocytes and alveolar macrophages of M. tuberculosis expressing green fluorescent protein. Neutralizing anti-CD14 antibodies did not affect bacillary uptake and the efficiency of bacillary entry was similar in THP-1 cells expressing low and high levels of CD14. However, most internalized bacteria were found in CD14+ but not in CD14- monocytes because M. tuberculosis infection upregulated CD14 expression. We conclude that: (1) CD14 does not mediate cellular entry by M. tuberculosis; (2) M. tuberculosis infection upregulates CD14 expression on mononuclear phagocytes, and this may facilitate the pathogen's capacity to modulate the immune response.

Cells, Cultured↗

PubMed2003

CD40 ligand trimer enhances the response of CD8+ T cells to Mycobacterium tuberculosis.

We investigated the effect of recombinant CD40 ligand trimer (CD40LT) on the functional capacity of peripheral blood CD8(+) T cells from healthy tuberculin reactors that were cultured with Mycobacterium tuberculosis-infected autologous monocytes. CD40LT enhanced the capacity of M. tuberculosis-responsive CD8(+) T cells to produce IFN-gamma by increasing the number of IFN-gamma-producing CD8(+) T cells and the amount of IFN-gamma produced per cell. CD40LT-induced IFN-gamma production was dependent on production of IL-12 and IL-18, but did not require IL-15. CD40LT up-regulated expression of the transcription factors phosphorylated CREB and c-Jun, both of which have been previously shown to stimulate IFN-gamma mRNA transcription by binding to the IFN-gamma promoter. CD40LT also enhanced the capacity of CD8(+) T cells to lyse M. tuberculosis-infected monocytes, and increased CTL activity was associated with higher expression of perforin and granulysin, but not of Fas ligand. We conclude that CD40LT can enhance CD8(+) T cell effector function in response to M. tuberculosis.

Adjuvants, Immunologic↗

PubMed2003

Spectrum of manifestations of Mycobacterium tuberculosis infection in primates infected with SIV.

To characterize the manifestations of coinfection with M. tuberculosis and SIV infection, we studied 12 SIV-infected rhesus monkeys, six of which were infected intrabronchially with a low dose of Mycobacterium tuberculosis H37Rv. In the six coinfected animals, M. tuberculosis antigen-stimulated lung and blood cells produced high concentrations of IFN-gamma but not IL-4 8-16 weeks after infection. Of the three coinfected animals with high levels of plasma viremia, two developed disseminated tuberculosis and the other died of bacterial peritonitis. Of three coinfected animals with moderate levels of plasma viremia, two had no clinical or radiographic evidence of tuberculosis or progressive SIV infection for 6 months after infection. At neuropsy, pulmonary granulomata were observed and acid-fast organisms or M. tuberculosis were present. These clinical, immunologic and pathologic findings are consistent with those in humans with latent tuberculosis infection (LTBI), and suggest that a model of LTBI in SIV-infected primates can be developed. Such a model will permit delineation of the immunologic and microbial factors that characterize LTBI in HIV-infected persons.

Animals↗

PubMed2002

Serum cytokine concentrations do not parallel Mycobacterium tuberculosis-induced cytokine production in patients with tuberculosis.

We measured serum cytokine concentrations and Mycobacterium tuberculosis-stimulated cytokine production by peripheral blood mononuclear cells (PBMCs) obtained from persons infected with M. tuberculosis. Serum interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) concentrations were elevated in patients with tuberculosis compared with healthy persons who had reactions to tuberculin skin tests, but IL-18 concentrations were not. In contrast, M. tuberculosis-stimulated PBMCs from patients with tuberculosis produced less IFN-gamma and IL-18 but similar amounts of IL-10, compared with PBMCs from healthy subjects who had reactions to tuberculin skin tests. Pretreatment of PBMCs from healthy subjects with reaction to tuberculin with serum from patients with tuberculosis inhibited IFN-gamma production in response to M. tuberculosis, and inhibition was blocked by anti-IL-10. Thus, serum concentrations of IFN-gamma, IL-18, and IL-10 do not parallel M. tuberculosis-induced cytokine levels, and increased IL-10 serum levels in patients with tuberculosis inhibit IFN-gamma production in response to mycobacterial antigens.

Cell Culture Techniques↗

PubMed2002

The NKp46 receptor contributes to NK cell lysis of mononuclear phagocytes infected with an intracellular bacterium.

We used human tuberculosis as a model to investigate the role of NK cytotoxic mechanisms in the immune response to intracellular infection. Freshly isolated NK cells and NK cell lines from healthy donors lysed Mycobacterium tuberculosis-infected monocytes to a greater extent than uninfected monocytes. Lysis of infected monocytes was associated with increased expression of mRNA for the NKp46 receptor, but not the NKp44 receptor. Antisera to NKp46 markedly inhibited lysis of infected monocytes. NK cell-mediated lysis was not due to reduced expression of MHC class I molecules on the surface of infected monocytes or to enhanced production of IL-18 or IFN-gamma. NK cell lytic activity against M. tuberculosis-infected monocytes and NKp46 mRNA expression were reduced in tuberculosis patients with ineffective immunity to M. tuberculosis compared with findings in healthy donors. These observations suggest that 1) the NKp46 receptor participates in NK cell-mediated lysis of cells infected with an intracellular pathogen, and 2) the reduced functional capacity of NK cells is associated with severe manifestations of infectious disease.

Cell Line↗

PubMed2002

Reduced expression of nuclear cyclic adenosine 5'-monophosphate response element-binding proteins and IFN-gamma promoter function in disease due to an intracellular pathogen.

Mycobacterium tuberculosis-induced IFN-gamma protein and mRNA expression have been shown to be reduced in tuberculosis patients, compared with healthy tuberculin reactors. To determine whether this decrease was associated with reduced activity of the IFN-gamma promoter, we first studied binding of nuclear proteins to the radiolabeled proximal IFN-gamma promoter (-71 to -40 bp), using EMSAs with nuclear extracts of freshly isolated peripheral blood T cells. Nuclear extracts of T cells from most tuberculosis patients showed markedly reduced expression of proteins that bind to the proximal IFN-gamma promoter, compared with findings in nuclear extracts of T cells from healthy tuberculin reactors. These DNA-binding complexes contained CREB proteins, based on competitive EMSAs, supershift assays, and Western blotting with an anti-CREB Ab. Transient transfection of PBLs with a luciferase reporter construct under the control of the IFN-gamma promoter revealed reduced IFN-gamma promoter activity in tuberculosis patients. Transient transfection of Jurkat cells with a dominant-negative CREB repressor plasmid reduced IFN-gamma promoter activity. These data suggest that reduced expression of CREB nuclear proteins in tuberculosis patients results in decreased IFN-gamma promoter activity and reduced IFN-gamma production.

Consensus Sequence↗