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Alan M Krensky

Publications and source records attributed to Alan M Krensky.

34 records · Page 2Linked to original sources
PubMed2004

Lesional T cells and dermal dendrocytes in psoriasis plaque express increased levels of granulysin.

Granulysin is a broad-spectrum potent antimicrobial peptide produced by the immunocytes. We determined granulysin levels in certain cutaneous inflammatory diseases and correlated expression of granulysin with the relative risks of secondary infections in these conditions. In immunohistochemistry stains a monoclonal antigranulysin antibody was used at 1:150 dilutions. Compared with atopic dermatitis and nummular eczema lesions where secondary infection with Staphylococcus aureus is very common, we found that a significantly increased number of granulysin-positive T cells (P < .01) were present in psoriatic plaques. Psoriasis plaques are heavily colonized with S aureus . It is a well-known observation that despite open cracks and fissures these plaques do not get infected. Increased levels of granulysin provide an explanation for relative immunity of psoriatic plaques against both gram-positive and gram-negative bacterial infections.

Antigens, Differentiation, T-Lymphocyte↗
PubMed2003

DQ 65-79, a peptide derived from HLA class II, mimics p21 to block T cell proliferation.

DQ 65-79, a peptide derived from residues 65-79 of the alpha-chain HLA class II molecule DQA03011, blocks T cell proliferation and induces T cell apoptosis. Using a yeast two-hybrid assay, we previously identified proliferating cell nuclear Ag (PCNA) as an intracellular ligand for DQ 65-79. In this study, we show that three regions of PCNA, residues 81-100, 121-140, and 241-261, interact with DQ 65-79. Residues 241-261 of PCNA also interact with the C terminus (residues 139-160) of the cell cycle regulator, p21, suggesting that DQ 65-79 and p21 might function similarly. We show here that DQ 65-79 competitively inhibits binding of p21 to PCNA and that both DQ 65-79 and p21 139-160 induce T cell apoptosis, suggesting that DQ 65-79 and p21 act similarly to inhibit cell growth.

3T3 Cells↗
PubMed2003

Intracellular mediators of granulysin-induced cell death.

Granulysin, a molecule present in the granules of CTL and NK cells, is cytolytic against microbes and tumors. Granulysin induces apoptosis of mammalian cells by damaging mitochondria and causing the release of cytochrome c and apoptosis-inducing factor, resulting in DNA fragmentation. Here we show that Ca2+ and K+ channels as well as reactive oxygen species are involved in granulysin-mediated Jurkat cell death. The Ca2+ channel blockers, nickel and econazole, and the K+ channel blockers, tetraethylammonium chloride, apamin, and charybdotoxin, inhibit the granulysin-induced increase in intracellular Ca2+ ([Ca2+](i)), the decrease in intracellular K+, and apoptosis. Thapsigargin, which releases Ca2+ from the endoplasmic reticulum, prevents a subsequent granulysin-induced increase in [Ca2+](i) in Jurkat cells, indicating that the initial increase in [Ca2+](i) is from intracellular stores. The rise in [Ca2+](i) precedes a decrease in intracellular K+, and elevated extracellular K+ prevents granulysin-mediated cell death. In granulysin-treated cells, electron transport is uncoupled, and reactive oxygen species are generated. Finally, an increase in intracellular glutathione protects target cells from granulysin-induced lysis, indicating the importance of the redox state in granulysin-mediated cell death.

Antigens, Differentiation, T-Lymphocyte↗
PubMed2003

Human NKT cells express granulysin and exhibit antimycobacterial activity.

Human NKT cells are a unique subset of T cells that express an invariant V alpha 24 TCR that recognizes the nonclassical Ag-presenting molecule CD1d. Activation of NKT cells is greatly augmented by the marine sponge-derived glycolipid alpha-galactosylceramide (alpha GalCer). Because human monocyte-derived cells express CD1d and can harbor the intracellular pathogen Mycobacterium tuberculosis, we asked whether the addition of alpha GalCer could be used to induce effector functions of NKT cells against infected monocytes, macrophages, and monocyte-derived dendritic cells. NKT cells secreted IFN-gamma, proliferated, and exerted lytic activity in response to alpha GalCer-pulsed monocyte-derived cells. Importantly, alpha GalCer-activated NKT cells restricted the growth of intracellular M. tuberculosis in a CD1d-dependent manner. NKT cells that exhibited antimycobacterial activity also expressed granulysin, an antimicrobial peptide shown to mediate an antimycobacterial activity through perturbation of the mycobacterial surface. Degranulation of NKT cells resulted in depletion of granulysin and abrogation of antimycobacterial activity. The detection of CD1d in granulomas of tuberculosis patients supports the potential interaction of NKT cells with CD1d-expressing cells at the site of disease activity. These studies provide evidence that alpha Gal Cer-activated CD1d-restricted T cells can participate in human host defense against M. tuberculosis infection.

Adjuvants, Immunologic↗
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

Granulysin.

Granulysin, a molecule expressed by human natural killer cells and activated T lymphocytes, exhibits cytolytic activity against a variety of microbes and tumors. Progress in understanding the structure, function and clinical relevance of granulysin over the past year encompasses three main areas: first, the solution of its crystal structure, providing new insights into its potential mechanism of target cell damage; second, inhibition of its function with small interfering RNA, indicating its relevance in microbial immunity; and third, association of granulysin expression in natural killer cells with good outcomes in cancer, indicating its potential utility as a diagnostic and suggesting its relevance to human disease.

Amino Acid Sequence↗
PubMed2003

Helicobacter pylori induces RANTES through activation of NF-kappa B.

Helicobacter pylori-infected gastric mucosa displays a conspicuous infiltration of mononuclear cells and neutrophils. RANTES (short for "regulated upon activation, normal T cell expressed and secreted") is a chemoattractant cytokine (chemokine) important in the infiltration of T lymphocytes and monocytes. RANTES may therefore contribute to the cellular infiltrate in the H. pylori-infected gastric mucosa. The aim of this study was to analyze the molecular mechanism responsible for H. pylori-mediated RANTES expression. We observed that gastric epithelial cells produced RANTES upon coculture with H. pylori. In addition, H. pylori induced RANTES mRNA expression and an increase in luciferase activity in cells which were transfected with a luciferase reporter construct derived from the RANTES promoter, in gastric epithelial cells, indicating that the induction of RANTES production occurred at the transcriptional level. Induction of RANTES was dependent on an intact cag pathogenicity island. Activation of the RANTES promoter by H. pylori occurred through the action of NF-kappa B. Transfection of kinase-deficient mutants of I kappa B kinase (IKK) and NF-kappa B-inducing kinase (NIK) inhibited H. pylori-mediated RANTES activation. In contrast, tumor necrosis factor alpha- or interleukin-1/Toll-like receptor signaling molecules-such as mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1, MyD88, and interleukin-1 receptor-associated kinase-did not play a role in RANTES activation by H. pylori. Collectively, H. pylori induced NF-kappa B activation through an intracellular signaling pathway that involved IKK and NIK, leading to RANTES gene transcription. RANTES induction by H. pylori may play an important role in gastric inflammation.

Antineoplastic Agents↗
PubMed2002

Selective depression of interferon-gamma and granulysin production with increase of proliferative response by Vgamma9/Vdelta2 T cells in children with tuberculosis.

Vgamma9/Vdelta2 T cells can contribute to protective immune response against Mycobacterium tuberculosis, although the extent to which and mechanisms by which they could actually protect against human tuberculosis remain unclear. We have previously reported that Vgamma9/Vdelta2 T cells from tuberculin purified protein derivative (PPD)-positive children, either healthy or affected by different clinical forms of tuberculosis, strongly proliferate to different phosphoantigens in vitro, whereas Vgamma9/Vdelta2 T cells from PPD-negative healthy subjects proliferate very poorly. We report here that Vgamma9/Vdelta2 T cells from tuberculous children have an increased proliferative activity, but decreased interferon (IFN)-gamma production and granulysin expression. After successful chemotherapy, the Vgamma9/Vdelta2 T cell proliferative response strongly decreased, whereas IFN-gamma and granulysin production consistently increased. Disease-associated changes in Vgamma9/Vdelta2 T cell effector functions in patients with tuberculosis are consistent with the possibility that these T cells may play a protective role in immune response against M. tuberculosis infection.

Adolescent↗
PubMed2002

CD8 T cell-mediated killing of Cryptococcus neoformans requires granulysin and is dependent on CD4 T cells and IL-15.

Granulysin is located in the acidic granules of cytotoxic T cells. Although the purified protein has antimicrobial activity against a broad spectrum of microbial pathogens, direct evidence for granulysin-mediated cytotoxicity has heretofore been lacking. Studies were performed to examine the regulation and activity of granulysin expressed by CD8 T cells using Cryptococcus neoformans, which is one of the most common opportunistic pathogens of AIDS patients. IL-15-activated CD8 T cells acquired anticryptococcal activity, which correlated with the up-regulation of granulysin. When granules containing granulysin were depleted using SrCl(2,) or when the gene was silenced using 21-nt small interfering RNA duplexes, the antifungal effect of CD8 T cells was abrogated. Concanamycin A and EGTA did not affect the antifungal effect, suggesting that the activity of granulysin was perforin independent. Following stimulation by the C. neoformans mitogen, CD8 T cells expressed granulysin and acquired antifungal activity. This activity required CD4 T cells and was dependent upon accessory cells. Furthermore, IL-15 was both necessary and sufficient for granulysin up-regulation in CD8 T cells. These observations are most consistent with a mechanism whereby C. neoformans mitogen is presented to CD4 T cells, which in turn activate accessory cells. The resultant IL-15 activates CD8 T cells to express granulysin, which is responsible for antifungal activity.

Adjuvants, Immunologic↗
PubMed2002

Identification of epitopes of Mycobacterium tuberculosis 16-kDa protein recognized by human leukocyte antigen-A*0201 CD8(+) T lymphocytes.

CD8(+) T cells could make an important contribution to protection against tuberculosis (TB), but the antigenic determinants recognized in the context of major histocompatibility complex class I molecules remain ill defined. Our aim was to identify nonamer peptides derived from the acr/16-kDa antigen. Two immunogenic peptides (p21-29 and p120-128) were identified by their ability to elicit cytotoxic CD8(+) T cells from juvenile patients recovering from TB. Epitope-specific recognition was demonstrated by the lysis of both Mycobacterium tuberculosis-infected and peptide-pulsed macrophages, the release of cytotoxic granules, and interferon-gamma and tumor necrosis factor-alpha production. CD8(+) T cell responses to p21-29 and p120-128 were detected ex vivo in freshly isolated peripheral blood mononuclear cells from patients with TB but not in those from healthy control subjects. Our data suggest that these antigenic peptides can play a critical role in effective immunity against mycobacterial infection and TB.

Antigens, Differentiation, T-Lymphocyte↗
PubMed2002

Functional domains and DNA-binding sequences of RFLAT-1/KLF13, a Krüppel-like transcription factor of activated T lymphocytes.

RFLAT-1/KLF13, a member of the Krüppel-like family of transcription factors, was identified as a transcription factor expressed 3-5 days after T lymphocyte activation. It binds to the promoter of the chemokine gene RANTES (regulated on activation normal T cell expressed and secreted) and regulates its "late" expression in activated T-cells. In this study, a series of experiments to define the functional domains of RFLAT-1/KLF13 were undertaken to further advance the understanding of the molecular mechanisms underlying transcriptional regulation by this factor. Using the GAL4 fusion system, distinct transcriptional activation and repression domains were identified. The RFLAT-1 minimum activation domain is localized to amino acids 1-35, whereas the repression domain resides in amino acids 67-168. Deletion analysis on the RFLAT-1 protein further supports these domain functions. The RFLAT-1 activation domain is similar to that of its closest family member, basic transcription element-binding protein 1. This domain is highly hydrophobic, and site-directed mutagenesis demonstrated that both negatively charged and hydrophobic residues are important for transactivation. The nuclear localization signal of RFLAT-1 was also identified using the RFLAT-1/green fluorescence protein fusion approach. RFLAT-1 contains two potent, independent nuclear localization signals; one is immediately upstream of the zinc finger DNA-binding domain, and the other is within the zinc fingers. Using mutational analysis, we also determined that the critical binding sequence of RFLAT-1 is CTCCC. The intact CTCCC box on the RANTES promoter is necessary for RFLAT-1-mediated RANTES transcription and is also required for the synergy between RFLAT-1 and NF-kappaB proteins.

3T3 Cells↗
PubMed2002

DQ 65-79, a peptide derived from HLA class II, induces I kappa B expression.

A synthetic peptide corresponding to residues 65-79 of the alpha helix of the alpha-chain of the class II HLA molecule DQA03011 (DQ 65-79) inhibits the proliferation of human T lymphocytes in an allele nonrestricted manner. By using microarray technology, we found that expression of 29 genes was increased or decreased in a human CTL cell line after treatment with DQ 65-79. This study focuses on one of these genes, IkappaB-alpha, whose expression is increased by DQ 65-79. IkappaB proteins, including IkappaB-alpha and IkappaB-beta, are increased in T cells treated with DQ 65-79. Nuclear translocation of the NF-kappaB subunits p65 and p50 is decreased in T cells after treatment with DQ 65-79, while elevated levels of p65 and p50 are present in cytosol. DQ 65-79 inhibits the degradation of IkappaB-alpha mRNA and inhibits the activity of IkappaB kinase. These findings indicate that the DQ 65-79 peptide increases the level of IkappaB proteins, thereby preventing nuclear translocation of the transcription factor, NF-kappaB, and inhibiting T cell proliferation.

Amino Acid Sequence↗
PubMed2002

A translational rheostat for RFLAT-1 regulates RANTES expression in T lymphocytes.

Activation of T lymphocytes by specific antigen triggers a 3- to 7-day maturation process. Terminal differentiation begins late after T cell activation and involves expression of effector genes, including the chemokine RANTES and its major transcriptional regulator, RANTES factor of late-activated T lymphocytes-1 (RFLAT-1). In this article we demonstrate that RFLAT-1 expression is translationally regulated through its 5'-UTR and in a cell type-specific manner. Overexpression of the translation initiation factor eIF4E increases RFLAT-1 protein, while inhibition of Mnk1, which phosphorylates eIF4E, reduces RFLAT-1 production, indicating cap-dependent translational regulation. These events are regulated by ERK-1/2 and p38 MAP kinases and allow T cells to rapidly adjust RANTES expression in response to changes in the cellular environment, such as stress and/or growth factors. These findings provide a molecular mechanism for a rheostat effect of increasing or decreasing RANTES expression at sites of inflammation. Memory T cells, already poised to make RANTES, are finely regulated by translational control of the major transcription factor regulating RANTES expression. This is the first example of such a mechanism regulating a chemokine, but it seems likely that this will prove to be a general way for cells to rapidly respond to stress, cytokines, and other proinflammatory factors in their local environment.

3T3 Cells↗
PubMed2001

A T cell-specific enhancer of the human CD40 ligand gene.

We observed that the human CD40 ligand (CD40L) gene 5'-flanking region conferred weak promoter activity in activated CD4 T cells, suggesting that additional regions are required for optimal CD40L gene transcription. We therefore examined a 3'-flanking segment of the CD40L gene, which contained a putative NF-kappaB/Rel cis-element, for its ability to enhance CD40L promoter function. This segment augmented CD40L promoter activity in an orientation-independent manner in CD4 T-lineage cells but not in human B cell or monocyte cell lines. Mapping of CD4 T-lineage cell nuclei identified a DNase I-hypersensitive site in the flanking region near the NF-kappaB/Rel sequence, suggesting a transcriptional regulatory role. This was further supported by truncation analysis and site-directed mutagenesis, which indicated that the CD40L 3'-flanking NF-kappaB/Rel cis-element was critical for enhancer function. Electrophoretic mobility shift assays showed that the cis-element preferentially bound the p50 form of the NF-kappaB1 gene contained in human T cell nuclear protein extracts. This binding also appeared to occur in vivo in CD4 T cells based on chromatin immunoprecipitation assays using NF-kappaB p50-specific antiserum. Together, these results suggest that the CD40L gene 3'-flanking region acts as a T cell-specific classical transcriptional enhancer by a NF-kappaB p50-dependent mechanism.

B-Lymphocytes↗