[Show abstract][Hide abstract] ABSTRACT: The binding of herpesvirus entry mediator (HVEM) to B and T lymphocyte attenuator (BTLA) is known to activate an inhibitory signaling cascade in effector T (Teff) cells, but we now report that the HVEM-BTLA pathway is also important to the suppressive function of regulatory T cells (Tregs). Although naive T cells up-regulated BTLA upon TCR activation, Treg expression of BTLA remained low, regardless of TCR activation. Moreover, BTLA(-/-) CD4(+)CD25(+) Tregs had normal suppressive activity, whereas BTLA(-/-) Teff cells were more resistant than wild-type Teff cells to suppression by Tregs, suggesting BTLA expression by Teff cells was required for their suppression by Tregs. In contrast to BTLA, HVEM expression was comparable in naive Tregs vs Teff cells, but after stimulation HVEM expression was quickly down-regulated by Teff cells, whereas HVEM was further up-regulated by Tregs. HVEM(-/-) Tregs had decreased suppressive activity as compared with wild-type Tregs, indicating that Treg expression of HVEM was required for optimal suppression. Consistent with this, T cells from Scurfy mice (FoxP3 mutant) lacked HVEM gene expression, and adoptively transferred wild-type but not HVEM(-/-) Tregs were able to control alloresponses in vivo by normal Teff cells. Our data demonstrate that Tregs can exert their effects via up-regulation of the negative costimulatory ligand HVEM, which upon binding to BTLA expressed by Teff cells helps mediate the suppressive functions of Tregs in vitro and in vivo.
The Journal of Immunology 06/2008; 180(10):6649-55. DOI:10.4049/jimmunol.180.10.6649 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Complex intracellular network interactions regulate gene expression and cellular behavior. Whether at the site of inflammation or within a tumor, individual cells are exposed to a plethora of signals. The transcription factor nuclear factor-kappaB (NF-kappaB) regulates genes that control key cellular activities involved in inflammatory diseases and cancer. NF-kappaB is regulated by several distinct signaling pathways that may be activated individually or simultaneously. Multiple ligands and heterologous cell-cell interactions have an impact on NF-kappaB activity. The G protein-coupled receptor (GPCR) superfamily makes up the largest class of transmembrane receptors in the human genome and has multiple molecularly distinct natural ligands. GPCRs regulate proliferation, differentiation, and chemotaxis and play a major role in inflammatory diseases and cancer. Both GPCRs and NF-kappaB have been, and continue to be, major targets for drug discovery. A clear understanding of network interactions between GPCR signaling pathways and those that control NF-kB may be valuable for the development of better drugs and drug combinations.
International Reviews Of Immunology 02/2008; 27(5):320-50. DOI:10.1080/08830180802262765 · 4.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: CCL1 is the predominant chemokine secreted from IgE-activated human and mouse mast cells in vitro, colocalizes to mast cells in lung biopsies, and is elevated in asthmatic airways. CCR8, the receptor for CCL1, is expressed by approximately 70% of CD4(+) T lymphocytes recruited to the asthmatic airways, and the number of CCR8-expressing cells is increased 3-fold in the airways of asthmatic subjects compared with normal volunteers. In vivo, CCL1 expression in the lung is reduced in mast cell-deficient mice after aeroallergen provocation. Neutralization of CCL1 or CCR8 deficiency results in reduced mucosal lung inflammation, airway hyperresponsiveness, and mucus hypersecretion to a similar degree as detected in mast cell-deficient mice. Adenoviral delivery of CCL1 to the lungs of mast cell-deficient mice restores airway hyperresponsiveness, lung inflammation, and mucus hypersecretion to the degree observed in wild-type mice. The consequences of CCR8 deficiency, including a marked reduction in Th2 cytokine levels, are comparable with those observed by depletion of CD4(+) T lymphocytes. Thus, mast cell-derived CCL1- and CCR8-expressing CD4(+) effector T lymphocytes play an essential role in orchestrating lung mucosal inflammatory responses.
The Journal of Immunology 09/2007; 179(3):1740-50. DOI:10.4049/jimmunol.179.3.1740 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent reports have challenged the notion that retroviruses and retroviral vectors integrate randomly into the host genome. These reports pointed to a strong bias toward integration in and near gene coding regions and, for gammaretroviral vectors, around transcription start sites. Here, we report the results obtained from a large-scale mapping of 572 retroviral integration sites (RISs) isolated from cells of 9 patients with X-linked SCID (SCID-X1) treated with a retrovirus-based gene therapy protocol. Our data showed that two-thirds of insertions occurred in or very near to genes, of which more than half were highly expressed in CD34(+) progenitor cells. Strikingly, one-fourth of all integrations were clustered as common integration sites (CISs). The highly significant incidence of CISs in circulating T cells and the nature of their locations indicate that insertion in many gene loci has an influence on cell engraftment, survival, and proliferation. Beyond the observed cases of insertional mutagenesis in 3 patients, these data help to elucidate the relationship between vector insertion and long-term in vivo selection of transduced cells in human patients with SCID-X1.
[Show abstract][Hide abstract] ABSTRACT: The NF-kappaB (NF-kappaB) transcription factor-signaling pathway has become a major focus for research aimed at understanding its role in development, maintenance and progression of disease. A large body of recent research suggests inhibition of NF-kappaB blocks inflammation, cancer development and progression, diabetes, stroke, muscle wasting, and other diseases. The enormous potential for the treatment of disease by inhibiting NF-kappaB lead to the development of inhibitory drugs that specifically target this pathway. At the same time offering great clinical potential, inhibition of NF-kappaB in vivo can be detrimental. NF-kappaB controls multiple functions in homeostasis including a functional immune response, cell cycle, and cell death. Genetic studies in mice and analysis of naturally occurring mutations in humans point to specific developmental and immune consequences due to altering NF-kappaB activity. The balance between therapeutic benefit and potential changes in normal cellular function and response during drug induced NF-kappaB inhibition will be one of the challenges in future clinical studies.
[Show abstract][Hide abstract] ABSTRACT: The transcription factor NF-kappaB plays a central role in regulating inflammation and apoptosis, making it a compelling target for drug development. We identified a small molecule inhibitor (ML120B) that specifically inhibits IKKbeta, an Ikappa-B kinase that regulates NF-kappaB. IKKbeta and NF-kappaB are required in vivo for prevention of TNFalpha-mediated apoptosis. ML120B sensitized mouse bone marrow progenitors and granulocytes, but not mature B cells to TNFalpha killing in vitro, and induced apoptosis in vivo in the bone marrow and spleen within 6 hours of a single oral dose. In vivo inhibition of IKKbeta with ML120B resulted in depletion of thymocytes and B cells in all stages of development in the bone marrow but did not deplete granulocytes. TNF receptor-deficient mouse thymocytes and B cells were resistant to ML120B-induced depletion in vivo. Surprisingly, surviving bone marrow granulocytes expressed TNFR1 and TNFR2 after dosing in vivo with ML120B. Our results show that inhibition of IKKbeta with a small molecule in vivo leads to rapid TNF-dependent depletion of T and B cells. This observation has several implications for potential use of IKKbeta inhibitors for the treatment of inflammatory disease and cancer.
[Show abstract][Hide abstract] ABSTRACT: Lipoprotein lipase (LPL) is a key regulator of triglyceride clearance. Its coordinated regulation during feeding and fasting is critical for maintaining lipid homeostasis and energy supply. Angiopoietin-like (Angptl)3 and Angptl4 are secreted proteins that have been demonstrated to regulate triglyceride metabolism by inhibiting LPL. We have taken a targeted genetic approach to generate Angptl4- and Angptl3-deficient mice as well as transgenic mice overexpressing human Angptl4 in the liver. The Angptl4 transgenic mice displayed elevated plasma triglycerides and reduced postheparin plasma (PHP) LPL activity. A purified recombinant Angptl4 protein inhibited mouse LPL and recombinant human LPL activity in vitro. In contrast to the transgenic mice, Angptl4-deficient mice displayed hypotriglyceridemia and increased PHP LPL activity, with greater effects in the fasted compared with the fed state. Angptl3-deficient mice also displayed hypotriglyceridemia with elevated PHP LPL activity, but these mice showed a greater effect in the fed state. Mice deficient in both Angptl proteins showed an additive effect on plasma triglycerides and did not survive past 2 months of age. Our results show that Angptl3 and Angptl4 function to regulate circulating triglyceride levels during different nutritional states and therefore play a role in lipid metabolism during feeding/fasting through differential inhibition of LPL.
[Show abstract][Hide abstract] ABSTRACT: Excessive mucus production by airway epithelium is a major characteristic of a number of respiratory diseases, including asthma, chronic bronchitis, and cystic fibrosis. However, the signal transduction pathways leading to mucus production are poorly understood. Here we examined the potential role of IkappaB kinase beta (IKKbeta) in mucus synthesis in vitro and in vivo. Tumor necrosis factor-alpha (TNF-alpha) or transforming growth factor-alpha stimulation of human epithelial cells resulted in mucus secretion as measured by MUC5AC mRNA and protein. TNF-alpha stimulation induced IKKbeta-dependent p65 nuclear translocation, mucus synthesis, and production of cytokines from epithelial cells. TNF-alpha, but not transforming growth factor-alpha, induced mucus production dependent on IKKbeta-mediated NF-kappaB activation. In vivo, TNF-alpha induced NF-kappaB as determined by whole mouse body bioluminescence. This activation was localized to the epithelium as revealed by LacZ staining in NF-kappaB-LacZ transgenic mice. TNF-alpha-induced mucus production in vivo could also be inhibited by administration into the epithelium of an IKKbeta dominant negative adenovirus. Taken together, our results demonstrated the important role of IKKbeta in TNF-alpha-mediated mucus production in airway epithelium in vitro and in vivo.
[Show abstract][Hide abstract] ABSTRACT: Macrophages exist as sentinels in innate immune response and react by expressing proinflammatory cytokines and up-regulating antigen-presenting and costimulatory molecules. We report a novel function for prokineticin-1 (PK1)/endocrine gland-derived vascular endothelial growth factor. Screening of murine tissue sections and cells for specific binding site leads to the identification of macrophages as an in vivo cellular target for PK1. We demonstrate PK1 induces differentiation of murine and human bone marrow cells into the monocyte/macrophage lineage. Human peripheral blood monocytes respond to PK1 by morphological changes and down-regulation of B7-1, CD14, CC chemokine receptor 5, and CXC chemokine receptor 4. Monocytes treated with PK1 have elevated interleukin (IL)-12 and tumor necrosis factor alpha and down-regulated IL-10 production in response to lipopolysaccharide. PK1 induces a distinct monocyte-derived cell population, which is primed for release of proinflammatory cytokines that favor a T helper cell type 1 response.
[Show abstract][Hide abstract] ABSTRACT: Ly108, a glycoprotein of the signaling lymphocytic activation molecule family of cell surface receptors expressed by T, B, NK, and APCs has been shown to have a role in NK cell cytotoxicity and T cell cytokine responses. In this study, we describe that CD4(+) T cells from mice with a targeted disruption of exons 2 and 3 of Ly108 (Ly108(DeltaE2+3)) produce significantly less IL-4 than wild-type CD4(+) cells, as judged by in vitro assays and by in vivo responses to cutaneous infection with Leishmania mexicana. Surprisingly, neutrophil functions are controlled by Ly108. Ly108(DeltaE2+3) mice are highly susceptible to infection with Salmonella typhimurium, bactericidal activity of Ly108(DeltaE2+3) neutrophils is defective, and their production of IL-6, IL-12, and TNF-alpha is increased. The aberrant bactericidal activity by Ly108(DeltaE2+3) neutrophils is a consequence of severely reduced production of reactive oxygen species following phagocytosis of bacteria. Thus, Ly108 serves as a regulator of both innate and adaptive immune responses.
The Journal of Immunology 06/2005; 174(10):5931-5. DOI:10.4049/jimmunol.174.10.5931 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The B7 homolog B7-H3 is important for the regulation of immune responses though its functions in vivo are controversial. We report the first clinical and experimental data concerning expression and function of B7-H3 in alloresponses. Immunohistological and molecular analyses showed B7-H3 expression by cells mediating rejection of human and mouse allografts. To analyze the significance of B7-H3 in rejecting allografts, we generated B7-H3-/- mice and showed that targeting of B7-H3 was synergistic with other forms of immune modulation; e.g. a regimen of rapamycin gave 12-14 days of survival in wild-type controls but led to permanent cardiac and islet allograft survival in B7-H3-/- mice. Cardiac allografts in treated B7-H3-/- mice showed markedly decreased production of key cytokine, chemokine and chemokine receptor mRNA transcripts as compared to wild-type controls. The incidence of chronic rejection in two different cardiac allograft models was also inhibited in B7-H3-/- as compared to wild-type recipients. Lastly, in addition to the expected antigen-presenting cell expression of B7-H3, CD4 and CD8 T cells showed B7-H3 induction upon cell activation, and both dendritic cell- and T cell-expressed B7-H3 each enhanced T cell proliferation in vitro and in vivo. We conclude that B7-H3 promotes T cell-mediated immune responses and the development of acute and chronic allograft rejection.
European Journal of Immunology 03/2005; 35(2):428-38. DOI:10.1002/eji.200425518 · 4.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A better understanding of inflammatory processes in endothelial cells (ECs) might reveal new ways of controlling inflammation and graft rejection. This study investigates EC genes regulated in response to human tumor necrosis factor (TNF)-alpha and xenogeneic natural antibodies (XNAs) that contribute to endothelial activation during transplantation.
Gene expression between resting and activated ECs was investigated by RNA differential display reverse-transcriptase polymerase chain reaction and confirmed by reverse-Northern blot.
Forty-five cDNA fragments corresponding to genes up-regulated in activated ECs were identified. Our findings show that TNF-alpha-mediated EC activation was associated with increased levels of mRNA for the adaptor protein Lnk, the nuclear protein RED, and the initiation factor eIF4G. We further show that Lnk and eIF4G were also up-regulated in response to XNA binding to ECs.
Our data suggest that TNF-alpha and XNAs could share common signaling pathways involving Lnk and eIF4G but may also drive specific transcriptional events.
[Show abstract][Hide abstract] ABSTRACT: RNA differential display (DD) RT-PCR is a useful method to identify and clone differentially expressed genes. However, the rate of false positives and redundancy associated with this PCR-based method as well as laborious downstream screening steps constitute major limitations. Here we present DD RT-PCR and reverse northern (RN) protocols allowing rapid and acurate identification of genes upregulated in porcine endothelial cells (EC) in response to TNFalpha.
The housekeeping gene beta-actin was used to investigate mispriming and to set up optimal conditions for DD-RT-PCR and RN. In this study DD was performed to compare resting and TNFalpha-activated ECs. Selection of DD-fragments was performed following 30-cycles of PCR using serial dilutions of template cDNA and regulation of 6 out of 17 candidates genes were first confirmed by semi-quantitative RN.
Using this protocol, 5 out of 6 DD-fragments were further confirmed to be upregulated by Northern blot, and 3 novel porcine cDNAs were cloned including the pro-apoptotic member of the Bcl-2 family, Noxa.
In this study we demonstrate that the combination of DD-RT-PCR and RN, which efficiently reduces the number of false positive candidates derived from mispriming at the screening step, allows a rapid identification of differentially expressed genes.
Molecular Medicine 10/2002; 8(9):559-67. · 4.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: LIGHT (TNFSF14), a tumor necrosis factor superfamily member expressed by activated T cells, binds to herpes virus entry mediator (HVEM) which is constitutively expressed by T cells and costimulates T cell activation in a CD28-independent manner. Given interest in regulating the effector functions of T cells in vivo, we examined the role of LIGHT-HVEM costimulation in a murine cardiac allograft rejection model. Normal hearts lacked LIGHT or HVEM mRNA expression, but allografts showed strong expression of both genes from day 3 after transplant, and in situ hybridization and immunohistology-localized LIGHT and HVEM to infiltrating leukocytes. To test the importance of LIGHT expression on allograft survival, we generated LIGHT-/- mice by homologous recombination. The mean survival of fully major histocompatibility complex-mismatched vascularized cardiac allografts in LIGHT-/- mice (10 days, P < 0.05) or cyclosporine A (CsA)-treated LIGHT+/+ mice (10 days, P < 0.05) was only slightly prolonged compared with LIGHT+/+ mice (7 days). However, mean allograft survival in CsA-treated LIGHT-/- allograft recipients (30 days) was considerably enhanced (P < 0.001) compared with the 10 days of mean survival in either untreated LIGHT-/- mice or CsA-treated LIGHT+/+ controls. Molecular analyzes showed that the beneficial effects of targeting of LIGHT in CsA-treated recipients were accompanied by decreased intragraft expression of interferon (IFN)-gamma, plus IFN-gamma-induced chemokine, inducible protein-10, and its receptor, CXCR3. Treatment of LIGHT+/+ allograft recipients with HVEM-Ig plus CsA also enhanced mean allograft survival (21 days) versus wild-type controls receiving HVEM-Ig (mean of 7 days) or CsA alone (P < 0.001). Our data suggest that T cell to T cell-mediated LIGHT/HVEM-dependent costimulation is a significant component of the host response leading to cardiac allograft rejection.
Journal of Experimental Medicine 04/2002; 195(6):795-800. · 12.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The SLAM family of human genes currently consists of seven related members of the immunoglobulin superfamily, membrane-associated proteins, including CD150 (SLAM), CD244 (2B4), CD84, CD229 ( Ly-9), BLAME, CD48, and 19A. These genes are expressed to varying degrees in subsets of immune cells (T, B, natural killer, and myeloid cells) and may function as ligands or receptors. This set of genes, related to CD2 and CD58 on Chromosome (Chr) 1p98, are found clustered close together in the human genome on Chr 1q22. Four of these family members (CD150, CD244, CD84, CD229) contain conserved tyrosine motifs in their cytoplasmic tails that enable them to bind intracellular signaling molecules SAP and EAT-2. SAP is mutated in human X-linked lymphoproliferative disease (XLP), and studies in XLP patients have shown that improper signaling via molecules that bind SAP contributes to the disease. We have identified two new members of the SLAM family (SF), which we term SF2000 and SF2001, which are expressed in immune cells and map in the SLAM gene cluster. SF2001 does not contain SAP-binding motifs in its short cytoplasmic tail. SF2000, which is co-expressed with SAP in T cells, binds both SAP and EAT-2. The data suggest that signaling through SF2000, together with CD150, CD244, CD84, and CD229, is controlled by SAP and therefore contributes to the pathogenesis of XLP.