Cyclooxygenase 2 inhibition promotes IFN-gamma-dependent enhancement of antitumor responses

In previous studies, we demonstrated an immune suppressive network in non-small cell lung cancer that is due to overexpression of tumor cyclooxygenase 2 (COX-2). In this study, we assessed the vaccination response to tumor challenge following either pharmacological or genetic inhibition of COX-2 in a murine lung cancer model. Treatment of naive mice with the COX-2 inhibitor, SC-58236, skewed splenocytes toward a type 1 cytokine response, inducing IFN-gamma, IL-12, and IFN-gamma-inducible protein 10, whereas the type 2 cytokines IL-4, IL-5, and IL-10 remained unaltered. Fifty percent of mice receiving SC-58236 and an irradiated tumor cell vaccine completely rejected tumors upon challenge. Those mice that did form tumors following challenge demonstrated a reduced tumor growth. In contrast, all mice either vaccinated with irradiated tumor cells alone or receiving SC-58236 alone showed progressive tumor growth. Studies performed in CD4 and CD8 knockout mice revealed a requirement for the CD4 T lymphocyte subset for the complete rejection of tumors. To determine the role of host COX-2 expression on the vaccination responses, studies were performed in COX-2 gene knockout mice. Compared with control littermates, COX-2(-/-) mice showed a significant tumor growth reduction, whereas heterozygous COX-2(-/+) mice had an intermediate tumor growth reduction following vaccination. In vivo depletion of IFN-gamma abrogated the COX-2 inhibitor-mediated enhancement of the vaccination effect. These findings provide a strong rationale for additional evaluation of the capacity of COX-2 inhibitors to enhance vaccination responses against cancer.     

Mutational analysis of the human 2B4 (CD244)/CD48 interaction: Lys68 and Glu70 in the V domain of 2B4 are critical for CD48 binding and functional activation of NK cells

Interaction between receptors and ligands plays a critical role in the generation of immune responses. The 2B4 (CD244), a member of the CD2 subset of the Ig superfamily, is the high affinity ligand for CD48. It is expressed on NK cells, T cells, monocytes, and basophils. Recent data indicate that 2B4/CD48 interactions regulate NK and T lymphocyte functions. In human NK cells, 2B4/CD48 interaction induces activation signals, whereas in murine NK cells it sends inhibitory signals. To determine the structural basis for 2B4/CD48 interaction, selected amino acid residues in the V domain of the human 2B4 (h2B4) were mutated to alanine by site-directed mutagenesis. Following transient expression of these mutants in B16F10 melanoma cells, their interaction with soluble CD48-Fc fusion protein was assessed by flow cytometry. We identified amino acid residues in the extracellular domain of h2B4 that are involved in interacting with CD48. Binding of CD48-Fc fusion protein to RNK-16 cells stably transfected with wild-type and a double-mutant Lys(68)Ala-Glu(70)Ala h2B4 further demonstrated that Lys(68) and Glu(70) in the V domain of h2B4 are essential for 2B4/CD48 interaction. Functional analysis indicated that Lys(68) and Glu(70) in the extracellular domain of h2B4 play a key role in the activation of human NK cells through 2B4/CD48 interaction.     

Regulation of rat basophilic leukemia-2H3 mast cell secretion by a constitutive Lyn kinase interaction with the high affinity IgE receptor (Fc epsilon RI)

Signaling through the high affinity IgE receptor is initiated by noncovalently associated Lyn kinase, resulting in the secretion of inflammatory mediators from mast cells. A fraction of the total cellular Lyn is associated via its N-terminal unique domain with the cytoplasmic domain of the Fc epsilonRI beta subunit before receptor aggregation. In the current study, we stably transfected the unique domain of Lyn into rat basophilic leukemia-2H3 mast cells and examined the consequences on Fc epsilonRI-induced signal transduction and mediator secretion to further define the role of the unique domain of Lyn in mast cell secretion. Tyrosine phosphorylation of Fc epsilonRI beta and gamma subunits was partially inhibited in the Lyn unique domain transfectants after Ag stimulation. Ag stimulation of Lyn unique domain transfectants was accompanied by enhanced phosphorylation of MEK and ERK-2, which are required for leukotriene C4 (LTC4) release, and production of LTC4 was increased 3- to 5-fold, compared with cells transfected with vector alone. Conversely, tyrosine phosphorylation of the adaptor protein Gab2, which is essential for mast cell degranulation, was inhibited after Ag stimulation of Lyn unique domain transfectants, and Ag-induced release of histamine was inhibited up to 48%. In rat basophilic leukemia-2H3 cells, Lyn thus plays a dual role by positively regulating Fc epsilonRI phosphorylation and degranulation while negatively regulating LTC4 production. This study provides further evidence that the constitutive interaction between the unique domain of Lyn and the Fc epsilonRI beta subunit is a crucial step in the initiation of Fc epsilonRI signaling and that Lyn is limiting for Fc epsilonRI-induced secretion of inflammatory mediators.     

IL-18 induces monocyte chemotactic protein-1 production in macrophages through the phosphatidylinositol 3-kinase/Akt and MEK/ERK1/2 pathways

Macrophages are activated during an inflammatory response and produce multiple inflammatory cytokines. IL-18 is one of the most important innate cytokines produced from macrophages in the early stages of the inflammatory immune response. Monocyte chemoattractant protein (MCP-1) is expressed in many inflammatory diseases such as multiple sclerosis and rheumatoid arthritis, and its expression is correlated with the severity of the disease. Both IL-18 and MCP-1 have been shown to be involved in inflammatory immune responses. However, it has been unclear whether IL-18 is involved in the induction of MCP-1. This investigation was initiated to determine whether IL-18 can induce MCP-1 production, and if so, by which signal transduction pathways. We found that IL-18 induced the production of MCP-1 in macrophages, which was IL-12-independent and was not mediated by autocrine cytokines such as IFN-gamma or TNF-alpha. We then examined signal transduction pathways involved in IL-18-induced MCP-1 production. We found that IL-18 did not activate the IkappaB kinase/NF-kappaB pathway, evidenced by no degradation of IkappaBalpha and no translocation of NF-kappaB p65 to the nucleus in IL-18-stimulated macrophages. Instead, IL-18 activated the PI3K/Akt and MEK/ERK1/2 pathways. Inhibition of either of these pathways attenuated MCP-1 production in macrophages, and inhibition of both signaling pathways resulted in the complete inhibition of MCP-1 production. On the basis of these observations, we conclude that IL-18 induces MCP-1 production through the PI3K/Akt and MEK/ERK1/2 pathways in macrophages.     

Identification of novel CD33 antigen-specific peptides for the generation of cytotoxic T lymphocytes against acute myeloid leukemia

Identification of immunogenic peptides for the generation of cytotoxic T lymphocytes (CTLs) may lead to the development of novel cellular therapies to treat disease relapse in acute myeloid leukemia (AML) patients. The objective of these studies was to evaluate the ability of unique HLA-A2.1-specific nonameric peptides derived from CD33 antigen to generate AML-specific CTLs ex vivo. We present data here on the identification of an immunogeneic HLA-A2.1-specific CD33(65-73) peptide (AIISGDSPV) that was capable of inducing CTLs targeted to AML cells. The CD33-CTLs displayed HLA-A2.1-restricted cytotoxicity against both mononuclear cells from AML patients and the AML cell line. The peptide- as well as AML cell-specificity of CD33-CTLs was demonstrated and the secretion of IFN-gamma by the CTLs was detected in response to CD33(65-73) peptide stimulation. The cultures contained a distinct CD33(65-73) peptide-tetramer(+)/CD8(+) population. Alteration of the native CD33(65-73) peptide at the first amino acid residue from alanine (A) to tyrosine (Y) enhanced the HLA-A2.1 affinity/stability of the modified CD33 peptide (YIISGDSPV) and induced CTLs with increased cytotoxicity against AML cells. These data therefore demonstrate the potential of using immunogenic HLA-A2.1-specific CD33 peptides in developing a cellular immunotherapy for the treatment of AML patients.     

Hydrogen peroxide mediates Rac1 activation of S6K1

We previously reported that hydrogen peroxide (H2O2) mediates mitogen activation of ribosomal protein S6 kinase 1 (S6K1) which plays an important role in cell proliferation and growth. In this study, we investigated a possible role of H2O2 as a molecular linker in Rac1 activation of S6K1. Overexpression of recombinant catalase in NIH-3T3 cells led to the drastic inhibition of H2O2 production by PDGF, which was accompanied by a decrease in S6K1 activity. Similarly, PDGF activation of S6K1 was significantly inhibited by transient transfection or stable transfection of the cells with a dominant-negative Rac1 (Rac1N17), while overexpression of constitutively active Rac1 (Rac1V12) in the cells led to an increase in basal activity of S6K1. In addition, stable transfection of Rat2 cells with Rac1N17 dramatically attenuated the H2O2 production by PDGF as compared with that in the control cells. In contrast, Rat2 cells stably transfected with Rac1V12 produced high level of H2O2 in the absence of PDGF, comparable to that in the control cells stimulated with PDGF. More importantly, elimination of H2O2 produced in Rat2 cells overexpressing Rac1V12 inhibited the Rac1V12 activation of S6K1, indicating the possible role of H2O2 as a mediator in the activation of S6K1 by Rac1. However, H2O2 could be also produced via other pathway, which is independent of Rac1 or PI3K, because in Rat2 cells stably transfected with Rac1N17, H2O2 could be produced by arsenite, which has been shown to be a stimulator of H2O2 production. Taken together, these results suggest that H2O2 plays a pivotal role as a mediator in Rac1 activation of S6K1.     

Specific excision of the selenocysteine tRNA[Ser]Sec (Trsp) gene in mouse liver demonstrates an essential role of selenoproteins in liver function

Selenium is essential in mammalian embryonic development. However, in adults, selenoprotein levels in several organs including liver can be substantially reduced by selenium deficiency without any apparent change in phenotype. To address the role of selenoproteins in liver function, mice homozygous for a floxed allele encoding the selenocysteine (Sec) tRNA([Ser]Sec) gene were crossed with transgenic mice carrying the Cre recombinase under the control of the albumin promoter that expresses the recombinase specifically in liver. Recombination was nearly complete in mice 3 weeks of age, whereas liver selenoprotein synthesis was virtually absent, which correlated with the loss of Sec tRNA([Ser]Sec) and activities of major selenoproteins. Total liver selenium was dramatically decreased, whereas levels of low molecular weight selenocompounds were little affected. Plasma selenoprotein P levels were reduced by about 75%, suggesting that selenoprotein P is primarily exported from the liver. Glutathione S-transferase levels were elevated in the selenoprotein-deficient liver, suggesting a compensatory activation of this detoxification program. Mice appeared normal until about 24 h before death. Most animals died between 1 and 3 months of age. Death appeared to be due to severe hepatocellular degeneration and necrosis with concomitant necrosis of peritoneal and retroperitoneal fat. These studies revealed an essential role of selenoproteins in liver function.     

Structures and analysis of highly homologous psychrophilic, mesophilic, and thermophilic adenylate kinases

The crystal structures of adenylate kinases from the psychrophile Bacillus globisporus and the mesophile Bacillus subtilis have been solved and compared with that from the thermophile Bacillus stearothermophilus. This is the first example we know of where a trio of protein structures has been solved that have the same number of amino acids and a high level of identity (66-74%) and yet come from organisms with different operating temperatures. The enzymes were characterized for their own thermal denaturation and inactivation, and they exhibited the same temperature preferences as their source organisms. The structures of the three highly homologous, dynamic proteins with different temperature-activity profiles provide an opportunity to explore a molecular mechanism of cold and heat adaptation. Their analysis suggests that the maintenance of the balance between stability and flexibility is crucial for proteins to function at their environmental temperatures, and it is achieved by the modification of intramolecular interactions in the process of temperature adaptation.