Marcelo Marcet-Palacios

CD8α is expressed by human monocytes and enhances FcγR-dependent responses

BackgroundCD8α enhances the responses of antigen-specific CTL activated through TCR through binding MHC class I, favoring lipid raft partitioning of TCR, and inducing intracellular signaling. CD8α is also found on dendritic cells and rat macrophages, but whether CD8α enhances responses of a partner receptor, like TCR, to activate these cells is not known. TCR and FcR, use analogous or occasionally interchangeable signaling mechanisms suggesting the possibility that CD8α co-activates FcR responses. Interestingly, CD8α+ monocytes are often associated with rat models of disease involving immune-complex deposition and FcR-mediated pathology, such as arthritis, glomerulonephritis, ischaemia, and tumors. While rat macrophages have been shown to express CD8α evidence for CD8α expression by mouse or human monocytes or macrophages was incomplete.ResultsWe detected CD8α, but not CD8β on human monocytes and the monocytic cell line THP-1 by flow cytometry. Reactivity of anti-CD8α mAb with monocytes is at least partly independent of FcR as anti-CD8α mAb detect CD8α by western blot and inhibit binding of MHC class I tetramers. CD8α mRNA is also found in monocytes and THP-1 suggesting CD8α is synthesized by monocytes and not acquired from other CD8α+ cell types. Interestingly, CD8α from monocytes and blood T cells presented distinguishable patterns by 2-D electrophoresis. Anti-CD8α mAb alone did not activate monocyte TNF release. In comparison, TNF release by human monocytes stimulated in a FcR-dependent manner with immune-complexes was enhanced by inclusion of anti-CD8α mAb in immune-complexes.ConclusionHuman monocytes express CD8α. Co-engagement of CD8α and FcR enhances monocyte TNF release, suggesting FcR may be a novel partner receptor for CD8α on innate immune cells.

The Transcription Factor Wilms Tumor 1 Regulates Matrix Metalloproteinase-9 through a Nitric Oxide-Mediated Pathway

Matrix metalloproteinase-9 (MMP-9) is released by human lung epithelial cells (LEC) in conditions such as asthma and chronic obstructive pulmonary disease and expression of MMP-9 correlates with the severity of these disorders. MMP-9 production has been reported to be regulated by a NO/soluble guanylate cyclase-dependent pathway. Transcriptional regulation of this enzyme, however, is poorly understood. Using phylogenetic analysis, we observed a highly conserved sequence in the 5′ flanking region of the MMP-9 gene containing binding sites for the transcription factor Wilms tumor 1 (WT1). We confirmed the presence of WT1 in human LEC and that treatment with TNF or a mixture containing LPS, PMA, and IFN-γ resulted in translocation of WT1 from the nucleus to the cytosol. This translocation coincided with increased expression of MMP-9 and could be blocked by inhibitors of the NO/soluble guanylate cyclase pathway. WT1 knockdown using small-interfering RNA up-regulated MMP-9 expression in the presence of the NO synthase inhibitor 1400W. Using either WT1 pulldown with probes for the conserved region of the MMP-9 promoter or chromatin immunoprecipitation, we confirmed WT1 binding to the MMP-9 promoter. These findings indicate WT1 is a repressor of MMP-9, regulated by a NO-mediated pathway in human LEC. To our knowledge, this is the first report of WT1 regulating MMP-9 expression. Further study is needed to determine whether clinical conditions exhibiting tissue remodeling, such as asthma and/or chronic obstructive pulmonary disease, demonstrate reduced levels of WT1 or its repressor activity.

Granzyme B inhibits vaccinia virus production through proteolytic cleavage of eukaryotic initiation factor 4 gamma 3.

Cytotoxic T lymphocytes (CTLs) are the major killer of virus-infected cells. Granzyme B (GrB) from CTLs induces apoptosis in target cells by cleavage and activation of substrates like caspase-3 and Bid. However, while undergoing apoptosis, cells are still capable of producing infectious viruses unless a mechanism exists to specifically inhibit viral production. Using proteomic approaches, we identified a novel GrB target that plays a major role in protein synthesis: eukaryotic initiation factor 4 gamma 3 (eIF4G3). We hypothesized a novel role for GrB in translation of viral proteins by targeting eIF4G3, and showed that GrB cleaves eIF4G3 specifically at the IESD1408S sequence. Both GrB and human CTL treatment resulted in degradation of eIF4G3 and reduced rates of translation. When Jurkat cells infected with vaccinia virus were treated with GrB, there was a halt in viral protein synthesis and a decrease in production of infectious new virions. The GrB-induced inhibition of viral translation was independent of the activation of caspases, as inhibition of protein synthesis still occurred with addition of the pan-caspase inhibitor zVAD-fmk. This demonstrated for the first time that GrB prevents the production of infectious vaccinia virus by targeting the host translational machinery.

Design and characterization of a novel human Granzyme B inhibitor

The intracellular roles of Granzyme B (GrB) in immune-mediated cell killing have been extensively studied. Recent data also implicate GrB in extracellular pathways of inflammation, cytokine activation and autoimmunity. Targeting (GrB) provides a new pharmaceutical agent for various inflammatory disorders. Serpina3n is a mouse extracellular inhibitor of GrB. There is no apparent equivalent in humans. In this study, we used a novel applied genetics approach to engineer a new extracellular GrB serpin. A chimeric protein was generated in which the reactive center loop (RCL) of human extracellular antichymotrypsin (ACT) was replaced with that of serpina3n. This serpin contained 27 amino acid residues from the serpina3n RCL and the remaining 395 residues from human ACT. The insertion converted human ACT into a GrB-inhibitory serpin. Several critical residues were identified by scanning mutagenesis on the chimera and serpina3n. Targeted mutagenesis was conducted on wild-type human ACT by specifically substituting those critical residues, creating a novel inhibitor that contains 99.3% human ACT sequence with only three point mutations. Wild-type human ACT had a kass for GrB of 2.26 × 10(4) M(-1) s(-1), whereas the novel inhibitor binds GrB with a kass of 7.65 × 10(5) M(-1) s(-1). This new drug candidate can be developed in animal models and further tested in clinical trials to help us understand the role of GrB in numerous disorders.

PROM-OOGLE: data mining and integration of on-line databases to discover gene promoters

The vast number of on-line biological and medical databases available can be a great resource for biomedical researchers. However, the different types of data and interfaces available can be overwhelming for many biomedical researchers to learn and make effective use of. Moreover, the available resources lack needed integration. Here we focus on an important task in medical research: to provide researchers with promoter analysis for a given gene. Prom-oogle is a web based data mining tool that provides a means for researchers to take a gene name of interest and obtain its promoter sequence in return after automatic integration of text databases. Additionally, the program is capable of returning multiple promoters from different genes allowing researchers to study how promoters regulate genes. This tool facilitates the process of acquiring information on a promoter and may lead to interesting discoveries.