Domenica Russo

An Anti-Inflammatory Role for Vα14 NK T cells in Mycobacterium bovis Bacillus Calmette-Guérin-Infected Mice

The possible contribution of NKT cells to resistance to Mycobacterium tuberculosis infection remains unclear. In this paper we characterized the Vα14 NKT cell population following infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG). BCG infection determined an early expansion of Vα14 NKT cells in liver, lungs, and spleen, which peaked on day 8 and was sustained until day 30. However, an NK1.1+ Vα14 NKT population preferentially producing IFN-γ predominated at an early stage (day 8), which was substituted by an NK1.1− population preferentially producing IL-4 at later stages (day 30). Despite the fact that Vα14 NKT cell-deficient mice eliminated BCG as did control mice, they had significantly higher numbers of granulomas in liver and lungs. Additionally, while control mice developed organized small granulomas, those in Vα14 NKT-deficient mice had signs of caseation, large cellular infiltrates, and some multinucleated macrophages, suggesting that Vα14 NKT cells may actually work as anti-inflammatory cells by limiting excessive lymphocyte influx and tissue pathology. In agreement, we found an increased spontaneous production and mRNA expression of TNF-α in liver and lungs of Vα14 NKT-deficient mice, whose neutralization in vivo by anti-TNF-α mAbs consistently reduced the number of granulomas in liver and lungs. Together, our results support a regulatory role for Vα14 NKT cells in the course of BCG infection through their ability to limit the extent of inflammatory response and point to an important role for this cell subset as a regulator of the balance between protective responses and immunopathology.

Immunoregulatory role of Jα281 T cells in aged mice developing lupus-like nephritis

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the emergence of autoreactive T cells. Humans and mice with SLE have reduced numbers of CD1d‐restricted invariant natural killer T (iNKT) cells, suggesting a key role for these cells in its immunopathogenesis. This subset uses an invariant TCR constituted by Vα14Jα281 chains paired with some Vβ domains. The regulatory role for iNKT cells in non‐autoimmune mice was suggested by our previous results showing that aged Jα281 knockout (KO) mice produce anti‐dsDNA. Here we show that old Jα281 KO mice have proteinuria and antibodies against dsDNA and cardiolipin. Histological analysis of Jα281 KO mice revealed glomeruli damage and deposition of C3c and IgG, mainly of the IgG3 subclass. In spleens of aged Jα281 KO mice there is an increase of activated marginal zone B cells. The evolution of lesions may depend on the age‐associated increase of autoantibodies production, preferentially IgG3, mainly secreted by marginal zone B cells. Our results provide the first evidence of a lupus‐like syndrome in non‐autoimmune mice, supporting an age‐related immunoregulatory role of Jα281+ cells, probably associated with the activation of marginal zone B cells.

Identification and phenotypic characterization of a subpopulation of T84 human colon cancer cells, after selection on activated endothelial cells

The extravasation of metastatic cells is regulated by molecular events involving the initial adhesion of tumor cells to the endothelium and subsequently the migration of the cells in the host connective tissue. The differences in metastatic ability could be attributed to properties intrinsic of the various primary tumor types. Thus, the clonal selection of neoplastic cells during cancer progression results in cells better equipped for survival and formation of colonies in secondary sites. A cell line (T84SF) exhibiting an altered phenotypic appearance was selected from a colon cancer cell line (T84) by repetitive plating on TNFα‐activated human endothelial cells and subsequent selection for adherent cells. Cell growth, motility, chemoinvasive abilities, tyrosine phosphorylation signaling, and the metastasis formation in nude mice of the two cell lines was compared. T84SF cells displayed in vitro an higher proliferation rate and a more invasive behavior compared to the parental cells while formed in vivo a greater number of metastatic colonies in nude mice. As concerns the signaling underlying the phenotypes of the selected cells, we examined the general tyrosine phosphorylation levels in both cell lines. Our results indicate that T84SF have an increased basal tyrosine phosphorylation of several proteins among which src kinase was identified. Treatment of cells with a specific inhibitor of src activity caused a greater in vitro inhibition of proliferation and invasive properties of T84 parental cells with respect to T84SF cells and diminished metastasis formation in vivo. Altogether, these data provide evidences that this new cell line may be valuable for identifying molecular mechanisms involved in the metastatic progression of colon cancer.

Different phenotypes of colon carcinoma cells interacting with endothelial cells: role of E-selectin and ultrastructural data

Abstract.Adhesion molecules are intimately involved in the process of tumour progression. Among them, E-selectin is an inducible endothelial cell adhesion molecule that plays a role in the interactions of neoplastic cells with the endothelium. These interactions are required for the trans-endothelial migration of tumour cells that leads to the growth at the new sites. Since the detailed events in the early phase of metastasis still remain poorly defined, our study has undertaken an electron-microscopic analysis of the interactions of human colon carcinoma cells with endothelial cells as well as an analysis of the effect of recombinant purified E-selectin in the cell signalling involved in colon cancer cell malignant phenotype. Results revealed that SW480 and T84 colon cancer cell lines show different features, different adhesion kinetics, a different cytoskeletal organization, and a different tyrosine phosphorylation pattern when seeded on an endothelial cell monolayer or recombinant E-selectin. In particular T84 cancer cells adhere more efficiently to the E-selectin and this interaction is associated with pronounced morphological changes, actin redistribution and filopodial processes, and an increase in tyrosine phosphorylation of different proteins. These data support the hypothesis that E-selectin ligand is not only a cell-cell adhesion molecule but also initiates a signalling transduction pathway inside the cells.

Comparative study of T84 and T84SF human colon carcinoma cells: in vitro and in vivo ultrastructural and functional characterization of cell culture and metastasis

To better understand the relationship between tumor heterogeneity, differentiation, and metastasis, suitable experimental models permitting in vitro and in vivo studies are necessary. A new variant cell line (T84SF) exhibiting an altered phenotype was recently selected from a colon cancer cell line (T84) by repetitive plating on TNF-α treated human endothelial cells and subsequent selection for adherent cells. The matched pair of cell lines provides a useful system to investigate the extravasation step of the metastatic cascade. Since analysis of morphological differences can be instructive to the understanding of metastatic potential of tumor cells, we compared the ultrastructural and functional phenotype of T84 and T84SF cells in vitro and in vivo. The reported ultrastructural features evidence differences between the two cell lines; selected cells showed a marked pleomorphism of cell size and nuclei, shape, and greater surface complexity. These morphological differences were also coupled with biochemical data showing a distinct tyrosine phosphorylation-based signaling, an altered localization of β-catenin, MAPK, and AKT activation, as well as an increased expression in T84SF cells of Bcl-XL, a major regulator of apoptosis. Therefore, these cell lines represent a step forward in the development of appropriate models in vitro and in vivo to investigate colon cancer progression.