ABSTRACT
The membrane-anchored form of the chemokine fractalkine (CX(3)CL1) has been identified as a novel adhesion molecule that interacts with its specific receptor (CX(3)CR1) expressed in monocytes, T cells and natural killer cells to induce adhesion. In addition, CX(3)CL1 can be cleaved from the cell membrane to induce chemotaxis of CX(3)CR1-expressing leucocytes. Recently, marked variations in CX(3)CR1 monocyte expression have been observed during several pathological conditions. Regulation of CX(3)CR1 in monocytes during basal or inflammatory/anti-inflammatory conditions is poorly understood. The aim of this study was therefore to examine CX(3)CR1 expression during monocyte maturation and the effect of soluble mediators on this process. We found that basal expression of CX(3)CR1 in fresh monocytes was reduced during culture, and that lipopolysacchairde accelerated this effect. In contrast, interleukin-10 and interferon-gamma treatment abrogated CX(3)CR1 down-modulation, through a phosphatidylinositol 3 kinase-dependent pathway. Most importantly, CX(3)CR1 membrane expression correlated with monocyte CX(3)CL1-dependent function. Taken together, our data demonstrate that CX(3)CR1 expression in monocytes can be modulated, and suggest that alterations in their environment are able to influence CX(3)CL1-dependent functions, such as chemotaxis and adhesion, leading to changes in the kinetics, composition and/or functional status of the leucocyte infiltrate.
Subject(s)
Interferon-gamma/pharmacology , Interleukin-10/pharmacology , Monocytes/drug effects , Monocytes/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Receptors, Chemokine/biosynthesis , CX3C Chemokine Receptor 1 , Cells, Cultured , Dose-Response Relationship, Drug , Humans , Interferon-gamma/immunology , Interleukin-10/immunology , Monocytes/immunology , Receptors, Chemokine/immunology , Time FactorsABSTRACT
Production of verocytotoxin or Shiga-like toxin (Stx), particularly Stx2, is the basis of hemolytic uremic syndrome, a frequently lethal outcome for subjects infected with Stx2-producing enterohemorrhagic Escherichia coli (EHEC) strains. The toxin is formed by a single A subunit, which promotes protein synthesis inhibition in eukaryotic cells, and five B subunits, which bind to globotriaosylceramide at the surface of host cells. Host enzymes cleave the A subunit into the A(1) peptide, endowed with N-glycosidase activity to the 28S rRNA, and the A(2) peptide, which confers stability to the B pentamer. We report the construction of a DNA vaccine (pStx2DeltaAB) that expresses a nontoxic Stx2 mutated form consisting of the last 32 amino acids of the A(2) sequence and the complete B subunit as two nonfused polypeptides. Immunization trials carried out with the DNA vaccine in BALB/c mice, alone or in combination with another DNA vaccine encoding granulocyte-macrophage colony-stimulating factor, resulted in systemic Stx-specific antibody responses targeting both A and B subunits of the native Stx2. Moreover, anti-Stx2 antibodies raised in mice immunized with pStx2DeltaAB showed toxin neutralization activity in vitro and, more importantly, conferred partial protection to Stx2 challenge in vivo. The present vector represents the second DNA vaccine so far reported to induce protective immunity to Stx2 and may contribute, either alone or in combination with other procedures, to the development of prophylactic or therapeutic interventions aiming to ameliorate EHEC infection-associated sequelae.
