Enrico Millo

Systemic sclerosis immunoglobulin G autoantibodies bind the human cytomegalovirus late protein UL94 and induce apoptosis in human endothelial cells

Systemic sclerosis is an autoimmune disease characterized by immunological and vascular abnormalities. Autoantibodies against intracellular antigens are associated with particular clinical features of the disease, whereas autoantibodies against cell surface antigens may be pathogenic by inducing endothelial cell damage, considered the primary event in the pathogenesis of the disease. Latent human cytomegalovirus infection may contribute to progression of systemic sclerosis through its ability to infect endothelial cells; however, direct links between human cytomegalovirus infection and systemic sclerosis are still lacking. Molecular mimicry is one of the mechanisms that account for the link between infection and autoimmunity. Here we have identified an immunodominant peptide using systemic sclerosis serum screening of a random peptide library; such peptide shares homology with autoantigens and with the human cytomegalovirus late protein UL94 (ref. 9). Immunoglobulin G antibodies against the peptide affinity-purified from the sera of patients with systemic sclerosis specifically recognized the viral product and autoantigens; moreover, such antibodies induced endothelial cell apoptosis through specific interaction with the cell surface integrin–NAG-2 protein complex. Our results provide evidence that antibodies against human cytomegalovirus cause apoptosis of endothelial cells, considered the initial pathogenic event of systemic sclerosis, and indicate a previously unknown mechanism for the etiological link between human cytomegalovirus infection and autoimmunity.

HLA-E-restricted recognition of cytomegalovirus-derived peptides by human CD8+ cytolytic T lymphocytes

HLA-E-restricted T cell receptor αβ+ CD8+ cytolytic T lymphocytes (CTLs) exist as monoclonal expansions in the peripheral blood of some individuals. Here, we show that they recognize, with high avidity, peptides derived from the UL40 protein of different human cytomegalovirus (CMV) strains. Recognition results in the induction of cytotoxicity, IFN-γ production and cell proliferation. Autologous cells pulsed with CMV-derived peptides become susceptible to lysis by HLA-E-restricted CTLs and induce their proliferation. The high avidity for CMV-derived peptides may explain how these cells are generated in vivo and suggest their possible role in the host defenses against CMV, a virus that evolved various mechanisms to down-regulate classical HLA class I molecules, thus escaping detection by conventional CTLs.

Abscisic acid is an endogenous cytokine in human granulocytes with cyclic ADP-ribose as second messenger

Abscisic acid (ABA) is a phytohormone involved in fundamental physiological processes of higher plants, such as response to abiotic stress (temperature, light, drought), regulation of seed dormancy and germination, and control of stomatal closure. Here, we provide evidence that ABA stimulates several functional activities [phagocytosis, reactive oxygen species and nitric oxide (NO) production, and chemotaxis] of human granulocytes through a signaling pathway sequentially involving a pertussis toxin (PTX)-sensitive G protein/receptor complex, protein kinase A activation, ADP-ribosyl cyclase phosphorylation, and consequent cyclic-ADP-ribose overproduction, leading to an increase of the intracellular Ca2+ concentration. The increase of free intracellular ABA and its release by activated human granulocytes indicate that ABA should be considered as a new pro-inflammatory cytokine in humans. This discovery is an intriguing example of conservation of a hormone and its signaling pathway from plants to humans and provides insight into the molecular mechanisms of granulocyte activation, possibly leading to the development of new antiinflammatory drugs.

IMMUNOGENICITY OF HUMAN MESENCHYMAL STEM CELLS IN HLA-CLASS I RESTRICTED T CELL RESPONSES AGAINST VIRAL OR TUMOR-ASSOCIATED ANTIGENS

Human mesenchymal stem cells (MSC) are immunosuppressive and poorly immunogenic but may act as antigen‐presenting cells (APC) for CD4+ T‐cell responses; here we have investigated their ability to serve as APC for in vitro CD8+ T‐cell responses. MSC pulsed with peptides from viral antigens evoked interferon (IFN)‐γ and Granzyme B secretion in specific cytotoxic T lymphocytes (CTL) and were lysed, although with low efficiency. MSC transfected with tumor mRNA or infected with a viral vector carrying the Hepatitis C virus NS3Ag gene induced cytokine release but were not killed by specific CTL, even following pretreatment with IFN‐γ. To investigate the mechanisms involved in MSC resistance to CTL‐mediated lysis, we analyzed expression of human leukocyte antigen (HLA) class I‐related antigen‐processing machinery (APM) components and of immunosuppressive HLA‐G molecules in MSC. The LMP7, LMP10, and ERp57 components were not expressed and the MB‐1 and zeta molecules were downregulated in MSC either unmanipulated or pretreated with IFN‐γ. Surface HLA‐G was constitutively expressed on MSC but was not involved in their protection from CTL‐mediated lysis. MSC supernatants containing soluble HLA‐G (sHLA‐G) inhibited CTL‐mediated lysis, whereas those lacking sHLA‐G did not. The role of sHLA‐G in such inhibition was unambiguously demonstrated by partial restoration of lysis following sHLA‐G depletion from MSC supernatants. In conclusion, human MSC can process and present HLA class I‐restricted viral or tumor antigens to specific CTL with a limited efficiency, likely because of some defects in APM components. However, they are protected from CTL‐mediated lysis through a mechanism that is partly sHLA‐G‐dependent.

The analysis of the natural killer-like activity of human cytolytic T lymphocytes revealed HLA-E as a novel target for TCR α/β-mediated recognition

Cytolytic T lymphocytes (CTL) are known to recognize antigen peptides in association with major histocompatibility complex (MHC) class I molecules expressed on target cells. However, a fraction of human CD8+ CTL has been shown to lyse certain natural killer (NK)‐susceptible target cells via still undefined mechanism(s). These CD8+ T cells, hereafter referred to as NK‐CTL, are frequently composed of cells expressing one single TCR Vβ expansion (different in different individuals), display a memory phenotype and express HLA class I‐specific inhibitory NK receptors. Here we show that cell populations or clones of NK‐CTL isolated from three healthy donors homogeneously expressed Vβ16, Vβ9 and Vβ3 TCR, respectively. Various clones isolated under limiting dilution conditions from Vβ16+ cells of donor 1 displayed identical TCR Vβ and Vα rearrangements, thus suggesting a substantial monoclonality of the NK‐CTL subset analyzed. NK‐CTL lysed a number of NK‐susceptible tumor target cells with the exception of those characterized by β2‐microglobulin (β2m) deficiency. However, the latter targets became susceptible to lysis upon β2m transfection. Using monoclonal antibodies specific for the relevant TCR Vβ or β2m we provide evidence suggesting that target cell lysis by NK‐CTL is mediated by the TCR itself upon recognition of β2m‐associated proteins. The cellular distribution of the potential β2m‐associated proteins in susceptible target cells suggested, as a likely candidate for TCR‐mediated recognition, the non‐classical HLA‐E molecule. The use, as target cells, of the murine TAP2‐deficient RMA‐S cells, either untransfected or transfected with HLA‐E, and loaded with an appropriate HLA‐E‐binding peptide, provided the direct demonstration that HLA‐E represents a ligand recognized by the TCR expressed by NK‐CTL. This is the first evidence that human TCR α/β can recognizeHLA‐E molecules, thus revealing a novel type of TCR‐mediated recognition, which may offer new insight in immune responses in both normal and disease conditions.

LANCL2 is necessary for abscisic acid binding and signaling in human granulocytes and in rat insulinoma cells

Abscisic acid (ABA) is a plant hormone regulating fundamental physiological functions in plants, such as response to abiotic stress. Recently, ABA was shown to be produced and released by human granulocytes, by insulin-producing rat insulinoma cells, and by human and murine pancreatic β cells. ABA autocrinally stimulates the functional activities specific for each cell type through a receptor-operated signal transduction pathway, sequentially involving a pertussis toxin-sensitive receptor/G-protein complex, cAMP, CD38-produced cADP-ribose and intracellular calcium. Here we show that the lanthionine synthetase C-like protein LANCL2 is required for ABA binding on the membrane of human granulocytes and that LANCL2 is necessary for transduction of the ABA signal into the cell-specific functional responses in granulocytes and in rat insulinoma cells. Co-expression of LANCL2 and CD38 in the human HeLa cell line reproduces the ABA-signaling pathway. Results obtained with granulocytes and CD38+/LANCL2+ HeLa transfected with a chimeric G-protein (Gαq/i) suggest that the pertussis toxin-sensitive G-protein coupled to LANCL2 is a Gi. Identification of LANCL2 as a critical component of the ABA-sensing protein complex will enable the screening of synthetic ABA antagonists as prospective new anti-inflammatory and anti-diabetic agents.

p38 MAP kinase mediates the cell death induced by prp106-126 in the sh-sy5y neuroblastoma cells

Prion diseases are neurodegenerative pathologies characterized by the accumulation in the brain of a protease-resistant form of the prion protein (PrP(c)), named PrP(Sc). A synthetic peptide homologous to residues 106-126 of PrP (PrP106-126) maintains many PrP(Sc) characteristics. We investigated the intracellular signaling responsible for the PrP106-126-dependent cell death of SH-SY5Y, a cell line derived from a human neuroblastoma. In this cell line, PrP106-126 induced apoptotic cell death and caused activation of caspase-3, although the blockade of this enzyme did not inhibit cell death. The p38 MAP kinase blockers, SB203580 and PD169316, prevented the apoptotic cell death evoked by PrP106-126 and Western blot analysis revealed that the exposure of the cells to the peptide induced p38 phosphorylation. Taken together, our data suggest that the p38 MAP kinase pathway can mediate the SH-SY5Y cell death induced by PrP106-126.

Identification of an import signal for, and the nuclear localization of, human lactoferrin

Many different unique functions have been attributed to lactoferrin (Lf), including DNA and RNA binding, and transport into the nucleus, where Lf binds to specific sequences and activates transcription. A pentapeptide, Gly‐Arg‐Arg‐Arg‐Arg, corresponding to a region of the N‐terminal portion of human Lf rich in basic amino acids, was synthesized and its intracellular localization was investigated. Peptide internalization was assayed using the rhodaminated form of the same molecule. This N‐terminal peptide sequence is able to be internalized within less than 10 min at concentration as low as 1 μM, and its intracellular localization is nuclear, mainly nucleolar. Similar behaviour was observed using peptides composed of either all l or d amino acids, the last one being a retro‐inverse peptide. The internalization process does not involve an endocytotic pathway, since no inhibition of the uptake was observed at 4 °C. The kinetics of peptide internalization was also evaluated. The internalization properties of such a short Lf pentapeptide have been assayed for its ability to transport peptide nucleic acids (PNAs) inside cells in order to improve their efficacy. The abundant transmembrane transport and nuclear localization of the proposed peptide, deriving from hLf and, for the first time, identified as a nuclear localization signal, could be used as an alternative strategy to tackle the unsolved problem of intracellular accumulation of antisense and antigene drugs and for the development of new pharmacological tools.

Abscisic Acid Released by Human Monocytes Activates Monocytes and Vascular Smooth Muscle Cell Responses Involved in Atherogenesis

Abscisic acid (ABA) is a phytohormone recently identified as a new endogenous pro-inflammatory hormone in human granulocytes. Here we report the functional activation of human monocytes and vascular smooth muscle cells by ABA. Incubation of monocytes with ABA evokes an intracellular Ca2+ rise through the second messenger cyclic ADP-ribose, leading to NF-κB activation and consequent increase of cyclooxygenase-2 expression and prostaglandin E2 production and enhanced release of MCP-1 (monocyte chemoattractant protein-1) and of metalloprotease-9, all events reportedly involved in atherogenesis. Moreover, monocytes release ABA when exposed to thrombin-activated platelets, a condition occurring at the injured vascular endothelium; monocyte-derived ABA behaves as an autocrine and paracrine pro-inflammatory hormone-stimulating monocyte migration and MCP-1 release, as well as vascular smooth muscle cells migration and proliferation. These results, and the presence of ABA in human arterial plaques at a 10-fold higher concentration compared with normal arterial tissue, identify ABA as a new signal molecule involved in the development of atherosclerosis and suggest a possible new target for anti-atherosclerotic therapy.

In chronic idiopathic urticaria autoantibodies against FcεRII/CD23 induce histamine release via eosinophil activation

Background Chronic idiopathic urticaria is a common skin disorder characterized by recurrent, transitory, itchy weals for more than 6 weeks. An autoimmune origin has been suggested based on the findings of auto‐antibodies (Abs) directed against either the α subunit of the high‐affinity IgE receptor or the IgE molecule in nearly half of the patients.