Daniele Chimenti

Monocyte/Macrophage-Derived Microparticles Up-Regulate Inflammatory Mediator Synthesis by Human Airway Epithelial Cells

Cell-derived microparticles (MP) are membrane fragments shed by virtually all eukaryotic cells upon activation or during apoptosis that play a significant role in physiologically relevant processes, including coagulation and inflammation. We investigated whether MP derived from monocytes/macrophages have the potential to modulate human airway epithelial cell activation. Monocytes/macrophages were isolated from the buffy coats of blood donors by Ficoll gradient centrifugation, followed by overnight culture of the mononuclear cell fraction. Adherent cells were washed and incubated with the calcium ionophore, A23187, or with histamine. The MP-containing supernatant was incubated with cells of the human bronchial epithelial line BEAS-2B and of the human alveolar line A549. IL-8, MCP-1, and ICAM-1 production was assessed by ELISA and by RT-PCR. In some experiments, monocytes/macrophages were stained with the fluorescent lipid intercalating dye PKH67, and the supernatant was analyzed by FACS. Stimulation of monocytes/macrophages with A23187 caused the release of particles that retain their fluorescent lipid intercalating label, indicating that they are derived from cell membranes. Incubation with A549 and BEAS-2B cells up-regulate IL-8 synthesis. Ultrafiltration and ultracentrifugation of the material abolished the effect, indicating that particulate matter, rather than soluble molecules, is responsible for it. Up-regulation of MCP-1 and ICAM-1 was also demonstrated in A549 cells. Similar results were obtained with histamine. Our data show that human monocytes/macrophages release MP that have the potential to sustain the innate immunity of the airway epithelium, as well as to contribute to the pathogenesis of inflammatory diseases of the lungs through up-regulation of proinflammatory mediators.

Cell surface expression of activating receptors and co-receptors on peripheral blood NK cells in systemic autoimmune diseases

Objectives: A defined role for natural killer (NK) cells and their activating receptors in autoimmunity has not been clearly established. The aim of this study was to evaluate the levels of the CD3–CD56+ NK cells and their expression of receptors and co-receptors in the peripheral blood of patients with systemic autoimmune disorders. Methods: Thirty-four subjects with systemic sclerosis (SSc), 14 with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), 14 with systemic lupus erythematosus (SLE), and 14 healthy donors were studied. The activating receptors NKp46, NKp44, NKp30, NKG2D, and DNAM-1 and the co-receptors NTB-A and 2B4 were analysed by flow cytometry on peripheral blood NK cells. Results: In SSc, AAV, and SLE we detected a significant decrease in the percentage of CD3–CD56+ NK cells compared to healthy controls. No differences in the expression of NKp46, NKp44, and NKp30 were identified. On the contrary, NKG2D and DNAM-1 expression was decreased in SLE, but not in SSc and AAV, NTB-A was decreased in SLE, and 2B4 in both SLE and SSc. No differences were detected between active and inactive SLE patients. In SSc, only patients affected by pulmonary arterial hypertension (PAH) and interstitial lung disease (ILD) had a low expression of DNAM-1, 2B4, and NKp30. Conclusions: These data demonstrate that patients with different systemic autoimmune diseases differ in the expression of activating receptors and co-receptors on CD3–CD56+ NK cells. The down-regulation of receptors and co-receptors in SSc with lung involvement suggests their possible role in this manifestation of the disease.

Effects of different LDL particles on inflammatory molecules in human mesangial cells.

Aims/hypothesisInflammation is a mechanism of glomerular damage in chronic glomerulopathies. LDL may increase the production of inflammatory cytokines in renal tissues. However, the relative role of native, oxidised and glycated LDL in promoting this process has been only partially elucidated.MethodsWe tested the inflammatory and proapoptotic effects of native, oxidised and glycated LDL in human mesangial cells (HMCs) by measuring levels of IL6, CD40 and macrophage migration inhibitory factor (MIF) genes, MIF protein, release of IL6, soluble CD40, fibronectin and laminin, early and late apoptosis, and extracellular regulated kinases (ERK) 1/2 and c-Jun N-terminal kinase (JNK) activation.ResultsIL6 and CD40 mRNA were dose-dependently upregulated by all three species; this was closely paralleled by their increased release. MIF mRNA was potently stimulated by modified LDL, as confirmed by immunostaining. Fibronectin and laminin release was stimulated by both oxidised and glycated, but not native, LDL. All LDL species induced some increase in late, but not early, apoptosis, and similarly activated JNK2/3 phosphorylation; in contrast, ERK1/2 phosphorylation was more strongly upregulated by oxidised than either native or glycated LDL.ConclusionsIn HMCs, the production and release of IL6 and CD40 is stimulated by both native and modified LDL, while MIF is more strongly stimulated by oxidised LDL. Regarding the pattern of mesangial expansion, fibronectin and laminin are upregulated by oxidised and glycated LDL. Apoptosis, if modest, is induced by all species. Intracellular signalling of native and modified LDL involves JNK2/3 and, perhaps more specifically, ERK1/2. Tight control of the lipid profile may be useful in preserving kidney function in patients with metabolic alterations.

Effect of Type I Interferon(s) on Cell Viability and Apoptosis in Primary Human Thyrocyte Cultures

BACKGROUND Interferon (IFN) therapy may induce a generalized activation of the immune system, hence triggering or exacerbating autoimmune disease. Apoptosis contributes to the development of hypothyroidism in autoimmune thyroiditis. IFN can affect all phases of the cell cycle and may induce apoptosis in several cell lines from varied histologies. To date, no data exist on the possible effect of type I IFN(s) on FAS/FASL system and cell apoptosis of human thyroid follicles. Therefore, we evaluated the effect of both IFN-alpha and -beta on apoptosis in primary human thyrocyte cultures and the potential role of the FAS/FASL pathway. METHODS Thyrocytes were cultured in monolayers and FAS, FASL, and Bcl-2 mRNA expression was determined by reverse transcriptase polymerase chain reaction after exposure to 10 mIU/mL bovine thyroid-stimulating hormone alone or in combination with increasing doses of IFN-alpha or -beta for 24, 48, and 72 hours. The percentage of apoptotic hypodiploid cells was evaluated by flow cytometry. RESULTS Thyroid-stimulating hormone significantly decreased FAS and increased Bcl-2 mRNA expression while reducing the percentage of hypodiploid cells. The concomitant addition of either IFN-alpha or -beta reduced cell viability and increased the number of hypodiloid cells, but only IFN-beta modulated the expression of FAS and Bcl-2 mRNA expression in a proapoptotic sense. CONCLUSIONS Both type I IFN(s) increase apoptosis in primary thyrocyte cultures, but only IFN-beta modulates FAS and Bcl-2 gene expression toward a proapoptotic pathway. Because apoptosis plays an important role in thyroid homeostasis and disease, this mechanism may contribute to the development and progression of type I IFN(s) therapy-associated thyroid disease.

A Deiminated Viral Peptide to Detect Antibodies in Rheumatoid Arthritis

Abstract: The data presented suggest that a deiminated viral peptide is specifically recognized by antibodies contained in rheumatoid arthritis (RA) sera. Antipeptide antibodies are not associated with the presence or severity of specific manifestations of RA, but are more frequent in subjects with erosive arthritis. Taking into account the association with rheumatoid factor and with erosive arthritis, we can conclude that antipeptide antibodies are markers of severe forms of RA. Our data also show familial aggregation of anticitrullinated peptide antibodies.

Endothelial Progenitor Cells in Mixed Cryoglobulinemia

Objective. Endothelial progenitor cells (EPC) are a rare population of circulating cells involved in vascular homeostasis. Our aim was to analyze EPC in patients with mixed cryoglobulinemia (MC). Methods. EPC were evaluated by cytometry according to guidelines of the International Society for Hematotherapy and Graft Engineering in 17 patients with MC and 36 controls. Results. Numbers of EPC were significantly increased in MC compared to controls and correlated with cryocrit, but not with clinical manifestations of the disease. Conclusion. The high number of EPC might indicate an intact vascular repair ability. Alternatively, EPC might be defective in homing ability and their increase may represent the attempt to restore vascular integrity.