Luciano Ottonello

Older patients are prone to multimorbidity and polypharmacy, with an inherent risk of adverse events and drug interactions. To the best of our knowledge, available information on the appropriateness of lipid-lowering treatment is extremely limited.

Background and Purpose: Non‐steroidal anti‐inflammatory drugs (NSAIDs) have been shown to induce PG‐independent anti‐inflammatory actions. Here, we investigated the role of three different NSAIDs (naproxen, ibuprofen and oxaprozin) on neutrophil responses to CXCL8 and C5a.Experimental Approach: Human neutrophils were isolated from healthy volunteers by dextran and Ficoll‐Hypaque density gradients. Neutrophils were pre‐incubated with different concentrations (1–100 µM) of NSAIDs or kinase inhibitors. Neutrophil degranulation into supernatants was tested by elisa and zymography. Neutrophil chemotaxis was determined using Boyden chambers. F‐actin polymerization was determined by Alexa‐Fluor 488‐conjugated phalloidin fluorescent assay. Integrin expression was assessed by flow cytometry. The phosphorylation of intracellular kinases was studied by Western blot.Key Results: Pretreatment with NSAIDs did not affect neutrophil degranulation, but inhibited neutrophil migration and polymerization of F‐actin, in response to CXCL8 and C5a. Pretreatment with different NSAIDs prevented C5a‐induced integrin (CD11b) up‐regulation, while only ibuprofen reduced CXCL8‐induced CD11b up‐regulation. Pre‐incubation with naproxen or oxaprozin, but not ibuprofen, inhibited the PI3K/Akt‐dependent chemotactic pathways. Both endogenous (released in cell supernatants) or exogenous (added to cell cultures) PGE₂ did not affect C5a‐ or CXCL8‐induced activities. Short‐term incubation with NSAIDs did not affect neutrophil PGE₂ release.Conclusion and Implications: Treatment with NSAIDs reduced C5a‐ and CXCL8‐induced neutrophil migration and F‐actin polymerization via different mechanisms. Inhibition by ibuprofen was associated with integrin down‐regulation, while naproxen and oxaprozin blocked the PI3K/Akt pathway. Both NSAID actions were independent of COX inhibition and PGE₂ release.

Several chemokines/chemokine receptors such as CCR7, CXCR4 and CXCR5 attract chronic lymphocytic leukemia (CLL) cells to specific microenvironments. Here we have investigated whether the CX₃CR1/CX₃CL1 axis is involved in the interaction of CLL with their microenvironment. CLL cells from 52 patients expressed surface CX₃CR1 and CX₃CL1 and released constitutively soluble CX₃CL1. One third of these were attracted in vitro by soluble CX₃CL1. CX₃CL1-induced phosphorylation of PI3K, Erk1/2, p38, Akt and Src was involved in induction of CLL chemotaxis. Leukemic B cells upregulated CXCR4 upon incubation with CX₃CL1 and this was paralleled by increased chemotaxis to CXCL12. Akt phosphorylation was involved in CX₃CL1-induced upregulation of CXCR4 on CLL. In proliferation centers from CLL lymph node and bone marrow, CX₃CL1 was expressed by CLL cells whereas CX₃CR1 was detected in CLL and stromal cells. Nurselike cells (NLCs) generated from CLL patient blood co-expressed surface CX₃CR1 and CX₃CL1, but did not secrete soluble CX₃CL1. Only half of NLC cell fractions were attracted in vitro by CX₃CL1. In conclusion, the CX₃CR1/CX₃CL1 system may contribute to interactions between CLL cells and tumor microenvironment by increasing CXCL12-mediated attraction of leukemic cells to NLC and promoting directly adhesion of CLL cells to NLC.

Metabolic syndrome is a proatherosclerotic condition clustering cardiovascular risk factors, including glucose and lipid profile alterations. The pathophysiological mechanisms favoring atherosclerotic inflammation in the metabolic syndrome remain elusive. Here, we investigated the potential role of the antilipolytic drug acipimox on neutrophil- and monocyte-mediated inflammation in the metabolic syndrome. Acipimox (500 mg) was orally administered to metabolic syndrome patients ( n = 11) or healthy controls ( n = 8). Serum and plasma was collected before acipimox administration ( time 0) as well as 2–5 h afterward to assess metabolic and hematologic parameters. In vitro, the effects of the incubation with metabolic syndrome serum were assessed on human neutrophil and monocyte migration toward the proatherosclerotic chemokine CCL3. Two to five hours after acipimox administration, a significant reduction in circulating levels of insulin and nonesterified fatty acid (NEFA) was shown in metabolic syndrome patients. At time 0 and 2 h after acipimox administration, metabolic syndrome serum increased neutrophil migration to CCL3 compared with healthy controls. No effect was shown in human monocytes. At these time points, serum-induced neutrophil migration positively correlated with serum levels of insulin and NEFA. Metabolic syndrome serum or recombinant insulin did not upregulate CCR5 expression on neutrophil surface membrane, but it increased intracellular JNK1/2 phosphorylation. Insulin immunodepletion blocked serum-induced neutrophil migration and associated JNK1/2 phosphorylation. Although mRNA expression of acipimox receptor (GPR109) was shown in human neutrophils, 5–500 μM acipimox did not affect insulin-induced neutrophil migration. In conclusion, results suggest that acipimox inhibited neutrophil proatherosclerotic functions in the metabolic syndrome through the reduction in circulating levels of insulin.

ARG1, expressed by human PMNs, inhibits T cell proliferation by depleting extracellular l-arginine. Here, we report that ARG1, released from gelatinase granules by PMNs, is inactive at physiological pH unless activated by factor(s) stored in azurophil granules. Whereas ARG1 exocytosis was induced by TNF-α or ionomycin, only the latter mediated the release of both granules, resulting in extracellular ARG enzyme activity at physiological pH. Furthermore, after fractionation of the different classes of granules, only the mixture of gelatinase and azurophil granules resulted in ARG1 activity at physiological pH. The use of protease inhibitors indicated the involvement of a PMSF- and leupeptin-susceptible serine protease in ARG1 processing and activation. Finally, the supernatant of viable PMNs undergoing frustrated phagocytosis, which mediates gelatinase and azurophil granule release, inhibited T cell proliferation through ARG-dependent mechanisms. In vivo, high ARG1 concentrations and increased ARG enzyme activity, sufficient to inhibit T cell proliferation, were observed in synovial fluids from RA. These findings suggest that PMNs, recruited at sites of immune complex deposition, induce ARG1-dependent immune suppression through concomitant exocytosis of gelatinase and azurophil granules.

Background and Purpose— The concept of “vulnerable plaque” has been extended to the more recent definition of the “cardiovascular vulnerable patient,” in which “intraplaque” and “systemic” factors contribute to the cumulative risk of acute cardiovascular events. Thus, we investigated the possible role of systemic and intraplaque inflammation in patients asymptomatic versus symptomatic for ischemic stroke. Methods— Regions upstream and downstream the blood flow were isolated from internal carotid plaques of patients asymptomatic (n=63) or symptomatic (n=18) for ischemic stroke. Specimens were analyzed for lipid, collagen, macrophage, lymphocyte, neutrophil, mast cell and smooth muscle cell content, and chemokine and cytokine mRNA expression. Chemokine receptors and adhesion molecules were assessed on circulating leukocytes by flow cytometry. Systemic inflammatory markers and biochemical parameters were measured on total blood, plasma, and serum. Results— Tumor necrosis factor-α and CCL5 serum levels as well as intercellular adhesion molecule-1 expression on circulating neutrophils were increased in symptomatic as compared with asymptomatic patients. Collagen content and smooth muscle cell infiltration were decreased in symptomatic plaques. In upstream regions of symptomatic plaques, lipid content and lymphocyte infiltration were increased. In downstream regions of symptomatic plaques, macrophage, neutrophil, and mast cell infiltration were increased. Intraplaque collagen content was positively correlated with smooth muscle cell infiltration and inversely correlated with macrophages, neutrophils, or serum tumor necrosis factor-α. Collagen reduction in downstream regions and serum tumor necrosis factor-α were independently associated with the likelihood of being symptomatic. Conclusions— Inflammatory mediators are increased in ischemic stroke. Despite statistically significant, the correlation between tumor necrosis factor-α serum level and intraplaque vulnerability was weak and probably of limited biological importance.

P>Background Coronary artery disease (CAD) represents the most relevant cause of death and morbidity in the adult population of developed and developing countries. During the last decades, a strong research effort has been performed to identify more selective markers and better assess the cardiovascular risk in both primary and secondary prevention. Materials and methods This review updates current knowledge regarding the pathophysiological relevance as possible markers of coronary calcification of the receptor activator of nuclear factor-kappa ligand (RANKL)/osteoprotegerin (OPG) system. Furthermore, the potential clinical use of both RANKL/OPG and coronary calcium score (CAC) to assess cardiovascular vulnerability has been discussed. Results Emerging evidence indicates that atherosclerotic plaque calcification is positively correlated with vulnerability. Several inflammatory mediators have been shown to modulate arterial calcification, thus increasing the risk of plaque rupture. Among these factors, RANKL/OPG axis might be of particular interest as a promising biomarker of plaque vulnerability in subjects with diffuse coronary calcification. Conclusion Together with clinical parameters of coronary calcification (such as CAC), circulating RANKL/OPG levels could contribute to better assess and predict cardiac events.

Fractalkine/CX₃CL1, a surface chemokine, binds to CX₃CR1 expressed by different lymphocyte subsets. Since CX₃CL1 has been detected in the germinal centres of secondary lymphoid tissue, in this study we have investigated CX₃CR1 expression and function in human naïve, germinal centre and memory B cells isolated from tonsil or peripheral blood. We demonstrate unambiguously that highly purified human B cells from tonsil and peripheral blood expressed CX₃CR1 at mRNA and protein levels as assessed by quantitative PCR, flow cytometry and competition binding assays. In particular, naïve, germinal centre and memory B cells expressed CX₃CR1 but only germinal centre B cells were attracted by soluble CX₃CL1 in a transwell assay. CX₃CL1 signalling in germinal centre B cells involved PI3K, Erk1/2, p38, and Src phosphorylation, as assessed by Western blot experiments. CX₃CR1⁺ germinal centre B cells were devoid of centroblasts and enriched for centrocytes that migrated to soluble CX₃CL1. ELISA assay showed that soluble CX₃CL1 was secreted constitutively by follicular dendritic cells and T follicular helper cells, two cell populations homing in the germinal centre light zone as centrocytes. At variance with that observed in humans, soluble CX₃CL1 did not attract spleen B cells from wild type mice. OVA immunized CX₃CR1⁻/⁻ or CX₃CL1⁻/⁻ mice showed significantly decreased specific IgG production compared to wild type mice. We propose a model whereby human follicular dendritic cells and T follicular helper cells release in the light zone of germinal centre soluble CX₃CL1 that attracts centrocytes. The functional implications of these results warrant further investigation.

BACKGROUND: Numerous mechanisms have been proposed to explain the beneficial action of intravenous immune globulin (IVIG) in autoimmune and systemic inflammatory disorders. Among others' data, an in vitro increase of intracellular TGF‐β expression when culturing CD4+ T lymphocytes in the presence of IVIG has been reported. As IVIG infusion involves administration of soluble contaminants likewise all hemoderivative preparations, we hypothesized that, besides several other immunomodulatory proposed mechanisms, the clinical effects of IVIG therapy might be, at least partly, due to contaminating soluble HLA Class I (sHLA‐I) molecules capable to exert pleiotropic immunomodulatory effects among which TGF‐β₁ modulation. STUDY DESIGN AND METHODS: Ex vivo and in vitro transcriptional and posttranscriptional modulation of TGF‐β₁ in CD8+ T lymphocytes and neutrophils after IVIG infusion was analyzed. RESULTS: Ex vivo analysis of cells drawn from 10 enrolled IVIG recipients pointed out a significant increase of TGF‐β₁ mRNA and intracellular TGF‐β₁ molecules in both leukotypes. In vitro comparable results were obtained incubating CD8+ T lymphocytes and neutrophils from healthy donors with IVIG. The immunodepletion of sHLA‐I and/or soluble Fas ligand (sFasL) abolished TGF‐β₁ modulation in both leukotypes. Coculture with human immunoglobulin (Ig)M monoclonal antibody or chimeric IgG (MabThera, Roche), whose manufacturing excludes “contamination,” did not exert any mRNA modulation. Finally, IgM or MabThera plus purified sHLA‐I molecules enhanced TGF‐β₁ mRNA in both white blood cells to levels comparable to those obtained with IVIG incubation. CONCLUSION: On the whole, these data lead us to speculate that the ability of IVIG administration to modulate TGF‐β₁ might be related to the immunomodulatory activities of sHLA‐I and sFasL molecules on activated CD8+ T lymphocytes and neutrophils.