Massimiliano Legnaro

Angiotensin II type 1 receptor expression on human leukocyte subsets: A flow cytometric and RT-PCR study

The renin-angiotensin system plays a key role in the regulation of cardiovascular functions and in particular angiotensin II type 1 receptor (AT1R)-operated pathways are involved in the modulation of inflammation in the vascular wall. In the present study we assessed the pattern of expression of AT1Rs on different human circulating leukocyte subsets. Venous blood was obtained from healthy male subjects. Leukocyte subsets were purified by immunomagnetic cell sorting or identified in whole blood using multiparametric cytometric analysis. RT-PCR analysis showed that AT1R mRNA was expressed in polymorphonuclear leukocytes (PMNs), monocytes, B-lymphocytes, and, to a lesser extent, T-lymphocytes. Flow cytometric analysis revealed that the frequency of expression of AT1Rs was: PMNs>monocytes>or=B-lymphocytes>>T-lymphocytes, while receptor density per positive cells was: PMNs>or=B-lymphocytes>T-lymphocytes>or=monocytes. AT1Rs are expressed on PMNs, monocytes, T- and B-lymphocytes, however the expression pattern is peculiar to each subset, possibly suggesting distinct roles in the various cell types. Investigating the expression and the functional role of AT1Rs on circulating leukocyte subsets, as well as their possible modifications in disease conditions before and after pharmacological treatments, is likely to provide novel clues to the comprehension of the mechanisms involved in the therapeutic efficacy of currently available agents.

Simvastatin treatment in subjects at high cardiovascular risk modulates AT1R expression on circulating monocytes and T lymphocytes.

Objective Angiotensin II, through the activation of angiotensin II type 1 receptors, plays a crucial role in atherosclerosis. Statins may interfere with the effects of angiotensin II. Methods We have investigated the expression of angiotensin II type 1 receptor, angiotensin II type 2 receptor and angiotensinogen on circulating monocytes and T-lymphocytes from subjects at high risk for vascular events before and during simvastatin treatment, and healthy controls. In-vitro experiments were also performed to assess the ability of simvastatin to interfere with angiotensin II signalling. Results In comparison with controls, high-risk subjects had similar angiotensin II type 1 receptor expression on the cell membranes but significantly higher angiotensin II type 1 receptor mRNA levels at least in monocyte subsets whereas their expression on T cells was similar. Angiotensin II type 2 receptor mRNA expression was higher than controls in both monocytes and T lymphocytes. No differences were observed in angiotensinogen expression on monocytes while T lymphocytes of high-risk subjects show higher expression. One-month treatment of high-risk subjects with simvastatin resulted in a reduction of angiotensin II type 1 receptor mRNA without affecting angiotensin II type 2 receptor whereas angiotensinogen mRNA expression was reduced at least in monocytes. Incubation in vitro with simvastatin reduces the expression of angiotensin II type 1 receptor mRNA levels on monocytes from untreated subjects. Conclusion Simvastatin induces down-regulation of the angiotensin II type 1 receptor, interferes with angiotensin II activity in immune cells and contributes to the anti-inflammatory profile of statins that can explain the therapeutic effects of these drugs.

Production of IL-8, VEGF and Elastase by Circulating and Intraplaque Neutrophils in Patients with Carotid Atherosclerosis.

Objectives Polymorphonuclear neutrophils (PMN) in atherosclerotic plaques have been identified only recently, and their contribution to plaque development is not yet fully understood. In this study, production of elastase, interleukin (IL)-8 and vascular endothelial growth factor (VEGF) by PMN was investigated in subjects with carotid stenosis undergoing carotid endarterectomy (CEA). Methods The study enrolled 50 patients (Pts) and 10 healthy subjects (HS). Circulating PMN (cPMN) isolated from venous blood (in both Pts and HS) and from plaques (pPMN, in Pts) were cultured, alone or with 0.1 μM fMLP. Elastase, IL-8 and VEGF mRNA were analyzed by real-time PCR. In CEA specimens, PMN were localized by immunohistochemistry. Results In both Pts cPMN and pPMN, IL-8 mRNA was higher at rest but lower after fMLP (P<0.01 vs HS), and VEGF mRNA was higher both at rest and after fMLP (P<0.01 vs HS), while elastase mRNA was not significantly different. On the contrary, protein production was always higher in cPMN of HS with respect to values measured in cells of Pts. In CEA specimens, CD66b+ cells localized to areas with massive plaque formation close to neovessels. Pts with soft and mix plaques, as defined by computed tomography, did not differ in cPMN or pPMN IL-8, VEGF or elastase mRNA, or in intraplaque CD66b+ cell density. However, Pts with soft plaques had higher white blood cell count due to increased PMN. Conclusions In Pts with carotid plaques, both circulating and intraplaque PMN produce IL-8, VEGF and elastase, which are crucial for plaque development and progression. These findings suggest mechanistic explanations to the reported correlation between PMN count and cardiovascular mortality in carotid ATH.

Dopaminergic Receptors on CD4+ T Naive and Memory Lymphocytes Correlate with Motor Impairment in Patients with Parkinson’s Disease

Parkinson’s disease (PD) is characterized by loss of dopaminergic neurons in substantia nigra pars compacta, α-synuclein (α-syn)-rich intraneuronal inclusions (Lewy bodies), and microglial activation. Emerging evidence suggests that CD4+ T lymphocytes contribute to neuroinflammation in PD. Since the mainstay of PD treatment is dopaminergic substitution therapy and dopamine is an established transmitter connecting nervous and immune systems, we examined CD4+ T naive and memory lymphocytes in PD patients and in healthy subjects (HS), with specific regard to dopaminergic receptor (DR) expression. In addition, the in vitro effects of α-syn were assessed on CD4+ T naive and memory cells. Results showed extensive association between DR expression in T lymphocytes and motor dysfunction, as assessed by UPDRS Part III score. In total and CD4+ T naive cells expression of D1-like DR decrease, while in T memory cells D2-like DR increase with increasing score. In vitro, α-syn increased CD4+ T memory cells, possibly to a different extent in PD patients and in HS, and affected DR expression with cell subset-specific patterns. The present results support the involvement of peripheral adaptive immunity in PD, and may contribute to develop novel immunotherapies for PD, as well as to better use of current dopaminergic antiparkinson drugs.

Isoflavone content and estrogenic activity of different batches of red clover (Trifolium pratense L.) extracts: An in vitro study in MCF-7 cells

The estrogenicity of different batches of red clover (Trifolium pratense L., Fabaceae; RCL) extracts and its relationship with the isoflavone content were assessed by measuring MCF-7 cell proliferation by flow cytometry and propidium iodide staining. RCL extracts were compared to estradiol (E2) and to the main RCL isoflavones biochanin A, daidzein, genistein and formononetin. Isoflavone content in the extracts was assayed by HPLC. E2 and isoflavones increased MCF-7 proliferation in a concentration-dependent fashion, with the following potency order: E2>>>genistein>biochanin A=daidzein>formononetin. Extracts increased MCF-7 proliferation with different potencies, which in four out of five extracts correlated with the ratios 5,7-dihydroxyisoflavones/7-hydroxyisoflavones. The efficacy of all extracts increased with decreasing genistein contents. A solution containing the main isoflavones at the average concentration of RCL extracts increased MCF-7 proliferation with higher potency and steeper concentration-response curve. The effects of E2, of RCL extracts and of the isoflavone solution were inhibited by the estrogen receptor antagonist 4-hydroxytamoxifen. Flow cytometric analysis of MCF-7 proliferation is a suitable bioassay for the estrogenicity of RCL extracts, thus expanding the characterization of individual batches beyond assessment of chemical composition and contributing to improved standardization of quality and activity.

Adrenergic modulation of migration, CD11b and CD18 expression, ROS and interleukin-8 production by human polymorphonuclear leukocytes

ObjectivesAdrenergic modulation of immunity has been extensively characterized, however, few information exist regarding polymorphonuclear leukocytes (PMN), despite their key role in immunity and inflammation. We investigated the effect of adrenergic agents on human PMN migration, CD11b and CD18 expression, reactive oxygen species (ROS) and interleukin (IL)-8 production, and on adrenoceptor (AR) expression.MethodsMigration was measured by the Boyden chamber assay, CD11b/CD18 expression was assessed by flow cytometry, intracellular ROS were detected by spectrofluorimetry, and IL-8 was quantitated by standard ELISA assay. AR mRNA levels were measured by real-time PCR and PMN morphology was studied by scanning electron microscopy.ResultsAdrenaline(A), noradrenaline and the β-AR agonist isoprenaline reduced N-formyl-Met-Leu-Phe (fMLP)-induced migration, CD11b/CD18 expression, and ROS production, without affecting IL-8. The effect of A on CD11b was antagonized by yohimbine and propranolol, and increased by prazosin. The effect on ROS production was completely abolished by propranolol. PMN expressed α1A-, α1B-, α1D-, α2A-, α2C-, β1-, β2-, and β3-AR mRNA. A prevented fMLP-induced morphological changes of PMN.ConclusionsAdrenergic agents reduced PMN responses mainly through β-AR, although α-AR may contribute at least to CD11b expression. AR-operated pathways in PMN should be investigated in disease conditions and in the response to therapeutic agents.

Angiotensin type 1 receptor expression and interleukin-8 production in polymorphonuclear leukocytes of patients with peripheral arterial disease.

We investigated angiotensin type 1 receptor (AT1R) expression and interleukin-8 (IL-8) productions in polymorphonuclear leukocytes obtained from patients with peripheral arterial disease (PAD) undergoing femoral endarterectomy. Subjects at high cardiovascular risk (high-risk subjects, HRS) and healthy controls (HC) were also enrolled. To this end, patients with PAD were studied 1 month before surgery, at the time of surgery, and 3 and 6 months after surgery. Polymorphonuclear leukocytes were obtained from venous blood and evaluated for AT1R expression at messenger RNA (mRNA) and protein level and IL-8 production (by means of enzyme-linked immunosorbent assay). At baseline, AT1R membrane expression was similar in cells from patients with PAD, HRS, and HC, whereas AT1R mRNA was similar in patients with PAD and HC and higher in HRS. During the follow-up period, AT1R expression progressively decreased both on the cell membrane and at the mRNA level. Both resting and stimulated production of IL-8 was lower in patients with PAD in comparison to HC and HRS and did not change during the follow up period. In PAD patients, femoral endarterectomy is associated with reduction of AT1R expression however with no apparent effect on IL-8 production. The relevance of such effects for cardiovascular protection deserves consideration.

Angiotensin II type 1 and type 2 receptor expression in circulating monocytes of diabetic and hypercholesterolemic patients over 3-month rosuvastatin treatment

BackgroundIn diabetes, a variety of pro-inflammatory cellular changes has been found in various cell types, including monocytes which are known to be involved in all the phases of atherogenesis. Angiotensin II (Ang II) type 1 receptor (AT1R) mediates the pro-atherogenic effects of Ang II whereas the type 2 receptor (AT2R) seems associated with atheroprotection. We sought to investigate the potential changes of AT1R-AT2R expression in human monocytes of type 2 diabetic- hypercholesterolemic patients and in hypercholesterolemic subjects, upon clinical treatment with rosuvastatin.MethodsThe AT1R membrane protein and mRNA AT1R and AT2R expression in monocytes were investigated in 10 type 2 diabetic-hypercholesterolemic patients and in 10 hypercholesterolemic subjects, before and after 3-month rosuvastatin treatment. Moreover, the serum cytokine levels of interferon-γ (IFN-γ) and interleukin-4 (IL-4) were detected.ResultsAs expected, rosuvastatin was associated with a change in the lipid profile in the two groups. Both the membrane protein (P = 0.008) and the AT1R mRNA expression (P = 0.038) were significantly reduced during treatment in the absence of AT2R expression change in diabetic-hypercholesterolemic patients whereas no significant difference was observed in hypercholesterolemic subjects. The serum IL-4 levels were increased during treatment whereas no change was observed in IFN-γ in diabetic-hypercholesterolemic patients. No cytokine change was observed in hypercholesterolemic subjects.ConclusionsOur study on monocytes of diabetic-hypercholesterolemic patients, showing a reduced AT1R but not AT2R expression during rosuvastatin treatment, suggests that statin therapy may modulate favorably the AT1-AT2 receptor balance in subjects with coexistent type 2 diabetes.

Dopaminergic receptors and adrenoceptors in circulating lymphocytes as putative biomarkers for the early onset and progression of multiple sclerosis

Clinically isolated syndrome (CIS) is a first, usually recovering, episode of neurological disturbance(s) suggestive of multiple sclerosis (MS). CIS subjects might benefit from early disease-modifying drugs, provided that those at high risk of developing MS can be identified. Gene expression for dopaminergic receptors (DR) and adrenoceptors (AR) is dysregulated in lymphocytes of MS patients and is affected by treatment with interferon (IFN)-β. In particular, lymphocyte DR D5 mRNA might be a marker of IFN-β response in MS patients. No information exists so far in CIS subjects. We investigated DR and AR gene expression in peripheral blood mononuclear cells (PBMC) and in CD4+ T effector (Teff) and regulatory (Treg) cells from CIS subjects, and assessed their relationship with MS progression after 12months. Expression of several DR and AR are upregulated in PBMC, Teff and Treg from CIS subjects. DR D3 and α2A-AR mRNA in PBMC, and DR D5 mRNA in Treg correlate with the risk of MS at 12months. Results show the involvement of dopaminergic and adrenergic pathways in CIS as well as in MS pathogenesis, supporting the evaluation of dopaminergic and adrenergic agents in MS.

Parkinson’s disease patients have a complex phenotypic and functional Th1 bias: cross-sectional studies of CD4+ Th1/Th2/T17 and Treg in drug-naïve and drug-treated patients

BackgroundParkinson’s disease (PD) affects an estimated 7 to 10 million people worldwide, and only symptomatic treatments are presently available to relieve the consequences of brain dopaminergic neurons loss. Neuronal degeneration in PD is the consequence of neuroinflammation in turn influenced by peripheral adaptive immunity, with CD4+ T lymphocytes playing a key role. CD4+ T cells may however acquire proinflammatory phenotypes, such as T helper (Th) 1 and Th17, as well as anti-inflammatory phenotypes, such as Th2 and the T regulatory (Treg) one, and to what extent the different CD4+ T cell subsets are imbalanced and their functions dysregulated in PD remains largely an unresolved issue.MethodsWe performed two cross-sectional studies in antiparkinson drug-treated and drug-naïve PD patients, and in age- and sex-matched healthy subjects. In the first one, we examined circulating Th1, Th2, Th17, and in the second one circulating Treg. Number and frequency of CD4+ T cell subsets in peripheral blood were assessed by flow cytometry and their functions were studied in ex vivo assays. In both studies, complete clinical assessment, blood count and lineage-specific transcription factors mRNA levels in CD4+ T cells were independently assessed and thereafter compared for their consistency.ResultsPD patients have reduced circulating CD4+ T lymphocytes, due to reduced Th2, Th17, and Treg. Naïve CD4+ T cells from peripheral blood of PD patients preferentially differentiate towards the Th1 lineage. Production of interferon-γ and tumor necrosis factor-α by CD4+ T cells from PD patients is increased and maintained in the presence of homologous Treg. This Th1-biased immune signature occurs in both drug-naïve patients and in patients on dopaminergic drugs, suggesting that current antiparkinson drugs do not affect peripheral adaptive immunity.ConclusionsThe complex phenotypic and functional profile of CD4+ T cell subsets in PD patients strengthen the evidence that peripheral adaptive immunity is involved in PD, and represents a target for the preclinical and clinical assessment of novel immunomodulating therapeutics.