Patrizia Piccioli

Glial and neuronal cells express functional chemokine receptor CXCR4 and its natural ligand stromal cell-derived factor 1

Abstract : Chemokines are a family of proteins that chemoattract and activate cells by interacting with specific receptors on the surface of their targets. The chemokine stromal cell‐derived factor 1, (SDF1), binds to the seventransmembrane G protein‐coupled CXCR4 receptor and acts to modulate cell migration, differentiation, and proliferation. CXCR4 and SDF1 are reported to be expressed in various tissues including brain. Here we show that SDF1 and CXCR4 are expressed in cultured cortical type I rat astrocytes, cortical neurons, and cerebellar granule cells. In cortical astrocytes, prolonged treatment with lipopolysaccharide induced an increase of SDF1 expression and a down‐regulation of CXCR4, whereas treatment with phorbol esters did not affect SDF1 expression and down‐modulated CXCR4 receptor expression. We also demonstrated the ability of human SDF1α (hSDF1α) to increase the intracellular calcium level in cultured astrocytes and cortical neurons, whereas in the same conditions, cerebellar granule cells did not modify their intracellular calcium concentration. Furthermore, in cortical astrocytes, the simultaneous treatment of hSDF1α with the HIV‐1 capside glycoprotein gp120 inhibits the cyclic AMP formation induced by forskolin treatment.

Stromal cell-derived factor-1α induces astrocyte proliferation through the activation of extracellular signal-regulated kinases 1/2 pathway

Stromal cell‐derived factor‐1 (SDF‐1), the ligand of the CXCR4 receptor, is a chemokine involved in chemotaxis and brain development that also acts as co‐receptor for HIV‐1 infection. We previously demonstrated that CXCR4 and SDF‐1α are expressed in cultured type‐I cortical rat astrocytes, cortical neurones and cerebellar granule cells. Here, we investigated the possible functions of CXCR4 expressed in rat type‐I cortical astrocytes and demonstrated that SDF‐1α stimulated the proliferation of these cells in vitro. The proliferative activity induced by SDF‐1α in astrocytes was reduced by PD98059, indicating the involvement of extracellular signal‐regulated kinases (ERK1/2) in the astrocyte proliferation induced by CXCR4 stimulation. This observation was further confirmed showing that SDF‐1α treatment selectively activated ERK1/2, but not p38 or stress‐activated protein kinase/c‐Jun N‐terminal kinase (SAPK/JNK). Moreover, both astrocyte proliferation and ERK1/2 phosphorylation, induced by SDF‐1α, were inhibited by pertussis toxin (PTX) and wortmannin treatment indicating the involvement of a PTX sensitive G‐protein and of phosphatidyl inositol‐3 kinase in the signalling of SDF‐1α. In addition, Pyk2 activation represent an upstream components for the CXCR4 signalling to ERK1/2 in astrocytes. To our knowledge, this is the first report demonstrating a proliferative effect for SDF‐1α in primary cultures of rat type‐I astrocytes, and showing that the activation of ERK1/2 is responsible for this effect. These data suggest that CXCR4/SDF‐1 should play an important role in physiological and pathological glial proliferation, such as brain development, reactive gliosis and brain tumour formation.

Expression of the chemokine receptor CXCR4 and its ligand stromal cell-derived factor 1 in human brain tumors and their involvement in glial proliferation in vitro

Abstract: Chemokines are a family of proteins that chemoattract and activate cells by interacting with specific receptors on the surface of their targets. They are grouped into four classes based on the position of key cysteine residues: C, CC, CXC, and CX3C. Stromal cell‐derived factor 1 (SDF1), the ligand of the CXCR4 receptor, is a CXC chemokine involved in chemotaxis and brain development that also acts as coreceptor for HIV‐1 infection. It has been proposed that CXCR4 is overexpressed and required for proliferation in human brain tumor cells. We previously demonstrated that CXCR4 and SDF1 are expressed in culture of cortical type I rat astrocytes, cortical neurons, and cerebellar granule cells. In this study, we analyzed the expression of CXCR4 and SDF1 in four human brain tumor tissues, showing that CXCR4 is expressed in all tumors analyzed, whereas SDF1 is expressed only in two tumor tissues. We also investigated the possible functions of CXCR4 expressed in rat type I cortical astrocytes, demonstrating that SDF1α stimulates the proliferation of these cells in vitro. Moreover, we studied by western blot the intracellular pathway involved in cell proliferation, demonstrating that SDF1α induces the ERK1/2 phosphorylation that is reduced by the PD98059 compound, an MEK inhibitor.

Inhibition of nuclear factor-κB activation induces apoptosis in cerebellar granule cells

The nuclear factor (NF)‐κB family of transcription factors plays important roles in the regulation of many activities of neuronal cells, such as synaptic transmission, inflammation, neuroprotection, and neurotoxicity. In resting cells, NF‐κB activity is present both in the cytoplasm, as an inducible‐inactive complex, and in the nucleus, as a constitutive form. Regulation of its inducible activity relies on processing of IκB(s), which occurs through the proteasome. Here we show that in cerebellar granule cells (CGC) the induction of apoptosis, by potassium withdrawal (5 mM KCl), decreases the amount of nuclear NF‐κB. To understand whether NF‐κB was required for CGC survival, these cells, maintained under depolarizing conditions (25 mM KCl and serum), were treated with proteasome inhibitors. The results show that these treatments reduce the nuclear amount of NF‐κB and increase p65 cytoplasmic levels, a process partially regulated via IκBα degradation. These events are also associated with an impairment in CGC survival, with changes in nuclear morphology, induction of DNA laddering, and oligonucleosome formation, consistent with apoptosis. According to the K+ deprivation model, PSI‐induced apoptosis is reversed by inhibitors of transcription and translation as well as by specific caspase inhibitors. Together our results show an important role for NF‐κB in maintaining CGC survival. Indeed, under conditions of mild depolarization (K25) necessary for CGC survival, NF‐κB is distributed between cytosol and nucleus, whereas, under apoptotic conditions (K5), it is depleted from the nucleus, such as after proteasome inhibitor treatment. Therefore, NF‐κB nuclear deprivation is involved in the induction of CGC apoptosis. J Neurosci. Res. 66:1064–1073, 2001.

The engagement of CTLA-4 on primary melanoma cell lines induces antibody-dependent cellular cytotoxicity and TNF-α production.

BackgroundCTLA-4 (Cytotoxic T lymphocyte antigen-4) is traditionally known as a negative regulator of T cell activation. The blocking of CTLA-4 using human monoclonal antibodies, such as Ipilimumab, is currently used to relieve CTLA-4-mediated inhibition of anti-tumor immune response in metastatic melanoma. Herein, we have analyzed CTLA-4 expression and Ipilimumab reactivity on melanoma cell lines and tumor tissues from cutaneous melanoma patients. Then, we investigated whether Ipilimumab can trigger innate immunity in terms of antibody dependent cellular cytotoxicity (ADCC) or Tumor Necrosis Factor (TNF)-α release. Finally, a xenograft murine model was set up to determine in vivo the effects of Ipilimumab and NK cells on melanoma.MethodsCTLA-4 expression and Ipilimumab reactivity were analyzed on 17 melanoma cell lines (14 primary and 3 long-term cell lines) by cytofluorimetry and on 33 melanoma tissues by immunohistochemistry. CTLA-4 transcripts were analyzed by quantitative RT-PCR. Soluble CTLA-4 and TNF-α were tested by ELISA. Peripheral blood mononuclear cells (PBMC), NK and γδT cells were tested in ADCC assay with Ipilimumab and melanoma cell lines. TNF-α release was analyzed in NK-melanoma cell co-cultures in the presence of ipilimumab. In vivo experiments of xenotransplantation were carried out in NOD/SCID mice. Results were analyzed using unpaired Student’s t-test.ResultsAll melanoma cell lines expressed mRNA and cytoplasmic CTLA-4 but surface reactivity with Ipilimumab was quite heterogeneous. Accordingly, about 2/3 of melanoma specimens expressed CTLA-4 at different level of intensity.Ipilimumab triggered, via FcγReceptorIIIA (CD16), ex vivo NK cells as well as PBMC, IL-2 activated NK and γδT cells to ADCC of CTLA-4+ melanoma cells. No ADCC was detected upon interaction with CTLA-4- FO-1 melanoma cell line. TNF-α was released upon interaction of NK cells with CTLA-4+ melanoma cell lines. Remarkably, Ipilimumab neither affected proliferation and viability nor triggered ADCC of CTLA-4+ T lymphocytes. In a chimeric murine xenograft model, the co-engraftment of Ipilimumab-treated melanoma cells with human allogeneic NK cells delayed and significantly reduced tumor growth, as compared to mice receiving control xenografts.ConclusionsOur studies demonstrate that Ipilimumab triggers effector lymphocytes to cytotoxicity and TNF-α release. These findings suggest that Ipilimumab, besides blocking CTLA-4, can directly activate the elimination of CTLA-4+ melanomas.

CTLA-4 is expressed by human monocyte— derived dendritic cells and regulates their functions

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is the major negative regulator of T-cell responses, although growing evidence supports its wider role as an immune attenuator that may also act in other cell lineages. Here, we have analyzed the expression of CTLA-4 in human monocytes and monocyte-derived dendritic cells (DCs), and the effect of its engagement on cytokine production and T-cell stimulatory activity by mature DCs. CTLA-4 was highly expressed on freshly isolated monocytes, then down-modulated upon differentiation toward immature DCs (iDCs) and it was markedly upregulated on mature DCs obtained with different stimulations (lipopolysaccharides [LPS], Poly:IC, cytokines). In line with the functional role of CTLA-4 in T cells, treatment of mDCs with an agonistic anti-CTLA-4 mAb significantly enhanced secretion of regulatory interleukin (IL)-10 but reduced secretion of IL-8/IL-12 pro-inflammatory cytokines, as well as autologous CD4+ T-cell proliferation in response to stimulation with recall antigen purified protein derivative (PPD) loaded-DCs. Neutralization of IL-10 with an anti-IL-10 antibody during the mDCs-CD4+ T-cell co-culture partially restored the ability of anti-CTLA-4-treated mDCs to stimulate T-cell proliferation in response to PPD. Taken together, our data provide the first evidence that CTLA-4 receptor is expressed by human monocyte-derived mDCs upon their full activation and that it exerts immune modulatory effects.

Proteasome Inhibitors Induce Cerebellar Granule Cell Death

Abstract: Many activities of neuronal cells, such as synaptic transmission, inflammation, neuroprotection, and neurotoxicity, are regulated by the activity of the transcription factor nuclear factor‐κB (NF‐κB). In resting cells, NF‐κB activity is present both in the cytoplasm, as an inducible‐inactive complex, and in the nucleus as a constitutive form. The activation of its inducible form is related to processing of IκB(s), which occurs through the proteasome. To understand whether NF‐κB is involved in the survival of cerebellar granule cells (CGCs) maintained under conditions of mild depolarization (25 mM KCl), these cells were treated with different proteasome inhibitors. The results presented show that these pharmacological tools reduce CGC survival with changes in nuclear morphology and induction of apoptosis. Furthermore, we demonstrate that PSI‐induced apoptosis is reverted by inhibitors of transcription and translation, as well as by specific caspase inhibitors. These issues are also associated with a redistribution of NF‐κB, in that a reduced amount of nuclear NF‐κB and an increased p65 cytoplasmic level have been observed. Finally, we propose that, at least in part, p65 metabolism could also be regulated by the ubiquitin‐proteasome complex. Altogether, the results presented define an important role for NF‐κB in maintainig CGC survival.

Pyrrolidinedithiocarbamate induces apoptosis in cerebellar granule cells: involvement of AP-1 and MAP kinases

Pyrrolidinedithiocarbamate (PDTC) is a compound displaying antioxidant, pro-oxidant and metal chelator properties in different cell types. It has been described that PDTC may exert either anti-apoptotic or apoptotic activity. Moreover it is known that this agent regulates the activity of redox-sensitive transcription factors, such as AP-1 and NF-kappaB. Using cerebellar granule cells (CGCs), a well-described model of neuronal primary cultures, we investigated the effects of different concentrations of this compound on cell viability and the intracellular mechanisms involved. PDTC used at concentrations, as low as 1 microM, exerts cytotoxic effects on CGC through the activation of the apoptotic machinery with a maximal efficacy for concentration of 10 microM. The PDTC-dependent apoptosis is correlated to a biphasic and long-lasting increase of AP-1 binding to the DNA, apparently without affecting the NF-kappaB whose activity was reduced only at much higher concentrations (100 microM). PDTC treatment enhanced ERK phosphorylation (maximal effect 1h) and p38 phosphorylation (maximal effect 7h) that was accompanied by an increase of both mRNA and protein of c-Jun. In conclusion the results presented show that PDTC exerts apoptotic effects on CGC, that are correlated to the activation of stress-pathways, involving mainly AP-1 and MAPKs.

Body mass index and circulating oestrone sulphate in women treated with adjuvant letrozole

Background:Obesity is an independent adverse prognostic factor in early breast cancer patients, but it is still controversial whether obesity may affect adjuvant endocrine therapy efficacy. The aim of our study (ancillary to the two clinical trials Gruppo Italiano Mammella (GIM)4 and GIM5) was to investigate whether the circulating oestrogen levels during treatment with the aromatase inhibitor letrozole are related to body mass index (BMI) in postmenopausal women with breast cancer.Methods:Plasma concentration of oestrone sulphate (ES) was evaluated by radioimmunoassay in 370 patients. Plasma samples were obtained after at least 6 weeks of letrozole therapy (steady-state time). Patients were divided into four groups according to BMI. Differences among the geometric means (by ANOVA and ANCOVA) and correlation (by Spearman’s rho) between the ES levels and BMI were assessed.Results:Picomolar geometric mean values (95% confidence interval, n=patients) of circulating ES during letrozole were 58.6 (51.0–67.2, n=150) when BMI was <25.0 kg m−2; 65.6 (57.8–74.6, n=154) when 25.0–29.9 kg m−2; 59.3 (47.1–74.6, n=50) when 30.0–34.9 kg m−2; and 43.3 (23.0–81.7, n=16) when ⩾35.0 kg m−2. No statistically significant difference in terms of ES levels among groups and no correlation with BMI were observed.Conclusions:Body mass index does not seem to affect circulating oestrogen levels in letrozole-treated patients.

Association of CTLA-4 gene variants with response to therapy and long-term survival in metastatic melanoma patients treated with ipilimumab: An Italian melanoma intergroup study

Ipilimumab (IPI) blocks CTLA-4 immune checkpoint resulting in T cell activation and enhanced antitumor immunity. IPI improves overall survival (OS) in 22% of patients with metastatic melanoma (MM). We investigated the association of CTLA-4 single nucleotide variants (SNVs) with best overall response (BOR) to IPI and OS in a cohort of 173 MM patients. Patients were genotyped for six CTLA-4 SNVs (−1661A>G, −1577G>A, −658C>T, −319C>T, +49A>G, and CT60G>A). We assessed the association between SNVs and BOR through multinomial logistic regression (MLR) and the prognostic effect of SNVs on OS through Kaplan–Meier method. Both −1577G>A and CT60G>A SNVs were found significantly associated with BOR. In particular, the proportion of responders was higher in G/G genotype while that of stable patients was higher in A/A genotype. The frequency of patients experiencing progression was similar in all genotypes. MLR evidenced a strong downward trend in the probability of responsiveness/progression, in comparison to disease stability, as a function of the allele A “dose” (0, 1, or 2) in both SNVs with reductions of about 70% (G/A vs G/G) and about 95% (A/A vs G/G). Moreover, −1577G/G and CT60G/G genotypes were associated with long-term OS, the surviving patients being at 3 years 29.8 and 30.8%, respectively, as compared to 12.9 and 14.4% of surviving patients carrying −1577G/A and CT60G/A, respectively. MM patients carrying −1577G/G or CT60G/G genotypes may benefit from IPI treatment in terms of BOR and long-term OS. These CTLA-4 SNVs may serve as potential biomarkers predictive of favorable outcome in this subset of patients.