Chiara Cioni

Norepinephrine and vasoactive intestinal peptide induce IL-6 secretion by astrocytes: Synergism with IL-1β and TNFα

Resident glial cells and invading inflammatory cells are responsible for cytokine production within the brain. Astrocytes are known to secrete a variety of cytokines upon stimulation with cytokines themselves, protein kinase C activators, bacterial or viral constituents. Astrocytes also have surface receptors for a wide number of neurotransmitters and neuropeptides and some of these substances affect astrocyte immune functions, such as major histocompatibility complex (MHC) class II antigen expression. To elucidate the activity of neuromediators on cytokine secretion by glial cells, we studied the secretion of interleukin-6 (IL-6) by cultured rat astrocytes after incubation with various neurotransmitters and neuropeptides. Norepinephrine (NE) and the β-adrenergic agonist isoproterenol (IPT) induced IL-6 secretion in a dose-dependent fashion. NE effect was predominantly mediated by β2-adrenergic receptors with a minor contribution of α1-adrenergic receptors. The induction of IL-6 release by dibutyryl-cAMP indicated that IL-6 secretion secondary to β2-adrenergic receptor activation probably occurs through cAMP signalling pathways. Vasoactive intestinal peptide (VIP) was the sole neuropeptide able to induce IL-6 secretion. NE and VIP promoted IL-6 mRNA synthesis and both substances synergized with interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) in inducing IL-6 release. Our findings provide further evidence that neurons modulate astrocyte cytokine production and thereby regulate central nervous system immune functions.

HIV-1 gp120 increases the permeability of rat brain endothelium cultures by a mechanism involving substance P.

Objective:To analyse whether an HIV-1 envelope protein might play a role in damaging the blood-brain barrier as a fundamental step in the early invasion of the central nervous system by HIV-1. Design:Analysis of permeability of rat brain endothelium cultures to albumin, to assess the functional integrity of the vascular component of the blood-brain barrier. Methods:Rat brain endothelium cultures prepared by cerebral microvessels were exposed to recombinant gp120IIIB on microporous membranes and passage of biotin-labelled albumin was analysed. Scanning electron microscopy was used to analyse cell culture morphology. Some cultures were preincubated with N-nitro-L-arginine methyl ester (L-NAME), a selective inhibitor of nitric oxide synthase, or with spantide, a selective substance P antagonist. Results:HIV-1 gp120 increased the permeability of rat brain endothelial cells to albumin in a dose-dependent manner. Scanning electron microscopy revealed profound gp120-induced alterations in cell morphology accounting for the increased permeability to macromolecules. These alterations were neutralized by anti-gp120 monoclonal antibody but not by isotype control antibody or L-NAME. By contrast, spantide and anti-substance P polyclonal antibody completely blocked the gp120-induced increase in albumin permeability. Control cultures exposed to measles virus nucleoprotein showed an increase in permeability that was not blocked by spantide. Brain endothelial cells, exposed to gp120, displayed cell surface immunoreactivity for substance P, suggesting that substance P is secreted by brain endothelium in response to gp120 stimulation and binds to brain endothelial cells through a receptor-mediated mechanism. Conclusions:These findings suggest a role for substance P in the gp120-induced increase in permeability of brain endothelium.

Substance P antagonist blocks leakage and reduces activation of cytokine-stimulated rat brain endothelium.

We recently demonstrated that substance P mediates increased permeability of brain endothelium exposed to HIV-1 gp120. To test whether substance P is involved in immune processes at the blood-brain barrier (BBB), we stimulated rat brain endothelial cultures prepared from cerebral microvessels with Interferon-gamma (IFN-gamma) and Tumor necrosis factor-alpha (TNF-alpha), two proinflammatory cytokines that alter the BBB and measured permeability to albumin and expression of adhesion molecule ICAM-1 and MHC class II antigen in the presence and absence of spantide, a powerful substance P antagonist. In a dose-dependent manner, spantide completely neutralized increased permeability induced by TNF-alpha and IFN-gamma and expression of MHC class II molecule induced by IFN-gamma and prevented associated cell morphological changes as revealed by scanning electron microscope. Spantide also reduced expression of ICAM-1 induced by TNF-alpha and IFN-gamma by 35% and 30%, respectively. Substance P mRNA was found in unstimulated brain endothelial cells and was upregulated after stimulation with TNF-alpha and IFN-gamma. These in vitro findings demonstrate that substance P plays a major pathogenetic role in damaging and activating the BBB vascular component in the presence of proinflammatory cytokines.

Circulating gp120 alters the blood-brain barrier permeability in HIV-1 gp120 transgenic mice.

The mechanism underlying invasion of the central nervous system by HIV-1 is unclear. We recently demonstrated blood-brain barrier changes in a model of HIV-1 gp120 transgenic mice. To test whether this alteration was intrinsic to the brain endothelium of transgenic mice or depended on circulating gp120, we used brain endothelial cultures from gp120 transgenic and non-transgenic mice and exposed them to serum from gp120 transgenic or non-transgenic mice. We measured permeability to albumin as a marker of functional endothelial integrity. A significant increase in permeability (up to 47%) was observed in transgenic and non-transgenic cultures exposed to serum samples from transgenic but not to those from non-transgenic mice. This permeability was neutralized after immunoabsorption of sera with anti-gp120 monoclonal antibody. These findings demonstrate that the blood-brain barrier alteration in HIV-1 gp120 transgenic mice is due to circulating gp120.

Enhanced secretion of substance P by cytokine-stimulated rat brain endothelium cultures

Substance P (SP) was analyzed in rat brain endothelium cultures after cytokine stimulation. SP secretion was found after stimulation with high doses of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha). High doses of interferon-gamma (INF-gamma) had no effect on this secretion. Elevated SP release was found to be associated with mRNA expression of beta-preprotachykinin (beta-PPT), precursor of SP, in the cells. Under cytokine stimulation, part of SP was bound to brain endothelial cell surface, suggesting the existence of an autocrine network for this neuropeptide. These findings suggest that SP may have an immunomodulatory action at the blood-brain barrier during inflammatory and autoimmune processes in the central nervous system.

Higher expression of BDNF receptor gp145trkB is associated with lower apoptosis intensity in T cell lines in multiple sclerosis

Conflicting data exist on expression of gp145trkB, the high affinity receptor for brain-derived neurotrophic factor (BDNF), on peripheral blood immunocompetent cells in multiple sclerosis (MS). We analyzed expression of gp145trkB by western blotting and flow cytometry in myelin basic protein (MBP)- and ovalbumin (OVA)-T cell lines prepared from 12 patients with relapsing-remitting MS and 12 normal healthy subjects (NHS) and correlated it with activation-induced apoptosis. We found a higher percentage of gp145trkB-expressing MBP-T cells in MS patients than in NHS (p=0.011). gp145trkB was mainly expressed by CD8(+) T cells to a higher extent in MS patients than in NHS (p=0.04). MBP-T cell lines from MS patients showed significantly lower apoptosis intensity than those from NHS (p=0.011). We found also a significant negative correlation between gp145trkB expression and apoptosis intensity in MS patients only (p=0.02). OVA-T cell lines showed a gp145trkB expression similar to that of MBP-T cell lines, with a higher expression in MS patients than NHS, and similar correlations with apoptosis intensity in MS. These findings suggest that gp145trkB is mainly expressed on T cell lines from MS patients and that the BDNF/gp145trkB axis is involved in the regulation of peripheral T cell apoptosis in MS.

Expression of Tight Junction and Drug Efflux Transporter Proteins in an in vitro Model of Human Blood–Brain Barrier

Interendothelial cell tight junctions (TJs) proteins contribute to maintain the structural and functional integrity of the blood–brain barrier (BBB) and several efflux transporters regulate transport of compounds across BBB. A unique double compartment-model of the BBB, consisting of cerebral endothelial cells isolated from cryopreserved human glial tumors, alone and in the presence of human astroglial cells derived from the same tissue preparation was established. Endothelial cell viability and transendothelial electrical resistance (TEER) were measured in this model and three representative TJ proteins – occludin (OCLN), zonula occludens-1 (ZO-1) and claudin-5 (CLN-5) – as well as several drug efflux transporters – P-glycoprotein (P-gp), multidrug resistance protein-1 and 2 (MRP-1 and MRP-2), organic anion-transporting polypeptide-1 and 3 (oatp1 and oatp3) were analyzed at both the protein and gene transcript level. Functional activity of P-gp and MRP-1 was also assessed. Endothelial cell viability as well as TEER significantly increased in the presence of glial cells. A significant increase of expression of OCLN, ZO-1, and CLN-5 proteins as well as of several drug transporter proteins except oatp3 and MRP-1, was also found in the presence of glial cells. All the gene transcripts protein analyzed were found to be significantly increased in the presence of glial cells. A suitable functional activity of P-gp and MRP-1 was also found. These results demonstrate that this brain endothelium culture system mimics a physiologically relevant situation and may therefore provide a new tool for studying the effects of biological fluids such as serum and cerebrospinal fluid from patients with neurological disorders underlying a BBB alteration in disease pathogenesis.

INTRATHECAL SYNTHESIS OF ANTI-MYELIN BASIC PROTEIN IGG IN HIV-1+ PATIENTS

Human immunodeficiency virus type 1 (HIV‐1)‐infected individuals frequently develop a broad spectrum of neurological syndromes, classified as HIV‐1‐associated cognitive/motor complex. Diffuse demyelination of hemispheric white matter is a commonly observed in HIV‐1 infected brain, but the events leading to myelin destruction are still obscure. Since oligodendrocyte infection by HIV‐1 is not proven as yet, myelin damage in HIV‐1 infection may result from indirect mechanisms such as the excessive release of myelinotoxic substances or the triggering of autoimmune responses directed to myelin constituents. To verify the latter hypothesis, we searched for elevated anti‐myelin basic protein (MBP) IgG levels in the cerebrospinal fluid (CSF) and serum of 25 patients with HIV‐1 infection, 12 with multiple sclerosis (MS), and 9 with non‐inflammatory neurological diseases (NIND). CSF, but not serum, anti‐MBP IgG levels were more frequently elevated in HIV‐1+ (16/25, 64%) than in MS (3/12, 25%) or NIND (0/9) patients. By using the anti‐MBP IgG index, the anti‐MBP IgG antibody specificity index (ASI), and the search for anti‐MBP oligoclonal IgG, we ascertained that anti‐MBP IgG were produced within the CNS in 13 of 25 (52%) HIV‐1+, in 6 of 12 (50%) MS, and in none of NIND patients. The incidence of increased CSF anti‐MBP IgG levels was higher among HIV‐1+ patients at stage II–III (4/4, 100%) or at stage IV B (7/9, 78%) than among those at stage IV C–IV D (5/12, 42%). Although our data indicate that intrathecal anti‐MBP IgG may occur early during HIV‐1 infection and that they are more common in patients with HIV‐1‐associated cognitive/motor complex, the possible demyelinating role of these antibodies remains to be demonstrated.

Expression of preprotachykinin-A mRNA isoforms and substance P production in T lymphocytes of human healthy subjects

The influence of sex and age on the expression pattern of preprotachykinin-A (PPT-A) mRNA isoforms encoding substance P and other tackykinins such as neurokinin A (NKA), neuropeptide K (NPK) and neuropeptide gamma (NPgamma) in human immunocompetent cells and the role of this pattern on SP production are unknown. To investigate these questions, we assessed PPT-A isoform expression and SP production in CD3+ lymphocytes of normal healthy subjects. There were no significant differences in PPT-A isoforms in relation to sex or age. The most frequently expressed isoforms were beta and gamma: after lymphocyte stimulation with phytohemagglutinin (PHA), there was a significant increase in their frequency (p<0.0001). Significantly higher SP levels were found in subjects expressing beta and gamma PPT-A than in those with beta PPT-A only (p=0.001). These findings provide evidence of a heterogeneous expression of PPT-A isoforms in CD3+ lymphocytes of normal healthy subjects.

Immunosuppressive monoclonal antibody to CD64 from patients with long-term stable multiple sclerosis

High intrathecal levels of anti-myelin basic protein (MBP) IgM were previously found to be significantly associated with early favorable course in a cohort of patients with multiple sclerosis (MS). A mAb to MBP 105-120 recognizing the 222-228 epitope of the extracellular domain of high affinity immunoglobulin gamma Fc-receptor I (CD64) was isolated from EBV(+) B cell clones of long-term stable RRMS patients. This mAb exerted immunosuppressive activity on MS-derived T cell lines through induction and release of high amounts of interleukin-10 and decreased levels of interleukin-12 from activated monocytes providing the biological basis for a potential new treatment for MS and other immune-mediated neurological disorders.