Yu-Min Huang

Lysophosphatidylcholine (LPC) induces proinflammatory cytokines by a platelet-activating factor (PAF) receptor-dependent mechanism

Oxidized low‐density lipoprotein (oxLDL) consists of both lipid components and apoprotein B100. OxLDL has both proinflammatory and cytotoxic properties. The present study was undertaken to investigate the effects of components in the lipid moiety of oxLDL on immune activation as determined by cytokine and immunoglobulin secretion. LPC induced interferon‐gamma (IFN‐γ) secretion in peripheral blood mononuclear leucocytes from healthy blood donors. The effect varied between individuals, and there were both responders and non‐responders. Furthermore, LPC induced enhanced antibody production, indicating B cell activation. None of eight oxysterols, arachidonic acid (AA), or 15‐lipoxygenase products of AA tested had immune stimulatory properties. We recently demonstrated that PAF and oxLDL induce IFN‐γ secretion by a common mechanism. LPC‐induced IFN‐γ secretion was inhibited by a specific PAF receptor antagonist, WEB 2170, indicating that the PAF receptor is involved in LPC‐induced immune activation. Both oxLDL‐ and LPC‐induced antibody formation was inhibited by WEB 2170. Furthermore LPC also induced tumour necrosis factor‐alpha secretion, and this effect was inhibited by WEB 2170. LPC is produced during lipid oxidation (as in oxLDL), but also by enzymes such as phospholipase A2. The findings indicate that LPC may play an important role in inflammatory reactions, including atherosclerosis.

Recruitment of dendritic cells to the cerebrospinal fluid in bacterial neuroinfections

Dendritic cells (DC) accumulate in the CNS during inflammation and may contribute to local immune responses. Two DC subsets present in human cerebrospinal fluid (CSF) are probably recruited from myeloid (CD11c(+)CD123(dim)) and plasmacytoid (CD11c(-)CD123(high)) blood DC. In bacterial meningitis and especially in Lyme meningoencephalitis, numbers of myeloid and plasmacytoid DC in CSF were increased, compared to non-inflammatory neurological diseases, and correlated with chemotactic activity of CSF for immature monocyte-derived DC (moDC). Multiple DC chemoattractants, including macrophage inflammatory protein (MIP)-1beta, monocyte chemotactic protein (MCP)-1, MCP-3, RANTES and stromal cell-derived factor (SDF)-1alpha were elevated in CSF in these two neuroinfections. Chemotaxis of immature moDC induced by these CSFs could be partially inhibited by mAbs against CXCR4, the receptor for SDF-1alpha, and CD88, the receptor for C5a. SDF-1alpha present in CSF also chemoattracted mature moDC, which in vivo could correspond to a diminished migration of antigen-bearing DC from the CSF to secondary lymphoid organs. Regulation of DC trafficking to and from the CSF may represent a mechanism of controlling the CNS inflammation.

Ischemic Stroke Is Associated With a Systemic Increase of Blood Mononuclear Cells Expressing Interleukin-8 mRNA

BACKGROUND AND PURPOSE Ischemic brain injury secondary to an arterial occlusion is characterized by acute local inflammation. Blood polymorphonuclear leukocytes (PMNL), primarily neutrophils, adhere to endothelial cells and rapidly invade the injured brain after the arterial occlusion. This neutrophilic invasion might correlate with the production of certain chemoattractants by blood mononuclear cells (MNC). We evaluated mRNA expression of the CXC chemokine interleukin (IL)-8, and the CC chemokines monocyte chemoattractant protein-1, macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta in blood MNC from patients with ischemic stroke. METHODS Peripheral blood was obtained at 8 AM on days 1 to 7 (mean, day 3) after onset of symptoms. In situ hybridization with radiolabeled synthetic oligonucleotide probes for the chemokines was adopted to measure chemokine mRNA expression in MNC. An enzyme-linked immunosorbent assay for IL-8 was used to measure IL-8 levels in plasma. RESULTS Most patients with ischemic stroke had high numbers of IL-8 mRNA expressing blood MNC, regardless of the time interval between onset of clinical symptoms and examination. There was a marked difference between patients with ischemic stroke and healthy subjects (median, 6228 versus 885 positive cells per 10(5) MNC; P<.0001). IL-8 levels in plasma correlated positively to IL-8 mRNA expression in examined patients (n=7) with ischemic stroke (r=.78, P<.05). In contrast, mRNA expression for the CC chemokines showed no significant difference between patients with ischemic stroke and healthy control subjects. CONCLUSIONS This study demonstrated a systemic increase of IL-8 mRNA expressing MNC and IL-8 levels in plasma from patients with ischemic stroke, suggesting that IL-8 could be involved in recruiting blood PMNL to the sites of cerebral ischemia.

Multiple sclerosis is associated with high levels of circulating dendritic cells secreting pro-inflammatory cytokines

Recent evidence emphasises a pivotal role for dendritic cells (DC) in the control of immunity by priming and tolerising T cells. DC capture and process antigens, express co-stimulatory molecules, migrate to lymphoid organs and secrete cytokines to initiate immune responses. In multiple sclerosis (MS), autoreactive T cells are proposed to play a pathogenic role by secreting pro-inflammatory cytokines, but studies on DC are lacking. To evaluate the involvement of DC in patients with MS, a modified procedure was used to prepare DC from blood of patients with MS and healthy subjects. DC were found to be potent stimulators of T cells in allogeneic and, to a lesser extent, in autologous mixed leukocyte reaction (MLR). Enzyme-linked immunospot (ELISPOT) assays were adopted to determine levels of IFN-gamma, TNF-alpha, IL-6 and IL-10 secreting DC vs. mononuclear cells (MNC). Proportionally more DC than MNC secreted IFN-gamma and IL-10 in both MS and healthy subjects. Patients with MS had higher levels of IFN-gamma, TNF-alpha and IL-6 secreting DC than healthy subjects. The differences for IFN-gamma and TNF-alpha secreting cells were confined to the subgroup of untreated MS patients and not observed in the subgroup examined during ongoing treatment with IFN-beta. Circulating DC secreting pro-inflammatory cytokines may represent another focus for the study of both immuno-pathogenesis and therapeutic interventions in MS.

IL-17 and IFN-γ mRNA expression is increased in the brain and systemically after permanent middle cerebral artery occlusion in the rat

Brain ischemia is characterized by local inflammation reflected by accumulation of inflammatory cells and a multitude of mediators. Among them, cytokines and chemokines may influence the inflammatory cascade that follows cerebral ischemia. Here we report on brain hemispheric and systemic increase of pro-inflammatory IL-17 and IFN-gamma, the anti-inflammatory cytokines IL-4 and IL-10, and the chemokines IP-10, IL-8 and MIP-2, 1 h to 6 days after permanent middle cerebral artery occlusion (pMCAO). IL-17 and IFN-gamma mRNA levels were elevated in the ischemic hemispheres of pMCAO-operated rats compared with corresponding hemispheres of sham-operated rats. Levels were slightly elevated at 1 h, and peaked at 6 days after pMCAO. IL-8 and MIP-2 levels in the ischemic hemispheres peaked at 24 h, whereas IP-10 showed a biphasic profile with two peaks at 6 h and 6 days after pMCAO. IL-4 peaked in the ischemic hemispheres at 6 h, when IL-10 levels were lower than in sham-operated rats, and IL-10 levels peaked at 2 days after pMCAO. Systemically, the numbers of IL-17 and IFN-gamma mRNA expressing blood mononuclear cells were elevated already at 1 h after pMCAO, preceding the changes in the ischemic hemispheres. Altered levels of IL-17 and IFN-gamma after pMCAO may affect outcome of brain ischemia.

Multiple sclerosis and optic neuritis: CCR5 and CXCR3 expressing T cells are augmented in blood and cerebrospinal fluid

Abstract. A role for chemokines as mediators of Th1 cell recruitment to the central nervous system (CNS) is probable in MS. Therefore we studied expression of Th1-related CCR5 and CXCR3 chemokine receptors in patients with MS and controls. Patients with untreated MS had elevated percentages of CCR5 and CXCR3 expressing T cells vs. healthy controls (HC) in blood, and vs. other non-inflammatory neurological diseases (OND) patients in CSF. Such elevation was not found in MS patients examined during ongoing treatment with IFN-β. Patients with optic neuritis (ON), a common first manifestation of MS, had elevated percentages of CXCR3 expressing T cells in blood compared with HC, and of CCR5 expressing T cells in CSF compared with OND patients. High chemokine receptor expression may be one prerequisite for Th1 cells to migrate to the CNS. Inhibition of chemokine receptor expression may constitute a potentially important therapeutic effect of IFN-β.

Dendritic cells exposed to estrogen in vitro exhibit therapeutic effects in ongoing experimental allergic encephalomyelitis

Immunomodulatory effects of estrogen have been demonstrated by clinical and experimental observations, but the mechanisms by which estrogen exhibits the effects remain to be defined. One possible mechanism by which estrogen inhibits the development of experimental allergic encephalomyelitis (EAE), a commonly used model of multiple sclerosis (MS) in humans, is over the functions of dendritic cells (DC). Here, we describe that splenic DC from Lewis rats obtained on day 12 post-immunization (p.i.) with myelin basic protein (MBP) encephalitogenic peptide 68-86+Freunds complete adjuvant (FCA), after being exposed in vitro 17beta-estradiol, exhibited therapeutic effects on acute EAE when injected subcutaneously on day 5 p.i. Blood mononuclear cells (MNC) were isolated from thus treated rats on day 12 p.i. Administration of estrogen-exposed DC prevented the expansion of CD4+ T cells and increased proportions of regulatory T cells producing IL-10 and CD4+CD28- suppressor T cells, accompanied with increased IL-10 and IFN-gamma, and reduced TNF-alpha production. Infiltrates of CD68+ macrophages within the central nervous system and MBP 68-86-induced T cell proliferation were inhibited in rats injected with estrogen-exposed DC compared to rats injected with naive DC. Estrogen up-regulated the expression of indoleamine 2,3-dioxygenase, which promotes tolerogenic properties of DC. The results suggest that in vitro exposure of DC to estrogen modulates DC functions and results in a therapeutic effect of DC.

Vaccination with autologous dendritic cells: from experimental autoimmune encephalomyelitis to multiple sclerosis

Autoimmune diseases such as multiple sclerosis (MS) are characterized by the loss of tolerance to self-determinants, activation of autoreactive lymphocytes and subsequent damage to single or multiple organs. The mechanisms by which autoimmune responses are triggered, and how activation of autoreactive lymphocytes is initiated and maintained, are not fully understood. Therapeutic approaches in autoimmune diseases have so far concentrated on antigens and T cells. Given the exceptional capacity of dendritic cells (DCs) to induce immunity in vivo, recent reports of the first successful clinical trials based on vaccination of tumor patients with autologous blood DCs pulsed in vitro with tumor antigen come as no surprise. The recent identification of tolerogenic subsets of DCs and their generation in culture may allow a novel approach to induce tolerance in autoimmune diseases. By selective in vitro manipulation of DCs and their subsequent reinfusion, DC-mediated tolerance has been achieved in animal models of human autoimmune diseases, including experimental autoimmune encephalomyelitis in Lewis rats and SJL/J mice and spontaneous diabetes in NOD mice. In vitro observations of human blood DCs are promising for DC-based treatment of MS and other diseases with an autoimmune component. Data from animal models and human materials suggest that DC-based immunotherapy could be beneficial at least as a complement to conventional therapy. Molecular-biological approaches to tolerogenic DCs could provide a rationale for designing immunotherapeutic strategies in autoimmune diseases.

Glatiramer acetate reduces lymphocyte proliferation and enhances IL-5 and IL-13 production through modulation of monocyte-derived dendritic cells in multiple sclerosis

Dendritic cells (DC), as the most effective antigen presenting cells, are protagonists of the complex immune network involved in multiple sclerosis (MS) lesion formation. Glatiramer acetate (GA), a synthetic random copolymer, is thought to exert its therapeutical effect in MS by favouring both Th2 cell development and IL‐10 production from peripheral lymphocytes as well as by systemically affecting the antigen presenting cells. In the present study we further analysed the mechanisms of action of GA by using an autologous DC‐lymphocytes (Ly) coculture system from 11 MS patients and 12 matched healthy controls (HC). We found that, in MS patients, pretreatment with GA significantly decreases the in vitro proliferative effect of DC on lymphocytes as compared to HC and to unpulsed or myelin basic protein (MBP)‐pulsed DC from MS patients (P < 0·05). In addition, GA‐treated DC from both MS patients and HC significantly increase the lymphocyte production of IL‐5 and IL‐13 as compared to MBP‐treated DC (P < 0·05). In conclusion our in vitro study may provide new therapeutical mechanisms of GA on lymphocytes, antiproliferative and Th2‐favouring effects, which are mediated by monocyte‐derived DC.

Dendritic cells derived from patients with multiple sclerosis show high CD1a and low CD86 expression

Dendritic cells (DC) are important antigen presenting cells (APC) and play a major role in initiating and orchestrating immune responses by priming T cells. Little is known about involvement of DC in multiple sclerosis (MS), where auto-aggressive T cells against myelin autoantigens are considered to contribute to inflammation and demyelination in the central nervous system. In this study, we compared phenotype and cytokine secretion of DC from patients with MS, other neurological diseases (OND) and healthy subjects. DC were generated from blood adherent mononuclear cells (MNC) by culture for 7 days with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). The yield and morphology of DC were similar in MS patients and controls. In both, the DC phenotype was that of immature myeloid lineage, comprising CD1a+ and CD11c+. The proportion of CD1a+ DC, being important for presentation of lipid antigens to T cells, was higher in MS patients compared to controls. The proportion of CD86+ DC, a co-stimulatory molecule that is assumed to promote Th2 differentiation, was low in MS. Low proportions of CD86+ DC were only observed in untreated MS patients but not in patients treated with IFN-b. Production of IL-10 and IL-12 p40 by DC did not differ in MS patients and controls. These findings indicate that alterations of functionally important surface molecules on DC are associated with MS.