Mathilde Kouwenhoven

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.

Multiple sclerosis is associated with an imbalance between tumour necrosis factor-alpha (TNF-alpha)- and IL-10-secreting blood cells that is corrected by interferon-beta (IFN-beta) treatment.

The up‐regulated B cell responses detectable in cerebrospinal fluid (CSF) and the augmented myelin antigen‐specific T cell responses observed in the CSF as well as systematically in patients with multiple sclerosis (MS) suggest the involvement of cytokines in disease development and perpetuation. Here we report on the parallel involvement of TNF‐α, IL‐6, IFN‐γ and IL‐10 in MS and controls, using enzyme‐linked immunospot (ELISPOT) assays to detect and enumerate cytokine‐secreting mononuclear cells (MNC) prepared from blood and, for IL‐6 and IL‐10, from CSF without in vitro stimulation. MS is associated with elevated levels of TNF‐α‐secreting blood MNC when compared with levels in groups of control patients with myasthenia gravis (MG) and other neurological diseases (OND) or healthy subjects. This elevation was confined to patients with untreated MS and not present in those examined during ongoing treatment with IFN‐β. Untreated patients with MS had lower numbers of IL‐10‐secreting blood MNC compared with the three control groups. In patients undergoing treatment with IFN‐β, numbers of IL‐10‐secreting cells were in the same range as in controls. Normalization of TNF‐α from elevated, and of IL‐10 from decreased levels could be one reason for the beneficial effects of IFN‐β in MS, although it remains to be shown whether these changes reflect phenomena primarily involved in MS pathogenesis or secondary changes. In CSF, levels of IL‐10‐secreting cells were higher than in blood in both MS and OND, with no difference between these groups. Systemic aberrations of IL‐6 and IFN‐γ and of IL‐6 in CSF in MS versus controls were only minor, irrespective of treatment with IFN‐β.

Multiple sclerosis: pro- and anti-inflammatory cytokines and metalloproteinases are affected differentially by treatment with IFN-beta.

Interferon-beta (IFN-beta) has a beneficial influence on the course of multiple sclerosis (MS) and has become standard treatment of this disease, though its mechanisms of action are incompletely understood. This study examines the effect of IFN-beta treatment on the cytokines IL-6, TNF-alpha, IFN-gamma and IL-10; the metalloproteinases MMP-3, -7 and -9 and the tissue inhibitor of metalloproteinase-1 (TIMP-1). IFN-beta treatment resulted in decreased numbers of mononuclear cells (MNC) secreting IL-6 and TNF-alpha and expressing mRNA of MMP-3 and MMP-9 compared to pretreatment levels. On the contrary, numbers of IL-10 secreting MNC and TIMP-1 mRNA expressing were augmented during IFN-beta therapy. Whether the down-regulatory effects on pro-inflammatory and upregulatory effects on anti-inflammatory molecules are a direct result of IFN-beta on the immune system or secondary to clinical stabilization of MS pathology induced by IFN-beta remains to be evaluated.

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.

Monocyte-derived dendritic cells express and secrete matrix-degrading metalloproteinases and their inhibitors and are imbalanced in multiple sclerosis

Dendritic cells (DC) are antigen-presenting cells (APC) that most efficiently initiate and control immune responses. Migration processes of blood DC are crucial to exert their professional antigen-presenting functions. Matrix-degrading metalloproteinases (MMP) are proteolytic enzymes, which are considered to be key enzymes in extracellular matrix (ECM) turnover and mediators of cell migration. Tissue inhibitors of metalloproteinases (TIMP) are important regulators of MMP activity. Here we investigate whether blood monocyte-derived immature DC (iDC) and mature DC (mDC) express, produce and secrete functionally active MMP-1, -2, -3 and -9 and their inhibitors TIMP-1 and -2, and examine their involvement in multiple sclerosis (MS). On mRNA level, we observed high numbers of MMP-2 and TIMP-2 mRNA expressing iDC in MS. On protein level, high percentages of MMP-1, -2 and -9 expressing iDC by flow cytometry, and high MMP-1 secretion by Western blot together with high MMP-2 and -9 activities in iDC supernatants as studied with zymography were observed. Similarly, MS is associated with high percentages of MMP-2 and -3 and of TIMP-1 expressing mDC by flow cytometry together with high MMP-3 secretion and high MMP-9 activity in culture supernatants. Spontaneous migratory capacity of both iDC and mDC over ECM-coated filters was higher in MS compared to healthy controls (HC). In conclusion, blood monocyte-derived iDC and mDC express, produce and secrete several MMP and TIMP. Alterations in these molecules as observed in MS may be functionally important for DC functioning.

Cytokines in multiple sclerosis: methodological aspects and pathogenic implications

Multiple sclerosis (MS) is one of the leading causes of disability among young adults of Caucasian origin. One hundred and fifty years after the first description of the disease, the cause of MS remains unknown. Ironically, the few hypotheses concerning MS pathogenesis that are valid today were first proposed over a hundred years ago. However, equipped with the advanced technology of molecular biology and imaging systems, we are at present progressively uncovering clues to understanding the pathogenesis of the disease. It is clearly evident that aberrant immune responses occur in MS, and it is likely that the spectrum of cytokines produced decisively influences disease outcome. The detrimental consequences of IFN-γ and the beneficial effects of IFN-βtreatment in MS support this hypothesis. However, there are still major gaps in our knowledge of the involvement of cytokines in MS. Numerous studies have addressed the question of cytokine levels in MS, often with conflicting results; elevated, normal and decreased levels of almost all cytokines have been reported. This scenario most probably reflects methodological dilemmas as well as the complex biology of cytokines. Here we focus on possible reasons for the discrepancies of results reported on cytokines in MS and summarize findings obtained in particular by the application of enzyme-linked immunospot (ELISPOT) assays to cytokine studies in MS.

Elevated expression of CCR5 by myeloid (CD11c+) blood dendritic cells in multiple sclerosis and acute optic neuritis

Myeloid and plasmacytoid dendritic cells (DC) are present in cerebrospinal fluid (CSF) in non‐inflammatory neurological diseases (NIND) and elevated in clinically definite multiple sclerosis (MS) and in early MS – acute monosymptomatic optic neuritis (ON). Here, we show that expression of CCR5, a chemokine receptor for regulated on activation, normal T cell expressed and secreted (RANTES) and macrophage inflammatory protein (MIP)‐1α/β, is elevated on blood myeloid (CD11c+) DC in MS and ON compared to non‐inflammatory controls. In contrast, expression of CXCR4, a receptor for stromal cell‐derived factor (SDF)‐1α, is similar in all groups. Blood myeloid DC from MS patients respond chemotactically to RANTES and MIP‐1β, which are expessed in MS lesions. In active MS and ON, expression of CCR5 by myeloid DC in blood correlates with numbers of these cells in CSF. Thus, elevation of CCR5 may contribute to recruitment of myeloid DC to CSF in MS and ON. Recruitment of plasmacytoid DC to CSF appears to be CCR5‐independent.

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.

Multiple sclerosis: a study of chemokine receptors and regulatory T cells in relation to MRI variables

Magnetic resonance imaging (MRI) remains the most valuable tool for monitoring disease activity and progression in patients with multiple sclerosis (MS), a chronic demyelinating disease of the central nervous system (CNS) with presumably autoimmune etiology. Chemokine receptors have been implicated in MS as key molecules directing inflammatory cells into the CNS. Regulatory (CD4+CD25+) T cells (Tr cells) are important in suppressing autoimmunity, and their absolute or functional deficit could be expected in MS. In the present study, venous blood was obtained from MS patients concurrent with MRI examination of the brain, and expression of chemokine receptors CCR1, CCR2, CCR5, CXCR3 and CXCR4 by CD4 T cells and monocytes, proportions of Tr cells, as well as expression of CD45RO, CD95, CTLA‐4, HLA‐DR and interleukin (IL)‐10 by Tr cells and non‐Tr (CD25−) CD4 T cells was analyzed by flow cytometry. Surface expression of CXCR3 by CD4 T cells was downregulated in the group of patients with high lesion load (LL) on T2‐weighted images and gadolinium (Gd)‐enhancing lesions on T1‐weighted images, compared to the group with high LL and no Gd‐enhancing lesions, and to the group with low LL, suggesting internalization of CXCR3 due to the release of its chemokine ligand (IP‐10/CXCL10) from active MS lesions. Proportions of Tr cells amongst all CD4 T cells, and expression of IL‐10 by Tr cells were increased in the patients with high LL and Gd‐enhancing lesions. These results suggest that there is correlation between MRI parameters, chemokine receptor expression and the status of circulating Tr cells in MS, but further studies need to discriminate between pathogenetically relevant and bystander phenomena.

Matrix Metalloproteinase and Cytokine Profiles in Monocytes over the Course of Stroke

Stroke is a common cause of death and disability in our society. Stroke is associated with changes in immune responses within the central nervous system as well as systemically. The cells contributing to such changes as well as the factors contributing to formation of the inflammatory infiltrate observed in stroke remain to be clarified. In this study, blood monocytes and corresponding mononuclear cells (MNC) were separated and examined in parallel within 4 days and 1–3 months after onset of ischemic stroke. Numbers of TNF-α-, IL-12-, IL-6-, and IL-10-secreting cells and of cells expressing mRNA for matrix metalloproteinase (MMP)-1, -2, -7, -9 and tissue inhibitor of MMP (TIMP)-1 were studied. The TNF-α-, IL-12-, and IL-6-secreting monocytes and MNC were elevated during the acute phase compared to healthy controls. Such differences were not observed when stroke patients were examined during convalescence. The IL-10-secreting monocytes did not change over the course of stroke. Levels of monocytes expressing MMP-1, MMP-7 and TIMP-1 mRNA were elevated in the acute phase of stroke patients compared to convalescence and healthy controls, as were levels of MMP-1, -2, -7, -9 and TIMP-1 mRNA expressing blood MNC. The MMP-2 and -9 activity as measured by zymography also was higher in MNC supernatants in the acute phase of stroke compared to convalescence. The high levels of proinflammatory cytokines and MMPs in blood monocytes and MNC further demonstrate the presence of systemic aberrations in the acute phase of stroke. Such changes may contribute to the influx of blood-borne cells into the ischemic lesions during the acute phase of stroke.