M.H.G. Rep

Recombinant Interferon-β blocks proliferation but enhances interleukin-10 secretion by activated human T-cells

Abstract Results from recent clinical trials have indicated that recombinant interferon-β (rIFN-β) is a promising drug for the treatment of Multiple Sclerosis (MS), a disease of supposed autoimmune etiology. To gain insight into the immunoregulatory properties of this cytokine, we analyzed effects of interferon-β (IFN-β) on T-cell functions in vitro. Interferon-β inhibited T-cell proliferation, as well as T-cell-dependent immunoglobulin secretion, in a dose-dependent manner. IFN-β did not inhibit upregulation of CD40L on activated T-cells, but blocked induction of CD25 on stimulated T-and B-lymphocytes. Secretion of interferon-gamma (IFN-y), tumour necrosis alpha (TNF-α) and IL-13 was inhibited by the addition of IFN-β, whereas IL-4 secretion was unaffected. Interestingly, IFN-β enhanced secretion of IL-2 about two-fold and secretion of IL-10 nearly four-fold. In summary, these findings suggest that IFN-β may exert direct effects on T-and B-cell function in vivo. In addition, enhanced secretion of IL-10 by activated T-cells may interfere with newly initiated and ongoing inflammatory immune reactions.

Interferon (IFN)-beta treatment enhances CD95 and interleukin 10 expression but reduces interferon-gamma producing T cells in MS patients

Abstract Interferon (IFN)-β has been shown to favorably alter the disease course of relapsing-remitting multiple sclerosis (RRMS) patients. Although its mode of action is still unclear, there is ample evidence from in vitro studies that IFN-β directly modulates the function of immune cells. We analyzed here the effects of IFN-β treatment on immune functions in vivo in a group of 25 RRMS patients who received IFN-β (8 MIU) on alternate days. At baseline and at 1, 3 and 6 months from the start of the treatment, parameters for differentiation and activation states of both monocytes and T lymphocytes were assessed. A transient increase was seen in plasma (p) interleukin (IL)-10 level whereas pIL-12 (p40) was not affected. A similar change was found in the ability of monocytes to secrete these cytokines in vitro. Notably, patients who in vitro readily responded to IFN-β with enhanced IL-10 production had the highest pIL-10 levels. Concerning T-cell differentiation, flowcytometric analysis of cytokine production showed that treatment with IFN-β moderately decreased the mean percentages of CD8pos T cells producing IL-2 and IFN-γ and CD8neg T cells producing IL-4 (p

Treatment with depleting CD4 monoclonal antibody results in a preferential loss of circulating naive T cells but does not affect IFN-gamma secreting TH1 cells in humans.

CD4(pos) TH1 T cells are considered to play a central role in a number of human autoimmune diseases such as rheumatoid arthritis (RA) and multiple sclerosis. Experimental treatment protocols aimed at selectively eliminating CD4(pos) T cells thus far have yielded disappointing clinical results. Here we analyzed phenotype and function of circulating T cells in multiple sclerosis patients treated with the chimeric CD4 mAb cM-T412 in a randomized, double-blind, placebo-controlled, magnetic resonance imaging-monitored phase II trial. Treatment resulted in a long-lasting depletion of CD4(pos) T cells but did not affect CD8(pos) T cell numbers. Analysis of CD4(pos) subpopulations showed that unprimed, CD45RA(pos)/R0(neg) lymphocytes were approximately three times more sensitive to the mAb than primed, CD45RA(neg)/R0(pos) T cells. Notably, within the CD45RA(pos) subset, T cells with phenotypic evidence of prior activation, i.e., expressing Fas, were relatively insensitive to cM-T412, compared with Fas(neg) cells. Remarkably, while a decrease in the number of IL-4-producing T helper 2 (TH2)-type cells in the anti-CD4 treated group was observed, numbers of IFN-gamma-producing T helper 1 (TH1)-type cells remained stable, resulting in a significant increase in the TH1/TH2 ratio. Our data show that treatment with depleting CD4 mAb does not eliminate the cells most strongly involved in the disease process, i.e., primed, IFN-gamma-producing TH1-type cells, and may therefore give an explanation for the lack of beneficial clinical effects of depleting CD4 mAb in human chronic autoimmune disease.

Seasonal variation in immune measurements and MRI markers of disease activity in MS

BackgroundThe exact mechanisms by which T cells contribute to MS progression are not known. Recently, the results of cross-sectional studies suggested seasonal variation of both interferon (IFN)-&ggr; production and the number of active MRI lesions in MS. ObjectiveTo investigate whether seasonal fluctuations of IFN-&ggr; and active MRI lesions could be confirmed and whether any correlations could be detected. MethodsData were analyzed from a group of 28 MS patients in whom detailed longitudinal monitoring of both immune function and MRI measurements had taken place. ResultsSignificant seasonal variation was observed in T-cell activation as measured by the ability of T cells to secrete the pro-inflammatory cytokines tumor necrosis factor-&agr; and IFN-&ggr;. Maximum values were found in samples obtained during autumn. Even though clear fluctuations were observed, no significant seasonal variation could be detected in the number of active MRI lesions. Fluctuations of in vitro IFN-&ggr; secretion correlated weakly with changes in active MRI lesions. ConclusionsThe finding of seasonal variation of immune function in serially MRI-monitored MS patients suggests an environmental role in T-cell activation.

Analysis of CD27 surface expression on T cell subsets in MS patients and control individuals

Within the peripheral blood, CD4+CD27- T cells only reside within the CD45RA- (memory or primed) T cell subset. Cells with this phenotype have characteristics of specialized effector T cells according to their cytokine secretion profiles and the expression of tissue-specific adhesion molecules. This subset was previously found to be increased in certain diseases that are associated with immune activation. Therefore we analyzed CD27 expression of peripheral blood and CSF T cells in MS patients. Within the CD4+ T cell subset no differences were seen between MS patients and controls in proportions of CD45RA-CD27- cells. However, when the CD3+ T cell compartment was analyzed, CD27- cells were also found within the CD45RA+ subset. These cells, most likely CD8+, are significantly reduced in PBL and CSF of MS patients as compared with OND patients. In MS and OND groups the level of CD27- cells in peripheral blood correlated significantly with that in CSF, indicating a balanced migration of CD27- cells between the two compartments. In OIND patients, however, this equilibrium was lost. The correlation of the level of CD27+ cells with the amount of intrathecally produced IgG in MS patients may suggest that CD27+ cells are responsible for B cell help in this disease.

Active MRI lesion appearance in MS patients is preceded by fluctuations in circulating T-helper 1 and 2 cells.

BACKGROUND The role of T cell subpopulations and their ability to produce immunoregulatory cytokines has been extensively studied in multiple sclerosis (MS). However, the exact mechanisms by which T cells and cytokines contribute to disease activity remain to be clarified. OBJECTIVES To analyze the longitudinal relation between markers of T cell activation and differentiation and disease activity in MS patients. METHODS During a period of 9 months, clinical disease activity was scored, monthly MRI scans were performed, and blood was taken for immune measurements in a group of 13 untreated clinically definite MS patients. RESULTS Disease activity, as measured by the occurrence of active MRI lesions, is associated with a significant transient decrease in both T cells producing interferon-gamma (IFN-gamma) and T cells producing interleukin (IL)-4. CONCLUSIONS Our results suggest that MRI-documented disease activity is associated with a transient decrease in circulating cytokine producing T cells, possibly due to the migration of activated T cells into the CNS.

A pilot study investigating the effects of orally administered pentoxifylline on selected immune variables in patients with multiple sclerosis

Multiple sclerosis is probably mainly mediated by T-helper 1 (TH1)-lymphocytes. TH1-function can be down-regulated in vitro and in animal experiments by pentoxifylline. Therefore, we included 20 multiple sclerosis patients in an open label pilot trial of pentoxifylline. Outcome parameter was the effect of treatment on levels of various cytokines and adhesion molecules in cerebrospinal fluid and serum, on production of TH1- and TH2-cytokines using cell stimulation assays, as well as on measures of T-cell activation and proliferation. Kurtzkes EDSS was a secondary efficacy parameter. A convincing and consistent effect of pentoxifylline could not be demonstrated.

Functional Capacities of High Endothelial Venules Appear not to be Controlled by Recirculating Lymphocytes

The influence of recirculating lymphocytes on the function and morphology of high endothelial venules (HEV) has been studied. Mice were depleted of lymphocytes by lethal (1200 cGy) total body irradiation; subsequently, the HEV in mesenteric and cervical lymph nodes were studied up to 7 days after irradiation for: 1) capacity to bind lymphocytes by using the in vitro HEV-binding assay, 2) for morphological aspects such as ultrastructure and endothelial height, 3) for presence of RNA (pyroninophylia) and MECA-325 expression. Although, commencing 3 days after irradiation, lymphocyte depletion was intense and no extravasation of lymphocytes was observed; HEV were capable of binding lymphocytes at normal levels. Also the ratio of B/T cell binding to HEV was comparable to normal. MECA-325 expression, pyroninophilia, and ultrastructure of high endothelial cells were not affected by lymphocyte depletion. However, the average height of endothelial cells, which is a measurement related to cell volume, declined during lymphocyte depletion, stabilizing at about 70% of normal levels from day 4. After intravenous injection of viable lymph node cells, endothelial cell height rapidly increased within a few hours in conjunction with lymphocyte extravasation and homing into the nodes. Restoration of endothelial cell height was not observed after infusion of thymocytes, lethally irradiated lymph node cells or supernatants rich in cytokines. We conclude that recirculating lymphocytes in blood and lymphoid tissues are not involved in controlling high endothelial cell activity including the specific function in lymphocyte extravasation. However, recirculating/extravasating lymphocytes contribute to the development of endothelial cell height. The significance of non-lymphoid (radioresistant) cells in the control of characteristic high endothelial function is suggested.

Regulation of Functional and Morphological Aspects of High Endothelium in Mouse

Lymphocyte migration and recirculation between lymphoid and also nonlymphoid tissues is essential for effective immunological surveillance. The entrance of blood-borne lymphocytes into peripheral lymphoid organs, such as lymph nodes and Peyer’s patches, occurs at particular vascular sites, so called post-capillary high endothelial venules (HEV;1,2). The endothelium of these HEV is unique in it’s capacity to mediate lymphocyte extravasation, and its characteristic cuboidal or ‘high’ cellular appearance. From recent investigations, it has become clear that subtile mechanisms are controlling a balanced and selective immigration of lymphocytes and lymphocyte subsets via HEV into mucosal and non-mucosal lymphoid tissues (3,4). Major contributions to studying lymphocyte extravasation and homing via interaction with high endothelium have been the production of monoclonal antibodies (MABs) against lymphocyte ‘homingreceptors’ for HEV (5,6,7), and the development of an elegant in vitro assays for lymphocyte-binding to HEV in frozen sections by Stamper and Woodruff (8). With these tools the nature and many aspects of the regulation of homing receptors on lymphocytes and lymphocyte subsets have been unraveled. However, little is known of the nature and regulation mechanisms of the specific sets of ligands on high endothelium involved in lymphocyte binding, nor about why this endothelium develops it’s characteristic high appearance and what mechanisms regulate this process. In this paper we will address mechanisms controlling specific functional and morphological qualities of high endothelium. We summarize some previous results obtained with HEV-specific MAB MECA-325, e.g. expression of the MECA-325 antigen in chronic inflammation and induction of the antigen in cultured endothelial cells by lymphokines (9,10). In addition, an in vivo model is described in which severe depletion of recirculating lymphocytes by irradiation affects endothelial height but not its lymphocyte-binding capacity or MECA-325 antigen expression. By injecting recirculating lymphocytes the characteristic high endothelial cell appearance can be quickly restored.

Analysis of CD27 surface expression on T-cell subsets in MS patients

Within the peripheral blood, CD4+CD27- T cells only reside within the CD45RA- (memory or primed) T cell subset. Cells with this phenotype have characteristics of specialized effector T cells according to their cytokine secretion profiles and the expression of tissue-specific adhesion molecules. This subset was previously found to be increased in certain diseases that are associated with immune activation. Therefore we analyzed CD27 expression of peripheral blood and CSF T cells in MS patients. Within the CD4+ T cell subset no differences were seen between MS patients and controls in proportions of CD45RA-CD27- cells. However, when the CD3+ T cell compartment was analyzed, CD27- cells were also found within the CD45RA+ subset. These cells, most likely CD8+, are significantly reduced in PBL and CSF of MS patients as compared with OND patients. In MS and OND groups the level of CD27- cells in peripheral blood correlated significantly with that in CSF, indicating a balanced migration of CD27- cells between the two compartments. In OIND patients, however, this equilibrium was lost. The correlation of the level of CD27+ cells with the amount of intrathecally produced IgG in MS patients may suggest that CD27+ cells are responsible for B cell help in this disease.