key: cord-0964287-r69961ao
authors: Wandinger, Klaus-Peter; Wessel, Karl; Neustock, Petra; Siekhaus, Aja; Kirchner, Holger
title: Diminished production of type-I interferons and interleukin-2 in patients with multiple sclerosis
date: 1997-07-01
journal: J Neurol Sci
DOI: 10.1016/s0022-510x(97)05383-5
sha: 44221b95d7931c51f83060d114e9c7fb43007971
doc_id: 964287
cord_uid: r69961ao

Several lines of evidence have supported the role of immunological mechanisms in the pathogenesis of multiple sclerosis (MS) and new immunomodulatory strategies for its treatment, e.g. subcutaneous application of interferon (IFN)-β, have emerged. We investigated the ability of peripheral blood mononuclear cells (PBMC) in 21 consecutive patients with clinically definite MS to produce interferons and lymphokines in response to viral or mitogenic stimulation. Ten patients showed clinical signs of disease activity (acute relapse) and 11 patients were in a stable condition. Additionally, white blood count, leukocyte differentiation and lymphocyte subtyping were performed. A group of age-related healthy blood donors served as control (n=20). There was no difference between patients and controls in the production of IFN-γ, tumor necrosis factor (TNF)-α and soluble interleukin (IL)-2 receptor. IFN-α and IFN-β responsiveness, however, was significantly lower in patients with stable disease than in patients with active disease and controls (p<0.001). Furthermore, secretion of IL-2 after stimulation was significantly diminished in both patient groups as compared to the control group (p<0.01). Analysis of T-cell subsets revealed a significantly lower amount of CD8(+) T-cells in patients with stable disease, leading to a significantly higher CD4/CD8 ratio in this group as compared to patients with active disease. Our study depicted an IL-2 deficiency in MS patients which is shared with other autoimmune diseases. In addition, our findings suggest that the ability to produce type-I IFNs, IFN-α and IFN-β, is primarily impaired in MS patients and changes in correlation to the course of disease activity.

tive T-lymphocytes in MS patients, and in particular on the usage of specific T-cell receptor elements for the recogni-Although the etiology of multiple sclerosis is still tion of immunodominant regions of autoantigens, such as unknown, there has been a considerable expansion in myelin basic protein (Wucherpfennig et al., 1990 ; Lodge et understanding the pathogenesis of the disease process al., 1994) . Others have assessed the role of cytokines, during the last few years (Poser, 1994) . Immunological peptidic or glycopeptidic mediators of the immune system, mechanisms, both cellular and humoral, seem to play a during the course of the disease. Beck et al. (1988) were central role in the inflammatory process which leads to the first to show that PBMC of individual MS patients demyelination and destruction of oligodendrocytes in the produced increased amounts of IFN-g and TNF-a at an central nervous system (CNS) (Raine, 1994; early stage of an exacerbation of the disease, thus preced-1995) .

ing the clinical manifestation of a relapse. These findings Some studies have focused on the presence of autoreac-were later confirmed for TNF-a on the mRNA level (Rieckmann et al., 1995) . TNF-a is understood to play a * crucial role in the upregulation of adhesion molecules on involved in T-cell migration to the sites of inflammation control group, we examined 20 age-matched healthy blood (Hartung et al., 1995) . Furthermore, high levels of expres-donors who fulfilled the criteria of the German regulations sion of proinflammatory as well as immunosuppressive for whole blood donation. cytokines were detected in MS lesions of postmortem CNS tissue by means of immunocytochemistry (Cannella and 2.2. Leukocyte count and lymphocyte subsets Raine, 1995) .

The understanding of the role of cytokines in multiple Peripheral venous blood samples were tested for whole sclerosis has become increasingly important since new blood and differential blood count by means of an autotreatment strategies for multiple sclerosis have emerged matic analyzer (Coulter STKS flow cytometer). Lympho- (McDonald, 1995) . Interferon beta-1b was reported to cyte subpopulation analysis was performed by flow cytomreduce exacerbation rates and the severity of exacerbations etry on a Coulter Epics XL / MCL flow cytometer (Coulter in patients with relapsing-remitting MS (The IFNB Multi-Electronics, Hileah, FL, USA). Following the instructions ple Sclerosis Study Group, 1993) . However, reports on the of the manufacturer, preparation and immunofluorescence ability to release type-I interferons upon viral stimulation staining of EDTA-anticoagulated blood samples were in MS patients are rare.

assisted by the Multi-Q-Prep Epics Immunology work Interferon responses of PBMC in general were shown to station (Coulter, Krefeld, Germany). For gating of monobe diminished in patients with MS (Neighbour et al., 1981;  nuclear lymphatic cells, we used forward and side light Salonen et al., 1982) , but the amount of IFN production scatter and immunostaining with anti-CD45 (CD45-FITC / could not be correlated with the clinical assessments of CD14-PE). For the determination of lymphocyte subpopu-1 1 disease activity. Other authors revealed completely normal lations such as T and B lymphocytes, CD4 and CD8 IFN-responses upon viral stimulation in MS patients T-cells, activated T-cells, as well as NK cells, the follow- (Tovell et al., 1983; Haahr et al., 1986) . Altogether, the ing antibodies were used: CD3-ECD/ CD4-PE / CD8-FITC, results were largely inconclusive, since different methods CD3-ECD, CD56-PE, CD16-FITC, CD3-FITC / CD19-PE, have been used for IFN-induction and detection.

CD3-FITC and CD25-PE. MsIgG2b-ECD/ MsIgG1-PE / The aim of this study was to establish a whole blood MsIgG1-FITC, MsIgG1-FITC and MsIgG2a-PE antibodies assay in order to investigate the ability of MS patients in served as controls (all antibodies were obtained from different stages of disease activity to produce interferons Coulter). and lymphokines in response to viral or mitogenic stimulation.

Heparinized blood was drawn by vein puncture from 2. Materials and methods patients and controls, stored at 4 8C and cultured in a whole blood assay within 4 h of collection according to a 2.1. Patients technique described previously (Kirchner et al., 1982) . For mitogenic induction of IL-2, sIL-2R and IFN-g, we used We investigated 21 consecutive patients (11 males and the plant lectin phytohemagglutinin (PHA, Murex Diag-10 females, ages 20-57, mean age6standard deviation nostik / Burroughs Wellcome, Dartford, UK) in a final 37610) fulfilling the diagnostic criteria of multiple concentration of 5 mg / ml. IFN-a and IFN-b production sclerosis (Poser et al., 1983) . Eleven patients had the was stimulated by Newcastle disease virus (NDV, strain relapsing-remitting course of MS, 8 patients were suffer-Kumarov, inactivated by UV-irradiation at 366 nm for 90 ing from the secondary-progressive subtype and 2 patients min, kindly provided by R. Zawatzky, German Cancer had the progressive-relapsing course of the disease accord-Research Center, Heidelberg) in a final concentration of ing to the standardized definitions (Lublin et al., 1996) . All 0.8 hemagglutinating units / ml. Lipopolysaccharide (LPS) patients were examined and classified independently by (Sigma, Deisenhofen, FRG), consisting of purified Estwo clinical neurologists and informed consent was ob-cherichia coli OIII:B4 endotoxin, was used for the inductained. 10 patients were seen during an acute episode of tion of TNF-a at a final concentration of 1 mg / ml. 100 ml new disease activity (acute relapse) and 11 patients were in whole blood was mixed with 850 ml culture medium a stable condition. An acute relapse was defined as (Seromed RPMI 1640, 20 mM Hepes; Biochrom, Berlin, significant worsening of preexisting symptoms as mea-Germany) supplemented with 2 mM L-glutamine, 100 U of sured by the Expanded Disability Status Scale (Kurtzke, penicillin per ml, and 100 ml of streptomycin per ml 1983) or appearance of new neurological deficits lasting (Gibco, Karlsruhe, Germany) in 5 ml PPN test tubes for at least 24 h. None of the patients had signs of systemic (Greiner, Nurtingen, Germany). 50 ml of the appropriate infections and plasma levels of CRP, a -macroglobulin and mitogen solution was added for the induction of cytokine 2 neopterin were within normal limits. The patients had not production. As negative control, we tested the cytokine been treated with immunosuppressive drugs or steroids production in unstimulated cultures. Tubes were covered during the preceding 3 months before blood sampling. As a and incubated in a humidified atmosphere of 5% CO at 2 37 8C. Supernates were harvested after 48 h (IL-2, TNF-a, control groups. p values,0.05 were considered to be IFN-a, IFN-b ) and 96 h (IFN-g and sIL-2R) and stored significant. frozen at 280 8C until assayed.

The concentrations of cytokines in the supernates of cell 3.1. Differential blood cell count and lymphocyte cultures were determined by means of quantitative en-subpopulations zyme-linked immunosorbent assay (ELISA). IFN-a was additionally determined in sera samples of the individual Differential blood cell counts showed no significant patients. ELISA kits for IFN-a (intraassay variance 3.7%, differences between the two patient groups (Table 1 ) and interassay variance 5.3%), IFN-g (intraassay variance were all within normal ranges. With regard to the lympho-5.2%, interassay variance 6.0%), TNF-a (intraassay vari-cyte subpopulations, we found a significantly lower 1 ance 3.5%, interassay variance 4.0%) and IL-2 (intraassay amount of CD8 T-cells in patients with stable disease, variance 3.9%, interassay variance 5.5%) were obtained leading to a significantly higher CD4 / CD8 ratio in this from Bio Source International (Camarillo, CA, USA). The patient group as compared to MS patients undergoing an ELISA kit for detection of sIL-2R (intraassay variance acute exacerbation ( p,0.03). 3.6%, interassay variance 2.0%) was obtained from R&D Systems (Minneapolis, MN, USA) and for IFN-b (intraas-3.2. Cytokine production after mitogenic stimulation say variance 4.1%, interassay variance 3.7%) from Medgenix Diagnostics (Ratingen, Germany). Serial dilu-Measurement of IFN-g, TNF-a and sIL-2R in the tions of samples and standards were incubated in microtiter supernates of mitogen-stimulated whole-blood cultures wells and the assays were performed as described in the revealed no significant differences in cytokine production manufacturer's protocol. The color intensity was measured between patient groups and controls ( Table 2 ). The results photometrically in an ELISA reader (Anthos Labtec, of the in vitro production of IL-2 after mitogenic stimula-Salzburg, Austria). Individual concentrations could be tion are shown in Fig. 1 . Interestingly, both patient groups determined from a standard curve. Minimal detectable showed a significantly ( p,0.005) decreased IL-2 prodoses were 5 pg / ml (TNF-a, IFN-a, IFN-g, IL-2, sIL-2R) duction in comparison to the control group. and 3 units / ml (IFN-b ) , respectively.

In contrast, NDV induced production of IFN-a showed highly significant differences in mean values between the 2.5. Statistics two patient groups. Patients with stable disease (156.6 pg / ml) produced significantly ( p,0.0001) lower amounts Statistical analysis was performed using commercially of IFN-a than controls (924.1 pg / ml) and patients with available software for personal computers (SPSS for disease activity (773.6 pg / ml). No influence of age and Windows 5:01, SPSS GmbH Software, Munich, Ger-sex was observed. Results are shown in Fig. 2 . No IFN-a many). The Mann-Whitney-U test was used to compare was detectable in sera samples of the patients. non-parametrically distributed data between patient and Detection of NDV induced IFN-b secretion revealed Values are given as means6standard deviations. * p,0.03. Table 2 Cytokine production in a whole-blood assay in MS patients with acute exacerbation (n510), with stable disease (n511) and controls (n520) (Fig. 3) . Overall, IFN-b was produced in termination of interleukin-2 in the supernates of (PHA)-stimulated cell cultures. The horizontal bars represent mean interleukin-2 levels in each lower amounts than IFN-a. However, the mean value in patient group. patients with stable disease (8.0 U / ml) was also significantly lower than in patients with disease activity (52.6 U / ml) and controls (29.6 U / ml).

No cytokines were detectable in the supernates of unstimulated cultures.

The present study revealed a remarkable decrease in production of IFN-a and IFN-b upon viral stimulation in PBMC of MS patients with stable disease and an impaired ability to secrete IL-2 in all MS patients irrespective of disease activity. Production of IFN-g and TNF-a did not differ from values observed in the control group, and titers of sIL-2R were not increased in the patient group either. Additionally, MS patients with stable disease showed a 1 significantly lower number of CD8 T-cells.

In previous publications, low IFN responses to various viral antigens have occasionally been described for MS patients (Neighbour et al., 1981; Salonen et al., 1982) . of the disease activity. Abb et al. (1983) showed an association of low IFN-a responsiveness with the MHC Columbia MS / MRI Analysis Group, 1995) could then allele HLA-DR2, which is over-represented in MS patients possibly be regarded as substitution of a preexisting deficit. (Hillert, 1994) . The defective IFN response could therefore

The second striking feature of this study was defective possibly be related to the genetic background of MS production of interleukin-2 in all MS patients regardless of patients. In the present study, however, diminished pro-their clinical state. In previous studies, an impairment of duction of IFN-a and IFN-b was exclusively observed in IL-2 production was predominantly described for MS MS patients with stable disease. Patients admitted to patients with stable disease (Selmaj et al., 1988) . hospital during an acute attack revealed normal IFN-a and Interleukin-2 is produced by activated helper T-cells and is IFN-b values.

required for appropriate lymphocyte function and prolifer-In response to viral stimulation, PBMC produce mainly ation (Smith, 1988) . Defective production of IL-2 therefore IFN-a and a small proportion of IFN-b (Berg et al., 1975) .

results in an impaired T-cell response and diminished The two interferons have similar biological and physico-protection against viral infections. Similar findings have chemical properties, are structurally related (Kirchner, been reported for a variety of autoimmune diseases, e.g. 1986) , and are, therefore, classified as type-I interferons.

diabetes mellitus (Zier et al., 1984; Roncarolo et al., 1988) However, there are striking homologies within the coding or Sjogren's syndrome (Miyasaka et al., 1984) and also for as well as flanking regions of the IFN-a and IFN-b genes, other neurological disorders like Parkinson's diseasë both located on chromosome 9 of the human genome. Our (Kluter et al., 1995) . Interleukin-2 further acts as an findings suggest a primary deficit in type-I IFN response in endogenous brain neurokine which is able to influence MS patients which may render them susceptible to factors central nervous functions (Haugen and Letourneau, 1990 ; causing exacerbations of the disease. Merrill, 1990) . Interleukin-2 mRNA has recently been Since interferons are the only cytokines with clear found to be present in human oligodendrocytes (Otero and antiviral potency, viruses might be responsible for trig -Merrill, 1995) , implicating a role for IL-2 in the developgering the pathophysiological processes leading to an ment of oligodendrocytes, their MBP content and reconstiexacerbation in MS. One possible mechanism could be tution (Benveniste and Merrill, 1986; Saneto et al., 1986 ; initiated by a virus with tropism to the central nervous Benveniste et al., 1987) . system, leading to destruction of myelin sheaths and Studies on cytokine patterns within postmortem CNS triggering off an immune response to released autoan-specimens, however, revealed high levels of IL-2 exprestigens, such as myelin basic protein (MBP). In this regard, sion in acute MS-lesions (Cannella and Raine, 1995) . coronavirus and human herpesvirus-6 are currently of Thus, decreased amounts of IL-2 in the supernates of interest on the basis of polymerase chain reaction and stimulated cell cultures could also result from increased immunohistochemical studies of MS brains (Challoner et binding of the cytokine to its receptor on activated T-cells, al., 1995) .

indicating higher in vitro proliferation upon mitogenic Molecular mimicry, i.e. an immune response against a stimulation in MS patients as compared to healthy conforeign antigen with some resemblance to a host protein, trols. pointed out that MBP specific T-cells from MS patients 1995). Our results support the hypothesis that CD8 T-cell crossreact with peptides from diverse viral agents (Richert mediated suppressor function is lost before and during MS et al., 1995; Wucherpfennig and Strominger, 1995) . attacks and recovers with remission (Antel et al., 1979) . The clinical observation that viral infections are fre-Interestingly, we did not find significant differences in quently associated with or followed by acute MS relapses the production of TNF-a and IFN-g between the two also suggests that an activation of the peripheral immune patient groups in this cross-sectional study. Since IFN-g compartment may contribute to an acceleration of disease and IL-2 are both produced by activated T-lymphocytes, progression (Sibley et al., 1985; Panitch, 1994) . Interest-the impaired IL-2 production may implicate a selective ingly, acyclovir, an antiviral drug, has recently been inhibitory mechanism that may downregulate IL-2 but not reported to significantly reduce exacerbation rates in MS IFN-g secretion of this T-cell subpopulation in MS pa- (Lycke et al., 1996) .

tients. However, an involvement of NK-cells, being known The question arises, however, as to what causes type-I as IL-2 producers, can not be excluded. IFN low responsiveness in MS patients and what are the Investigating CNS specimens from MS patients, Cannelmechanisms involved in upregulation of IFN-a and IFN-b la and Raine (1995) found high expression of TNF-a in gene expression in the course of a relapse. Serial studies in acute and chronic plaques, thus stressing its role in the individual patients are required to reveal if type-I IFN pathogenesis of the disease. In their study, however, TNFproduction increases at the beginning of an attack or if it a was mainly produced by microglial cells and by foamy leads to its remission. The positive effect of treatment with macrophages within the lesions, i.e. cells which are not IFN-b and IFN-a (Durelli et al., 1994;  The IFNB Multiple detectable in the peripheral blood. Sclerosis Study Group and the University of British

In conclusion, we found abnormalities in the IFN-a and Hartung, H.-P., Reiners, K., Archelos, J.J., Michels, M., Seeldrayers, P.,

Heidenreich, F., Pflughaupt, K.W. and Toyka, K. V. (1995) Circulating activity and in their ability to produce IL-2. The etiology adhesion molecules and tumor necrosis factor receptor in multiple of these immunological deficits is unknown. We conclude sclerosis: Correlation with magnetic resonance imaging. Ann. Neurol., that the selective immunological abnormalities in MS 38: 186-193. patients provide the background for disease susceptibility, Haugen, P.K. and Letourneau, P.C. (1990) Interleukin-2 enhances chick and rat sympathetic, but not sensory neurite outgrowth. J. Neurosci.

progression and exacerbation, possibly triggered by diverse Res., exogenous factors, e.g. viruses. The individual production Hillert, J. (1994) Human leukocyte antigen studies in multiple sclerosis.

of endogenous type-I IFNs in MS patients may be a useful Ann. Neurol., tool for monitoring disease activity and for studying the 

idiopathic Parkinson's disease

Rating neurologic impairment in multiple sclerosis: Association of human leucocyte low responsiveness to inducers of An expanded disability status scale (EDSS). Neurology, 33: 1444-interferon alpha with HLA-DR2

Myelin function in multiple sclerosis: Correlation with clinical disease activibasic protein peptide specificity and T-cell receptor gene usage of ty

Defining the clinical course of precedes clinical manifestation in multiple sclerosis: Do cytokines multiple sclerosis: Results of an international survey

proliferation and maturation by interleukin-2

Acyclovir treatment

Myelin basic of relapsing-remitting multiple sclerosis: A randomized, placeboprotein-specific RNA levels in interleukin-2-stimulated oligodencontrolled, double-blind study

New treatments for multiple sclerosis may delay Berg

37: interferons on sepharose-bound antibodies

Interleukin-2 effects in the central nervous system

The adhesion molecule and cytokine Ann

leukin 2 deficiencies in rheumatoid arthritis

Interferon re-G.C. (1995) Plaque-associated expression of human herpesvirus 6 in sponses of leukocytes in multiple sclerosis

Molecular cloning of IL-2Ra

Alterations in levels of CD28 / CD8 suppressor cell line: Presence of IL-2R mRNAs in the human central nervous system

Influence of infection on exacerbations of multiple Immunol

Chronic systemic high-dose recombinant New diagnostic criteria for multiple sclerosis: Guidelines for research interferon alpha-2a reduces exacerbation rate, MRI signs of disease protocols

The Dale E. McFarlin memorial lecture: The Interferon induction, 29-59-oligo A synthetase and lymphocyte subimmunology of the multiple sclerosis lesion

Myelin basic protein-reactive human

Stimulation by diverse microbial antigens. Ann. NY interleukin-2 production by peripheral blood mononuclear cells in Acad

Tumor necrosis infections and multiple sclerosis. Lancet, 1: 1313-1315. factor-alpha messenger RNA expression in patients with relapsing-Smith, K.A. (1988) Interleukin-2: Inception, impact and implications. remitting multiple sclerosis is associated with disease activity

Interferon production by lymphocytes from multiple sclerosis

usage to immunodominant regions of myelin basic protein. Science, (1982) PPD-, PWM-and PHA-induced interferon in stable multiple 248: 1016-1019. sclerosis: Association with the HLA-Dw2 antigen and clinical vari-Wucherpfennig

Decreased progenitor cell proliferation in vitro