Increased interleukin-1 production by peripheral blood mononuclear cells in patients with multiple sclerosis

Journal of the Neurological Sciences. 102(1991) 100-t04

t00

Elsevier

JNS 03493

Increased interleukin-1 production by peripheral blood mononuclear cells in patients with multiple sclerosis Masayuki Matsuda, Naoyuki Tsukada, Koichi Miyagi and Nobuo Yanagisawa Department of Neurology and Health Medical Center, Shinshu University, 3-1-I Asahi Matsumoto 390 (Japan)

(Received 8 July, 1990) (Revised, received 30 October, 1990) (Accepted 5 November, 1990) Key words: Multiple sclerosis; Interleukin-1; Mononuclear cells; Enzyme-linked immunosorbent assay

Summary The production ofinterleukin-1 (IL-1) by peripheral blood mononuclear cells (MNC) was assessed in patients with relapsing multiple sclerosis (MS) in both the active and inactive phase, in chronic progressive MS patients, in other neurological diseases, and in healthy subjects. Production was determined by measuring the IL-1 concentration in cultures with MNC supernatants using enzyme-linked immunosorbent assay (ELISA). IL-1 in sera of MS patients and healthy subjects also was investigated. MNC IL-I~ production was significantly higher in MS patients (180.2 + 177.5 pg/ml) than in healthy subjects (66.2 + 66.0 pg/ml) (P < 0.05). RelapsingMS patients in the active phase had significantly higher MNC IL- 1~ concentrations (360.1 _+ 130.0 pg/ml) than normal subjects (P < 0.001), but MNC IL-I~ production in patients with relapsing MS in the inactive phase (65.3 + 52.8 pg/ml) or chronic progressive MS (80.9 + 7t:9 pg/ml) was not increased significantly. MNC IL-1/3production in MS patients was not elevated significantly. IL-17 and -18 were not detected in sera of MS patients. The correlation between increased IL-1 ~ production and the clinical course of MS suggests that activated MNC may play a role in the pathogenesis of MS.

Introduction Many studies have shown abnormalities of humoral and cellular immunity in patients with multiple sclerosis (MS). Reduction in the number of suppressor T lymphocytes (Reinherz et al. 1980) and increased IgG levels in cerebrospinal fluid (CSF) have been demonstrated (Tourtellotte 1970). Failure to detect specific antibody production in CSF, however, indicates that abnormalities in humoral immunity are due to disturbances in regulatory processes (Paterson and Whitacre 1981). Recent reports have provided data on T lymphocyte activation in patients with MS (Noronha et al. 1980; Golaz et al. 1983). One indication of T lymphocyte activation is the expression of interleukin-2 (IL-2) receptors. Interleukins are factors responsible for T-lymphocyte growth and differentiation. Interleukin-1 (IL-1)is secreted by stimulated antigen-presenting macrophages and provides the second signal for helper T cells after antigen recognition Correspondence to: Dr. M. Matsuda, Department of Neurology, Shinshu University, School of Medicine, 3-1-1 Asahi, Matsumoto 390, Japan. Tel: 0263(35)4600 ext. 2304.

(Oppenheim et al, 1986). IL-2 is secreted by activated T cells and mediates T lymphocyte growth and clonal expansion (Morgan et al. 1976). The generation of IL-1 and IL-2 provides the appropriate conditions for T and B cell activation and the initiation of immune response. IL-1 is also produced by stimulated astrocytes in the central nervous system (CNS) (Fontana and Grob 1984) and regulates a variety of functions, including body temperature and hyperpyrexia (Bernheim 1986). Tumor necrosis factor (TNF) is another endogenous pyrogen which induces production of fever (Dinaretlo et al. 1986). Either or both of these mediators may play a role in MS directly by myelin and oligodendrocytes damage (Robbins et al. 1987; Brosnan et al. 1988) or indirectly by inducing fever (Bernheim 1986; Dinarello et al. 1986), as the neurologic status in MS patients worsens during febrile episodes (Waxman 1987). In this study, we investigate the presence of 1L-1 in the sera of patients with MS and measure its production in cultures of peripheral blood mononuclear cells (MNC) using an enzyme-linked immunosorbent assay (ELISA).

0022-510X/91/$03.50 © 1991 Elsevier Science Publishers B.V. (BiomedicalDivision)

101 Materials and methods

Patients Peripheral blood samples were collected from 22 patients clinically diagnosed as having definite MS using the criteria of Rose et al. (1976), including 13 women and 9men, ranging in age from 24 to 62 years (mean age 42.7 years). These patients were divided into chronic progressive type and relapsing type using the categorization of Schumacher et al. (1965). Inflammatory lesions were comfirmed by magnetic resonance imaging (MRI) or computed tomography (CT) in the CNS in all patients. Eight had chronic progressive MS, while 14 patients had relapsing disease (8 in the active phase, and 6 in the inactive phase). No anti-human T-lymphotropic virus type 1 antibodies were found in the sera of patients with MS. All patients had been offsteroid or immunosuppressive therapy for at least 6 months at the time of sampling. Serum samples were stored at - 7 0 °C until analyzed. Other neurological diseases Samples were obtained from 8 patients with GuillainBarr6 syndrome, and 11 patients with degenerative neurological disease including spinocerebellar degeneration and Parkinson's disease for comparison.

body was added to each well on a plate, and washed 3 times with phosphate buffered saline (PBS). Following this, the reaction with PBS supplemented with condensed blocking solution was allowed to proceed for 4 h at room temperature, each well was washed three times with PBS, and the samples and standard human recombinant IL-I~ (or lfi) were allowed to react overnight at room temperature. After 3 washings with PBS, anti-human IL-I~ (or lfl) rabbit antisera was added and allowed to react for 2 h at room temperature before being washed 3times with PBS. Peroxidase labeled anti-rabbit IgG antibody was added to each well and allowed to react for 2 h at room temperature. o-Phenylenediamine solution was added at room temperature, and the reaction was stopped after 20 rain by the addition of 1 N H 2 S O 4. The absorbance of each well was measured by a Beckman LS-5800 spectrophotometer. A standard curve was constructed, and the IL-I:~ (or lfl) concentration in each sample was determined. The IL-I~ (or lfi) standard curve, control specimens and test samples were assayed in triplicate.

IL-1 a induced by cultured mononuclear cells (pg/m-O) 500

Normal controls Peripheral blood samples from 10 healthy individuals were examined for comparison. These healthy controls were between the ages of 27 and 60 years (mean age 40.5 years). Culture of peripheral blood MNC Blood specimens were collected in heparinized tubes. The MNC were isolated using the Ficoll-Hypaque method. The cells were washed three times with Hank's balanced salt solution (HBSS, Gibco, Grand Island, NY), absorbed through a nylon fiber column to remove B lymphocytes, and suspended in 199 medium (Gibco) supplemented with 10 % heat-inactivated fetal bovine serum (Gibco). The number of viable ceils was determined by trypan blue exclusion and adjusted to a concentration of 2 x 106/ml. The solutions containing MNC were seeded in 100/d/well in Falcon microtiter plates and incubated in 37 °C for 72 h. Then, they were centrifuged at 1700 × g for 30 rain, and their supernatants were harvested and stored at 4 ° C until the IL- 1 assay. IL-1 ELISA We determined the concentration of IL-la and -lfl in each sample using a commercially available ELISA test kit for the quantitation of human IL-1 (Ohtsuka, Tokyo, Japan). Anti-human IL-1~ (or lfl)mouse monoclonal anti-

P < O.05 l

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400

300

200

100

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Multiple sclerosis p a t i e n t s

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Normal c o n t r o l s

Fig. 1. Mean level of IL-I:~ production from MS patients was significantly increased compared to that of controls (P < 0.05).

102

IL-I# induced by cultured mononuclear cells

Results

(pg/me)

IL-I~ and 1# levels in culture MNC Supernatants from cultured MNC from MS patients and normal controls had IL-I concentrations of 180.2 + 177.5 and 66.2 + 66.0 pg/ml(mean _+ SD), respectively (Fig. 1). The concentration in MS patients was significantly higher than normal controls (P < 0.05). Patients with MS were divided into 3 groups" active and inactive relapsing type, and chronic progressive type, with IL-I~ concentrations of 360.1 _+ 130, 65.3 _+ 52.8 and 80.9 + 71.9 pg/ml, respectively (Fig. 2). The concentration during the active phase of relapsing MS only was significantly higher than that of normal controls (P < 0.001). The concentration of IL-I# produced by cultured MNC from MS patients (active, inactive and chronic progressive) and normalcontrols were48.2 + 45.6, 32.2 + 17.8, 49.0 + 56.2 and 16.2 + 17.4 pg/ml, respectively. No significant difference in the concentrations of IL- 1 was present between any group of MS patients and normal controls (Fig. 3). Significant increased IL-I~ levels were also detected in the concentration of patients with Guillain-Barr6 syndrome

50O

400

300

200

100

IL-1 a induced by cultured mononuclear cells (pg/m,~) 500

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active phase inactive phase

P <0.0(]I

' Relapsing type

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type

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Norma~ controls

Multiple sclerosis patients

Fig. 3. No significant difference in the concentrations of IL,1/~ was present between any group of MS patients and normal controls. 400 IL 1Q Induced

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300

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300 200

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200 100 100

. active phase l inactive phaseJ chronic 1 Relapsing type I progressivetype

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Fig. 2. The mean level of IL-I~ production during the active phase of relapsing MS was significantly increased compared to that of controls (P < 0.001 ).

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Fig. 4. Significant increased IL-I~ levels were detectable in the concentrations of patients with Guillain,Barr6 Syndrome (P < 0.001). OND: other neurological diseases,

103 (Fig. 4). No significant differences were found between non-inflammatory degenerative disease and normal controls. Serum leve& of IL-1 and 1 No significant increase in the serum concentration of either IL-I:~ or lfl was demonstrated in patients with MS.

Discussion This study demonstrated a significant increase in IL- 1 production by cultured peripheral blood MNC from patients with MS, with patients in the active phase of relapsing MS showing the greatest increase in IL-I~ production. However, IL-lfi production was not increased, and neither IL-I~ nor lfiwas detected in serum specimens. Changes in IL-2 in patients with MS have been investigated. Elevated levels of serum IL-2 and the released form of the IL-2 receptor have been demonstrated in patients with chronic progressive MS (Trotter etal. 1988; Greenberg et al. 1988). A sensitive ELISA method has been employed to measure IL-2 levels in CSF and sera from patients with MS, and detectable levels of IL-2 were found in samples obtained from patients in the active phase of relapsing MS (Gallo et al. 1988). These results suggest that an active immune mechanism involving IL-2 production takes place within the CNS and that an activated cellular state parallels the evolution of the demyelinating pathologic process. IL-1 also is a type of cytokine believed to be important as a second signal necessary for IL-2 production (Robb 1984). The first signal is antigen presentation to T cells in the context of MHC molecules. IL-1 has been shown to enhance IL-2 production by antigen-activated lymphocytes (Larsson 1980). Changes in the status of IL-1 in patients with MS, however, are not known. Selmaj and colleagues have examined the production of IL-1 and IL-2 by peripheral blood MNC in patients with MS using a biologic assay (Selmaj et al. 1988) but did not find any significant change in IL-1 production. On the other hand, Merrill et al. (1989) investigated inflammatory products involving IL-1 produced by macrophages from blood and CS F of patients with MS and reported that peripheral blood macrophages spontaneously produced elevated levels of IL-I in 40". of MS patients examined. IL-1 is produced mainly by tnacrophages and monocytes. There are several lines of evidence suggesting that macrophages are activated in MS and that they contribute to demyelination (Merrill et al. 1989). The production of IL-1 and IL-2 by peripheral blood MNC stimulated with human myelin basic protein was assessed in vitro and a significant increase in production was confirmed (Selmaj et al. 1988).

Macrophages are activated and produce prostaglandin E and IL-1 in situ in the demyelinating lesion (Hofman et al. 1986). Our data support the idea that systemic activation of MNC involving macrophages occurs in patients with MS. Recently, increased levels of anti-endothelial cell antibodies have been shown in the sera of patients with MS (Tanaka et al. 1987; Tsukada et al. 1989), and attention has focused on the hypothesis that cerebral vascular endothelial cells may play an important role in the initiation and development of immune reactions in CNS. An antigen may be presented to T helper cells in association with Ia determinants on antigen-presenting vascular endothelial cells. Although the effect of IL-1 on vascular endothelial cells is not understood clearly, IL-1 has been reported to make endothelial cells more adhesive to lymphocytes (Pober et al. 1987). Changes in the adhesiveness of the endothelial cell surface also may be correlated with changes in antigen expression on endothelial cells after the interaction between these cells and activated lymphocytes. The results in this present study indicate that increased IL- 1~ levels are clearly not specific for MS, since they were also frequently found in Guillain-Barr6 Syndrome, which is also a demyelinating disease, but it may be produced during an active phase of relapsing MS. The correlation between increased IL-1~ production and the clinical course of MS indicates that activated MNC may play a pathogenetic role in the MS lesions. Acknowledgements This study was supported by grants No. 61570385 and 63570363 from the Ministry of Education and the Intractable Disease Division; Public Health and Welfare of Japan.

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