Decreased IL-3 production by peripheral blood mononuclear cells in patients with multiple sclerosis

Journal of the Neurologtcal Sctences, 118 (1993) 79-82

79

© 1993 Elsevter Science Pubhshers B V All rights reserved 0022-510X/93/$06.00 JNS 04076

Decreased IL-3 production by peripheral blood mononuclear cells in patients with multiple sclerosis M. H u b e r m a n a, F. Shalit b, I. R o t h - D e r i b, B. G u t m a n a, E. Kott a a n d B. Sredni b a Department of Neurology, Metr Hospital, Kfar Sara, Israel, and b C A I R Instttute, Department ofLtfe Sctences, Bar llan Umverstty, Ramat Gan 52900, Israel

(Received 13 November, 1992) (Revised, received 15 February, 1993) (Accepted 8 March, 1993) Key words. Multiple sclerosis, Interleukin-3, Interleukm-2; Tumor necrosis factor, y-Interferon

Summary The production of interleukin-3 by peripheral blood mononuclear cells (MNC) was assessed in patients with relapsing multiple sclerosis (MS) in both the active and the stable state, and in healthy controls IL-3 levels were compared to levels of production of interleukln-2 (IL-2), tumor necrosis factor (TNF) and T-interferon (-~-IFN). No significant differences in IL-3 levels were observed between stable-state patients and controls When levels of cytokme production of patients in the inactive phase were compared to those of the same patments during relapse a sigmficant decrease m IL-3 levels was observed, as opposed to significant increases m 3,-IFN and TNF levels, and an increase, though a non-slgmficant, m IL-2 levels. The functional slgmficance of lowered IL-3 production is unknown. However, the findings support the hypothesis of a highly complex interaction of overlapping regulatory influences within the cytokine network which parallels MS &sease activity

Introduction Multiple sclerosis, an inflammatory and demyelinating disease of the central nervous system (CNS) has been associated with aberrations in both humoral and cellular immunity. Much evidence has accumulated showing that cytokine secretion by mononuclear cells may be directly related to MS disease activity. Studies have documented a relationship between exacerbations and mitogen-driven production of T-IFN and IL-2 (secreted by activated T lymphocytes) (Beck et al. 1988; Selmaj et al. 1988), prostaglandins and I L - l - a (secreted by activated monocytes) (Dore-Duffy et al. 1986; Matsuda et al. 1991). A later study investigated spontaneous secretion of IL-1/3, T N F - a and P G E 2 and showed that spontaneous cytokine production differed significantly between patients with active and stable MS (Rudick and Ransohoff 1992). In this study we set out to test production levels of IL-3 in MS patients during active and inactive stages, and to compare their levels of production to those of IL-2, T N F and ~/-IFN This human IL-3 was tested by

Correspondence to Dr_ M Huberman, Department of Neurology, Melr Hospital, Kfar Sava, 44281 Israel Tel (52) 900-116, Fax- (52) 900-873

the method of Fishman (Fishman et al. 1990) and is referred to as interleukin-3-1ike activity (IL-3-LA). This human growth factor is spontaneously produced by monocytes and lymphocytes and has been shown to be biologically and blochemically similar to IL-3 As IL-3 is known to control the differentiation of pluripotent stem cells, it was interesting to see whether variation in cytokine production by mature leukocytes In MS disease may stem from differences in IL-3 production levels during the active and inactive stages of the disease.

Patients and methods Patients

Peripheral blood samples were collected from 20 patients clinically diagnosed as having definite MS with a relapsing-remitting disease course using the criteria of Poser et al. (1983), including 12 women and 8 men, ranging m age from 20 to 50 years (mean age 35 7 years). Age- and sex-matched normal control subjects were selected from volunteers. None of the patients or controls were on lmmunosuppressive or steroid therapy. A differential blood count was carried out routinely on all patients and displayed normal values." Each patient investigation included a complete neuro-

80 logical examination, an assessment of the degree of disability using the Expanded Dlsabdlty Status Scale (EDSS) and cytokine secretion studies Eleven patients subsequently developed an acute exacerbation and were reinvestigated within 2 weeks of the relapse

Culture of pertpheral blood MNC H u m a n mononuclear cells (MNC) were separated from fresh heparlnized blood of healthy donors and MS patients by Ficoll-Hypaque density gradient centrffugation (Pharmacia Fine Chemicals, Uppsala, Sweden), as described by Boyum (1969). IL-2 producuon and quanttftcatton H u m a n MNC, 1 5 × 106/ml, were suspended in enriched RPMI-1640 culture medium (Gibco, Grand Island, NY), supplemented with 10% heat reactivated fetal calf serum (FCS) (Biological Sciences, Belt Haemek, Israel), 2 mM L-glutamine, 10 m M non-essential amino acids, 3 m M sodium pyruvate, 5 × 10 -5 2-mercaptoethanol (2-ME), and incubated at 37°C in the presence of 1 / z g / m l phytohemagglutinin (PHA-M; Glbco) for 48 h. Supernatants were collected and stored at 4°C until they were assayed for IL-2 activity. The ability of the supernatant fraction to support the growth of the IL-2-dependent C T L L clone was used to assay IL-2 production (Gilhs and Smith 1977) C T L L cells (10 4 per well) were seeded in tnphcate in culture medmm, with or without dilutions of the supernatant fractions After 48 h, [3H]thymidme uptake was determined m a liquid scintillation counter. One unit of IL-2 activity was defined as the reciprocal log 2 dilution required to give 50% of the maximal proliferation of 104 IL-2 dependent murine C T L L after 48 h of culture IL-3 production and assay H u m a n M N C cells, 3 x 106, were suspended m RPMI-1640 supplemented with 10% FCS and incubated for 48 h at 37°C. After the incubation period, cell viablhty was evaluated using the trypan blue exclusion test. Cell viability was > 90%, with no significant differences among experimental groups. Supernatants were collected and IL-3 activity was assayed by its abihty to stimulate the prohferatlon of the IL-3 responsive cell hne 32-Dc123 as described by Fishman et al (1990). Briefly, 0.1 ml of 32-Dc123 cells (104 per well) were seeded in triplicate in culture medium with or without dilution of the supernatant fraction. Cultures were incubated for 24 h at 37 °. Each well was pulsed with 1 ~Cl [3H]thymldine for the final 6 h of culture Thymidine uptake was determined in a hquid scintillation counter. One unit of IL-3 actwity was defined as the reciprocal log 2 dilution required to gwe 50% of the maximum proliferation of 10 4 32Dc123 cells after

48 h of culture The standard was recombinant murlnc IL-3 (Genzyme, Boston, MA)

TNF secretton and btoassay H u m a n MNC, 1.5 × 10~'/ml, were suspended in enriched culture medium and incubated for 96 h at 37°C in the presence of 1 ~zg/ml PHA. The cytotoxlc activity of the supernatants was tested in the presence of actmomycin-D (Sigma, St. Louis, MO, USA; final concentratlon 1 / z g / m l ) on L929 cells, as reported (Flick and Gifford 1984). T N F levels were defined as the reciprocal of the dilution resulting in 50% cell cytotoxloty IFN-y secretton and btoassay IFN-y levels were tested in supernatants prepared by the same method as that for human TNF. Supernatants were titrated on HeP 2 monolayers by a cytopathogen inhibition test using vesicular stomatitis virus as a challenge. IFN nters were expressed m international units (IU) using natural IFN-y as a reference Stattsttcal analysis Median values were compared by the (nonparametrlc) Wilcoxon test.

Results

Cytokme levels tn cultured MNC Levels of IL-3 production by steady state MS patients were compared to those of age- and sex-matched healthy controls. As can be seen in Fig. 1, no s~gnificant differences were found between levels of IL-3

COMPARISON OF IL-3 PRODUCTION FROM MNC OF CONTROLS AND STABLE STATE MS PATIENTS

40"

30' ¢ 20'

10

Control

Fig

Stable State

1 N o s i g n i f i c a n t d i f f e r e n c e s m IL-3 p r o d u c t i o n w e r e f o u n d b e t w e e n M N C o f c o n t r o l s a n d M S p a t i e n t s in t h e s t a b l e s t a t e

81

(82%, P < 0.01). IL-2 production levels showed an increase of 80%, however the increase was not found to be statistically significant.

IL-3 PRODUCTION FROM MNC OF MS PATIENTS OF THE RELAPSING REMITrlNG TYPE

40

Discussion 30 P=0.05

¢ 20

10

Stable state

Relapse

Fig. 2 Level of IL-3 production was significantly decreased in MS patients during relapse as compared to those in stable state

(p

=

0 05)

secretion in controls versus patients in the steady state (28.0 _+ 3.5 vs. 33.6 _ 6.1 U / m l ) . Comparison of IL-3 production by leukocytes from groups of patients with MS in the active and stable stage of the disease were made. As shown in Fig. 2, there was a significant decrease in IL-3 production level during relapse (33.6_+ 6.1 vs 15.6 5:6.8 U / m l , P = 0.05). Relapse IL-3 levels were significantly different from those of the controls ( P < 0.05). Longitudinal evaluaUon of 11 patients with MS during stable and active phases of the disease (Fig. 3), showed a significant decrease relative to stable state in IL-3 production (64%, P = 0.05) in contrast to a significant increase in y - I F N (127%, P = 0.02) and T N F

In the past few years studies have demonstrated aberrant cytokine production In MS disease related to disease activity. Longitudinal studies have shown a significant increase in y - I F N and T N F - a levels preceding clinical manifestations of exacerbations in MS disease (Beck et al. 1988; Olsson 1992). In accordance with these findings the investigators postulated that T N F a n d / o r y - I F N may trigger exacerbations of MS. Recent studies have indeed shown that these lymphoklnes play a role in the pathogenesis of the disease (Sharief et al. 1991). These data suggest that determination of y - I F N and T N F production capacity could be a means of monitoring disease activity and, more importantly, in predicting the recurrence of relapse in MS patients Our study investigated levels of IL-3 secretion in MS disease as compared to levels of secretion of IL-2, T N F and y-IFN. We demonstrated that there is no significant change m spontaneous IL-3 secretion between controls and steady state patients, as has previously been shown to be the case with the other three lymphokines studied (Beck et al. 1988; Selmaj et al. 1988) However, IL-3 secretion from M N C of patients in relapse was significantly lower than in patients with actwe MS. Although this finding seemed at first to contradict our Intuition, the results are compatible with previous studies which show that monocytes from patients with active disease spontaneously secreted less

COMPARISON OF CYTOKINE SECRETION BETWEEN MS PATIENTS AT THE STABLE STATE AND AT RELAPSE 1501

•

P=0.02

[

100' P
so.

E o g

==

0-

o -50 P=O 05 -100 4

,-I IL-2

IL-3

T-IFN

TNF

Fig 3 Significant decrease m IL-3 production in longitudinal study of MS patients during stable and active states (p = 0 05) as opposed to increase m y-IFN (p = 0 02) and TNF (p < 0 01) production. Change in IL-2 production was not statistically significant

82 TNF and PGE 2 as compared to patients wtth stable MS (Dore-Duffy 1986; Merrill et al. 1983; Rudlck and Ronsohoff 1992). Preliminary study of IL-3 production levels in 5 patients measured after recovery from relapse indicated an increase In levels of IL-3 secretion during recovery with values approaching control levels (data not shown). These results strengthen the observation of decreased IL-3 levels during relapse as compared to inactive disease. Results indicate an inverse relationship between 1L-3 production and that of y-IFN and TNF during relapse. Two explanations may account for th~s: - IL-3 supernatant are spontaneously derived, whereas TNF, IL-2 and y-IFN require the addition of m~togen in wtro Thus it would seem that IL-3 levels could represent the physiological state of the MNC. - it could be that an increase in IL-3 receptors on the activated T lymphocytes during relapse would result in an increased binding of IL-3 to these cells thus causing an artificially reduced level of lymphokmes in the supernatant. Our results confirm that MS disease follows a recurrent course associated with immunological aberrations which can be correlated with disease stage. This suggests that not only TNF and y-IFN, but also IL-3 is an important effector molecule in MS. A deeper understanding of the h~ghly complex interaction of regulatory influences within the cytokme network may enable us to use levels of cytokme secretion as markers of disease activity and to shed new hght on the pathophyslology of multiple sclerosis. Acknowledgement This work was partly sponsored by the Dorsha Wallman Cancer Research Endowment

References Beck, J, P Rondot, L Catmot, E Falcoff, H Klrchner and J Wletzerbm (1988) Increased production of interferon gamma and tumor necrosis factor precedes chnlcal manifestation m multiple sclerosis Do cytoklnes trigger off exacerbations ~ Acta Neurol S c a n d , 7 8 318-323 Boyum, A (1969) Separation of leukocytes from blood and bone marrow Scand J Chn_ Lab Invest., 21 77-80 Dore-Duffy, P , J O Donaldson, T Koff, M. Longo and W Terry (1986) Prostaglandm release m multiple sclerosis_ Correlation with disease activity Neurology, 36:1587-1590 Flshman, P, M Dialdettl, Y Sofer, S Grossman and B Srednl (1990) Spontaneous release of a factor with mterleukm-3-hke activity by human lymphocytes and monocytes Nat Immun Cell Growth Regul, 9 341-345 Fhck, D and G Glfford (1984) Comparison of in vitro cell cytotoxlc assays for tumor necrosis factor J Immunol Methods, 68 167 175 Gllhs, S and K A Smith (1977) Long term culture of tumor specific cytotoxtc T cells Nature, 268 154-156 Matsuda, M , T Naoyukl, K Mlyagl and N Yanaglsawa (1991) Increased tnterleukln-I production by peripheral blood mononuclear cells m patlent~ with multiple sclerosis J Neurol Sct, 102 100-104 Merrdl, J E , R H . Gerner, L W Myers and G W Elllson (1983) Regulation of natural killer cell cytotoxlclty by prostaglandm E m the peripheral blood and cerebrospmal fluid of pattents w~th multiple sclerosis and other neurological diseases J Neurotmmunol, 4 223-237 Olsson, T (1992) Immunology of multiple sclerosts Curr Opinion Neurol Neurosurg, 5 195-202 Poser, C M , D W Paty and L Schelnberg (1983) New diagnostic criteria for multiple sclerosis gmdelmes for research protocols Ann Neurol, 13_ 227-231 Rudlck, R A and R M Ransohoff (1992) Cytokme secretion by multiple sclerosis monocytes. Arch Neurol, 49 265-270 Selmal, K , Z Nowak and H Tchorzewskl (1988) Interleukm-1 and mterleukm-2 production by peripheral blood mononuclear cells m multiple sclerosis patients_ J Neurol Scl, 85 67-76 Sharlef, F, M Phil and R Hentges (1991) Association between tumor necrosis factor-~ and disease progression in patients w~th multiple sclerosis N Engl J Med, 325 467-472