doi:10.1006/cyto.2001.0881, available online at http://www.idealibrary.com on SHORT COMMUNICATION EXPRESSION OF TNF-
mRNA BY PERIPHERAL BLOOD MONONUCLEAR CELLS OF MULTIPLE SCLEROSIS PATIENTS TREATED WITH IFN- 1A Paola Sarchielli,1 Adriana Critelli,1 Laura Greco1 Eva Sokola,1 Ardesio Floridi,2 Virgilio Gallai1 The aim of the present study was to verify the expression of tumour necrosis factor (TNF)-
mRNA by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in unstimulated peripheral blood mononuclear cells (MNCs) of 15 relapsing-remitting multiple sclerosis (MS) patients who underwent treatment with IFN- 1a (6 millions of international units (MIU) i.m. once a week) and in 15 untreated MS patients matched for age and expanded disability status score (EDSS). At the same time the expression of TNF-
mRNA was assessed in 10 healthy age-matched control subjects. All MS patients were assessed at the basal time and after 6 months. At the basal time, the band of TNF-
mRNA was detectable in 12 out of the 15 untreated patients and in 13 out of the 15 patients who underwent IFN- 1a treatment. The higher TNF-
mRNA was evident in patients with gadolinium-enhancing lesions. At the 6-month follow-up, 13 out of the 15 untreated patients still had detectable values of TNF-
mRNA and no significant difference emerged when compared with basal time. On the contrary, the expression of TNF-
mRNA was absent at the same time in nine out of the 15 patients treated with IFN- 1a. A longitudinal analysis carried out monthly in eight MS patients (four untreated and four treated) revealed a transient increase in TNF-
mRNA expression in MNCs of all four treated patients in the first 3 months, supporting previous findings of an early immunoenhancing effect of IFN- 1a. This early activation is followed by an inhibitory effect of IFN- 1a on TNF-
mRNA expression in about 2/3 of treated MS patients when assessed at 6 months. Further long-term studies are needed to confirm this immunomodulatory effect of IFN- 1a not only on TNF-
but also on other cytokines of Th1 and Th2 types.  2001 Academic Press Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system characterized by a series of immuno-mediated events culminating in the destruction of myelin and olygodendrocytes in the white matter.1 Among pro-inflammatory cytokines, a pivotal role in MS pathophysiology is played by tumour necrosis factor (TNF)-
as demonstrated by in vitro and in vivo From the 1Department of Neuroscience, Neurological Clinic and 2 Department of Internal Medicine, Laboratory of Biochemistry, University of Perugia, Italy Correspondence to: Paola Sarchielli, Neurological Clinic, Department of Neuroscience, Policlinico Monteluce, Via E. Dal Pozzo 06126 Perugia, Italy. E-mail: neuro@netemedia.net Received 9 August 2000; received in revised form 24 January 2001; accepted for publication 18 March 2001  2001 Academic Press 1043–4666/01/110294+05 $35.00/0 KEY WORDS: multiple sclerosis/TNF-
/mRNA/IFN- 1a 294 studies in the experimental allergic encephalomyelitis and in MS. This 17 kiloDalton (kDA) cytokine is produced by many cell types including macrophages, T and B cells. The pleiotropic effects of TNF-
include myelinotoxicity, upregulation of MHC class II molecules in a wide range of cell types, even astrocytes, and induction of adhesion molecules on endothelial cells. TNF-
exerts these effects in concert with other cytokines such as interferon (IFN)- contributing to mediate the inflammatory process underlying demyelinating lesions in the central nervous system (CNS) of MS patients. Although there are conflicting data on the presence of soluble cytokines in serum and cerebrospinal fluid of MS patients, several observations indicate an increased production of TNF-
in both these body fluids in the active phase of the disease.2,3 CYTOKINE, Vol. 14, No. 5 (7 June), 2001: pp 294–298 TNF-
mRNA in IFN- 1a-treated MS patients / 295 More recent research showed increased expression of TNF-
and lymphotoxin mRNA by mononuclear cells (MNCs) and MBP+ T cell clones in MS patients, particularly those in relapse or with a severe and progressive form compared to those with a stable clinical disease.4–7 Treatment with recombinant IFN- both 1a and 1b has been demonstrated to reduce the frequency and severity of relapse and lessen the lesional load in patients with MS. The immunomodulating effects of IFN- in patients with multiple sclerosis involves many aspects of the immune function, but the results on cytokine profile variations, including that of TNF-
, are contradictory. The present research was focused on the effects of IFN- 1a on TNF-
during the first 6 months of treatment. TNF-
mRNA expression was assessed in peripheral blood MNCs of 15 MS patients with relapsing-remitting (R-R) form, using RT-PCR comparing the results with those of a group of 15 untreated R-R MS patients, matched for age and disability score. RESULTS In 12 out of the 15 untreated patients and 13 out of the 15 patients who underwent IFN- 1a treatment, the band of TNF-
was detectable at the basal time. Only five control subjects showed TNF-
expression in their MNCs without evidence of infections or other possible causes of induction of cytokine secretion (Fig. 1). The semiquantitative analysis of PCR products revealed significantly higher levels of TNF-
in untreated and treated patients (14.25.4 and 16.86.3 relative units) compared to the control subjects showing detectable values of TNF-
mRNA (2.33.4 relative units) (P<0.0001). In both treated and untreated MS patient groups there was a significant difference, at the basal time, in the mRNA expression pattern between patients with gadolinium enhancing lesions and patients without active lesions (treated MS patients: 21.66.7 vs 7.43.2, two untreated MS patients: 4.37.9 vs 9.83.6, P<0.0001 and P<0.0002 respectively). Thirteen out of 15 untreated patients showed detectable values of TNF-
mRNA at the 6-month follow-up without significant difference compared with basal time (16.16.9). On the other hand, in nine out of the 15 patients treated with IFN- 1a the expression of TNF-
mRNA was absent at the 6-month examination (Fig. 2). In the remaining six patients treated with IFN- 1a the values of relative units of TNF-
were significantly lower (9.43.5) than those found in the same patients at the basal time (22.67.4) (P<0.0001) but they were, in S CA CT PA PT 760 bp 363 bp Figure 1. Ethidium bromide-stained gel showing the expression of TNF-
mRNA in an untreated MS patient with active lesions at MRI. Both in the patient and control a 760 bp band is present corresponding to -actin. In the patient a 363 bp band is evident, corresponding to TNF-
mRNA. S=standard; CA=control subject: -actin; CT=control subject: TNF-
; PA=untreated MS patient: -actin; PT=untreated MS patient: TNF-
. S CT PT CA PA 760 bp Figure 2. Ethidium bromide-stained gel showing the expression of TNF-
mRNA in an MS patient treated with IFN- 1a. Both in the treated patient and control subject a 760 bp band is present, corresponding to -actin, whereas the 363 bp band corresponding to TNF-
mRNA is not evident. S=standard; CA=control subject: -actin; CT=control subject: TNF-
; PA=MS patient treated with IFN- 1a: -actin; PT=MS patient treated with IFN- 1a: TNF-
. 296 / Sarchielli et al. CYTOKINE, Vol. 14, No. 5 (7 June, 2001: 294–298) TABLE 1. Mean values (SD) of TNF-
mRNA expression in PBMCs of four untreated MS patients, four treated MS patients and four control subjects Untreated MS patients MS patients treated with IFN--1a Control subjects 1st month 2nd month 3rd month 4th month 5th month 6th month 28.26.4 25.28.5 3.31.2 27.27.6 29.67.5 3.21.4 28.87.4 37.49.6 3.31.5 28.96.6 24.47.2 3.11.6 27.55.9 15.54.2 2.91.1 27.75.2 10.63.4 3.01.3 All untreated patients had gadolinium enhancing lesions at the basal time. Three of these patients had active scans at the 6-month MRI follow-up. One patient had a clinical relapse between the 3rd and the 4th months and an increase in the value of TNF-
mRNA expression in PBMCs, revealed in the 3rd month (at least 15 days prior the clinical relapse). Three out of the four patients who underwent treatment with IFN- 1a had active lesions on MRI at the basal time. A significant increase in the levels of TNF-
mRNA expression in PBMCs was observed in the 2nd, and to a greater extent in the 3rd month in all treated patients even if none of them had an acute clinical relapse. These levels tended to progressively decrease in the following months reaching values significantly lower than those found at the basal time (P<0.01). DISCUSSION The results of the present research confirm the expression of TNF-
mRNA in the MNCs of MS patients at the basal time and its relationship to disease activity evidenced by enhancing lesions even if no clinical relapse was present. This expression was also maintained at the 6-month follow-up in 1/3 of patients, who showed gadolinium-enhancing lesions at MRI. As well as the effect of treatment with IFN- 1a on the TNF-
mRNA expression in MNCs of MS patients, we found a temporary increase in the expression of mRNA of TNF-
in a subgroup of IFN-treated patients during the first phase of the treatment compared with the basal time. This can be interpreted as the expression of an early rise in MS activity which 45 40 TNF-α mRNA (relative units) any case, significantly higher than those detected in the control group (2.31.4, P<0.0002). Five of these patients had gadolinium enhancing lesions at the magnetic resonance imaging (MRI) treatment at the start of monitoring. A longitudinal analysis of TNF-
was performed monthly in four untreated MS patients, in four patients who underwent treatment with IFN- 1a and in four control patients who had detectable mRNA of TNF-
. One out of four untreated patients had a clinical relapse during the longitudinal monitoring. An increase in the expression of TNF-
mRNA was detected in this patient, evident just 2 weeks before the clinical relapse (at the time of blood drawing). This expression returned to previous values in the month following the recovery from relapse. This finding could be representative of the raised amount of activated cells expressing the mRNA for this pro-inflammatory cytokine in peripheral blood, which can be stimulated via external triggers, enter in the brain, recruit other immunocompetent cells and induce the immune cascade involved in the formation of demyelinating lesions. All treated patients had active lesions at the MRI examination at the beginning of monitoring. None of these patients had a clinical relapse during the treatment period. In the first 3 months all treated MS patients showed a transient increase in TNF-
relative units compared to the basal time. These values lowered after this period but did not reach the value of the control subjects (Table 1). The variations in mRNA of TNF-
expression in untreated and treated MS patients and in control subjects examined monthly are shown in Fig. 3. The stimulation of MNCs with LPS induced the expression of mRNA of TNF-
both in the control subjects and in untreated and treated patients who did not show the amplification product of TNF-
before stimulation. 35 30 25 20 15 10 5 0 –5 1 2 3 4 Time (months) 5 6 Figure 3. Plot of means of relative units of TNF-
mRNA in four IFN- 1a-treated MS patients, four untreated patients and four control subjects examined monthly during the entire period of the study. Untreated MS patients, — — IFN--treated MS patients, — —; control subjects, — —. TNF-
mRNA in IFN- 1a-treated MS patients / 297 was also suggested in the first months of treatment by both MR spectroscopic study11 and immunological data.12 The long-term effect of the IFN- 1a treatment consists of the inhibition of TNF-
mRNA expression in at least 2/3 of MS patients treated. This effect could be due to an inhibiting action exerted by IFN- 1a on IFN- production, which is known to induce TNF-
production both in peripheral blood and brain. In addition, the decrease in the expression of TNF-
mRNA after stimulation with lypopolysaccarides in MS patients treated for 6 months with IFN- could be ascribed to an inhibition of the LPS receptor expression on the membrane of peripheral blood MNCs or, alternatively, to the drastic reduction of the activated blood cells expressing the TNF-
mRNA in peripheral blood. Whether this effect is secondary to other events involving the complex circuitry of immune dysregulation in MS remains to be established. The profile of other cytokines can be modified by long-term treatment with IFN-, including an increase in immunoregulatory cytokines, but this finding has not been consistently demonstrated in studies carried out on this topic. The conflicting results could be due to different methods and to the different in vitro and in vivo approaches. The latter could be more expressive of changes of immune function in MS patients due to immunomodulatory treatment and should be preferred for future research. standardised procedures suggested by the guidelines of the MS/ MR Steering Committee of Concerted Action on MS.10 The presence and number of Gaololinium–diethylenetriominepentacetic acid (Gd-DTPA)-enhancing lesions was recorded. RT- PCR for mRNA expression of TNF-
Blood was drawn at the basal time and after 6 months in both treated and untreated MS patients, and in the former at least 72 h after the weekly administration of IFN- 1a, thereby avoiding acute effects on TNF-
mRNA expression in MNCs. Four IFN- 1a-treated patients, four untreated patients and four controls underwent a monthly longitudinal analysis of TNF-
mRNA expression during the entire period of the study. RNA extraction, reverse transcription and polymerase chain reaction (PCR) were carried out using the method described by Rieckmann et al.5 with minor modifications. Primers from published sequences which covered a splice junction and amplified only cDNA and not genomic DNA were used. The sequences of the primers are: -actin: 5 -ACGGGTCACCCACATGTG-3 3 -CTAGAAGCATT GCGGTGGACGATG-5 TNF-
: 5 -TGAGCACTGAAA GCATGATC-3 3 -TTATCTCTCAGCTCCACGCC-5 . Values were given as relative units and were calculated as density of TNF-
and actin amplification products ratio 100. Positive controls (LPS-stimulated cells) and negative controls (Buffer alone and RNA) were included in each experiment. The expression of TNF-
mRNA was also evaluated in 10 age-matched healthy control subjects (five males and five females, mean age: 31.34.8 yrs). Statistical analysis MATERIALS AND METHODS Statistical analysis included Tukey test and Fisher’s least significant difference (LSD). Five per cent for two-sided tests was chosen as a minimum level of statistical significance. We Patients and control individuals thank Nella Perugini for the technical assistance. The protocol was approved by the local ethical committee and patients gave their written informed consent. The study was performed on 30 patients with definite MS according to Poser et al.’s8 criteria for at least 2 years and a baseline EDSS 9 between 1.0 to 3.5 inclusive. Fifteen R-R MS patients (five males and 10 females, mean age: 30.44.2 years) underwent treatment with IFN 1a, the other 15 patients (5 males and 10 females, mean age: 32.15.6 years) were untreated. The duration of disease was: 6.013.0 years in the untreated group and 5.513.5 years in the treated group. None of the patients had undergone immunosuppressant therapy within the 6 months prior to the study, nor adrenocorticotrophin hormone or corticosteroid treatment within 2 months prior to the beginning of the study. In the treated group, IFN- 1a (Avonex, Dompé Biotec SpA, Milan, Italy), glycosylated recombinant IFN- analogous to the natural sequence, was administered intramuscularly at the dosage of 30 g (6.0 millions of international units) (MIU) weekly, for up to 24 weeks. Both treated and untreated MS patients underwent clinical examination and MRI at the basal time and also during any occurring relapses. Brain MRI was performed according to the REFERENCES 1. Steinman L (1996) Multiple sclerosis: a coordinated immunological attack against myelin in the central nervous system. Cell 85:299–302. 2. Beck J, Rendot P, Catinot L, Falcoff E, Kirchner H, Wietzerbin J (1988) Increased production of interferon- and tumor necrosis factor precedes clinical manifestation in multiple sclerosis: Do cytokines trigger off exacerbations? Acta Neurol Scand 78: 318–323. 3. Sharif MK, Hentgens R (1991) Association between tumor necrosis factor-
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