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Immunopharmacology and Immunotoxicology, 30:915–923, 2008Copyright © Informa UK, Ltd.ISSN: 0892-3973 print / 1532-2513 onlineDOI: 10.1080/08923970802302753
LIPI0892-39731532-2513Immunopharmacology and Immunotoxicology, Vol. 1, No. 1, July 2008: pp. 1–15Immunopharmacology and ImmunotoxicologyInterleukin-10 and Interleukin-12 Modulation in Patients with Relapsing-Remitting Multiple Sclerosis on Therapy with Interferon-beta 1a: Differences in Responders and Non RespondersCytokine Profile in MS after IFNbeta-1a TherapyP.B. Carrieri et al.
Pietro B. Carrieri,1 Paolo Ladogana,2 Gaetano Di Spigna,2 Maria Fulvia de Leva,1 Maria Petracca,1 Silvana Montella,1 Luigi Buonavolontà,2 Ciro Florio,3 and Loredana Postiglione2
1Department of Neurological Sciences, University of Naples “Federico II” , Italy2Department of Cellular and Molecular Biology and Pathology, University of Naples“Federico II”, Italy3Antonio Cardarelli Hospital, Naples, Italy
We examined the effects of interferon (IFN)beta-1a on interleukin (IL)-12p70 andIL-10 secretion in 27 Relapsing Remitting Multiple Sclerosis (RRMS) patients, dividedin responders and non-responders. In responders, IFNbeta-1a does not change theIL-12p70 concentrations, but it leads to a remarkable increase in the IL-10 production.Besides, a high IL-10/IL-12 ratio is demonstrated during the first six months oftherapy. In non-responders, there were not significant alterations in the cytokineprofile. We suggest that IFNbeta-1a effect in RRMS patients could be explained by itsmodifying effect on cytokine pattern. Moreover, we propose a possible role of IL-10/IL-12 ratio as a serum marker predictive of favorable clinical course.
Keywords Multiple Sclerosis, Cytokines, Interferon-Beta.
Address correspondence to Pietro B. Carrieri, M.D., Dipartimento di Scienze Neurolog-iche, University of Naples “Federico II,” Via S. Pansini 5, 80131 Naples, Italy; E-mail:[email protected]
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INTRODUCTION
The pathogenesis of Multiple Sclerosis (MS), the most common central nervoussystem (CNS) demyelinating disease, includes immunomediated as well asneurodegenerative processes. The former are characterized by increase of pro-inflammatory T-helper1 (Th1) cytokines such as tumor necrosis factor (TNF)-α,interleukin (IL)-2, IL-12 and interferon (IFN)-gamma(1,2) and, conversely, down-regulation of anti-inflammatory Th2 ones, such as IL-4 and IL-10.(3)
In MS, IL-12 acts as an inflammatory cytokine inducer through activationand maturation of Th1 cells secreting IFN-γ;(4) increased IL-12 expression hasbeen found to correlate with clinical disability measured by Expanded DisabilityStatus Scale (EDSS)(5) and disease activity assessed by the presence of gado-linium (Gd)-enhancing lesions on brain MRI.(6) IL-10 is a major suppressor ofTh1 cytokines and acts to decrease inflammatory cytokines production bymacrophages.(7) Patients with high IL-10 production have a significant lowerdisability and a lower T2-weighted MRI lesion load.(8)
IFNbeta is a remarkably pleiotropic molecule: antiviral, pro- and anti-proliferative, pro- and anti-apoptotic, and several immunoregulatory activitieshave been described. It has a proven effect on relapsing-remitting (RR) MS,presumably through its influence on T-cell immunity.(9) It down regulates Th1cytokines and causes a shift toward the Th2 phenotype,(10–12) signalingthrough the STAT4 pathway;(13) it also induces IL-10.(14)
The aim of our work is to better characterize the effects of IFNbeta-1a oncytokines in RRMS patients. Therefore, we examined IL-12p70 and IL-10in vitro peripheral blood mononuclear cells (PBMCs) production at the begin-ning of therapy, following it up one year, because IFNbeta-1a effect in RRMSpatients could be explained by its modifying effect on cytokine pattern.
MATERIALS AND METHODS
SubjectsTwenty-nine RRMS subjects (11 men; 18 women; age 20–48 years, mean
36.4), according to McDonald criteria,(15) were enrolled. The pre-treatmentEDSS score was between 0 and 3.5. All patients had experienced two or morerelapses during the two years prior to the study. None had received immuno-suppressant/immunomodulatory therapy in the previous 6 months. Pregnant,breastfeeding, or with other neurological, autoimmune, or infective diseasespatients were excluded. All subjects performed a complete history, neurologicalexamination, serum samples and MRI scanning at baseline, then they weretreated with IFNbeta-1a 22 μg subcutaneously three times weekly.Neurological examination was also obtained 2, 4, 6, 8, 10, and 12 months afterthe beginning of treatment, while MRI scanning was performed at 6 and
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12 months of therapy and in case of appearance of new neurological symptomsor clinical relapses (new or worsening of pre-existing symptoms, lasting ≥ 24hours) during the study course.
MRI scanning was performed and evaluated for T1, T2 and T1 post-Gdsequences by an investigator blinded to clinical and serum data. Methylpredniso-lone (1 g daily iv for three days and 500 mg for other two days) was administeredat the discretion of the evaluating neurologist for relapses with objective findings.
The study population was divided into two groups, responders and non-responders. We considered non-responders patients with new lesions or newGd-enhanced plaques during the study course. Informed consent was obtainedwith the approval of the Ethical Committee of Federico II University MedicineSchool, Naples.
Blood SamplesBlood samples for PBMCs isolation and cytokines determination were
collected prior to the IFNbeta-1a therapy ( just before the first injection) andafter 2, 4, 6, 8, 10, and 12 months, at 8:00 a.m. to avoid daily variation.Samples were coded and stored at −70°C until analyze. PBMCs were isolatedfrom heparinized venous blood by room temperature 20 min 1900 g centrifu-gation over Ficoll-hypaque (Nycomed, Oslo, Norway) (volume 1:1).
Cells were cultured in dish 60 × 15 mm style (Becton Dickinson NJ, USA)plates at a density of 1 × 106/ml in RPMI-1640 containing 2 mM L-glutamine(Life Technologies, Rockville, MD, USA), 50 mM gentamicin, and 5% human ABserum (Sigma, St. Louis, MO, USA), for 72 hours. Supernatants, after roomtemperature 5min 1500g centrifugation, were harvested and stored at −20°C.
Supernatants were measured for IL-10 and IL-12 by enzyme-linkedimmunosorbent assay (ELISA) using the high sensitivity procedure (R&DSystems Minneapolis, MN, USA). The intra-kit variability coefficient was < 5%.450 nm absorbance was measured by a spectrophotometer.
StatisticsStudent’s paired t-test was used to compare IL-12p70 and IL-10 values
and IL-10/IL-12 ratio at different time during IFNbeta-1a therapy. Theobserved differences were considered statistically significant at p < 0,05.
RESULTS
PatientsTwo patients dropped out of the study because of the appearance of side
effects (flu-like symptoms). Seven patients developed new MRI T2 lesions orT1 Gd-enhanced lesions during the study course and they were included in the
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non-responder group (3 men, 4 women; age range: 22 to 50 years, mean 34.5 ys).The responder group was of 20 people (8 men, 12 women; age range: 20 to43 years, mean 35.9 ys).
The mean of pre-treatment EDSS score in the responders was 2.7 at thebeginning and 2.9 at the end of the study. Among the non-responders, instead,the mean of pre-treatment EDSS score was 2.8 at the beginning and 3.4 at theend of the study. The EDSS score at the end of the study was not statisticallydifferent from the initial one in both groups.
IL-10As it is shown in Figure 1, we found in the responders a significant
increase in the IL-10 production assessed 2, 4 and 6 months after the begin-ning of therapy with IFNbeta-1a. The highest values were reached after sixmonths of treatment (305,9 ± 106,3, p < 0,05 respect to basal values). Notably,at the 8th, 10th, and 12th month of therapy IL-10 concentrations were notsignificantly different from the baseline. In the non-responders there were notsignificant differences from the baseline at any time.
IL-12p70In the responders, as shown in Figure 2, IFNbeta-1a did not produce
statistically significant effects on IL-12p70 productive capacity of PBMCs
Figure 1: IL-10 production (pg/ml) from PBMCs, measured by ELISA. The vertical bars representthe means ± SD of 27 RRMS patients (20 responders and 7 non-responders).
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from patients with MS at any time. Also in the non-responders, the level ofIL-12p70 was not significantly different in comparison with baselinevalue.
IL-10/IL-12 RATIOIn the responders, highest values were reached at the fourth (m ± SD,
136,06 ± 195,37; p < 0,05 respect to baseline value) and at the sixth monthof therapy (m ± SD, 225,38 ± 401,04; p < 0,05 respect to baseline value),when compared with baseline values. In the responders group there was notsignificant difference in comparison with baseline values at any time.See Figure 3.
DISCUSSION
MS is thought to be related to the dysregulation of Th1 inflammatorycytokines and Th2 anti-inflammatory cytokines and we found a differentcytokine profile in patients with a stable or active phase of MS.(16) The causeof this imbalance is nowadays unknown, but several hypothesis have beenconsidered.
IFNbeta use in MS patients has been proven to improve clinical andMRI outcomes but, to date, its effective mechanisms in MS are poorlyunderstood. An increasing body of literature supports the hypothesis that
Figure 2: IL-12p70 production (pg/ml) from PBMCs, measured by ELISA. The vertical bars representthe means ± SD of 27 RRMS patients (20 responders and 7 non-responders).
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IFNbeta related changes in IL-12 and IL-10 profile are involved in itstherapeutic benefits.(17) Other authors, moreover, do not find altered IL-10/IL-12 levels.(18,19) IL-10 is an immunosuppressive cytokine essential in themodulation of the immune response, while IL-12 is an inflammatory cytok-ine, acting by inducing a predominant Th1 cytokines.(20) In particular, therole of IL-10 was evaluated in vitro experiments on dendritic cells (DCs).DCs activated in the presence of IL-10 fail to stimulate T cell proliferation,whose injection reduces the severity of experimental autoimmune diseases.IL-10 seems to act by controlling the kinetics and the quality of DC activa-tion.(21) These results might offer significant therapeutic promises: IgGtreatment may help to prevent relapses of MS by controlling DCs matura-tion, and, consequently, inhibiting invasion of immune cells into the CNSand affecting the cytokine profile.(22) It is interesting to note that treatmentwith glatiramer acetate as well, a drug approved for MS, in a mouse modelpromoted development of anti-inflammatory type II monocytes, character-ized by increased secretion of IL-10, and decreased production of IL-12 andTNF.(23)
Our results could help to elucidate IFNbeta-1a action mechanism in MS.In our work, we analyzed cytokine profile in relation to clinical outcome. Ourdata indicate that the treatment with IFNbeta-1a induces a progressiveupregulation of IL-10 until the sixth month of treatment, then decreasingslowly to baseline values, but only in responders. In the group of non-responders,
Figure 3: IL-10/IL-12p70 ratio. The vertical bars represent the means ± SD of 27 RRMS patients(20 responders and 7 non-responders).
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there is no significant increase in IL-10 values during the course of treatment.IFNbeta-1a therapy does not affect IL-12 production, either in responders orin non-responders. These results are in contrast with Graber et al., who foundan increase in IL-10 levels over the course of treatment with IFNbeta, butwith a different kinetic: this increase, in fact, remains constant overthe time.(24)
A significant alteration in the IL-10/IL-12 ratio is associated with differ-ent diseases of likely autoimmune origin, such as wheezing or colitis, orinfectious diseases: a low IL-10/IL-12 ratio may be associated with a severeclinical course.(25) Our data show the increase of IL-10/IL-12 ratio in respond-ers, but not in non-responders, also for MS.
According to our data, we suggest that IFNbeta-1a outcome in RRMSpatients could be explained by its modifying effect on cytokine pattern onlyduring the first months of therapy; later, other mechanisms are probablyinvolved, yet not understood. Leptin, i.e., an adipocyte-derived hormone actingon CD4+CD25+ regulatory T cells (Tregs), whose depletion is associated withautoimmunity also in MS.(26) One of us already demonstrated an inverse rela-tionship between leptin secretion and a reduced function and/or generation ofTreg, suggesting a possible role for leptin in MS pathogenesis.(27) It is thoughtto induce the TNF-alpha, IL-6, and IL-10 production by PBMCs of patients inan acute phase of disease, but not in patients in a stable phase or in healthycontrols.(28)
In conclusion, although the progressively increasing number in literatureof works regarding the cytokine pattern in MS, their role has not been fullyestablished. Our data show that both IL-10 and IL-10/IL-12 ratio increase,due to IFNbeta-1a treatment, could be associated to a favorable clinical out-come; otherwise IL-12 is not influenced by IFNbeta-1a. The association of highvalues of IL-10/IL-12 ratio in MS with more favorable clinical course couldunderline its role as a prognostic marker in MS patients. Nevertheless, fur-ther studies are needed to establish the relationship between cytokine profileand clinical features in different MS stages and subtypes, thus allowing thebest therapeutic choice as possible.
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