Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) in which the cytokines and the inflammatory process are key factors. Cytokines like interleukin (IL)-4, IL-2, or interferon gamma (IFN-γ) have been widely studied in MS because of their immunomodulatory role. A critical step in the inflammatory process underlying MS pathology is immune cell trafficking across the blood–brain barrier, and this is associated with worsening clinical symptoms. Natalizumab (Tysabri^®, Biogen Idec, CA), a monoclonal antibody designed against the α4 subunit of α4β1 (very late antigen-4; VLA-4) and α4β7 receptors located on the lymphocyte surface, has demonstrated efficacy in reducing disease activity and relapse frequency in MS patients and is widely accepted as a drug for the relapsing–remitting subtype of multiple sclerosis (RRMS)[¹–³]. The humanized IgG4 specifically binds to the surface of mononuclear circulating blood cells presenting α4, thus inhibiting the adhesion to the VCAM at the endothelium and decreasing extravasation to adjacent tissues. In the context of MS, which is usually considered a T cell autoimmune disease affecting the CNS, the beneficial effects observed with the treatment have been attributed to immunosuppression by a blockade on T cell trafficking through the endothelium [⁴, ⁵].

The clinical improvement and efficacy observed in natalizumab treatment are counter-weighted by important side effects including opportunistic infections and also cases of fatal progressive multifocal leukoencephalopathy (PML) detected during the clinical trials [¹]. Some questions still remain unresolved as the effect of natalizumab in the first days and months of treatment when allergic reactions and acute infusion reactions can occur [⁶]. Antibody binding to the VLA-4 receptor may not only play a role in inhibiting the inflammation process but also have effects on the immune system itself.

Although the mechanism of action of the drug is supposed to be clear, the biological effects of integrin α4 blockade or T cell traffic inhibition are still unknown. Already existing drugs for MS like interferon-beta or glatiramer acetate seem to modify CD4⁺ CD25⁺ Foxp3 regulatory T cell activity [⁷–¹⁰] or promote the anti-inflammatory cytokine pathway [¹¹–¹⁴]. The goal of our study was to gain insight into the immunological effects of natalizumab treatment in RRMS patients over a period of 20 months with a focus on pro-inflammatory and anti-inflammatory cytokine protein levels as well as regulatory T cell frequency and suppressor function.

This is the first study to show the effects of natalizumab over a period of 20 months with a special focus on the first hours, days, and months after initiating treatment. At the protein level, we have detected that natalizumab therapy is associated with an increase of pro-inflammatory and anti-inflammatory cytokines within the first 2 months of treatment. This affects IFN-γ and IL-12p70 and also the anti-inflammatory IL-4 or IL-10. Interestingly, important cytokines for MS such as IL-2, IL-17, or IL-1β were only elevated after 1 year of treatment, suggesting different immunologic mechanisms. It would be interesting to further analyze whether the reduction to basal levels in the cytokines after the first 2 months of treatment observed in our study might be associated with the recently described reduction in CD49d antigen expression and therefore a possible reduction of antibody binding in circulating cells [¹⁸]. A recent work has reported increased levels of T cells secreting pro-inflammatory cytokines in blood circulation in patients after 1 year of natalizumab treatment [¹⁷]. In agreement with these results, we observed elevated serum levels of pro-inflammatory IL-2, IL-17, and IL-1β after 1 year.

In our study of cytokines based on serum samples, we have also taken into account the first hours and days after the first administration of the antibody, when we were able to detect differences in cytokine levels such as IFN-γ, IL-4, or IL-10. A recent paper studying cytokines in patients with MS at baseline and after 1 year did not address some of the cytokines included in our study such us IL-17, IL-12p70, or IL-13 [¹⁹]. Our study has detected increased levels for IL-1β, IL-2, and also IL17 in the long-term treatment. It would be interesting to analyze in the future if the IL-23, implicated in Th17 cell induction, would also be increased with natalizumab. Another recent paper has reported increased levels of CD4⁺ T cells producing pro-inflammatory IFN-γ, tumor necrosis factor (TNF), and interleukin (IL)-17 upon stimulation in vitro with anti-CD3 in blood samples from patients after 6 months treatment with natalizumab [²⁰]. Those authors have also found that the CD4⁺ markers CD25, HLA-DR, and CCR6, as well as the frequency of CD8⁺ T cells producing IL-2 and IL-17, were increased. In agreement with some of these observations, our results also suggest elevated IL-17 levels at the long-term treatment with natalizumab. Although our observations require replication with larger numbers of patients for confirmation, Natalizumab treatment did induce some modifications in cytokine patterns in our cohort of RRMS patients. Furthermore, these responses seemed to be different at several stages; there was a rapid rise in cytokines after the first administration when rejection and allergic reaction might occur, whereas other cytokines were only elevated at the long-term treatment when the risk of opportunistic infections or PML risk is higher.

Natalizumab was not designed to have biological effects upon binding to α4 but rather to physically interfere with the endothelial attachment of VLA-4⁺ cells [¹]. However, previous studies in rats suggested that antibody binding to VLA-4 might mediate VLA-4⁺ cell stimulation [²¹, ²²]. As we observed different cytokine response patterns in patients, we decided to test whether natalizumab exposure might affect mononuclear cells in vitro. Previous groups have addressed this question for CD4+ T cells [¹⁷]. Since the VLA-4 antigen is present in many blood cell subtypes, we co-cultured total purified PBMCs in the presence of physiological concentrations of natalizumab. We did not detect significant effects on proliferation, and we did not observe differences in cytokine patterns when the antibody was present. Therefore, if natalizumab does have any effect on blood cells, these effects might be more than just on proliferation or cytokine production and should be further addressed. It would be important to mention that this effect (if it exists) might depend on the subject or patient studied (note that we only used healthy donors because of the heterogeneity of RRMS patients).

In accordance with recent studies, we found that natalizumab does not affect regulatory T cell function. A recent study showed that the capacity to migrate across the blood brain barrier and suppressive function as well as foxp3+ cell frequency seem to not be affected by natalizumab [¹⁸]. At this time, there are contradictory results on the effect of natalizumab on the frequency of circulating regulatory T cells and another group has reported a relative decrease in CD4+ CD25^high cells [²³]. However, none of these studies have found any effect on the suppressor function of the cells [¹⁸, ²³]. In our case, we detected a relative tendency to a decrease in the frequency of CD4+ CD25 FoxP3+ cells within the first month (not significant); however, we also did not obtain any difference in the suppressor function of these cells in comparison with baseline levels. It would also be interesting to consider other regulatory T cell markers in our study like the ILR-7 receptor (CD127) or the CD39 ectonucleotidase [²⁴], but, unfortunately, this was not considered at the beginning of our study. Already existing drugs for MS like interferon-beta and glatiramer acetate seem to modify CD4⁺ CD25 ⁺ Foxp3 regulatory T cell activity [⁷–¹⁰]. Regulatory T cells have been described as important players in controlling immune response and balancing tolerance versus autoimmune responses [²⁵, ²⁶]. Our results, in accordance with some of the previous reports suggest that natalizumab seems to exert its beneficial properties independently of affecting regulatory T cell function.

In summary, treatment with natalizumab in RRMS patients induces changes in the inflammatory mechanism. While IFN-γ, IL-12p70, IL-4, and IL-10 were elevated just a few hours or days after first IV infusion, pro-inflammatory cytokines like IL-1β, IL-2, and IL-17 were elevated only at the long-term treatment suggesting different immune mechanisms. We have found that globally the treatment with the antibody does not modify regulatory T cell function. All these results suggest other immunological effects of natalizumab in addition to VLA-4 interaction and CNS extravasation inhibition. If these changes in the cytokine equilibrium are related to beneficial or negative side effects associated to natalizumab is as yet unknown and requires further investigation.

This work was supported by a grant from Biogen Idec. We thank the Neurology Department and Neuroscience Research Laboratory staff at La Paz University Hospital and La Paz Research Institute (Idipaz). We also want to thank all the MS patients and healthy donors who voluntarily and generously participated in the study.

Conflict of Interest Statement E. Diez-Tejedor has collaborated as clinical advisor investigator in clinical trials as speaker with the following companies: Astra-Zeneca, Bayer, Bristol-Myers Squibb, Boehringer Ingelheim, Cellerix, Ferrer Grupo, Knoll, Lilly, Parke-Davis, Pfizer, Sanofi-Synthelabo, Servier, UCB Pharma, Uriach, and EVER Neuro Pharma. C. Oreja-Guevara has collaborated as consultant and in clinical trials with Biogen Idec, Merck Serono, Teva, Bayer-Schering, and Novartis.

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