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NK cells and their receptors in naive and rituximab-treated patients with anti-MAG polyneuropathy
Journal of the Neurological Sciences 331 (2013) 86–89
Contents lists available at SciVerse ScienceDirect
Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
NK cells and their receptors in naive and rituximab-treated patients with anti-MAG polyneuropathy Luana Benedetti a,⁎, Monica Facco b, Diego Franciotta c, Chiara Dalla Torre d, Marta Campagnolo d, Marta Lucchetta d, Elisa Boscaro b, Mario Ermani d, Massimo Del Sette a, Tamara Berno b, Laura Candiotto b, Renato Zambello b, Chiara Briani d a
Department of Neurology, Osp. S. Andrea, La Spezia, Italy Department of Medicine-DIMED, University of Padova, Italy c Laboratory of Neuroimmunology, IRCCS, National Institute of Neurology ‘C. Mondino’, Pavia, Italy d Department of Neurosciences, University of Padova, Italy b
a r t i c l e
i n f o
Article history: Received 17 January 2013 Accepted 10 May 2013 Available online 10 June 2013 Keywords: Natural killer (NK) cells CD94/NKG2A Myelin associated glycoprotein Anti-MAG antibodies IgM Monoclonal gammopathy Rituximab
a b s t r a c t Background: Natural killer (NK) cells can bridge innate and acquired immunity, and play a role in autoimmunity. A few studies evaluated the distribution of NK cells and the expression of their receptors in chronic immune-mediated demyelinating polyneuropathies. We investigated NK cell distribution and NK cell receptor expression in 20 naïve patients with anti-MAG polyneuropathy (MAG-PN). Methods: Using flow cytometry, we analysed NK cells and a series of NK cell receptors in the peripheral blood of patients with MAG-PN, and, as controls, in patients with chronic inflammatory demyelinating peripheral polyradiculoneuropathy (CIDP) and in healthy subjects. Six MAG-PN patients were also tested after rituximab treatment. Results: At baseline the percentage of NK cells did not differ among the groups. KIR2DL2 receptor expression in MAG-PN patients was higher, andCD94/NKG2A receptor expression in both MAG-PN and CIDP patients was lower than in healthy controls. These abnormalities did not correlate with any clinical or demographic variable. No modification was found after rituximab therapy. Conclusions: The data suggest that MAG-PN shows abnormalities in NK cell receptors that characterise other autoimmune diseases, and cannot help in differential diagnosis with CIDP. The impairment of the relevant CD94/NKG2A inhibitory pathway, which might play a central role in the development and perpetuation of MAG-PN, warrants further functional investigations. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Chronic demyelinating polyneuropathy associated with anti-myelinassociated glycoprotein (MAG) antibodies and IgM paraproteinemia (MAG-PN) is a disease characterised by prominent sensory signs and symptoms, which is considered different from chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), notwithstanding the two diseases share an immune-mediated pathogenesis [1]. Dissimilarly from CIDP, MAG-PN usually manifests with a slowly progressive course, and poorly responds to conventional immunomodulating treatments [2]. MAG is a glycoprotein expressed in the myelin of the peripheral nervous system (PNS), whose HNK-1 extracellular epitope is the target of the anti-MAG antibodies [2]. Interestingly, HNK-1 is broadly expressed in the central nervous system (CNS) grey matter, and it is also a marker of human natural killer (NK) cells [3]. ⁎ Corresponding author at: Department of Neurology, Osp. S. Andrea, Via Vittorio Veneto, La Spezia, Italy. Tel.: +39 0187533311–3498110936; fax: +39 0187533024. E-mail address: [email protected] (L. Benedetti). 0022-510X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jns.2013.05.015
NK cells are lymphocytes that bridge innate and acquired immunity. They contribute to both effector and regulatory functions of innate immunity through the cytotoxic activity and the production of pro- and anti-inflammatory cytokines and chemokines [3]. The latter function mainly accounts for their regulatory role in acquired immunity. To prevent autoimmune reactivities, NK cell functions are tightly regulated by the integration of signals coming from inhibitory and activating receptors [3]. Among the inhibitory receptors, the killer cell immunoglobulin-like inhibitory receptors (KIR) bind some classical MHC class I molecules, and the CD94/NKG2A receptors bind the non-classical MHC class I molecules, whereas the NK cell protein 30 (NKp30), NKp44, and NKp46 represent the main receptors with activating functions [3]. NK cells seem to act as a two edged weapon, playing opposite roles with both regulatory and inducer activity even in the same disease; accordingly, a defined role for such cells and their receptors in autoimmunity has not been clearly established, and discordant data on their modifications in systemic and CNS autoimmune diseases have been reported [4,5]. Conversely, only few data are available in the PNS autoimmune diseases [6].
L. Benedetti et al. / Journal of the Neurological Sciences 331 (2013) 86–89
We studied NK cell distribution and NK cell receptor expression in patients with MAG-PN, and, as controls, in patients with CIDP and in healthy subjects. Some MAG-PN patients were followed up to evaluate both NK cell and receptor modifications after treatment with rituximab, a B-cell depleting drug, whose therapeutic action could be partially mediated by NK cells too [7]. 2. Materials and methods 2.1. Patients and controls Twenty, naïve to therapy patients with MAG-PN (15 men, 5 women, mean age 70.1 ± 9.9 years; range, 50–89), whose clinical and demographic characteristics are shown in the table (Supplementary material), were studied. The diagnosis was based on accurate clinical history, neurophysiologic studies, and high-titer positivity of anti-MAG antibodies [8]. All patients underwent neurological evaluation, and severity of the disease was scored through the inflammatory neuropathy cause and treatment (INCAT) scales for arm and leg disabilities [9]. Sex and agematched controls included 9 patients with CIDP [10], and 12 healthy subjects (HS). Six MAG-PN patients, who were treated with rituximab, 375 mg/sq for four consecutive weekly infusions, were prospectively followed up for one year; in these patients, neurological assessment, peripheral blood white cell immunophenotype and NK receptor characterization were repeated 6 and 12 months after rituximab treatment. The local ethical committee approved the study. 2.2. Immunophenotype of NK cells, and expression of NK cell receptors Peripheral blood mononuclear cells (PBMCs) were obtained by centrifugation on a Ficoll–Hypaque gradient. A panel of commercially available florescein isothiocyanate- (FITC), phycoerythrin- (PE), PE Cy5 and allophycocyanin-conjugated (APC) mouse anti-human CD3, anti-CD4, anti-CD57, anti-CD8, anti-CD56, anti-CD16 mAbs, with the specific isotype-matched control reagents (BD Pharmingen San Diego, CA, USA; R&D Systems, Toronto, Canada; Caltag Laboratories; Immunotech, Marseille, France) was used. The following mAbs to NK receptors were kindly provided by Drs. A. Moretta and M. Vitale (National Institute for Cancer Research, Genoa, Italy): EB6 (IgG1, anti-KIR2DL1, and anti-KIR2DS1),GL183 (IgG1, anti-KIR2DL2, antiKIR2DL3, and anti-KIR2DS2), FES172 (IgG2a, anti-KIR2DS4), Z27 (IgG1, anti-KIR3DL1, and anti-KIR3DS1),Q66 (IgM, anti-KIR3DL2), XA185 (IgG1, anti-CD94), and Z199 (IgG2b,anti-NKG2A). The expression of the studied antigens on PBMCs was analysed by flow cytometry, with direct or indirect immunofluorescence assays, as previously reported [11]. The analyses were performed on freshly recovered lymphocytes. Briefly, cells were stained with the appropriate mAbs, either unlabeled or labelled; staining with unlabeled mAb was followed by PE- or FITC-conjugated isotype-specific goat anti-mouse second reagent (Southern Biotechnology, Birmingham, AL, or Caltag, Burlingame, CA). A PE-Cy5 CD16 was set, and the status of the antigens of interest was analysed only on CD16+ gated cells. For fluorescenceactivated cell sorter analysis, 1 × 104 cells were acquired, and the analyses were determined by overlaying the histograms of samples stained with the different reagents. Stained cells were scored using a FACSCalibur analyser (Becton Dickinson). Samples were analysed by four colour cytofluorimetric analysis, and data were processed using CELLQuest software (Becton Dickinson). Values were given as % positive staining after subtraction of isotype control. 2.3. Statistical analysis Kruskal–Wallis with Dunn's post test was used to compare the between-group mean differences, Friedman with Dunn's multiple comparison test for repeated measures, and Spearman test for
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correlations. Data are reported as means ± SD. p b 0.05 was considered as significant. 3. Results The frequencies of CD16+ NK cells were similar in the three studied groups (MAG-PN, 16.7% ± 6.4; CIDP, 14.2% ± 5.4; HS,18.2% ± 5.9), although a trend for higher NK cell percentages, at baseline, was observed in about half of the MAG-PN patients; these patients' clinical, demographic and serological features, which included the anti-MAG antibody titers, did not differ from those of the patients with lower percentages (data not shown). In the rituximab-treated patients, there were no significant differences in the NK cell proportions, at baseline vs at 6 or 12 months after the therapy (data not shown); all but one of the 6 patients responded to rituximab, and therefore a comparison between responders vs non-responders was not applicable. Among the analysed receptors, KIR2DL2, KIR3DS1, and CD94/NKG2A were consistently detected in all the patients and controls. KIR3DS1 receptor was not differently expressed in the three studied groups (MAG-PN, 25.0% ± 23.7; CIDP, 23.4% ± 23.4; HS, 24.8% ± 7.6). KIR2DL2 receptor expression in MAG-PN patients (29.3% ± 16.8) and in CIDP patients (24.7% ± 15.7) was higher than in HS (15.5% ± 11.1), but the difference was statistically significant for the MAG-PN patients only (p = 0.012) (Fig. 1, panel a). CD94/NKG2A receptor expression in both MAG-PN (31.8% ± 14.8) and CIDP (29.2% ± 22.3) patients was lower than in HS (60.4% ± 23.5; p = 0.0030 for both groups) (Fig. 1, panel b). The percentages of the NK cell receptors did not correlate with the clinical, demographic and serological variables, or, in the rituximab-treated patients, at baseline vs at 6 or 12 months after the therapy (data not shown); a responder vs non-responder comparison was not feasible (see above). A representative flow cytometric dot plot showing CD94/NKG2A + CD16+ cells is shown in Fig. 1 (panel c). 4. Discussion The loss of the immunological tolerance to peripheral nerve components characterises chronic immune-mediated peripheral neuropathies. The immune attack is exerted by activated T cells, macrophages, complement, and autoantibodies [1]. NK cells likely contribute to such attack, due to their known participation in autoimmunity by acting at nearly all its pathogenetic steps, through activation of autoreactive T cells and other innate cells, cytokine production, and direct cytotoxicity [12]. NK cell activity is shaped by the integration of signals from inhibitory and activation receptors, with a predominant role usually played by the inhibitory receptors [5]. While data have been produced on NK cells in the CNS inflammatory demyelination [13–15], in the PNS inflammatory demyelination only one study reported similar values of peripheral blood NK cell percentages and activity in polyneuropathic patients, with or without serum anti-MAG antibodies, and in healthy controls [6]. In line with this study, we found no differences in NK cell percentages between patients with MAG-PN, or with CIDP, and healthy controls. In multiple sclerosis, NK cell blood counts were found lower than those in healthy subjects [14], whereas Rauch et al. reported normal NK cell activity [13]. As a whole, studies in humans support a protective role for NK cells in demyelinating pathologies, though it is likely that such cells can either enhance or ameliorate the disease, as suggested by in vitro studies and animal models [12,16]. A decrease in the percentage of NK cells compared to healthy controls has been reported in autoimmune connective diseases, with between-disease differences likely due to the heterogeneous NK receptor expression [4]. Therefore, a univocal interpretation of data on NK cells is difficult, as the balance of afferent signals and the modulation of responses by individual genetic backgrounds are barely predictable [12]. NK cells may also promote B cell responses by enhancing the production of autoantibodies [17]. We found no correlation between blood NK cell counts and anti-MAG antibody titers.
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L. Benedetti et al. / Journal of the Neurological Sciences 331 (2013) 86–89
Fig. 1. KIR2D2 and CD94/NKG2A expression in patients with chronic demyelinating polyneuropathy associated with anti-myelin-associated glycoprotein (MAG) antibodies and with IgM paraproteinemia (MAG-PN), patients with chronic inflammatory demyelinating polyneuropathy (CIDP), and healthy subjects (HS). KIR2D2 receptor expression in MAG-PN patients was higher than in HS (a); CD94/NKG2A receptor expression in both MAG-PN and CIDP patients was lower than in HS (b). Horizontal bars indicate median values, with the respective standard errors. Representative flow cytometric dot plot showing CD94/NKG2A + CD16+ cells (c).
Studying NK cell receptors can help better understand the role of NK cells in autoimmunity. Particularly, the inhibitory receptors are crucial to promote NK cell-mediated self-tolerance, and prevent autoimmune responses [4,5]. We found that KIR2DS2 receptor expression in MAG-PN patients was higher than in healthy subjects, whereas CIDP patients showed a trend for higher values vs controls that did not reach statistical significance. Interestingly, high KIR2DS2 receptor expression, which entails a diminished NK cell inhibitory activity, is associated with systemic sclerosis, rheumatoid vasculitis, type 1 diabetes, and systemic lupus erythematosus [18,19]. Analogously, the reducedCD94/NKG2A expression that we found in both MAG-PN and CIDP patients is considered to favour autoimmune processes [4,5]. Consistent data indicate that CD94/NKG2A is strongly hypo-expressed in autoimmune diseases, such as multiple sclerosis [15], and psoriasis [20], whereas Mitsuo et al. reported its hyper-expression in rheumatic diseases [21]. These discrepancies are somewhat expected, and could depend on different methodologies, timing of sampling, or both [5]. Our results suggest that CD94/NKG2A might work as a fundamental checkpoint in the development and maintenance of pathological processes underlying the most common immune-mediated peripheral neuropathies too. Indeed, similar to what is reported in patients with long-lasting type 1 diabetes [22], we found that its expression was reduced even in patients with long disease duration, up to 24 years. These findings are potentially important from a therapeutic perspective, since the administration of antibodies that activate NK cells by blocking the CD94/NKG2A receptor, favours the elimination of pathogenic T cells, and stops disease progression in experimental collagen-induced arthritis [23]. Such CD94/NKG2A receptor-blocking antibodies ameliorate experimental autoimmune encephalomyelitis too [24]. Rituximab is an anti-CD20 humanised monoclonal antibody that has been used in MAG-PN with dubious benefit [25]. Among the different mechanisms of action of the drug, antibody-dependent cellular cytotoxicity is a major mechanism that involves CD16-mediated activation of NK cells (9). Lurati et al. reported higher NK cell percentages, which predicted clinical responses, in patients with rheumatoid arthritis [26]. In our longitudinal observation of rituximab-treated MAG-PN patients, we found neither modifications of both NK cell counts and NK
cell receptors after therapy, nor correlations of these parameters with the clinical and serological variables. The relatively small number of our case series weakens the consistency of these findings. In conclusion, our data suggest that MAG-PN shows abnormalities in some NK cell receptors that are broadly typical of other autoimmune diseases. Such abnormalities allow no clear differentiation with the other most common immune-mediated polyneuropathy, CIDP, notwithstanding that the two peripheral nerve diseases seem to be characterised by divergent cytokine profiles, namely MAG-PN by interleukin-10 hyperproduction, and CIDP by an increase in pro-inflammatory Th1 cytokines [27]. Over the disease course persistent down-regulation of the functionally fundamental CD94/NKG2A inhibitory pathway might play a central role in the development and perpetuation of MAG-PN. Functional studies on such inhibitory pathway are needed to better understanding its impact on NK cell role in MAG-PN and CIDP, with the perspective of new therapeutic approaches. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jns.2013.05.015. Conflicts of interest None declared. Acknowledgements The authors wish to thank: Drs. A. Moretta and M. Vitale (University of Genova, Italy) for providing the mAbs for NKRs. References [1] Dalakas MC. Clinical trials in CIDP and chronic autoimmune demyelinating polyneuropathies. J Peripher Nerv Syst 2012;17(Suppl. 2):34–9. [2] Latov N, Sherman WH, Nemni R, Galassi G, Shyong JS, Penn AS, et al. Plasma cell dyscrasia and peripheral neuropathy with a monoclonal antibody to peripheral nerve myelin. N Engl J Med 1980;303:618–21. [3] Abo T, Balch CM. A differentiation antigen of human NK and K cells identified by a monoclonal antibody (HNK-1). J Immunol 1981;127:1024–9.
L. Benedetti et al. / Journal of the Neurological Sciences 331 (2013) 86–89 [4] Puxeddu I, Bongiorni F, Chimenti D, Bombardieri S, Moretta A, Bottino C, et al. Cell surface expression of activating receptors and co-receptors on peripheral blood NK cells in systemic autoimmune diseases. Scand J Rheumatol 2012;41:298–304. [5] Subleski JJ, Jiang Q, Weiss JM, Wiltrout RH. The split personality of NKT cells in malignancy, autoimmune and allergic disorders. Immunotherapy 2011;10:1167–84. [6] Della Casa Alberighi O, Nobile Orazio E, Bonara P, Hu C, Spagnol G, Radelli L, et al. NK cells in patients with peripheralneuropathy and IgM monoclonalproteinreacting with myelin-associatedglycoprotein (MAG). J Neuroimmunol 1988;18:207–16. [7] Fischer L, Penack O, Gentilini C, Nogai A, Muessig A, Thiel E, et al. The antilymphoma effect of antibody-mediated immunotherapy is based on an increased degranulation of peripheral blood natural killer (NK) cells. Exp Hematol 2006;34: 753–9. [8] Joint Task Force of the EFNS and the PNS. Guideline on management of paraproteinemic demyelinating neuropathies. Report of a Joint Task Force of the European Federation of Neurological Societies and the Peripheral Nerve Society–first revision. J Peripher Nerv Syst 2010;15:185–95. [9] Merkies IS, Schmitz PI, van der Meché FG, Samijn JP, van Doorn PA, Inflammatory Neuropathy Cause and Treatment (INCAT) group. Clinimetric evaluation of a new overall disability scale in immune mediated polyneuropathies. J Neurol Neurosurg Psychiatry 2002;72:596–601. [10] Joint Task Force of the EFNS and the PNS. European Federation of Neurological Societies/Peripheral Nerve Society Guideline on management of chronic inflammatory demyelinating polyradiculoneuropathy: report of a joint task force of the European Federation of Neurological Societies and the Peripheral Nerve Society–First Revision. J Peripher Nerv Syst 2010;15:1–9. [11] Baesso I, Pavan L, Boscaro E, Miorin M, Facco M, Trentin L, et al. T-cell type lymphoproliferative disease of granular lymphocytes (LDGL) is equipped with a phenotypic pattern typical of effector cytotoxic cells. Leuk Res 2007;31:371–7. [12] Mayo L, Quintana FJ, Weiner HL. The innate immune system in demyelinating disease. Immunol Rev 2012;248:170–87. [13] Rauch HC, Montgomery IN, Kaplan J. Natural Killer cell activity in multiple sclerosis and myasthenia gravis. Immunol Invest 1985;14:427–34. [14] Kreuzfelder E, Shen G, Bittorf M, Scheiermann N, Thraenhart O, Seidel D, et al. Enumeration of T, B and natural killer peripheral blood cells of patients with multiple sclerosis and controls. Eur Neurol 1992;32:190–4.
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[15] O'Keeffe J, Gately CM, Counihan T, Hennessy M, Leahy T, Moran AP, et al. T-cells expressing natural killer (NK) receptors are altered in multiple sclerosis and responses to alpha-galactosylceramide are impaired. J Neurol Sci 2008;275:22–8. [16] Kaur G, Trowsdale J, Fugger L. Natural killer cells and their receptors in multiple sclerosis. Brain Jun 25 2012 [Epub ahead of print]. [17] Yuan D, Thet S, Zhou XJ, Wakeland EK, Dang T. The role of NK cells in the development of autoantibodies. Autoimmunity 2011;44:641–51. [18] Perricone R, Perricone C, De Carolis C, Shoenfeld Y. NK cells in autoimmunity: a two-edg’d weapon of the immune system. Autoimmun Rev 2008;7:384e90. [19] Pellett F, Siannis F, Vukin I, Lee P, Urowitz MB, Gladman DD. KIRs and autoimmune disease: studies in systemic lupus erythematosus and scleroderma. Tissue Antigens 2007;69(Suppl. 1):106e8. [20] Son SW, Kim EO, Ryu ES, Kim TJ, Kim JN, Choi JE, et al. Upregulation of Fas and downregulation of CD94/NKG2A inhibitory receptors on circulating natural killer cells in patients with new-onset psoriasis. Br J Dermatol 2009;161:281–8. [21] Mitsuo A, Morimoto S, Nakiri Y, Suzuki J, Kaneko H. Decreased CD161 + CD8+ T cells in the peripheral blood of patients suffering from rheumatic diseases. Rheumatology 2006;45:1477–84. [22] Rodacki M, Svoren B, Butty V, Besse W, Laffel L, Benoist C, et al. Altered natural killer cells in type 1 diabetic patients. Diabetes 2007;56:177–85. [23] Leavenworth JW, Wang X, Wenander CS, Spee P, Cantor H. Mobilization of natural killer cells inhibits development of collagen-induced arthritis. Proc Natl Acad Sci U S A 2011;108:14584–9. [24] Leavenworth JW, Schellach C, Kim HJ, Lu L, Spee P, Cantor H. Analysis of the cellular mechanism underlying inhibition of EAE after treatment with anti-NKG2A F(ab’)2. Proc Natl Acad Sci U S A 2010;107:2562–7. [25] Lunn MP, Nobile-Orazio E. Immunotherapy for IgM anti-myelin-associated glycoprotein paraprotein-associated peripheral neuropathies. Cochrane Database Syst Rev 2012;5:CD002827. [26] Lurati A, Bertani L, Marrazza M, Re KA, Bompane D, Scarpellini M. NK cell count as predictor of clinical response in patients with rheumatoid arthritis treated with rituximab. Biologics 2012;6:83–7. [27] Gironi M, Saresella M, Marventano I, Guerini FR, Gatti A, Antonini G, et al. Distinct cytokine patterns associated with different forms of chronic dysimmune neuropathy. Muscle Nerve 2010;42:864–70.
Contents lists available at SciVerse ScienceDirect
Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
NK cells and their receptors in naive and rituximab-treated patients with anti-MAG polyneuropathy Luana Benedetti a,⁎, Monica Facco b, Diego Franciotta c, Chiara Dalla Torre d, Marta Campagnolo d, Marta Lucchetta d, Elisa Boscaro b, Mario Ermani d, Massimo Del Sette a, Tamara Berno b, Laura Candiotto b, Renato Zambello b, Chiara Briani d a
Department of Neurology, Osp. S. Andrea, La Spezia, Italy Department of Medicine-DIMED, University of Padova, Italy c Laboratory of Neuroimmunology, IRCCS, National Institute of Neurology ‘C. Mondino’, Pavia, Italy d Department of Neurosciences, University of Padova, Italy b
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Article history: Received 17 January 2013 Accepted 10 May 2013 Available online 10 June 2013 Keywords: Natural killer (NK) cells CD94/NKG2A Myelin associated glycoprotein Anti-MAG antibodies IgM Monoclonal gammopathy Rituximab
a b s t r a c t Background: Natural killer (NK) cells can bridge innate and acquired immunity, and play a role in autoimmunity. A few studies evaluated the distribution of NK cells and the expression of their receptors in chronic immune-mediated demyelinating polyneuropathies. We investigated NK cell distribution and NK cell receptor expression in 20 naïve patients with anti-MAG polyneuropathy (MAG-PN). Methods: Using flow cytometry, we analysed NK cells and a series of NK cell receptors in the peripheral blood of patients with MAG-PN, and, as controls, in patients with chronic inflammatory demyelinating peripheral polyradiculoneuropathy (CIDP) and in healthy subjects. Six MAG-PN patients were also tested after rituximab treatment. Results: At baseline the percentage of NK cells did not differ among the groups. KIR2DL2 receptor expression in MAG-PN patients was higher, andCD94/NKG2A receptor expression in both MAG-PN and CIDP patients was lower than in healthy controls. These abnormalities did not correlate with any clinical or demographic variable. No modification was found after rituximab therapy. Conclusions: The data suggest that MAG-PN shows abnormalities in NK cell receptors that characterise other autoimmune diseases, and cannot help in differential diagnosis with CIDP. The impairment of the relevant CD94/NKG2A inhibitory pathway, which might play a central role in the development and perpetuation of MAG-PN, warrants further functional investigations. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Chronic demyelinating polyneuropathy associated with anti-myelinassociated glycoprotein (MAG) antibodies and IgM paraproteinemia (MAG-PN) is a disease characterised by prominent sensory signs and symptoms, which is considered different from chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), notwithstanding the two diseases share an immune-mediated pathogenesis [1]. Dissimilarly from CIDP, MAG-PN usually manifests with a slowly progressive course, and poorly responds to conventional immunomodulating treatments [2]. MAG is a glycoprotein expressed in the myelin of the peripheral nervous system (PNS), whose HNK-1 extracellular epitope is the target of the anti-MAG antibodies [2]. Interestingly, HNK-1 is broadly expressed in the central nervous system (CNS) grey matter, and it is also a marker of human natural killer (NK) cells [3]. ⁎ Corresponding author at: Department of Neurology, Osp. S. Andrea, Via Vittorio Veneto, La Spezia, Italy. Tel.: +39 0187533311–3498110936; fax: +39 0187533024. E-mail address: [email protected] (L. Benedetti). 0022-510X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jns.2013.05.015
NK cells are lymphocytes that bridge innate and acquired immunity. They contribute to both effector and regulatory functions of innate immunity through the cytotoxic activity and the production of pro- and anti-inflammatory cytokines and chemokines [3]. The latter function mainly accounts for their regulatory role in acquired immunity. To prevent autoimmune reactivities, NK cell functions are tightly regulated by the integration of signals coming from inhibitory and activating receptors [3]. Among the inhibitory receptors, the killer cell immunoglobulin-like inhibitory receptors (KIR) bind some classical MHC class I molecules, and the CD94/NKG2A receptors bind the non-classical MHC class I molecules, whereas the NK cell protein 30 (NKp30), NKp44, and NKp46 represent the main receptors with activating functions [3]. NK cells seem to act as a two edged weapon, playing opposite roles with both regulatory and inducer activity even in the same disease; accordingly, a defined role for such cells and their receptors in autoimmunity has not been clearly established, and discordant data on their modifications in systemic and CNS autoimmune diseases have been reported [4,5]. Conversely, only few data are available in the PNS autoimmune diseases [6].
L. Benedetti et al. / Journal of the Neurological Sciences 331 (2013) 86–89
We studied NK cell distribution and NK cell receptor expression in patients with MAG-PN, and, as controls, in patients with CIDP and in healthy subjects. Some MAG-PN patients were followed up to evaluate both NK cell and receptor modifications after treatment with rituximab, a B-cell depleting drug, whose therapeutic action could be partially mediated by NK cells too [7]. 2. Materials and methods 2.1. Patients and controls Twenty, naïve to therapy patients with MAG-PN (15 men, 5 women, mean age 70.1 ± 9.9 years; range, 50–89), whose clinical and demographic characteristics are shown in the table (Supplementary material), were studied. The diagnosis was based on accurate clinical history, neurophysiologic studies, and high-titer positivity of anti-MAG antibodies [8]. All patients underwent neurological evaluation, and severity of the disease was scored through the inflammatory neuropathy cause and treatment (INCAT) scales for arm and leg disabilities [9]. Sex and agematched controls included 9 patients with CIDP [10], and 12 healthy subjects (HS). Six MAG-PN patients, who were treated with rituximab, 375 mg/sq for four consecutive weekly infusions, were prospectively followed up for one year; in these patients, neurological assessment, peripheral blood white cell immunophenotype and NK receptor characterization were repeated 6 and 12 months after rituximab treatment. The local ethical committee approved the study. 2.2. Immunophenotype of NK cells, and expression of NK cell receptors Peripheral blood mononuclear cells (PBMCs) were obtained by centrifugation on a Ficoll–Hypaque gradient. A panel of commercially available florescein isothiocyanate- (FITC), phycoerythrin- (PE), PE Cy5 and allophycocyanin-conjugated (APC) mouse anti-human CD3, anti-CD4, anti-CD57, anti-CD8, anti-CD56, anti-CD16 mAbs, with the specific isotype-matched control reagents (BD Pharmingen San Diego, CA, USA; R&D Systems, Toronto, Canada; Caltag Laboratories; Immunotech, Marseille, France) was used. The following mAbs to NK receptors were kindly provided by Drs. A. Moretta and M. Vitale (National Institute for Cancer Research, Genoa, Italy): EB6 (IgG1, anti-KIR2DL1, and anti-KIR2DS1),GL183 (IgG1, anti-KIR2DL2, antiKIR2DL3, and anti-KIR2DS2), FES172 (IgG2a, anti-KIR2DS4), Z27 (IgG1, anti-KIR3DL1, and anti-KIR3DS1),Q66 (IgM, anti-KIR3DL2), XA185 (IgG1, anti-CD94), and Z199 (IgG2b,anti-NKG2A). The expression of the studied antigens on PBMCs was analysed by flow cytometry, with direct or indirect immunofluorescence assays, as previously reported [11]. The analyses were performed on freshly recovered lymphocytes. Briefly, cells were stained with the appropriate mAbs, either unlabeled or labelled; staining with unlabeled mAb was followed by PE- or FITC-conjugated isotype-specific goat anti-mouse second reagent (Southern Biotechnology, Birmingham, AL, or Caltag, Burlingame, CA). A PE-Cy5 CD16 was set, and the status of the antigens of interest was analysed only on CD16+ gated cells. For fluorescenceactivated cell sorter analysis, 1 × 104 cells were acquired, and the analyses were determined by overlaying the histograms of samples stained with the different reagents. Stained cells were scored using a FACSCalibur analyser (Becton Dickinson). Samples were analysed by four colour cytofluorimetric analysis, and data were processed using CELLQuest software (Becton Dickinson). Values were given as % positive staining after subtraction of isotype control. 2.3. Statistical analysis Kruskal–Wallis with Dunn's post test was used to compare the between-group mean differences, Friedman with Dunn's multiple comparison test for repeated measures, and Spearman test for
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correlations. Data are reported as means ± SD. p b 0.05 was considered as significant. 3. Results The frequencies of CD16+ NK cells were similar in the three studied groups (MAG-PN, 16.7% ± 6.4; CIDP, 14.2% ± 5.4; HS,18.2% ± 5.9), although a trend for higher NK cell percentages, at baseline, was observed in about half of the MAG-PN patients; these patients' clinical, demographic and serological features, which included the anti-MAG antibody titers, did not differ from those of the patients with lower percentages (data not shown). In the rituximab-treated patients, there were no significant differences in the NK cell proportions, at baseline vs at 6 or 12 months after the therapy (data not shown); all but one of the 6 patients responded to rituximab, and therefore a comparison between responders vs non-responders was not applicable. Among the analysed receptors, KIR2DL2, KIR3DS1, and CD94/NKG2A were consistently detected in all the patients and controls. KIR3DS1 receptor was not differently expressed in the three studied groups (MAG-PN, 25.0% ± 23.7; CIDP, 23.4% ± 23.4; HS, 24.8% ± 7.6). KIR2DL2 receptor expression in MAG-PN patients (29.3% ± 16.8) and in CIDP patients (24.7% ± 15.7) was higher than in HS (15.5% ± 11.1), but the difference was statistically significant for the MAG-PN patients only (p = 0.012) (Fig. 1, panel a). CD94/NKG2A receptor expression in both MAG-PN (31.8% ± 14.8) and CIDP (29.2% ± 22.3) patients was lower than in HS (60.4% ± 23.5; p = 0.0030 for both groups) (Fig. 1, panel b). The percentages of the NK cell receptors did not correlate with the clinical, demographic and serological variables, or, in the rituximab-treated patients, at baseline vs at 6 or 12 months after the therapy (data not shown); a responder vs non-responder comparison was not feasible (see above). A representative flow cytometric dot plot showing CD94/NKG2A + CD16+ cells is shown in Fig. 1 (panel c). 4. Discussion The loss of the immunological tolerance to peripheral nerve components characterises chronic immune-mediated peripheral neuropathies. The immune attack is exerted by activated T cells, macrophages, complement, and autoantibodies [1]. NK cells likely contribute to such attack, due to their known participation in autoimmunity by acting at nearly all its pathogenetic steps, through activation of autoreactive T cells and other innate cells, cytokine production, and direct cytotoxicity [12]. NK cell activity is shaped by the integration of signals from inhibitory and activation receptors, with a predominant role usually played by the inhibitory receptors [5]. While data have been produced on NK cells in the CNS inflammatory demyelination [13–15], in the PNS inflammatory demyelination only one study reported similar values of peripheral blood NK cell percentages and activity in polyneuropathic patients, with or without serum anti-MAG antibodies, and in healthy controls [6]. In line with this study, we found no differences in NK cell percentages between patients with MAG-PN, or with CIDP, and healthy controls. In multiple sclerosis, NK cell blood counts were found lower than those in healthy subjects [14], whereas Rauch et al. reported normal NK cell activity [13]. As a whole, studies in humans support a protective role for NK cells in demyelinating pathologies, though it is likely that such cells can either enhance or ameliorate the disease, as suggested by in vitro studies and animal models [12,16]. A decrease in the percentage of NK cells compared to healthy controls has been reported in autoimmune connective diseases, with between-disease differences likely due to the heterogeneous NK receptor expression [4]. Therefore, a univocal interpretation of data on NK cells is difficult, as the balance of afferent signals and the modulation of responses by individual genetic backgrounds are barely predictable [12]. NK cells may also promote B cell responses by enhancing the production of autoantibodies [17]. We found no correlation between blood NK cell counts and anti-MAG antibody titers.
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Fig. 1. KIR2D2 and CD94/NKG2A expression in patients with chronic demyelinating polyneuropathy associated with anti-myelin-associated glycoprotein (MAG) antibodies and with IgM paraproteinemia (MAG-PN), patients with chronic inflammatory demyelinating polyneuropathy (CIDP), and healthy subjects (HS). KIR2D2 receptor expression in MAG-PN patients was higher than in HS (a); CD94/NKG2A receptor expression in both MAG-PN and CIDP patients was lower than in HS (b). Horizontal bars indicate median values, with the respective standard errors. Representative flow cytometric dot plot showing CD94/NKG2A + CD16+ cells (c).
Studying NK cell receptors can help better understand the role of NK cells in autoimmunity. Particularly, the inhibitory receptors are crucial to promote NK cell-mediated self-tolerance, and prevent autoimmune responses [4,5]. We found that KIR2DS2 receptor expression in MAG-PN patients was higher than in healthy subjects, whereas CIDP patients showed a trend for higher values vs controls that did not reach statistical significance. Interestingly, high KIR2DS2 receptor expression, which entails a diminished NK cell inhibitory activity, is associated with systemic sclerosis, rheumatoid vasculitis, type 1 diabetes, and systemic lupus erythematosus [18,19]. Analogously, the reducedCD94/NKG2A expression that we found in both MAG-PN and CIDP patients is considered to favour autoimmune processes [4,5]. Consistent data indicate that CD94/NKG2A is strongly hypo-expressed in autoimmune diseases, such as multiple sclerosis [15], and psoriasis [20], whereas Mitsuo et al. reported its hyper-expression in rheumatic diseases [21]. These discrepancies are somewhat expected, and could depend on different methodologies, timing of sampling, or both [5]. Our results suggest that CD94/NKG2A might work as a fundamental checkpoint in the development and maintenance of pathological processes underlying the most common immune-mediated peripheral neuropathies too. Indeed, similar to what is reported in patients with long-lasting type 1 diabetes [22], we found that its expression was reduced even in patients with long disease duration, up to 24 years. These findings are potentially important from a therapeutic perspective, since the administration of antibodies that activate NK cells by blocking the CD94/NKG2A receptor, favours the elimination of pathogenic T cells, and stops disease progression in experimental collagen-induced arthritis [23]. Such CD94/NKG2A receptor-blocking antibodies ameliorate experimental autoimmune encephalomyelitis too [24]. Rituximab is an anti-CD20 humanised monoclonal antibody that has been used in MAG-PN with dubious benefit [25]. Among the different mechanisms of action of the drug, antibody-dependent cellular cytotoxicity is a major mechanism that involves CD16-mediated activation of NK cells (9). Lurati et al. reported higher NK cell percentages, which predicted clinical responses, in patients with rheumatoid arthritis [26]. In our longitudinal observation of rituximab-treated MAG-PN patients, we found neither modifications of both NK cell counts and NK
cell receptors after therapy, nor correlations of these parameters with the clinical and serological variables. The relatively small number of our case series weakens the consistency of these findings. In conclusion, our data suggest that MAG-PN shows abnormalities in some NK cell receptors that are broadly typical of other autoimmune diseases. Such abnormalities allow no clear differentiation with the other most common immune-mediated polyneuropathy, CIDP, notwithstanding that the two peripheral nerve diseases seem to be characterised by divergent cytokine profiles, namely MAG-PN by interleukin-10 hyperproduction, and CIDP by an increase in pro-inflammatory Th1 cytokines [27]. Over the disease course persistent down-regulation of the functionally fundamental CD94/NKG2A inhibitory pathway might play a central role in the development and perpetuation of MAG-PN. Functional studies on such inhibitory pathway are needed to better understanding its impact on NK cell role in MAG-PN and CIDP, with the perspective of new therapeutic approaches. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jns.2013.05.015. Conflicts of interest None declared. Acknowledgements The authors wish to thank: Drs. A. Moretta and M. Vitale (University of Genova, Italy) for providing the mAbs for NKRs. References [1] Dalakas MC. Clinical trials in CIDP and chronic autoimmune demyelinating polyneuropathies. J Peripher Nerv Syst 2012;17(Suppl. 2):34–9. [2] Latov N, Sherman WH, Nemni R, Galassi G, Shyong JS, Penn AS, et al. Plasma cell dyscrasia and peripheral neuropathy with a monoclonal antibody to peripheral nerve myelin. N Engl J Med 1980;303:618–21. [3] Abo T, Balch CM. A differentiation antigen of human NK and K cells identified by a monoclonal antibody (HNK-1). J Immunol 1981;127:1024–9.
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