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Features of intrathecal immunoglobulins in patients with multiple sclerosis
Journal of the Neurological Sciences 288 (2010) 147–150
Contents lists available at ScienceDirect
Journal of the Neurological Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n s
Features of intrathecal immunoglobulins in patients with multiple sclerosis Franziska Di Pauli a, Viktoria Gredler a, Bettina Kuenz a, Andreas Lutterotti a, Rainer Ehling a, Claudia Gneiss a, Michael Schocke b, Florian Deisenhammer a, Markus Reindl a, Thomas Berger a,⁎ a b
Clinical Department of Neurology, Innsbruck Medical University, Anichstrasse 35, A-6020 Innsbruck, Austria Department of Radiology, Innsbruck Medical University, Anichstrasse 35, A-6020 Innsbruck, Austria
a r t i c l e
i n f o
Article history: Received 28 July 2009 Received in revised form 15 September 2009 Accepted 17 September 2009 Available online 13 October 2009 Keywords: Multiple sclerosis Immunoglobulins Subclasses Cerebrospinal fluid Progression Oligoclonal bands Disease course B cells
a b s t r a c t We have analyzed immunoglobulin (Ig) isotypes and IgG subclasses in cerebrospinal fluid (CSF) and serum of patients with multiple sclerosis (MS) and other neurological diseases to determine whether different Ig isotype patterns correlate with clinical or paraclinical findings and CSF B cell populations. Intrathecal IgG1 synthesis was elevated in MS patients. An increased intrathecal IgM production was found in patients with a higher cerebral MRI lesion burden, whereas other clinical and paraclinical parameters were not associated with a specific Ig isotype or subclass profile. Finally, intrathecal IgG production (IgG1 and IgG3) correlated with the presence of mature B cells and plasma blasts. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Multiple sclerosis (MS) is a devastating demyelinating disorder thought to be mainly driven by an autoimmune attack [1,2]. Components of the immune system, including both cellular and humoral arm, are involved in the pathogenesis of MS, which is neuropathologically characterized by inflammation, demyelination and axonal loss. Clinically the immune response is reflected by the nearly mandatory detection of an intrathecal immunoglobulin (Ig) synthesis and oligoclonal bands (OCB) during (differential-) diagnostic procedures [3]. Recent results showed a high degree of concordance between the Ig proteome and B cell transcriptome in the cerebrospinal fluid (CSF) of MS patients, thus considering that the CSF B cells are the source of OCBs [4]. Since clonal B cell expansion and antibody production are established disease characteristics [3,5,6] and Ig and complement are found in MS certain lesions [7], a pathogenetic role of antibodies in MS has been suggested. The predominant subclasses in the CSF of MS patients are IgG1 and IgG3 [8]. Binding of various IgG subclasses to preferred Fc receptors (FcR) initiates a broad variety of executive responses in effector cells, such as degranulation, phagocytosis, antibody-dependent cell-mediated cytotoxicity (ADCC), production of multiple inflammatory cytokines and other mediators in mast cells, monocytes or macrophages [9,10], but FcR stimulation can also attenuate those functions [11,12].
⁎ Corresponding author. Tel.: +43 512 504 26277; fax: +43 512 504 24260. E-mail address: [email protected] (T. Berger). 0022-510X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2009.09.016
Since each isotype and subclass of Ig's has distinct biological functions, the preponderance of a specific subclass could be crucial for the outcome of a disease [13,14]. However, in MS correlations of immunoglobulins and autoantigenic profiles with disease evolution and severity are still under debate. In this study we aimed to study possible differences of Ig isotype patterns with regard to different MS disease activity parameters. Therefore we have analyzed the total Ig isotype and IgG subclass content and distribution in CSF and serum samples from patients at the onset of disease (CIS), relapsing–remitting, chronic progressive MS and other neurological diseases (OND) to correlate different Ig isotype patterns with the disease courses, MRI findings and CSF B cell populations. Further, we were interested whether Ig isotype patterns may predict an early conversion to CDMS. Additionally, in a subgroup of patients we analyzed CSF B cell subpopulations and their association with the Ig profile. 2. Methods 2.1. Patients Patients were recruited prospectively between 2004 and 2008 from the Clinical Department of Neurology, Innsbruck Medical University, Austria [15]. This study was approved by the ethical committee of Innsbruck Medical University (study Nr. oUN2045, 217/4.12, 07.07.2004) and all patients gave written informed consent. CIS and MS patients were included, if: (1) clinical symptoms and characteristic MRI findings according to the original “McDonald Criteria”
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[16] and (2) presence of intrathecal Ig synthesis (elevated Ig indices and/ or oligoclonal IgG bands) [17] Thirty-four patients with a CIS, 17 patients with RRMS and 11 patients with CPMS (2 with secondary progressive-SP and 9 with primary progressive-PP MS) were included. Patients were examined by a neurologist (F.D., A.L., R.E., C.G., and T.B.), including assessment of the expanded disability status scale (EDSS) and confirmation of relapse/disease progression during a follow-up period of at least 12 months. CDMS was diagnosed if a new relapse occurred. The control group consisted of 24 patients with other neurological diseases (OND). The OND group included 17 patients with noninflammatory OND (pseudotumor cerebri, migraine, psychogenic neurological symptoms, sinus venous thrombosis, cavernoma, vascular leukoencephalopathy, seizure, herniated vertebral disc, transient ischemic attack, spastic paraparesis, multiple system atrophy, neuropathic pain syndrome, cerebellar infarction, brain tumor, focal dystonia, and ischemic transverse spinal cord syndrome) and 7 patients with inflammatory OND (myelitis, ventriculitis, vasculitis, systemic lupus erythematosus, antiphospholipid syndrome, sarcoidosis and Guillain–Barré syndrome). In all patients lumbar puncture and MRI have been performed for diagnostics reasons.
2.4. Characterization of CSF cell populations by flow cytometry The staining of CSF cell populations (CD3+ T cells, CD56+ NK cells, CD19+CD138− mature B cells, CD19+CD138+ plasma blasts and CD19−CD138+ plasma cells) and FACS analysis was performed in 58 patients as described earlier [15]. 2.5. Statistics Statistical analysis was performed using SPSS (release 14.0, SPSS Inc., USA) software and GraphPad Prism 5 (GraphPad, San Diego, USA). Clinical, demographic and CSF data were compared using Dunn's multiple comparison test, Chi-Square test, Kruskal–Wallis test, Fisher's exact test or Mann–Whitney-U test. Statistical significance was defined as p-value < 0.05. Correlations were described by Spearman's nonparametric correlation. Statistical significance was defined as two-sided p-value<0.01. 3. Results Demographic, clinical and CSF characteristics of all prospective collected MS patients and neurological controls are shown in Table 1.
2.2. Sample collection CSF and serum samples (Sarstaedt Monovettes, Nuembrecht, Germany) were collected during standard diagnostic lumbar puncture and peripheral vein puncture. CSF samples were analyzed for CSF cell populations within 30 min after lumbar puncture and cell-free supernatants were stored at −80 °C for CSF subclasses analysis. Serum was prepared by centrifugation, 10 min at 3000 rpm, and stored at −20 °C until further analyses were performed. 2.3. Determination of CSF and serum levels of IgG subclasses CSF and serum samples were analyzed for total IgG, IgM and IgA levels using a Beckman clinical nephelometer. Total IgG1–4 CSF and serum levels were measured by ELISA using a commercially available test kit (Invitrogen, Carlsbad, CA, USA) according to manufacturer's guidelines (human IgG subclass profile ELISA kit, Catalog No. 99-1000, Zymed Laboratories, Carlsbad, CA). CSF was analyzed at a dilution of 1:20 and serum samples were analyzed at a dilution of 1:2500. The Ig indices were calculated according to the Delpech and Lichtblau protein quotient (IgGCSF/IgGserum:albuminCSF/albuminserum.).
3.1. IgG subclasses in MS patients and controls CSF IgG1 levels were significantly elevated in patients with a CIS (p < 0.01), RRMS (p < 0.001) and CPMS (p < 0.01) patients compared with OND. In contrast CSF IgG2, IgG3 and IgG4 subclass levels did not differ in any of the groups (Fig. 1). Further, there were no significant differences between IgG1, IgG2, IgG3 and IgG4 serum levels between the MS patients and controls. The comparison between the intrathecal synthesis of IgM, IgA, total IgG and each of its subclasses revealed similar results. We found significantly increased total IgG indices in patients with a CIS (p<0.001) and RRMS (p<0.01), which were predominantly due to increased intrathecal IgG1 synthesis (Table 1 and Fig. 1). Further, intrathecal IgM synthesis was significantly higher in MS patients than in OND (Table 1). 3.2. Correlation of IgG subclasses with clinical and paraclinical parameters Additionally, it was analyzed whether MRI activity, defined by the number of lesions on T2-weighted MRI (<9 or≥9T2 lesions) and the presence of gadolinium enhancing lesions on T1 weighted MRI (no or ≥1 lesions), were correlated with CSF levels or indices of IgM, IgA, IgG
Table 1 Demographic, clinical and CSF data of analyzed patients.
n Females (%) Age (y)1 EDSS1 Acute relapse MRI Gd + lesions ≥ 9 MRI T2 lesions OCB CSF cells/µl1 IgG index1 IgM index1 IgA index1 Q-Alb1 IgG 1 index1 IgG 2 index1 IgG 3 index1 IgG 4 index1
CIS
RRMS
CPMS
OND
34 26 (76%) 30 (18–49) # 1.8 (0–3.5) 32 (94%) 24 (73%) # 13 (38%) # 34 (100%) # 8 (1–76) # 0.83 (0.06–3.15) 0.13 (0.00–1.59) 0.36 (0.00–4.83) 4.46 (2.3–76.61) 0.94 (0.36–4.01) 0.56 (0.17–1.68) 0.65 (0.20–2.48) 0.42 (0–9.00)
17 14 (82%) 32 (16–63) # 2.0 (0–4.0) 12 (71%) 9 (69%) # 6 (36%) # 15 (88%) # 7 (1–17) # 0.86 (0.42–2.01) # 0.15 (0.07–0.46) # 0.40 (0.25–2.45) 5.28 (2.84–12.23) 0.93 (0.38–3.76) # 0.39 (0.09–0.79) 0.50 (0.17–2.93) 0.32 (0.15–0.99)
11 3 (27%) 47 (34–63) 6.0 (3.5–7.0) 1 (9%) 4 (44%) # 3 (33%) # 9 (82%) # 4 (0–20) 0.74 (0.43–4.46) 0.13 (0.05–0.76) 0.34 (0.26–1.03) 6.83 (2.30–10.90) 0.78 (0.35–4.29) 0.40 (0.21–1.92) 0.35 (0.19–4.65) 0.31 (0.07–0.60)
24 16 (67%) 43 (22–68)
# # # #
0 (0%) 0 (0%) 2/20 (10%) 2 (0–211) 0.50 (0.39–1.09) 0.07 (0–0.46) 0.29 (0.19–0.82) 4.95 (2.31–17.11) 0.52 (0.39–1.97) 0.50 (0–0.90) 0.47 (0.15–2.32) 0.40 (0–1.02)
p-value 0.0112 <0.0013 <0.0013 <0.0013 <0.0012 0.0162 <0.0012 <0.0013 <0.0013 0.0063 0.0143 0.1603 0.0013 0.1663 0.0493 0.5403
Data are shown as median (range), p-value: groups were compared using 2Chi-Square test and 3Kruskal–Wallis test, # statistically different from OND control group. Abbreviations: n = number of patients, y = years, EDSS = expanded disability status score, MRI Gd + lesions = presence of lesions on gadolinium-enhanced T1-weighted MRI (neg = negative, pos = positive), ≥9 T2 MRI lesions = ≥9 lesions on T2-weighted MRI, OCB = oligoclonal bands, and Q-Alb = albumin quotient.
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Fig. 1. IgG1–4 subclasses in CSF and sera of patients with a CIS, RRMS, CPMS and OND (outlier not shown).
and its subclasses in MS patients. Only an increased intrathecal IgM synthesis was significantly associated with a higher lesion load on T2weighted MRI with a median IgM index of 0.16, range 0.06–1.59 in the group with more than 9 lesions versus 0.11, range 0–0.76 (p<0.01). During an acute relapse of MS CSF cells were significantly increased (median 23.5, range 2–227 versus median 13.0, range 1–60, p = 0.003). Furthermore, a tendency to an elevated intrathecal IgG3 synthesis was shown in patients with a relapse as compared to those without a relapse (median 0.64, range 0.17–2.94 versus median 0.42, range 0.19–4.65, p = 0.035). There were no significant differences regarding the other Ig isotype/subclass indices between patients with or without acute disease activity. A subgroup analyses was performed in 31 CIS patients with a followup of at least 12 months to determine a possible predictive value of a
specific immunoglobulin isotype pattern for an early conversion to CDMS. Neither CSF levels and indices of IgM or IgA nor of total IgG were associated with an early conversion to CDMS within given timepoints of one or three years (Table 2). No specific subclass pattern showed a prognostic value for an early conversion to CDMS. 3.3. IgG subclasses and CSF B cells In 58 patients we analyzed CSF B (mature B cells, plasma blasts and plasma cells) and T cells and their correlations with different immunoglobulin isotype pattern. Mature B cells (CD19+CD138−) and plasma blasts (CD19+CD138+) were associated with intrathecal IgM (R=0.356, p<0.001, and R=0.491, p<0.001 respectively) and total IgG synthesis (R=0.416, p<0.001, and R=0.592, p<0.001 respectively). Furthermore,
Table 2 Correlation of CSF findings and conversion to CDMS after a CIS within 3 years.
CSF cells/µl1 Q-Alb1 IgG index1 IgM index1 IgA index1 IgG1 index1 IgG2 index1 IgG3 index1 IgG4 index1
CIS
CDMS
Hazard ratio
95% CI
p-value
7 (1–76) 4.69 (2.82–76.61) 0.85 (0.49–2.05) 0.12 (0–0.82) 0.34 (0–0.75) 0.81 (0.37–2.77) 0.52 (0.31–0.81) 0.59 (0.20–0.87) 0.42 (0–1.00)
9 (3–52) 4.46 (2.30–8.94) 0.87 (0.06–3.15) 0.13 (0.06–1.59) 0.38 (0–4.83) 1.20 (0.36–4.01) 0.66 (0.17–1.68) 0.73 (0.31–2.48) 0.43 (0–9.00)
1.013 0.930 0.677 0.460 1.258 0.405 1.496 6.432 0.949
0.996–1.029 0.610–1.417 0.085–5.374 0.101–2.102 0.725–2.181 0.055–3.007 0.192–11.641 1.018–40.644 0.605–1.491
0.126 0.736 0.712 0.316 0.414 0.377 0.700 0.048 0.822
Data are shown as 1median (range), p-value: groups were compared using Cox proportional-hazards model.
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the IgG subclasses analyses showed a significant correlation of the IgG1 index with mature B cells (R=0.456, p<0.001) and plasma blasts (R=0.616, p<0.001) and of the IgG3 index with the same B cell subclasses (R=0.340, p<0.01, and R=0.382, p<0.01 respectively), whereas CSF T cells, NK cells and plasma cells were not correlated with any of the analyzed CSF findings. 4. Discussion This study was conducted to determine the role of immunoglobulin subclass patterns in patients with different courses of MS and ONDs. The detected levels are similar to those of other populations [18]. We found significantly elevated CSF IgG1 levels in CIS, RRMS and CPMS patients and increased IgG1 indices in CIS and RRMS patients compared with controls. These results are partly in accordance with previous studies which show either elevated IgG1 levels alone or elevated IgG1 and IgG3 levels in MS patients [8,18–21]. Raknes et al. did not find any associations of total IgG or IgG subclass levels and the initial disease course [22], also in our study cohort there were no significant correlations between the disease course and the CSF findings. Furthermore, we could confirm an increase in total IgG, IgM and IgA indices in MS patients compared with controls ([23,29]), but there were no significant differences between CIS, RRMS and CPMS patients. We found a significant association of cerebral MRI lesion burden with an increased intrathecal IgM synthesis, which is in contrast with the findings of Sharief and Thompson [23]. A correlation of the MRI activity with the IgG index was described [24,25], which was not confirmed by our results. No correlations between CSF parameters and other clinical or paraclinical features were described in previous studies [26–28]. Until now no study to our knowledge investigated whether a special immunoglobulin subclass phenotype can predict an early conversion to CDMS. There were no significant correlations in our 31 CIS patients during the follow-up of 36 months, a final conclusion of these questions is limited due the small sample size. Since the negative results in the different clinical and paraclinical measurements, it is likely that the immunoglobulin subclasses play only a minor role in the outcome of MS. However, it is possible that relevant autoantibodies are reduced in the CSF because of being bound to their antigen in the tissue and therefore they may not be detected with classical methods. In a subgroup analysis we could detect a correlation between mature B cells and plasma blasts in the CSF and CSF IgG1 levels and intrathecal IgG1 and IgG3 synthesis. These results suggest that the CSF B cells are the source of the intrathecal IgGs and that the clonal expanded B cells produce mainly IgG1. Recently the link between subject-specific immunoglobulin transcriptomes to the proteomes in diagnostic CSF samples from MS patients was demonstrated and supports the pathogenetic relevance of CSF antibodies [4]. The role of the CSF B cells and the intrathecal produced immunoglobulins in the disease development and progression is less clear. Although, it is known that the cytokine milieu during B cell differentiation decides about the mainly produced antibody subtypes, the exact regulation of the subclass switch remains an open question. In other diseases with preferential Th1 immunity such as type 1 diabetes and Lyme's disease IgG1 dominance is well known. In conclusion, our results reveal a preponderance of IgG 1 also in the humoral immune response of MS and, therefore, supports the hypothesis of a mainly Th1 driven autoimmune disease. Acknowledgements This study was supported by a research grant of the Gemeinnuetzige Hertiestiftung (Frankfurt, Germany). The authors wish to thank
Carolyn Rainer, Ingrid Gstrein and Karin Steinlechner for excellent technical assistance. References [1] Hafler DA, Slavik JM, Anderson DE, O'Connor KC, De Jager P, Baecher-Allan C. Multiple sclerosis. Immunol Rev 2005;204:208–31. [2] Sospedra M, Martin R. Immunology of multiple sclerosis. Annu Rev Immunol 2005;23: 683–747. [3] Kabat EA, Glusman M, Knaub V. Quantitative estimation of the albumin and gammaglobulin in normal and pathological cerebrospinal fluid by immunochemical methods. Am J Med 1948;4:653–62. [4] Obermeier B, Mentele R, Malotka J, Kellermann J, Kumpfel T, Wekerle H, et al. Matching of oligoclonal immunoglobulin transcriptomes and proteomes of cerebrospinal fluid in multiple sclerosis. Nat Med 2008;14:688–93. [5] McDonald WI, Compston A, Edan G, Goodkin D, Hartung HP, Lublin FD, et al. Recommended diagnostic criteria for multiple sclerosis: guidelines from the International Panel on the diagnosis of multiple sclerosis. Ann Neurol 2001;50:121–7. [6] Cepok S, Jacobsen M, Schock S, Omer B, Jaekel S, Boddeker I, et al. Patterns of cerebrospinal fluid pathology correlate with disease progression in multiple sclerosis. Brain 2001;124:2169–76. [7] Lucchinetti C, Bruck W, Parisi J, Scheithauer B, Rodriguez M, Lassmann H. Heterogeneity of multiple sclerosis lesions: implications for the pathogenesis of demyelination. Ann Neurol 2000;47:707–17. [8] Losy J, Mehta PD, Wisniewski HM. Identification of IgG subclasses' oligoclonal bands in multiple sclerosis CSF. Acta Neurol Scand 1990;82:4–8. [9] Hulett MD, Hogarth PM. Molecular basis of Fc receptor function. Adv Immunol 1994;57: 1–127. [10] Jefferis R, Lund J. Interaction sites on human IgG-Fc for FcgammaR: current models. Immunol Lett 2002;82:57–65. [11] Ravetch JV, Bolland S. IgG Fc receptors. Annu Rev Immunol 2001;19:275–90. [12] Gerber JS, Mosser DM. Stimulatory and inhibitory signals originating from the macrophage Fcgamma receptors. Microbes Infect 2001;3:131–9. [13] Garraud O, Perraut R, Riveau G, Nutman TB. Class and subclass selection in parasitespecific antibody responses. Trends Parasitol 2003;19:300–4. [14] Dhandayuthapani S, Izumi S, Anandan D, Bhatia VN. Specificity of IgG subclass antibodies in different clinical manifestations of leprosy. Clin Exp Immunol 1992;88: 253–7. [15] Kuenz B, Lutterotti A, Ehling R, Gneiss C, Haemmerle M, Rainer C. Cerebrospinal fluid B cells correlate with early brain inflammation in multiple sclerosis. PLoS ONE 2008;3:e2559. [16] Polman CH, Reingold SC, Edan G, Filippi M, Hartung HP, Kappos L, et al. Diagnostic criteria for multiple sclerosis: 2005 revisions to the “McDonald Criteria”. Ann Neurol 2005;58:840–6. [17] Freedman MS, Thompson EJ, Deisenhammer F, Giovannoni G, Grimsley G, Keir G, et al. Recommended standard of cerebrospinal fluid analysis in the diagnosis of multiple sclerosis: a consensus statement. Arch Neurol 2005;62:865–70. [18] Greve B, Magnusson CG, Melms A, Weissert R. Immunoglobulin isotypes reveal a predominant role of type 1 immunity in multiple sclerosis. J Neuroimmunol 2001;121: 120–5. [19] Vartdal F, Vandvik B. Multiple sclerosis: subclasses of intrathecally synthesized IgG and measles and varicella zoster virus IgG antibodies. Clin Exp Immunol 1983;54: 641–7. [20] Grimaldi LM, Maimone D, Reggio A, Raffaele R. IgG1, 3 and 4 oligoclonal bands in multiple sclerosis and other neurological diseases. Ital J Neurol Sci 1986;7:507–13. [21] Palmer DL, Minard BJ, Cawley LP. Letter: IgG subgroups in cerebrospinal fluid in multiple sclerosis. N Engl J Med 1976;294:447–8. [22] Raknes G, Fernandes Filho JA, Pandey JP, Myhr KM, Ulvestad E, Nyland H, et al. IgG allotypes and subclasses in Norwegian patients with multiple sclerosis. J Neurol Sci 2000;175:111–5. [23] Sharief MK, Thompson EJ. Intrathecal immunoglobulin M synthesis in multiple sclerosis. Relationship with clinical and cerebrospinal fluid parameters. Brain 1991;114 (Pt 1A):181–95. [24] Brandao CO, Ruocco HH, Farias AS, Oliveira C, Cendes F, Damasceno BP, et al. Intrathecal immunoglobulin G synthesis and brain injury by quantitative MRI in multiple sclerosis. Neuroimmunomodulation 2006;13:89–95. [25] Milanese C, Savoiardo M, La Mantia L, Campi A, Visciani A, Salmaggi A, et al. Magnetic resonance imaging in multiple sclerosis: diagnostic value and clinical correlations. Ital J Neurol Sci 1988;9:127–34. [26] Vrethem M, Fernlund I, Ernerudh J, Ohman S. Prognostic value of cerebrospinal fluid IgA and IgG in multiple sclerosis. Mult Scler 2004;10:469–71. [27] Baum K, Nehrig C, Girke W, Brau H, Schorner W. Multiple sclerosis: relations between MRI and CT findings, cerebrospinal fluid parameters and clinical features. Clin Neurol Neurosurg 1990;92:49–56. [28] Lambin P, Gervais A, Levy M, Defendini E, Dubarry M, Lebon P, et al. Intrathecal synthesis of IgG subclasses in multiple sclerosis and in acquired immunodeficiency syndrome (AIDS). J Neuroimmunol 1991;35:179–89. [29] Henriksson A, Kam-Hansen S, Link H. IgM, IgA and IgG producing cells in cerebrospinal fluid and peripheral blood in multiple sclerosis. Clin Exp Immunol 1985;62(1):176–84. PMID: 4064372.
Contents lists available at ScienceDirect
Journal of the Neurological Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n s
Features of intrathecal immunoglobulins in patients with multiple sclerosis Franziska Di Pauli a, Viktoria Gredler a, Bettina Kuenz a, Andreas Lutterotti a, Rainer Ehling a, Claudia Gneiss a, Michael Schocke b, Florian Deisenhammer a, Markus Reindl a, Thomas Berger a,⁎ a b
Clinical Department of Neurology, Innsbruck Medical University, Anichstrasse 35, A-6020 Innsbruck, Austria Department of Radiology, Innsbruck Medical University, Anichstrasse 35, A-6020 Innsbruck, Austria
a r t i c l e
i n f o
Article history: Received 28 July 2009 Received in revised form 15 September 2009 Accepted 17 September 2009 Available online 13 October 2009 Keywords: Multiple sclerosis Immunoglobulins Subclasses Cerebrospinal fluid Progression Oligoclonal bands Disease course B cells
a b s t r a c t We have analyzed immunoglobulin (Ig) isotypes and IgG subclasses in cerebrospinal fluid (CSF) and serum of patients with multiple sclerosis (MS) and other neurological diseases to determine whether different Ig isotype patterns correlate with clinical or paraclinical findings and CSF B cell populations. Intrathecal IgG1 synthesis was elevated in MS patients. An increased intrathecal IgM production was found in patients with a higher cerebral MRI lesion burden, whereas other clinical and paraclinical parameters were not associated with a specific Ig isotype or subclass profile. Finally, intrathecal IgG production (IgG1 and IgG3) correlated with the presence of mature B cells and plasma blasts. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Multiple sclerosis (MS) is a devastating demyelinating disorder thought to be mainly driven by an autoimmune attack [1,2]. Components of the immune system, including both cellular and humoral arm, are involved in the pathogenesis of MS, which is neuropathologically characterized by inflammation, demyelination and axonal loss. Clinically the immune response is reflected by the nearly mandatory detection of an intrathecal immunoglobulin (Ig) synthesis and oligoclonal bands (OCB) during (differential-) diagnostic procedures [3]. Recent results showed a high degree of concordance between the Ig proteome and B cell transcriptome in the cerebrospinal fluid (CSF) of MS patients, thus considering that the CSF B cells are the source of OCBs [4]. Since clonal B cell expansion and antibody production are established disease characteristics [3,5,6] and Ig and complement are found in MS certain lesions [7], a pathogenetic role of antibodies in MS has been suggested. The predominant subclasses in the CSF of MS patients are IgG1 and IgG3 [8]. Binding of various IgG subclasses to preferred Fc receptors (FcR) initiates a broad variety of executive responses in effector cells, such as degranulation, phagocytosis, antibody-dependent cell-mediated cytotoxicity (ADCC), production of multiple inflammatory cytokines and other mediators in mast cells, monocytes or macrophages [9,10], but FcR stimulation can also attenuate those functions [11,12].
⁎ Corresponding author. Tel.: +43 512 504 26277; fax: +43 512 504 24260. E-mail address: [email protected] (T. Berger). 0022-510X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2009.09.016
Since each isotype and subclass of Ig's has distinct biological functions, the preponderance of a specific subclass could be crucial for the outcome of a disease [13,14]. However, in MS correlations of immunoglobulins and autoantigenic profiles with disease evolution and severity are still under debate. In this study we aimed to study possible differences of Ig isotype patterns with regard to different MS disease activity parameters. Therefore we have analyzed the total Ig isotype and IgG subclass content and distribution in CSF and serum samples from patients at the onset of disease (CIS), relapsing–remitting, chronic progressive MS and other neurological diseases (OND) to correlate different Ig isotype patterns with the disease courses, MRI findings and CSF B cell populations. Further, we were interested whether Ig isotype patterns may predict an early conversion to CDMS. Additionally, in a subgroup of patients we analyzed CSF B cell subpopulations and their association with the Ig profile. 2. Methods 2.1. Patients Patients were recruited prospectively between 2004 and 2008 from the Clinical Department of Neurology, Innsbruck Medical University, Austria [15]. This study was approved by the ethical committee of Innsbruck Medical University (study Nr. oUN2045, 217/4.12, 07.07.2004) and all patients gave written informed consent. CIS and MS patients were included, if: (1) clinical symptoms and characteristic MRI findings according to the original “McDonald Criteria”
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[16] and (2) presence of intrathecal Ig synthesis (elevated Ig indices and/ or oligoclonal IgG bands) [17] Thirty-four patients with a CIS, 17 patients with RRMS and 11 patients with CPMS (2 with secondary progressive-SP and 9 with primary progressive-PP MS) were included. Patients were examined by a neurologist (F.D., A.L., R.E., C.G., and T.B.), including assessment of the expanded disability status scale (EDSS) and confirmation of relapse/disease progression during a follow-up period of at least 12 months. CDMS was diagnosed if a new relapse occurred. The control group consisted of 24 patients with other neurological diseases (OND). The OND group included 17 patients with noninflammatory OND (pseudotumor cerebri, migraine, psychogenic neurological symptoms, sinus venous thrombosis, cavernoma, vascular leukoencephalopathy, seizure, herniated vertebral disc, transient ischemic attack, spastic paraparesis, multiple system atrophy, neuropathic pain syndrome, cerebellar infarction, brain tumor, focal dystonia, and ischemic transverse spinal cord syndrome) and 7 patients with inflammatory OND (myelitis, ventriculitis, vasculitis, systemic lupus erythematosus, antiphospholipid syndrome, sarcoidosis and Guillain–Barré syndrome). In all patients lumbar puncture and MRI have been performed for diagnostics reasons.
2.4. Characterization of CSF cell populations by flow cytometry The staining of CSF cell populations (CD3+ T cells, CD56+ NK cells, CD19+CD138− mature B cells, CD19+CD138+ plasma blasts and CD19−CD138+ plasma cells) and FACS analysis was performed in 58 patients as described earlier [15]. 2.5. Statistics Statistical analysis was performed using SPSS (release 14.0, SPSS Inc., USA) software and GraphPad Prism 5 (GraphPad, San Diego, USA). Clinical, demographic and CSF data were compared using Dunn's multiple comparison test, Chi-Square test, Kruskal–Wallis test, Fisher's exact test or Mann–Whitney-U test. Statistical significance was defined as p-value < 0.05. Correlations were described by Spearman's nonparametric correlation. Statistical significance was defined as two-sided p-value<0.01. 3. Results Demographic, clinical and CSF characteristics of all prospective collected MS patients and neurological controls are shown in Table 1.
2.2. Sample collection CSF and serum samples (Sarstaedt Monovettes, Nuembrecht, Germany) were collected during standard diagnostic lumbar puncture and peripheral vein puncture. CSF samples were analyzed for CSF cell populations within 30 min after lumbar puncture and cell-free supernatants were stored at −80 °C for CSF subclasses analysis. Serum was prepared by centrifugation, 10 min at 3000 rpm, and stored at −20 °C until further analyses were performed. 2.3. Determination of CSF and serum levels of IgG subclasses CSF and serum samples were analyzed for total IgG, IgM and IgA levels using a Beckman clinical nephelometer. Total IgG1–4 CSF and serum levels were measured by ELISA using a commercially available test kit (Invitrogen, Carlsbad, CA, USA) according to manufacturer's guidelines (human IgG subclass profile ELISA kit, Catalog No. 99-1000, Zymed Laboratories, Carlsbad, CA). CSF was analyzed at a dilution of 1:20 and serum samples were analyzed at a dilution of 1:2500. The Ig indices were calculated according to the Delpech and Lichtblau protein quotient (IgGCSF/IgGserum:albuminCSF/albuminserum.).
3.1. IgG subclasses in MS patients and controls CSF IgG1 levels were significantly elevated in patients with a CIS (p < 0.01), RRMS (p < 0.001) and CPMS (p < 0.01) patients compared with OND. In contrast CSF IgG2, IgG3 and IgG4 subclass levels did not differ in any of the groups (Fig. 1). Further, there were no significant differences between IgG1, IgG2, IgG3 and IgG4 serum levels between the MS patients and controls. The comparison between the intrathecal synthesis of IgM, IgA, total IgG and each of its subclasses revealed similar results. We found significantly increased total IgG indices in patients with a CIS (p<0.001) and RRMS (p<0.01), which were predominantly due to increased intrathecal IgG1 synthesis (Table 1 and Fig. 1). Further, intrathecal IgM synthesis was significantly higher in MS patients than in OND (Table 1). 3.2. Correlation of IgG subclasses with clinical and paraclinical parameters Additionally, it was analyzed whether MRI activity, defined by the number of lesions on T2-weighted MRI (<9 or≥9T2 lesions) and the presence of gadolinium enhancing lesions on T1 weighted MRI (no or ≥1 lesions), were correlated with CSF levels or indices of IgM, IgA, IgG
Table 1 Demographic, clinical and CSF data of analyzed patients.
n Females (%) Age (y)1 EDSS1 Acute relapse MRI Gd + lesions ≥ 9 MRI T2 lesions OCB CSF cells/µl1 IgG index1 IgM index1 IgA index1 Q-Alb1 IgG 1 index1 IgG 2 index1 IgG 3 index1 IgG 4 index1
CIS
RRMS
CPMS
OND
34 26 (76%) 30 (18–49) # 1.8 (0–3.5) 32 (94%) 24 (73%) # 13 (38%) # 34 (100%) # 8 (1–76) # 0.83 (0.06–3.15) 0.13 (0.00–1.59) 0.36 (0.00–4.83) 4.46 (2.3–76.61) 0.94 (0.36–4.01) 0.56 (0.17–1.68) 0.65 (0.20–2.48) 0.42 (0–9.00)
17 14 (82%) 32 (16–63) # 2.0 (0–4.0) 12 (71%) 9 (69%) # 6 (36%) # 15 (88%) # 7 (1–17) # 0.86 (0.42–2.01) # 0.15 (0.07–0.46) # 0.40 (0.25–2.45) 5.28 (2.84–12.23) 0.93 (0.38–3.76) # 0.39 (0.09–0.79) 0.50 (0.17–2.93) 0.32 (0.15–0.99)
11 3 (27%) 47 (34–63) 6.0 (3.5–7.0) 1 (9%) 4 (44%) # 3 (33%) # 9 (82%) # 4 (0–20) 0.74 (0.43–4.46) 0.13 (0.05–0.76) 0.34 (0.26–1.03) 6.83 (2.30–10.90) 0.78 (0.35–4.29) 0.40 (0.21–1.92) 0.35 (0.19–4.65) 0.31 (0.07–0.60)
24 16 (67%) 43 (22–68)
# # # #
0 (0%) 0 (0%) 2/20 (10%) 2 (0–211) 0.50 (0.39–1.09) 0.07 (0–0.46) 0.29 (0.19–0.82) 4.95 (2.31–17.11) 0.52 (0.39–1.97) 0.50 (0–0.90) 0.47 (0.15–2.32) 0.40 (0–1.02)
p-value 0.0112 <0.0013 <0.0013 <0.0013 <0.0012 0.0162 <0.0012 <0.0013 <0.0013 0.0063 0.0143 0.1603 0.0013 0.1663 0.0493 0.5403
Data are shown as median (range), p-value: groups were compared using 2Chi-Square test and 3Kruskal–Wallis test, # statistically different from OND control group. Abbreviations: n = number of patients, y = years, EDSS = expanded disability status score, MRI Gd + lesions = presence of lesions on gadolinium-enhanced T1-weighted MRI (neg = negative, pos = positive), ≥9 T2 MRI lesions = ≥9 lesions on T2-weighted MRI, OCB = oligoclonal bands, and Q-Alb = albumin quotient.
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Fig. 1. IgG1–4 subclasses in CSF and sera of patients with a CIS, RRMS, CPMS and OND (outlier not shown).
and its subclasses in MS patients. Only an increased intrathecal IgM synthesis was significantly associated with a higher lesion load on T2weighted MRI with a median IgM index of 0.16, range 0.06–1.59 in the group with more than 9 lesions versus 0.11, range 0–0.76 (p<0.01). During an acute relapse of MS CSF cells were significantly increased (median 23.5, range 2–227 versus median 13.0, range 1–60, p = 0.003). Furthermore, a tendency to an elevated intrathecal IgG3 synthesis was shown in patients with a relapse as compared to those without a relapse (median 0.64, range 0.17–2.94 versus median 0.42, range 0.19–4.65, p = 0.035). There were no significant differences regarding the other Ig isotype/subclass indices between patients with or without acute disease activity. A subgroup analyses was performed in 31 CIS patients with a followup of at least 12 months to determine a possible predictive value of a
specific immunoglobulin isotype pattern for an early conversion to CDMS. Neither CSF levels and indices of IgM or IgA nor of total IgG were associated with an early conversion to CDMS within given timepoints of one or three years (Table 2). No specific subclass pattern showed a prognostic value for an early conversion to CDMS. 3.3. IgG subclasses and CSF B cells In 58 patients we analyzed CSF B (mature B cells, plasma blasts and plasma cells) and T cells and their correlations with different immunoglobulin isotype pattern. Mature B cells (CD19+CD138−) and plasma blasts (CD19+CD138+) were associated with intrathecal IgM (R=0.356, p<0.001, and R=0.491, p<0.001 respectively) and total IgG synthesis (R=0.416, p<0.001, and R=0.592, p<0.001 respectively). Furthermore,
Table 2 Correlation of CSF findings and conversion to CDMS after a CIS within 3 years.
CSF cells/µl1 Q-Alb1 IgG index1 IgM index1 IgA index1 IgG1 index1 IgG2 index1 IgG3 index1 IgG4 index1
CIS
CDMS
Hazard ratio
95% CI
p-value
7 (1–76) 4.69 (2.82–76.61) 0.85 (0.49–2.05) 0.12 (0–0.82) 0.34 (0–0.75) 0.81 (0.37–2.77) 0.52 (0.31–0.81) 0.59 (0.20–0.87) 0.42 (0–1.00)
9 (3–52) 4.46 (2.30–8.94) 0.87 (0.06–3.15) 0.13 (0.06–1.59) 0.38 (0–4.83) 1.20 (0.36–4.01) 0.66 (0.17–1.68) 0.73 (0.31–2.48) 0.43 (0–9.00)
1.013 0.930 0.677 0.460 1.258 0.405 1.496 6.432 0.949
0.996–1.029 0.610–1.417 0.085–5.374 0.101–2.102 0.725–2.181 0.055–3.007 0.192–11.641 1.018–40.644 0.605–1.491
0.126 0.736 0.712 0.316 0.414 0.377 0.700 0.048 0.822
Data are shown as 1median (range), p-value: groups were compared using Cox proportional-hazards model.
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the IgG subclasses analyses showed a significant correlation of the IgG1 index with mature B cells (R=0.456, p<0.001) and plasma blasts (R=0.616, p<0.001) and of the IgG3 index with the same B cell subclasses (R=0.340, p<0.01, and R=0.382, p<0.01 respectively), whereas CSF T cells, NK cells and plasma cells were not correlated with any of the analyzed CSF findings. 4. Discussion This study was conducted to determine the role of immunoglobulin subclass patterns in patients with different courses of MS and ONDs. The detected levels are similar to those of other populations [18]. We found significantly elevated CSF IgG1 levels in CIS, RRMS and CPMS patients and increased IgG1 indices in CIS and RRMS patients compared with controls. These results are partly in accordance with previous studies which show either elevated IgG1 levels alone or elevated IgG1 and IgG3 levels in MS patients [8,18–21]. Raknes et al. did not find any associations of total IgG or IgG subclass levels and the initial disease course [22], also in our study cohort there were no significant correlations between the disease course and the CSF findings. Furthermore, we could confirm an increase in total IgG, IgM and IgA indices in MS patients compared with controls ([23,29]), but there were no significant differences between CIS, RRMS and CPMS patients. We found a significant association of cerebral MRI lesion burden with an increased intrathecal IgM synthesis, which is in contrast with the findings of Sharief and Thompson [23]. A correlation of the MRI activity with the IgG index was described [24,25], which was not confirmed by our results. No correlations between CSF parameters and other clinical or paraclinical features were described in previous studies [26–28]. Until now no study to our knowledge investigated whether a special immunoglobulin subclass phenotype can predict an early conversion to CDMS. There were no significant correlations in our 31 CIS patients during the follow-up of 36 months, a final conclusion of these questions is limited due the small sample size. Since the negative results in the different clinical and paraclinical measurements, it is likely that the immunoglobulin subclasses play only a minor role in the outcome of MS. However, it is possible that relevant autoantibodies are reduced in the CSF because of being bound to their antigen in the tissue and therefore they may not be detected with classical methods. In a subgroup analysis we could detect a correlation between mature B cells and plasma blasts in the CSF and CSF IgG1 levels and intrathecal IgG1 and IgG3 synthesis. These results suggest that the CSF B cells are the source of the intrathecal IgGs and that the clonal expanded B cells produce mainly IgG1. Recently the link between subject-specific immunoglobulin transcriptomes to the proteomes in diagnostic CSF samples from MS patients was demonstrated and supports the pathogenetic relevance of CSF antibodies [4]. The role of the CSF B cells and the intrathecal produced immunoglobulins in the disease development and progression is less clear. Although, it is known that the cytokine milieu during B cell differentiation decides about the mainly produced antibody subtypes, the exact regulation of the subclass switch remains an open question. In other diseases with preferential Th1 immunity such as type 1 diabetes and Lyme's disease IgG1 dominance is well known. In conclusion, our results reveal a preponderance of IgG 1 also in the humoral immune response of MS and, therefore, supports the hypothesis of a mainly Th1 driven autoimmune disease. Acknowledgements This study was supported by a research grant of the Gemeinnuetzige Hertiestiftung (Frankfurt, Germany). The authors wish to thank
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