Free light chain monomer–dimer patterns in the diagnosis of multiple sclerosis

Journal of Immunological Methods 390 (2013) 74–80

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Research paper

Free light chain monomer–dimer patterns in the diagnosis of multiple sclerosis Batia Kaplan a,⁎, Sizilia Golderman a, Gilad Yahalom b, Regina Yeskaraev c, Tamar Ziv d, Boris M. Aizenbud e, Ben-Ami Sela c, f, Avi Livneh c, f a

Heller Institute of Medical Research, Sheba Medical Center, Tel-Hashomer, Israel Department of Neurology, Sheba Medical Center, Tel-Hashomer, Israel c Institute of Chemical Pathology, Sheba Medical Center, Tel-Hashomer, Israel d Smoler Proteomics Center, Department of Biology, Technion, Israel Institute of Technology, Israel e Signal Processing Division, Elta Systems (IAI), Ashdod, Israel f Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel b

a r t i c l e

i n f o

Article history: Received 10 January 2013 Received in revised form 24 January 2013 Accepted 25 January 2013 Available online 31 January 2013 Keywords: Multiple sclerosis Diagnosis Free light chains Cerebrospinal fluid Western blotting Mass spectrometry

a b s t r a c t In our search of new biomarkers for multiple sclerosis (MS), we aimed to characterize the immunoglobulin (Ig) free light chains (FLC) in patients' cerebrospinal fluid (CSF) and serum, and to evaluate the diagnostic utility of FLC monomer–dimer patterns for MS. FLC were analyzed by Western blotting and mass spectroscopy. CSF and serum samples were examined for the presence of oligoclonal Ig bands by a conventional laboratory test for MS. Three distinct pathological FLC monomer–dimer patterns, typical of MS but not of other neurological diseases, were revealed. In 31 out 56 MS patients the highly increased CSF levels of κ monomers and dimers were demonstrated. In 18 MS patients, the increased κ-FLC levels were accompanied by highly elevated λ dimers. Five MS cases showed no significant elevation in κ-FLC, but they displayed abnormally high λ dimer levels. The intensity of the immunoreactive FLC bands was measured to account for κ and λ monomer and dimer levels and their ratios in the CSF and serum. Combined usage of different FLC parameters allowed the determination of the appropriate FLC threshold values to diagnose MS. The developed method showed higher sensitivity and specificity (96% and 90%, respectively), as compared to those of the conventional OCB test (82% and 70%, respectively). Our study highlights the role of the differential analysis of monomeric and dimeric κ- and λ-FLC for the precise diagnosis of MS. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Multiple sclerosis (MS) is a putatively autoimmune inflammatory disease, leading to demyelination, axonal damage and

Abbreviations: FLC, free light chains; MS, multiple sclerosis; CSF, cerebrospinal fluid; OCB, oligoclonal bands; Ig, immunoglobulin; MRI, magnetic resonance imaging; SDS, sodium dodecyl sulphate; SB, sample buffer; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid. ⁎ Corresponding author at: Heller Institute of Medical Research, Sheba Medical Center, Tel-Hashomer 52621, Israel. Tel.: +972 3 530 3363. E-mail address: [email protected] (B. Kaplan). 0022-1759/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jim.2013.01.010

neuronal loss. Since clinical manifestations in MS overlap with other neurological diseases, magnetic resonance imaging (MRI) and the laboratory tests are used to improve diagnostic specificity. In the most commonly used laboratory test, the demonstration of oligoclonal immunoglobulin (Ig) bands (OCB) indicates an intrathecal production of Igs and supports the diagnosis of MS. Yet, oligoclonal Igs might be seen in other inflammatory and infectious CNS diseases (Jenkins et al., 2001), making the differentiation of MS from other neurological diseases difficult. Intrathecal production of not only intact Igs, but also Ig free light chains (FLC), is now regarded as an important

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immunological response developing in the CNS of MS patients. Normally, light chains are produced by B-cells in excess over heavy chains, and the majority of light chains bound to heavy chains. The physiological levels of unbound light chains, i.e. FLC, are low. FLC exist as two isotypes, kappa (κ) or lambda (λ). Both κ and λ FLC are found in different body fluids, including serum and cerebrospinal fluid (CSF), where they present in two major molecular forms, namely, the monomers and dimers. Although in a healthy state the amounts of secreted FLC are low, under pathological conditions the levels of FLC might be abnormally high. The finding of increased CSF levels of Ig FLC in MS was first described in the late 70s (Vandvik, 1977; Perini et al., 1979; Link and Laurenzi, 1979). However, the methods applied to detect FLC, mainly the electrophoretic techniques, were basically qualitative and of questionable specificity in the diagnosis of MS (Rudick et al., 1986; Bracco et al., 1987; Sindic and Laterre, 1991; Kolar et al., 1980; Cavuoti et al., 1998). Recent achievements in FLC detection and quantification, especially the development of the highly sensitive nephelometric FLC assays, revived the significance of FLC analysis in MS diagnosis. An increased level of κ-FLC was demonstrated in the CSF of MS patients and considered as an important marker of this disease (Fischer et al., 2004; Desplat-Jego et al., 2005; Presslauer et al., 2008). Yet, the specificity of this nephelometric test remained lower than that of the OCB test (Presslauer et al., 2008) and even lower than the IgG index (Desplat-Jego et al., 2005). The finding that λ-FLC may also increase significantly in MS (Arneth and Birklein, 2009), raised expectations that a combined analysis of κ and λ-FLC may increase the accuracy of MS detection. Indeed, we have developed a diagnostic method based on a numerical evaluation of the κ- and λ-FLC monomers in the patient's CSF and serum (Kaplan et al., 2010), and showed it to be of higher specificity and sensitivity for MS detection than the commonly used OCB test. We have also noted that while CSF κ-FLC levels were significantly elevated in most MS patients, CSF levels of λ dimer varied widely. The present study was aimed to further characterize λ dimer levels in MS and evaluate the role of FLC monomer–dimer patterns in the diagnosis of MS. 2. Materials and methods 2.1. Patients and samples CSF and serum samples, collected from patients, were stored at −30 °C until used. The patients' records were reviewed and three patient groups were defined. The MS group included 56 patients with definite MS diagnosis, based on the McDonald criteria (McDonald et al., 2001). The non-MS group consisted of 39 patients with other neurological diseases where the diagnosis of MS was excluded. This group included vasculitis (n=5), lupus (n=1), cavernoma (n=1), astrocytoma (n=1), cerebral ischemic events (n=1), Guillain–Barré syndrome (n=1), stroke (n=2), neuromyelitis optica (NMO) (n=1), internuclear ophthalmoplegia (INO) (n=1), migraine (n=1), myelitis (n= 5), optic neuritis (n=4), and the non-MS cases with no definite clinical diagnosis (n=15). The third patient group contained 8 patients showing no evidence for demyelinating, inflammatory/ infectious and other known neurological diseases. The study was approved by the institutional review board.

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2.2. Oligoclonal banding test A standard procedure for the detection of oligoclonal IgG bands was employed, using high-resolution agarose gel electrophoresis (Hydragel 6 CSF), coupled with immunofixation (Sebia, Moulineaux, France). 2.3. Sample preparation for Western blot analysis 2.3.1. CSF samples Twenty μL of the tested CSF sample was dried in a SpeedVac and stored at −30 °C until used. The dried sample was redissolved in 30 μL of sample buffer SBx2 (containing 0.2 mol/L sucrose, 6% SDS, 125 mmol/L Tris, 4 mmol/L Na2EDTA, pH 6.8) prior to the electrophoretic run. Alternatively, CSF sample was applied directly onto the gel following its dilution with SBx5 (1/0.5, vol/vol). CSF sample preparation, whether with or without drying, did not affect the obtained results. 2.3.2. Serum samples Five μL of serum sample was diluted with 0.9% sodium chloride solution, 1/80. Ten μL aliquots of the obtained solution were dried and stored at −30 °C until used. The dried serum sample was re-dissolved in 40 μL of SBx2 prior to the electrophoretic run. Alternatively, serum samples were applied directly onto the gel (without sample drying). For this purpose, the diluted sample was mixed with SBx5 (1/3), and then subjected to electrophoresis. 2.3.3. CSF positive control samples Positive control represented a mixture of CSF samples obtained from 15 patients with definite MS diagnosis. The obtained mixture was divided into 20 μL aliquots, dried and stored at −30 °C until used. The dry residue was re-dissolved in 30 μL SBx2 prior to electrophoresis. Positive control samples were included in each electrophoretic run. 2.4. Western blotting CSF and serum samples were analyzed using Western blotting as described (Kaplan et al., 2010). Briefly, electrophoresis was performed on high resolution 10–20% Nu-Sep Tris-Tricine gels (Gradipore Frenchs Forest, Australia) under non-reducing conditions. The electrophoretically separated proteins were blotted onto nitrocellulose (Schleicher and Schuell, Dassel, Germany) with a Gradipore LongLife Transfer buffer. FLC bands were immunodetected with rabbit antibodies to human immunoglobulin κ and λ light chains (DAKO, Carpinteria, CA, USA). Immunodetection procedure was performed using Bench Pro 4100 Card Processing station (Invitrogen), allowing automation of manual processing steps. Proteins were visualized with Super-Signal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA). 2.5. Western blot data analysis A software developed by us previously (Kaplan et al., 2010) was used for quantitative evaluation of the intensity of FLC-κ and FLC-λ immunoreactive bands, as a measure of FLC levels in the CSF and serum. The obtained immunoreactivity value of the FLC band in the tested sample (Asample) was

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normalized relatively to that in the positive control sample (Acontr). To analyze the monomeric FLC, 3 diagnostic FLC tests, developed by us earlier (Kaplan et al., 2010), were applied to determine FLC indices accounting for CSF λ- and κ-monomer levels (T0 and T1, respectively), for CSF κ/λ monomer ratio values (T2), and for differences in κ/λ monomer ratios in the serum versus CSF of the same patient (T3): T0 T1 T2 T3

Acontr λ monomer/Asample λ monomer in CSF Acontr κ monomer/Asample κ monomer in CSF, Acontr κ/λ monomer/Asample κ/λ monomer in CSF, Asample κ/λ monomer ratio in the serum divided to that in the CSF.

Mathematical multiplication of the tests T1, T2 and T3 values was used to construct the Combined Test Index for monomers (CTIm): CTIm = T1 × T2 × T3. For the quantitative assessment of the dimeric λ FLC, three new additional tests were applied in the present study to determine FLC indices accounting for λ dimer level in the CSF (T4), for λ monomer/dimer ratio in the CSF (T5), and for differences in λ monomer/dimer ratios in the CSF versus serum of the same patient (T6): T4 T5 T6

Acontr λ monomer/Asample λ dimer in CSF, Asample λ monomer in CSF/Asample λ dimer in CSF, Asample λ monomer/Asample λ dimer in the CSF divided by that in the serum.

To construct the Combined Test Index for dimers (CTId), multiplication of the tests 4, 5 and 6 values was used: CTId = T4 × T5 × T6. 2.6. Mass spectroscopy 2.6.1. Preparation of FLC-enriched CSF fractions for protein identification by mass spectrometry Preparation of CSF fractions enriched with FLC monomers (25 kDa CSF fraction) and FLC dimers (50 kDa CSF fraction) was performed by micro-preparative electrophoresis as described earlier (Kaplan et al., 2008, 2009). The CSF samples were run on Nu-Sep 10–20% polyacrylamide Tris-Tricine gels (Gradipore, Frenchs Forest, Australia) under non-reducing conditions. After completion of the electrophoretic run, the proteins of interest were eluted from gel using elution buffer containing 0.1% SDS, 0.05 mol/L Tris–HCl (pH 7.0), 0.1 mmol/L ethylenediaminetetraacetic acid (EDTA), and 0.2 mol/L NaCl. The eluted proteins containing either FLC monomers or disulfide bound FLC dimers were dialyzed using VISKINGdialysis tubing, MW cut off 12 kDa (Serva, Heidelberg, Germany). Finally, the FLC-enriched CSF samples were run on Nu-Sep 10–20% polyacrylamide gels and stained with Coomassie Blue. The gel slices containing the bands of interest were excised for protein identification by mass spectrometry.

temperature for 30 min) and digested in 10% acetonitrile and 10 mmol/L ammonium bicarbonate with modified trypsin (Promega) overnight at 37 °C. The resulting tryptic silica capillaries (J&W Scientific, USA) packed with Reprosil reversed phase material (Dr Maisch GmbH, Germany). The peptides were eluted with linear 95 min gradients of 7 to 40% and 8 min at 95% acetonitrile with 0.1% formic acid in water at flow rates of 0.25 μL/min. Mass spectrometry was performed by an ion-trap mass spectrometer (Orbitrap, Thermo) in a positive mode using repetitively full mass spectra scan followed by collision induced dissociation of the 7 most dominant ion selected from the first mass spectra scan. The mass spectrometry data was analyzed using the Sequest 3.31 software (J. Eng and J. Yates, University of Washington and Finnigan, San Jose) searching against the human section Uniprot database. 2.7. Statistical analysis The nonparametric Mann–Whitney test for comparing two unmatched samples (method I — for n ≥ 5 values per group) was applied to compare CTI values in different MS patient groups. Outlying values were determined (at p b 0.01) using Grubbs' test. 3. Results 3.1. Pathological FLC profiles in MS The CSF samples of 56 patients with definite MS diagnosis were subjected to Western blot analysis as described above. The obtained data were in concordance with our previous study (Kaplan et al., 2010): most MS patients (49 out of 56) demonstrated abnormally high levels of κ monomers and dimers in comparison to control patients and to most patients with other neurological diseases (Fig. 1). In addition to high κ levels, 18 out of 49 MS patients demonstrated highly increased levels of λ dimers. Other MS patients with the abnormally high FLC-κ levels (31 out of 49 MS cases) displayed the relatively low levels of both monomeric and dimeric λ FLC. Unexpectedly, 5 out of 56 MS patients showed no significant elevation in the monomeric κ-FLC, but these MS patients displayed abnormally high λ dimer levels. Thus, three distinct pathological FLC patterns were revealed in MS: “κ-type” pattern (about 55% of MS patients) showing high κ and low λ levels, “mixed κ–λ” type pattern (35%) where increased κ levels were accompanied by high levels of λ dimers, and “λ-type” pattern (10%) where the major pathological feature was increased dimerization of λ FLC. We also found that FLC profile in the CSF was different from that in the serum of the same MS patient (Fig. 1). In the “κ-type” and “mixed type”, κ/λ monomer ratio in the CSF was high compared to that in the serum. In the “mixed type” and “λ-type”, λ dimer/monomer ratio in the CSF was high compared to that in the serum. 3.2. FLC indices in MS

2.6.2. In gel proteolysis and mass spectrometry analysis The proteins in each gel slice were reduced with 2.8 mmol/L DTT (60 °C for 30 min), modified with 8.8 mmol/L iodoacetamide in 100 mmol/L ammonium bicarbonate (in the dark, room

A specially developed software (Kaplan et al., 2010) was applied for the quantification of the intensity of immunoreactive κ and λ bands and for the determination of the FLC

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well as “κ-type” versus “λ-type” groups. Taken together, MS patients were different with respect to either CTIm or CTId values, or to both of them, suggesting that there might be a correlation with different clinical settings of this disease. 3.3. FLC indices in non-MS cases FLC indices of most non-MS patients differed from those of MS patients. Thirty four out of 39 non-MS patients showed high CTIm values (from 10 to > 200) compared to “κ type” and “mixed type” MS patients (CTIm from 0.002 to 12). These non-MS patients also demonstrated high CTId values (from about 0.1 to 20) compared to MS patients with “mixed” and “λ type” (CTId from 0.0001 to 0.009). λ monomer levels in these non-MS patients (T0 from 0.15 to 10) were similar to λ monomer levels in the MS patients (T0 from 0.13 to 5.0). 3.4. FLC criteria to support MS

Fig. 1. Western blot analysis of CSF-serum pairs demonstrating three pathological FLC patterns typically observed in the MS patients. Samples were run on 10–20% Nu-Sep gels under non-reducing conditions, blotted and immunostained using anti-kappa and anti-lambda antibodies to detect the monomeric (25 kDa) and dimeric (50 kDa) FLCs. In the “κ-type” FLC pattern, the CSF of MS patient shows the abnormally high FLC-κ levels without significant elevation of λ type FLCs. The CSF of MS patient with a “mixed κ–λ type” FLC pattern is characterized by high κ levels together with significantly elevated λ dimers. In a less frequent “λ-type” FLC pattern, MS patient shows the abnormally high CSF λ dimer level without significantly increased κ-FLCs. None of these pathological FLC patterns was observed in the non-MS patient with stroke.

indices. Combined indices for FLC monomers (CTIm) were calculated to account for the κ monomer levels and for κ/λ monomer ratio in the patient's CSF and serum. Combined indices for λ dimers (CTId) were calculated to account for λ dimer level and λ dimer/monomer ratio in the patient's CSF and serum. The CTIm and CTId values for MS patients with 3 different FLC patterns (“κ type”, “mixed κ–λ type” and “λ type”) are presented in Table 1. CTIm values in the MS patients with “κ type” and “mixed type” differed significantly from those with “λ-type” (Table 2). The statistically significant differences in the CTId values were found when comparing “κ-type” group versus “mixed type” groups, as

The obtained data showed that the diagnosis of MS on a basis of a single FLC parameter might be imprecise. We found that combined use of different FLC indices was effective to characterize FLC patterns and to differentiate MS from other non-MS neurological diseases. Based on analysis of the obtained CTIm, CTId and T0 values, FLC threshold values were determined to support or to exclude the diagnosis of MS. We showed that in the MS cases with “κ” type FLC pattern and in most MS cases with “mixed” type FLC pattern, the CTIm values were below 4. In the MS cases with “mixed” and “λ” type FLC patterns, the CTId values were below 0.01. Taken together, FLC data support MS diagnosis when (a) CTIm b 4, or/and (b) CTId b 0.01. In both cases, the λ monomer index was T0 >0.1. 3.5. The developed FLC method versus the OCB test The results of OCB and FLC analyses of MS and non-MS group patients were compared. Forty six out of 56 MS cases were OCB-positive, while the rest of the MS cases were either OCB-negative (n= 6) or questionable (n=4). Data of FLC analysis supported the diagnosis of MS in 54 out of 56 MS patients (Table 3). In the non-MS group, 25 out of 36 cases were OCB-negative, 11 cases were OCB-positive. The 11 oligopositive cases in this patient group included patients with clinical diagnoses of myelitis (n= 3), optic neuritis (n= 3), and INO (n= 1), as well as 4 non-MS patients with no definite clinical diagnosis. The data of FLC analysis excluded MS diagnosis in 34 out of 39 non-MS cases, while in 4 cases the obtained FLC data supported the diagnosis of MS. The latter cases included patients with clinical diagnoses of optic neuritis (n= 2),

Table 1 FLC pattern types in the MS patients.

FLC pattern type in MS Patient number n (%) CTIm mean (range) CTId mean (range) a b

Without outlier (19.2). Without outlier (11.5).

κ-Type

Mixed κ–λ type

31 (57%) 0.67 (0.002–1.8)

18 (34%) 2.5 (0.1–19.2) 1.57 (0.1–6.0)a 0.003 (0.0001–0.009)

0.86 (0.011–11.5) 0.32 (0.011–3.76)b

λ-Type 5 (9%) 55.5 (43.2–79.3) 0.003 (0.0001–0.0052)

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Table 2 Comparison of CTI values of MS patients with different FLC pattern types.

Table 4 Mass spectroscopy-based identification of FLC κ and λ constant region peptides in the CSF of non-MS patients.

p value “κ-Type” CTIm versus “mixed type” CTIm — with outliers “κ-Type” CTIm versus “mixed type” CTIm — without outliers “κ-Type” CTId versus “mixed type” CTId — with outliers “κ-Type” CTId versus “mixed type” CTId — without outliers “κ-Type” CTIm versus “λ-type” CTIm “κ-Type” CTId versus “λ-type” CTId — with outliers “κ-Type” CTId versus “λ-type” CTId — without outliers “Mixed type” CTIm versus “λ-type” CTIm — with outliers “Mixed type” CTIm versus “λ-type” CTIm — without outliers “Mixed type” CTId versus “λ-type” CTId

≤0.08 ≤0.13 ≤1.1 × 10−7 ≤1.5 × 10−7 ≤0.0004 ≤0.0007 ≤3.7 × 10−5 ≤5.9 × 10−5 ≤7.6 × 10−5 ≤0.85

myelitis (n=1), and INO (n= 1). As result, our diagnostic FLC test demonstrated specificity of 90% and sensitivity of 96%, while the conventional OCB test showed specificity of 70% and sensitivity of 82%. Thus, although our FLC test takes more time to perform (7 h) compared to the oligoclonality test (2.5 h), it was more efficient in respect to specificity and sensitivity.

3.6. Protein identification by mass spectrometry supports Western blot data CSF samples obtained from 8 MS and 2 non-MS patients were subjected to FLC identification by mass spectrometry. For this purpose, the 25 and 50 kDa CSF fractions containing monomeric and dimeric FLC, respectively, were prepared according to the procedure described in the Materials and methods section. Sequences of Ig light chains, as well as of other CSF proteins of a similar molecular mass were identified. The FLC constant domain sequences typically detected in the non-MS and MS disorders are presented in the Tables 4 and 5, respectively.

3.7. Distribution of κ and λ subtypes in MS patients Protein identification by mass spectrometry revealed sequences belonging to κ and λ variable domains (Table 6). Different κ subtypes (V-I, V-II, V-III, V-IV) were identified in the CSF of 8 tested MS patients. Distribution of κ-subtypes in MS was similar to that in the non-MS cases. λ variable domain sequences were identified in 5 out of 8 tested MS cases, and found to be mainly restricted to V-III subtype. No λ variable domain sequences were detected in the non-MS cases.

Table 3 FLC versus OCB test.

MS patients, n (+/−/?)a Non-MS patients, n (+/−/?) Specificity, % Sensitivity, % a

OCB test

FLC test

56 (46/6/4) 36 (11/25/0) 70% 82%

56 (54/2/0) 39 (4/35/0) 90% 96%

Support MS/does not support MS/questionable.

Non-MS patient # 539 with stroke # 539: FLC monomer peptides

# 539: FLC dimer peptides

Ig kappa chain C region HKVYACEVTHQGLSSPVTK SGTASVVCLLNNFYPR TVAAPSVFIFPPSDEQLK VDNALQSGNSQESVTEQDSK VYACEVTHQGLSSPVTK Ig lambda-like polypeptide AGVETTKPSK ANPTVTLFPPSSEELQANK.A SYSCQVTHEGSTVEK Ig lambda-2 chain C region AAPSVTLFPPSSEELQANK AGVETTTPSK

Ig kappa chain C region SFNRGEC SGTASVVCLLNNFYPR TVAAPSVFIFPPSDEQLK VDNALQSGNSQESVTEQDSK VYACEVTHQGLSSPVTK Ig lambda-like polypeptide SYSCQVTHEGSTVEK YAASSYLSLTPEQWK

Non-MS patient # 858 with astrocytoma # 858: FLC monomer peptides

# 858: FLC dimer peptides

Ig kappa chain C region SFNRGEC SGTASVVCLLNNFYPR TVAAPSVFIFPPSDEQLK VDNALQSGNSQESVTEQDSK VYACEVTHQGLSSPVTK Ig lambda-like polypeptide AGVETTKPSK SYSCQVTHEGSTVEK Ig lambda-2 chain C region AAPSVTLFPPSSEELQANK AGVETTTPSK

Ig kappa chain C region SFNRGEC SGTASVVCLLNNFYPR TVAAPSVFIFPPSDEQLK VYACEVTHQGLSSPVTK Ig lambda-2 chain C region AGVETTTPSK

4. Discussion In the present study we applied a quantitative Western blot technique to evaluate the FLC κ and λ monomer and dimer levels in the CSF and sera of MS and non-MS patients. The FLC method, developed by us, was shown to be highly efficient in the diagnosis of MS yielding 96% sensitivity and 90% specificity. We also found that MS patients may be allocated into 3 subsets according to their FLC patterns: “κ-type” (increased κ level, 57% of patients), “λ-type” (increased λ dimer level, 9%), and “mixed κ–λ type” (34%). This study highlights the role of the differential analysis of monomeric and dimeric κ- and λ-FLC for precise diagnosis of MS. Further studies are needed to understand whether these differences in immunological response observed by us in MS patients are related to different clinical settings of this disease, its prognosis and response to treatment. Increased λ light chain dimerization observed in the CSF of MS patients is a new finding which merits attention. High levels of disulfide-bound dimeric FLCs were found in the plasma of patients with monoclonal diseases, such as multiple myeloma and amyloidosis AL (Kaplan et al., 2009, 2011), insinuating a possible role for light chain subtype restriction in the pathogenesis of MS as well. To date, it is still unknown whether each of the oligoclonal FLC bands detected in the CSF of MS patients (Sindic and Laterre, 1991; Goffette et al., 2004; Zeman et al., 2012) contains monoclonal proteins. Protein identification by mass spectrometry applied in this study displayed different κ subtypes (V-I, V-II, V-III, V-IV) in the CSF

B. Kaplan et al. / Journal of Immunological Methods 390 (2013) 74–80 Table 5 Mass spectroscopy-based identification of FLC κ and λ constant region peptides in the CSF of MS patients. MS patient # 127 # 127: FLC monomer peptides

# 127: FLC dimer peptides

Ig kappa chain C region DSTYSLSSTLTLSK SFNRGEC SGTASVVCLLNNFYPR TVAAPSVFIFPPSDEQLK VDNALQSGNSQESVTEQDSK VYACEVTHQGLSSPVTK

Ig kappa chain C region DSTYSLSSTLTLSK HKVYACEVTHQGLSSPVTK SFNRGEC SGTASVVCLLNNFYPR TVAAPSVFIFPPSDEQLK VDNALQSGNSQESVTEQDSK VYACEVTHQGLSSPVTK Ig lambda-like polypeptide AGVETTKPSK ANPTVTLFPPSSEELQANK SYSCQVTHEGSTVEK VTVLGQPK YAASSYLSLTPEQWK

Ig lambda-like polypeptide AGVETTKPSK ANPTVTLFPPSSEELQANK SYSCQVTHEGSTVEK Ig lambda-2 chain C region AAPSVTLFPPSSEELQANK AGVETTTPSK

Ig lambda-2 chain C region AAPSVTLFPPSSEELQANK AGVETTTPSK MS patient # 130 # 130: FLC monomer peptides

# 130: FLC dimer peptides

Ig kappa chain C region DSTYSLSSTLTLSK HKVYACEVTHQGLSSPVTK SFNRGEC SGTASVVCLLNNFYPR TVAAPSVFIFPPSDEQLK VDNALQSGNSQESVTEQDSK VYACEVTHQGLSSPVTK Ig lambda-like polypeptide AGVETTKPSK ANPTVTLFPPSSEELQANK SYSCQVTHEGSTVEK Ig lambda-2 chain C region AAPSVTLFPPSSEELQANK AGVETTTPSK

Ig kappa chain C region DSTYSLSSTLTLSK HKVYACEVTHQGLSSPVTK SFNRGEC SGTASVVCLLNNFYPR TVAAPSVFIFPPSDEQLK VDNALQSGNSQESVTEQDSK VYACEVTHQGLSSPVTK Ig lambda-like polypeptide AGVETTKPSK SYSCQVTHEGSTVEK YAASSYLSLTPEQWK Ig lambda-2 chain C region AAPSVTLFPPSSEELQANK AGVETTTPSK

of MS patients, with a distribution comparable to non-MS cases. In contrast, λ variable domain sequences were mainly restricted to V-III subtype. Although the limitations of mass spectrometry-based identification of variable light chain domains should be accounted for (Lavatelli and Vrana, 2011), the obtained preliminary data may point to the presence of clonally restricted λ FLC dimers in MS. These data raise a

Table 6 Distribution of κ- and λ-FLC subtypes in the CSF of MS patients based on κ and λ variable region peptide identification by mass spectrometry. Patient

Diagnosis

κ variable domains

λ variable domains

# 127 # 130 # 162 # 156 # 25 # 125 # 131 #159 # 858 # 539

MS MS MS MS MS MS MS MS Astrocytoma Stroke

V-I, V-II, V-III V-I, V-II, V-III V-I, V-II, V-III V-I, V-II, V-III V-I, V-III V-III V-III V-I, V-III V-I, V-III V-I, V-II, V-III

V-III V-III n.d.a V-III n.d. n.d. V-III V-IV n.d. n.d.

a

Not detected.

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question whether the presence of clonally restricted dimeric λ FLC may be related to disease aggressivity. Comparison of clinical data of MS patients with and without increased λ dimerization might be helpful in this respect. The exact mechanisms for the increased formation of FLC dimers and their pathophysiologic relevance are still insufficiently understood. Ig light chains possess five cysteine residues, four of which are involved in the two disulfide bonds that stabilize the variable (V) and constant (C) domain, respectively, and a carboxyl-terminal cysteine that is responsible for the intermolecular disulfide bond between light chain and immunoglobulin heavy chain or between two light chains. It is generally believed that formation of disulfide bonds in proteins occurs intracellularly and is catalyzed by a family of oxireductases. Activity of these enzymes depends on redox potential within different compartments of the cell, thus affecting the ability of the cell to perform oxidation, i.e., formation of S–S bridge, or reduction of S–S bond to two thiol groups (SH) (Wilkinson and Gilbert, 2004; Xiao et al., 2005). Under pathological conditions, such as oxidative stress, shift in redox potential may occur and affect the subtle balance between dithiol and disulfide states. Of note, oxidative stress markers were found to be elevated in monoclonal gammopathies and autoimmune/inflammatory diseases, including MS (Ferretti et al., 2006; Migrino et al., 2009; Sharma et al., 2009). These findings suggest that both the structural peculiarities of FLC, as well as the diseaserelated environmental conditions play a role in FLC dimerization. In conclusion, the data obtained in the present study showed abnormal FLC patterns highly specific for MS. Of note are the elevated λ dimer/monomer ratios in more than 40% of MS patients. Although pathophysiology of increased light chain dimerization is yet unclear, the abnormal FLC dimer/monomer ratios should be taken into account in the FLC-based diagnosis of MS. Acknowledgment Thanks are due to the Smoler Proteomics Center at the Technion (Israel) for performing protein identification by mass spectrometry. References Arneth, B., Birklein, F., 2009. High sensitivity of free lambda and free kappa light chains for detection of intrathecal immunoglobulin synthesis in cerebrospinal fluid. Acta Neurol. Scand. 119, 39–44. Bracco, F., Gallo, P., Menna, R., Battistin, L., Tavolato, B., 1987. Free light chains in the CSF in multiple sclerosis. J. Neurol. 234, 303–307. Cavuoti, D., Baskin, L., Jialal, I., 1998. Detection of oligoclonal bands in cerebrospinal fluid by immunofixation electrophoresis. Am. J. Clin. Pathol. 109, 585–588. Desplat-Jego, S., Feuillet, L., Pelletier, J., Bernard, D., Cherif, A., Boucraut, J., 2005. Quantification of immunoglobulin free light chains in cerebrospinal fluid by nephelometry. J. Clin. Immunol. 25, 338–345. Ferretti, G., Bacchetti, T., DiLudovico, F., Viti, B., Angeleri, V.A., Danni, M., Provinciali, L., 2006. Intracellular oxidative activity and respiratory burst of leucocytes isolated from multiple sclerosis patients. Neurochem. Int. 48, 87–92. Fischer, Ch., Arneth, B., Kehler, L., Lotz, J., Lackner, K.J., 2004. Kappa free light chains in cerebrospinal fluid as markers of intrathecal immunoglobulin synthesis. Clin. Chem. 50, 1809–1813. Goffette, S., Schlueo, M., Henry, H., Duprez, T., Sindic, C.J., 2004. Detection of oligoclonal free kappa chains in the absence of oligoclonal IgG in the CSF of

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