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Increased anti-KIR4.1 antibodies in multiple sclerosis: Could it be a marker of disease relapse? Livnat Brill, Lotem Goldberg, Arnon Karni, Panayiota Petrou, Oded Abramsky, Haim Ovadia, Tamir Ben-Hur, Dimitrios Karussis and Adi Vaknin-Dembinsky Mult Scler published online 12 November 2014 DOI: 10.1177/1352458514551779 The online version of this article can be found at: http://msj.sagepub.com/content/early/2014/10/25/1352458514551779
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research-article2014
MSJ0010.1177/1352458514551779Multiple Sclerosis JournalL Brill, L Goldberg
MULTIPLE SCLEROSIS MSJ JOURNAL
Research Paper
Increased anti-KIR4.1 antibodies in multiple sclerosis: Could it be a marker of disease relapse?
Multiple Sclerosis Journal 1–8 DOI: 10.1177/ 1352458514551779 © The Author(s), 2014. Reprints and permissions: http://www.sagepub.co.uk/ journalsPermissions.nav
Livnat Brill, Lotem Goldberg, Arnon Karni, Panayiota Petrou, Oded Abramsky, Haim Ovadia, Tamir Ben-Hur, Dimitrios Karussis and Adi Vaknin-Dembinsky
Abstract Background: Screening of putative autoimmune targets in multiple sclerosis (MS) revealed a proportion of patients carrying antibodies (Abs) against KIR4.1, a potassium channel that shares functional properties with AQP4. Both are localized at the perivascular astrocytic processes. Aims: To measure anti-KIR4.1 Abs in the serum of MS and neuromyelitis optica (NMO) patients, and to identify the clinical and laboratory characteristics of patients harboring anti-KIR4.1 Abs. Methods: We measured anti-KIR4.1 Abs in serum, using the peptide KIR4.1 (83–120) enzyme-linked immunosorbent assay (ELISA). Results: Serum levels of anti-KIR4.1 Abs were significantly higher in MS and NMO patients than in healthy controls (HCs); with Abs detected in 21 of 80, 10 of 45, and 2 of 32 individuals, respectively (MS versus HC, p < 0.05). The level of anti-KIR4.1 Abs was significantly higher during MS relapse, versus remission (p = 0.04). The clinical characteristics of our study patients did not vary based on KIR4.1 positivity. Conclusions: Anti-KIR4.1 Abs were found in similar proportions of patients with MS and NMO, at a significantly higher level than observed in HCs; consequently, the presence of Abs does not discriminate between these demyelinating diseases. However, anti-KIR4.1 Ab levels differed in MS patients during relapse and remission; as such, they may represent a marker of disease exacerbation.
Keywords: Autoimmune disease, biomarker, immunoassay, immunology, KIR4.1, multiple sclerosis, neuromyelitis optica, potassium channel, relapse Date received: 20 January 2014; accepted: 26 August 2014 Introduction Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease that affects the central nervous system (CNS) and is characterized by demyelination, astrogliosis and damage to oligodendrocytes. MS leads to substantial disability affecting sensation, motor, autonomic, and neurocognitive functions.1,2
T cells, while histopathological studies reveal the prominent deposition of immunoglobulins (Igs) and complement activation in acute MS demyelinating lesions.3–6 Moreover, new therapies that deplete B cells have shown beneficial effects. 7,8 Therefore, specific autoantibodies may represent potential candidate biomarkers for MS.
There is an urgent need to identify biomarkers that provide information on disease activity, as well as early pathogenesis, progression and disease type.
Neuromyelitis optica (NMO) is an idiopathic demyelinating disease of the CNS that had been considered as a severe variant of MS. The discovery of NMOimmunoglobulin G (IgG) against the water channel aquaporin-4 (AQP4) in the serum of patients with NMO was a breakthrough in understanding demyelination in CNS autoimmune diseases.9,10 The exact role of
The involvement of immune cells in the pathogenesis of MS has been described. Early investigations indicate that disease progression is driven by CD4 effector
Correspondence to: Adi Vaknin-Dembinsky Neurology Department, Multiple Sclerosis and Immunobiology Research, Hadassah-Hebrew University Hospital, Ein Karem, PO Box 12000, Jerusalem 91120, Israel. [email protected] Livnat Brill Lotem Goldberg Panayiota Petrou Oded Abramsky Haim Ovadia Tamir Ben-Hur Dimitrios Karussis Adi Vaknin-Dembinsky Department of Neurology and Laboratory of Neuroimmunology, and the Agnes-Ginges Center for Neurogenetics, HadassahHebrew University Medical Center Jerusalem, Israel Arnon Karni Neuroimmunology Laboratory, Department of Neurology, Tel Aviv Sourasky Medical Center, and 2Sackler’s Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
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Multiple Sclerosis Journal antibodies against AQP4 in the immunopathogenic cascade of NMO is not clear: There are still patients with classical NMO disease whom are seronegative to AQP4.11,12 Recently, it was shown that KIR4.1 is a target of the autoantibody response in a subgroup of patients with MS.13 KIR4.1 (also known as KCNJ10) is a potassium channel subunit expressed by astrocytes and it is involved in spatial K(+) buffering, regulating neural activity.14–17 KIR4.1 channels are shown to co-localize with AQP4, the water channel expressed on astrocytes’ foot processes.18,19 Furthermore, a protein complex consisting of KIR4.1, AQP4 and several members of the dystrophin glycoprotein family was identified using an immunoprecipitation assay, supporting that there is a functional interaction between the two proteins.20
with OND (51 female and 38 male; average age 30.5 ± 8.8; ONDs were: three individuals with pseudo-tumor cereberi, eight with amyotrophic lateral sclerosis (ALS), four with stroke and one with migraine). We obtained written informed consent from all patients and controls. We measured anti-nuclear antibodies (ANA-Ab) using an indirect immunofluorescence assay (IFA-ANA), as previously described.21 Our MS and NMO patients had been diagnosed according to the 2010 McDonald criteria22 for MS and the NMO diagnostic criteria, respectively.23 The patients with CIS had at least one episode compatible with a relapse of MS, and two or more lesions on magnetic resonance imaging (MRI). The MS patient sera (n = 80) obtained at the Hadassah MS Center were negative for anti-AQP4 antibodies.
Srivastava et al.13 recently identified a specific serum IgG directed against KIR4.1 in 47% of patients with MS. The serum levels of anti-KIR4.1 antibodies significantly higher in the MS group than in patients with other neurological diseases (OND) or healthy controls (HCs). No correlation was established between anti-KIR4.1 antibodies and disease subtype, severity nor oligoclonal bands (OCBs) in the cerebrospinal fluid (CSF).
At the time of sampling, there were 43 patients with MS treated with interferon-beta (IFNβ), seven with glatiramer acetate, five with azathioprine, three with fingolimod and three with natalizumab; while 47 patients were not receiving immunomodulatory treatment and no treatment information was available for seven of our study patients. Out of the 45 NMO patients, seven were being treated with azathioprine and none received steroid treatment for the 2 months prior to testing.
To examine the possibility of using anti-KIR4.1 antibodies as a biomarker for MS, we measured their levels in patients with MS, clinically-isolated syndrome (CIS) and NMO, and compared those antibody levels during the relapse and remission phases of MS. We also assessed the clinical and laboratory characteristics of MS and NMO patients whom were positive for anti-KIR4.1 antibodies.
Serum samples obtained from 12 patients with MS were drawn at two time points that were 3–6 months apart, during their disease relapse and remission. The samples drawn during MS relapses were taken prior to receipt of intravenous (IV) steroid therapy. We used our samples to assess anti-KIR4.1 antibodies in an enzyme-linked immunosorbent assay (ELISA), as described below.
Methods
Anti-KIR4.1 antibody testing by ELISA and Western blot Serum anti-KIR4.1 antibody titers were measured by ELISA, using KIR4.1 peptide 83–120 as the capture antigen, as described by Srivastava et al.13 Based on those findings, which showed comparable results with the whole KIR4.1 protein and the KIR4.1 83– 120 peptide,13 KIR4.1 peptide 83–120 was established as the immunogenic KIR4.1 peptide. ELISA plate (Nunc Immunoplate, Thermo Scientific) wells were coated overnight with 6 µg/ml KIR4.1 83–120 peptide or Kcnq4 119–131 as the control antigen (potassium voltage-gated channel, KQT-like subfamily, Member 4, expressed in the human brain), in coating buffer (BioLegend, San Diego, CA, USA). As described by Srivastava et al.,13 an additional negative
Patients Our patient cohort included 115 patients with MS (83 female and 32 male; mean age 38 ± 12.5 years; disease duration 4.1 ± 4.1 years; expanded disability status scale (EDSS) 2 ± 1.8); 45 patients with NMO (34 female and 11 male; average age 39.4 ± 14 years; disease duration 5.3 ± 5 years); and 16 patients with CIS (12 female and 4 male; average age 38.2 ± 10.1 years), whom were seen at the Hadassah and Sourasky MS centers in Israel. We collected clinical data from the patient’s files, in order to investigate the correlation between clinical characteristics and the presence of KIR4.1 antibodies. An age- and sex-matched control group consisted of 73 HCs, plus a group of 16 patients
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L Brill, L Goldberg et al. solubilized by boiling in sample buffer for 10 min before loading onto 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels. Western blot was performed with eight serum samples from anti-KIR 4.1 seropositive and seronegative patients, diluted 1:500, or anti-KIR4.1 antibody as a positive control (#APC-035, Alomone Lab, Israel), anti-human IgG (H+L), APC conjugate antibody diluted 1:7000 or anti-rabbit IgG (H+L), APC conjugate antibody 1:30K (Promega). Figure 1. Anti-KIR4.1 antibody detection blocked with increasing concentrations of KIR4.1 peptide in a KIR4.1 peptide 83–120 competitive assay. Serum samples from seven individuals with anti-KIR4.1-positive antibodies were incubated with increasing concentrations of KIR 4.1 peptide (0, 6, 30, 60 and 120 µg/ml) prior to adding the samples to the ELISA plate. Results are displayed as mean OD for each concentration of the peptide. ELISA: Enzyme-linked immunosorbent assay; OD: optical density
control comprising plates that were coated with bovine serum albumin was included. Following a blocking step, serum samples, diluted 1:1000 in ELISA assay diluents (BioLegend), were added and incubated overnight at 4°C. Anti-human IgG antibody (Dako), diluted 1:5000, was added to each well for 2 hrs at room temperature. Plates were developed with 100 µl of substrate solution (TMB One Component, Southern Biotech), and 100 µl of sulfuric acid (2 M H2SO4) was added to terminate the reaction. We measured optical density (OD) at 450 nm. Serum was considered to be anti-KIR4.1 positive if the OD was more than 2 standard deviations (SDs) above the mean value for the normal control samples. To validate the specific binding of serum anti-KIR4.1 antibody to KIR4.1, we performed a competitive assay. Serum samples from seven individuals with anti-KIR4.1-positive titers were incubated with increasing concentrations of KIR4.1 peptide (0, 6, 30, 60 and 120 µg/ml) for 12 hrs prior to adding the samples to the ELISA plate. We measured the titer of serum anti-KIR4.1 antibody using ELISA, as described above. As shown in Figure 1, there was significant blocking by KIR4.1 peptide, confirming the specificity of the KIR4.1 ELISA assay. We further correlated the results obtained by the KIR4.1 peptide ELISA assay with serum reactivity to the whole KIR4.1 protein, using western blotting. Extracts of KIR4.1 that were over-expressed in Xenopus oocytes (as previously described)24 were
Validation Analyses were confirmed by a validation assay using 78 serum samples: 35 were from MS patients and 43 from HCs. Serum samples were obtained from the Sourasky MS Center in Israel and were delivered on dry ice to the neuroimmunology laboratory at the Hadassah Medical Center; the sample recipients were blinded as to the disease status of the patient providing samples. Statistical analysis Statistical significance was calculated using T-test, Pearson’s chi square test and Fisher’s exact test. Results Anti-KIR4.1 antibodies can be detected in a proportion of MS and CIS patients First, we analyzed serum specimens from 80 patients with MS, 16 CIS patients, 32 HCs and 16 patients with OND. In this initial analysis of patients from Hadassah MS Center, we detected antibodies to KIR4.1 in 21 of 80 patients with MS (26%); in 4 of 16 patients with CIS (25%); in 2 of 16 with OND (12.5%) and in 1 of the 32 HCs (3%) (Figure 2(a)). Noting that this proportion of patients with MS with antibodies to KIR4.1 was considerably lower than that described by Srivastava et al., we then analyzed an additional 78 serum samples from Sourasky MS Center. Only 5 out of 35 patients with MS (14%) and 3 out of 43 HCs (7%) had antibodies to KIR4.1 (Figure 2(b)). Combining the results from both MS centers, the total antibody titers against KIR4.1 were significantly higher in the serum samples from patients with MS than in those from HCs and patients with OND (p = 0.002), as seen in Figure 2. There was no significant difference between CIS patients and HCs in the prevalence of anti-KIR4.1 antibody seropositivity in this cohort (p = 0.13). The combination of CIS and MS
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Multiple Sclerosis Journal
Figure 2. (a) Anti KIR4.1 antibodies can be detected in a proportion of MS and CIS patients. Serum anti-KIR4.1 antibody levels were measured by ELISA using purified peptide KIR4.1 83–120 as the capture antigen. We found that 21 of 80 patients with MS (26%) were seropositive for anti-KIR4.1 antibodies; as well as 4 of 16 patients with CIS (25%), 2 of 16 with OND (12.5%) and 1 of the 32 HCs (3%). (b) Validation analysis. Validation analysis of 78 blinded serum samples drawn at a different MS center showed that 5 out of 35 (14%) patients with MS were seropositive for anti-KIR4.1 antibodies, as were 3 out of 43 HCs. Combining the results from both MS centers, the total antibody titers against KIR4.1 were significantly higher in the serum samples from patients with MS than in those from HCs and patients with OND (p = 0.002). (c) Anti-KIR4.1 antibodies cannot discriminate between patients with NMO and MS. We found that 10 out of 45 (22%) NMO patients were seropositive for anti-KIR4.1 antibodies (NMO versus HCs, p = 0.005; NMO versus MS, p = 0.6). In addition, 9 out of 37 (24%) patients who were seropositive for anti-AQP4 antibodies (NMO-SP) were also seropositive for anti-KIR4.1 antibodies; 1 out of 8 (12.5%) patients who were seronegative for anti-AQP4 antibodies (NMO-SN) was also seropositive for anti-KIR4.1 antibodies (p = 0.9). CIS: clinically-isolated syndrome; ELISA: enzyme-linked immunosorbent assay; HC: healthy control; MS: multiple sclerosis; NMO: neuromyelitis optica; OD: optical density; OND: other neurological disease; SN: seronegative; SP: seropositive
resulted in antibody titers against KIR4.1 that were significantly higher than that of healthy controls (26%, p = 0.005).
Anti-KIR4.1 antibodies do not define a specific disease subtype and cannot discriminate between patients with NMO and MS The majority of patients with NMO have serum anti-AQP4 antibodies.11 AQP4 co-localizes and might interact with KIR4.1.25 We found that 10 of 45 (22%) patients with NMO were seropositive for anti-KIR4.1 antibodies (Figure 2(c)). Serum levels of anti-KIR4.1 antibodies were significantly higher in the serum specimens from patients with NMO than in those from HCs and from patients with OND (p = 0.005). Anti-KIR4.1 antibody seropositivity was observed in patients whom were positive and negative for anti-AQP4 antibodies, with no significant difference between the groups. The prevalence of anti-KIR4.1 antibody seropositivity was not significantly different in patients with MS and NMO (p = 0.6). To investigate whether patients with anti-KIR4.1 antibody seropositivity have unique clinical or laboratory characteristics, we compared age, sex, EDSS, disease
duration, disease progression, CSF OCBs and ANA-Ab in our cohort. As seen in Table 1, no correlation was observed between KIR4.1 antibody positivity and age, sex, EDSS, OCBs, disease progression nor disease duration among patients with MS or NMO; however, there was a positive correlation between anti-KIR4.1 antibodies and detection of ANA-Ab in patients with NMO (p = 0.007). We analyzed whether receipt of MS therapy influenced anti-KIR4.1 antibody seropositivity. As is shown in Supplementary Figure 1 in the Supplementary Appendix, we found no difference between untreated MS patients and patients treated with IFNβ (p = 0.3). Of the 26 MS patients (from both MS centers) with positive antiKIR4.1 antibodies, 14 were untreated; 7 were treated with IFNβ; 2 with natalizumab; and 1 each with glatiramer acetate, fingolimod and azathioprine.
Increased anti-KIR 4.1 antibodies in MS relapse In 12 patients, we obtained blood samples and tested for anti-KIR4.1 antibodies during MS relapse and remission. In the majority of patients, the first blood test was obtained during the MS relapse (before
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L Brill, L Goldberg et al. Table 1. Clinical and laboratory characterization of patients with anti-KIR4.1 antibodies. KIR4.1 antibody positivity in patients with MS and NMO did not correlate with age, sex, disease duration, disease progression, EDSS, nor the occurrence of CSF OCBs. A positive correlation was found between anti-KIR4.1 antibodies and the detection of ANA-Ab, only in patients with NMO (p = 0.007). Data presented as mean ± SD.
Mean age Mean EDSS Mean disease duration (yrs) Mean disease progression (EDSS/disease duration) ANA-Ab OCB AQP4-Ab
MS KIR4.1(+) n = 26 (23%)
MS KIR4.1(-) n = 89 (77%)
p value
NMO KIR4.1(+) n = 10 (22%)
NMO KIR4.1(-) n = 35 (78%)
p value
10.6±37.5 2±2.2 4.2±5 0.45±0.3
13±38 1.7 ± 2 4±3.7 0.72 ±0.86
0.89 0.79 0.08 0.1
16±33.6 – 2.8±3.2 –
13.8±41 – 5.3±6 –
0.1 – 0.1 –
5.9% 31% ND
2.7% 44% ND
0.09 0.3 –
50% 20% 80%
8.7% 13% 84%
0.007 0.6 0.9
Ab: antibody; ANA-Ab: antinuclear antibodies; AQP: aquaporin; CSF: cerebrospinal fluid; EDSS: Expanded Disability Status Scale; MS: multiple sclerosis; ND: not determined; NMO: neuromyelitis optica; OCB: oligoclonal bands; yrs: years
therapy with intravenous (IV) steroids). As is shown in Figure 3, sera obtained during the MS relapses had significantly higher reactivity to KIR4.1, compared with sera obtained during remission (p = 0.04). Analysis of samples obtained from NMO patients 3–6 months apart revealed no correlation between antiKIR 4.1 levels, AQP4 positivity, disease relapse nor progression (data not shown). In order to further study patients with positive antiKIR4.1 antibodies, we examined the clinical outcome of CIS and patients with very high reactivity to antiKIR4.1 antibodies (OD > 1.2). Patients with CIS were followed for more than 2 years. The four CIS patients that were seropositive to KIR4.1 had no further clinical disease activity, whereas 2 of the 12 seronegative CIS patients had a second MS relapse, fulfilling the diagnosis of clinically definite MS (CDMS). Four patient blood samples exhibited very high reactivity to KIR4.1. Compared to those with lower reactivity and those seronegative to KIR4.1, no differences were observed with respect to age, sex, disease duration, disease progression nor severity. Accordingly, we saw no unique characteristics in the MS patients with high reactivity to KIR4.1 nor CIS patients with positive KIR4.1 reactivity, although the patient groups studied were small.
Discussion In the current study, we detected serum anti-KIR4.1 antibodies in a subgroup of MS patients, although the proportion was considerably lower than the 47% described by Srivastava et al.13 The proportion of MS and NMO patients with anti-KIR4.1 antibodies was comparable. Patients with serum anti-KIR4.1 antibodies were not characterized by unique clinical nor
Figure 3. Increased anti-KIR 4.1 antibodies in MS relapse. Blood samples of MS patients were obtained at two time points, 3–6 months apart: Once during MS relapse and once during remission. In the majority of patients, the first blood tests were drawn during a MS relapse (before therapy with IV steroids). Compared with serum drawn during remission, the serum obtained during a relapse had significantly higher reactivity to KIR4.1 (p = 0.04). IV: intravenous; MS: multiple sclerosis; OD: optical density
laboratory findings; however, reactivity to KIR4.1 was significantly higher during MS relapses. The lower proportion of seropositivity in our study, compared with that of Srivastava et al.13 could be attributed to different factors. Also recently, Watanabe et al.,26 Nerrant et al.27 and Brickshawana et al.28 found antiKIR4.1 antibody positivity rates of 3.9%, 7.5% and 1%, respectively, in their MS patients cohorts; which was lower than our observation (23%) and as described by Srivastava et al.13 (46.9%). The genetic and/or environmental background can affect the prevalence of serum autoantibodies; other autoimmune diseases tend to have a distinct prevalence, depending on the geographical
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Multiple Sclerosis Journal origin of the patients. For example, in patients with myasthenia gravis (MG) the frequency of anti-MuSK antibody-positivity varies according to latitude (low value is consistent with the northerly latitude).29,30 A second explanation for the lower prevalence of anti-KIR4.1 antibodies in our cohort could relate to the percentage of patients treated with disease-modifying therapy; however, in both our study and the Srivastava et al.13 study, the prevalence of anti-KIR 4.1 antibodies was equal in treated and untreated MS patients, and was the same as that detected in patients with CIS; consequently, this effect is likely to have been minor. Finally, the method used to detect antiKIR 4.1 antibodies might have affected the seropositivity. We used ELISA directed toward the 83–120 KIR4.1 peptide, whereas Srivastava et al.13 also used the whole KIR4.1 protein ELISA. In a recently published work from the same group, the authors mentioned that a whole protein ELISA might be more suitable, given the need for oligomerization and posttranslational modification of the KIR4.1 protein.31 However, Srivastava et al.13 demonstrated comparable results when using the 83–120 KIR4.1 peptide and the whole KIR4.1 protein, suggesting that this factor is unlikely to have had a major impact. In Figure 1 we demonstrated that increasing concentrations of 83–120 KIR4.1 peptide in the ELISA plate in our studies completely blocked detection of serum anti-KIR 4.1 antibodies, confirming that the antibody interaction is specific. Moreover, we validated the reactivity of representative sera from eight patients that were positive in the peptide ELISA assay, using Western blotting (data not shown). In the second part of our work, we measured anti-KIR 4.1 antibodies in patients with NMO. We demonstrated that the proportion of NMO patients with serum antiKIR4.1 antibodies was comparable to that in MS patients. The presence of a highly specific serum autoantibody, the NMO-IgG against the water channel AQP4, is the main biomarker that distinguishes NMO from MS.9,32 Autoantibodies that bind to AQP4 have been found in the majority (~80%) of NMO patients11,33 and are believed to play a role in the pathogenesis of the disease.34,35 Both AQP4 and KIR4.1 are channels expressed along the astroglial foot processes, and might mediate coupling between water homeostasis and potassium regulation in the CNS.25,36 It is therefore possible that in patients with NMO, astrocytic damage initiated by anti-AQP4 antibodies and followed by complement activation could mediate exposure of KIR4.1 protein to the immune system, thereby triggering production of specific anti-KIR4.1 antibodies. The
finding that anti-KIR4.1 antibody positivity is comparable in NMO and MS patients may support the hypothesis that different initial insults occur in CNS demyelinating diseases, causing the release or exposure of identical proteins. Therefore, anti-KIR4.1 antibodies may be present as a secondary event (epiphenomenon) following neuronal tissue and blood brain barrier destruction. We also observed increased ANA-Ab in NMO patients with anti-KIR4.1 antibody positivity. The detection of autoimmune antibodies in patients with an antibody-mediated disease such as NMO is not surprising. Patients with NMO are known to have increased serum anti-nuclear antibodies and Sjögren syndrome A (SSA) antibodies. 37 We and others have described increased comorbidity of NMO in patients with MG.38, 39 The presence of unique autoantibodies in patients with CNS demyelinating diseases is likely to provide a major contribution, both as a diagnostic tool, and in advancing our understanding of the pathogenesis of these diseases. In the last decades, considerable effort has been invested in the search for specific MS autoantibodies. Several autoantibodies have been detected, mainly against myelin antigens such as myelin associated glycoprotein (MBP), proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG)40,41; however, their roles are still controversial and most of the existing data represent indications of involvement, rather than proofs for primary and direct causation of anti-myelin antibodymediated tissue damage. It remains to be established whether anti-KIR4.1 antibodies can cause demyelination. This was partially achieved by the transfer of the antibodies to an animal model. Srivastava et al.13 injected KIR4.1 serum IgG into the cisternae magnae of mice, which led to a profound reduction of KIR4.1 expression, altered expression of glial fibrillary acidic protein (GFAP) in astrocytes, and activation of the complement cascade at sites of KIR4.1 expression. Comparing patients with anti-KIR4.1 antibody seropositivity and seronegativity revealed no differences in age, sex, disease duration, disease progression, EDSS, nor the occurrence of CSF OCBs. There was also no difference in anti-KIR4.1 antibody positivity between untreated and IFNβ-treated patients. The only positive laboratory correlation was a significantly increased ANA-Ab in NMO patients with positive anti-KIR4.1 antibodies. Recently, Kraus et al.31 found that children with MS, CIS or demyelinating encephalomyelitis (comprising acute disseminated and multiphasic
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L Brill, L Goldberg et al. disseminated encephalomyelitis) all have more than 50% positivity to KIR4.1. These findings support our data and suggest that anti-KIR 4.1 antibodies cannot be used as a discriminative biomarker for CNS demyelinating diseases. While we found no unique characteristics of MS patients with anti-KIR4.1 antibodies, we did observe increased KIR4.1 autoantibody levels during MS relapse. Further validation and exploration of these findings could expand our understanding of the antigenic triggers of NMO and MS, and might lead to the use of anti-KIR4.1 antibodies as a biomarker for MS relapse. Acknowledgements We want to thank Alomone Lab Israel for providing us with the human KIR4.1 protein (expressed in Xenopus oocytes) and antibody, as well as for their technical support. Conflict of interest The authors declare that there are no conflicts of interest. Funding This research received no specific grant from any funding agency in the public, commercial, or not-forprofit sectors.
References 1. Noseworthy JH, Lucchinetti C, Rodriguez M, et al. Multiple sclerosis. N Engl J Med 2000; 343: 938–952. 2. Compston A and Coles A. Multiple sclerosis. Lancet 2008; 372: 1502–1517. 3. McFarland HF and Martin R. Multiple sclerosis: A complicated picture of autoimmunity. Nat Immunol 2007; 8: 913–919. 4. Storch MK, Piddlesden S, Haltia M, et al. Multiple sclerosis: In situ evidence for antibody- and complement-mediated demyelination. Ann Neurol 1998; 43: 465–471. 5. Lassmann H, Bruck W and Lucchinetti CF. The immunopathology of multiple sclerosis: An overview. Brain Pathol 2007; 17: 210–218. 6. Breij EC, Brink BP, Veerhuis R, et al. Homogeneity of active demyelinating lesions in established multiple sclerosis. Ann Neurol 2008; 63: 16–25. 7. Hauser SL, Waubant E, Arnold DL, et al. B-cell depletion with rituximab in relapsing–remitting multiple sclerosis. N Engl J Med 2008; 358: 676–688.
8. Giacomini PS, Darlington PJ and Bar-Or A. Emerging multiple sclerosis disease-modifying therapies. Curr Opin Neurol 2009; 22: 226–232. 9. Lennon VA, Wingerchuk DM, Kryzer TJ, et al. A serum autoantibody marker of neuromyelitis optica: Distinction from multiple sclerosis. Lancet 2004; 364: 2106–2112. 10. Lennon VA, Kryzer TJ, Pittock SJ, et al. IgG marker of optic-spinal multiple sclerosis binds to the aquaporin-4 water channel. J Exp Med 2005; 202: 473–477. 11. Jiao Y, Fryer JP, Lennon VA, et al. Updated estimate of AQP4-IgG serostatus and disability outcome in neuromyelitis optica. Neurology 2013; 81: 1197–204. 12. Kitley J, Leite MI, Kuker W, et al. Longitudinally extensive transverse myelitis with and without aquaporin 4 antibodies. JAMA Neurol 2013; 70: 1375–1381. 13. Srivastava R, Aslam M, Kalluri SR, et al. Potassium channel KIR4.1 as an immune target in multiple sclerosis. N Engl J Med 2012; 367: 115–123. 14. Butt AM and Kalsi A. Inwardly rectifying potassium channels (Kir) in central nervous system glia: A special role for Kir4.1 in glial functions. J Cell Mol Med 2006; 10: 33–44. 15. Verkhratsky A and Steinhauser C. Ion channels in glial cells. Brain Res Brain Res Rev 2000; 32: 380–412. 16. Kofuji P and Newman EA. Potassium buffering in the central nervous system. Neuroscience 2004; 129: 1045–1056. 17. Newman EA. High potassium conductance in astrocyte endfeet. Science 1986; 233: 453–454. 18. Nagelhus EA, Horio Y, Inanobe A, et al. Immunogold evidence suggests that coupling of K+ siphoning and water transport in rat retinal Muller cells is mediated by a coenrichment of Kir4.1 and AQP4 in specific membrane domains. Glia 1999; 26: 47–54. 19. Tait MJ, Saadoun S, Bell BA, et al. Water movements in the brain: Role of aquaporins. Trends Neurosci 2008; 31: 37–43. 20. Connors NC, Adams ME, Froehner SC, et al. The potassium channel Kir4.1 associates with the dystrophin-glycoprotein complex via alphasyntrophin in glia. J Biolog Chem 2004; 279: 28387–28392. 21. Monce NM Jr, Bogusky RT and Cappel NN. An enzyme immunoassay screening test for the detection of total antinuclear antibodies. J Clin Lab Anal 1991; 5: 439–442.
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Multiple Sclerosis Journal 22. Polman CH, Reingold SC, Banwell B, et al. Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria. Ann Neurol 2011; 69: 292–302. 23. Wingerchuk DM, Lennon VA, Pittock SJ, et al. Revised diagnostic criteria for neuromyelitis optica. Neurology 2006; 66: 1485–1489. 24. Garcia-Caballero A, Dang Y, He H, et al. ENaC proteolytic regulation by channel-activating protease 2. J General Physiol 2008; 132: 521–535. 25. Masaki H, Wakayama Y, Hara H, et al. Immunocytochemical studies of aquaporin 4, Kir4.1 and alpha1-syntrophin in the astrocyte endfeet of mouse brain capillaries. Acta Histochem Cytochem 2010; 43: 99–105. 26. Watanabe M, Yamasaki R, Kawano Y, et al. Anti-KIR4.1 antibodies in Japanese patients with idiopathic central nervous system demyelinating diseases. Clin Exp Neuroimmunol 2013; 4: 241–242. 27. Nerrant E, Salsac C, Charif M, et al. Lack of confirmation of anti-inward rectifying potassium channel 4.1 antibodies as reliable markers of multiple sclerosis. Mult Scler 2014.
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32. Weinshenker BG. Neuromyelitis optica is distinct from multiple sclerosis. Arch Neurol 2007; 64: 899–901. 33. Jarius S, Probst C, Borowski K, et al. Standardized method for the detection of antibodies to aquaporin-4 based on a highly sensitive immunofluorescence assay employing recombinant target antigen. J Neurolog Sci 2010; 291: 52–56. 34. Cayrol R, Saikali P and Vincent T. Effector functions of antiaquaporin-4 autoantibodies in neuromyelitis optica. Ann NY Acad Sci 2009; 1173: 478–486. 35. Hinson SR, Roemer SF, Lucchinetti CF, et al. Aquaporin-4-binding autoantibodies in patients with neuromyelitis optica impair glutamate transport by down-regulating EAAT2. J Exp Med 2008; 205: 2473–2481. 36. Wen H, Nagelhus EA, Amiry-Moghaddam M, et al. Ontogeny of water transport in rat brain: Postnatal expression of the aquaporin-4 water channel. Eur J Neurosci 1999; 11: 935–945. 37. Pittock SJ, Lennon VA, De Seze J, et al. Neuromyelitis optica and non organ-specific autoimmunity. Arch Neurol 2008; 65: 78–83.
28. Brickshawana A, Hinson SR, Romero MF, et al. Investigation of the KIR4.1 potassium channel as a putative antigen in patients with multiple sclerosis: A comparative study. Lancet Neurol 2014; 13: 795–806.
38. Vaknin-Dembinsky A, Abramsky O, Petrou P, et al. Myasthenia gravis-associated neuromyelitis opticalike disease: An immunological link between the central nervous system and muscle? Arch Neurol 2011; 68: 1557–1561.
29. Niks EH, Kuks JB and Verschuuren JJ. Epidemiology of myasthenia gravis with anti-muscle-specific kinase antibodies in The Netherlands. J Neurol Neurosurg Psychiatry 2007; 78: 417–418.
39. Leite MI, Coutinho E, Lana-Peixoto M, et al. Myasthenia gravis and neuromyelitis optica spectrum disorder: A multicenter study of 16 patients. Neurology 78: 1601–1607.
30. Yeh JH, Chen WH, Chiu HC, et al. Low frequency of MuSK antibody in generalized seronegative myasthenia gravis among Chinese. Neurology 2004; 62: 2131–2132.
40. O’Connor KC, Lopez-Amaya C, Gagne D, et al. Antimyelin antibodies modulate clinical expression of childhood multiple sclerosis. J Neuroimmunol 2010; 223: 92–99.
31. Kraus V, Srivastava R, Kalluri SR, et al. Potassium channel KIR4.1-specific antibodies in children with acquired demyelinating CNS disease. Neurology 2014; 82: 470–473.
41. Tomassini V, De Giglio L, Reindl M, et al. Antimyelin antibodies predict the clinical outcome after a first episode suggestive of MS. Mult Scler 2007; 13: 1086–1094.
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Increased anti-KIR4.1 antibodies in multiple sclerosis: Could it be a marker of disease relapse? Livnat Brill, Lotem Goldberg, Arnon Karni, Panayiota Petrou, Oded Abramsky, Haim Ovadia, Tamir Ben-Hur, Dimitrios Karussis and Adi Vaknin-Dembinsky Mult Scler published online 12 November 2014 DOI: 10.1177/1352458514551779 The online version of this article can be found at: http://msj.sagepub.com/content/early/2014/10/25/1352458514551779
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551779
research-article2014
MSJ0010.1177/1352458514551779Multiple Sclerosis JournalL Brill, L Goldberg
MULTIPLE SCLEROSIS MSJ JOURNAL
Research Paper
Increased anti-KIR4.1 antibodies in multiple sclerosis: Could it be a marker of disease relapse?
Multiple Sclerosis Journal 1–8 DOI: 10.1177/ 1352458514551779 © The Author(s), 2014. Reprints and permissions: http://www.sagepub.co.uk/ journalsPermissions.nav
Livnat Brill, Lotem Goldberg, Arnon Karni, Panayiota Petrou, Oded Abramsky, Haim Ovadia, Tamir Ben-Hur, Dimitrios Karussis and Adi Vaknin-Dembinsky
Abstract Background: Screening of putative autoimmune targets in multiple sclerosis (MS) revealed a proportion of patients carrying antibodies (Abs) against KIR4.1, a potassium channel that shares functional properties with AQP4. Both are localized at the perivascular astrocytic processes. Aims: To measure anti-KIR4.1 Abs in the serum of MS and neuromyelitis optica (NMO) patients, and to identify the clinical and laboratory characteristics of patients harboring anti-KIR4.1 Abs. Methods: We measured anti-KIR4.1 Abs in serum, using the peptide KIR4.1 (83–120) enzyme-linked immunosorbent assay (ELISA). Results: Serum levels of anti-KIR4.1 Abs were significantly higher in MS and NMO patients than in healthy controls (HCs); with Abs detected in 21 of 80, 10 of 45, and 2 of 32 individuals, respectively (MS versus HC, p < 0.05). The level of anti-KIR4.1 Abs was significantly higher during MS relapse, versus remission (p = 0.04). The clinical characteristics of our study patients did not vary based on KIR4.1 positivity. Conclusions: Anti-KIR4.1 Abs were found in similar proportions of patients with MS and NMO, at a significantly higher level than observed in HCs; consequently, the presence of Abs does not discriminate between these demyelinating diseases. However, anti-KIR4.1 Ab levels differed in MS patients during relapse and remission; as such, they may represent a marker of disease exacerbation.
Keywords: Autoimmune disease, biomarker, immunoassay, immunology, KIR4.1, multiple sclerosis, neuromyelitis optica, potassium channel, relapse Date received: 20 January 2014; accepted: 26 August 2014 Introduction Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease that affects the central nervous system (CNS) and is characterized by demyelination, astrogliosis and damage to oligodendrocytes. MS leads to substantial disability affecting sensation, motor, autonomic, and neurocognitive functions.1,2
T cells, while histopathological studies reveal the prominent deposition of immunoglobulins (Igs) and complement activation in acute MS demyelinating lesions.3–6 Moreover, new therapies that deplete B cells have shown beneficial effects. 7,8 Therefore, specific autoantibodies may represent potential candidate biomarkers for MS.
There is an urgent need to identify biomarkers that provide information on disease activity, as well as early pathogenesis, progression and disease type.
Neuromyelitis optica (NMO) is an idiopathic demyelinating disease of the CNS that had been considered as a severe variant of MS. The discovery of NMOimmunoglobulin G (IgG) against the water channel aquaporin-4 (AQP4) in the serum of patients with NMO was a breakthrough in understanding demyelination in CNS autoimmune diseases.9,10 The exact role of
The involvement of immune cells in the pathogenesis of MS has been described. Early investigations indicate that disease progression is driven by CD4 effector
Correspondence to: Adi Vaknin-Dembinsky Neurology Department, Multiple Sclerosis and Immunobiology Research, Hadassah-Hebrew University Hospital, Ein Karem, PO Box 12000, Jerusalem 91120, Israel. [email protected] Livnat Brill Lotem Goldberg Panayiota Petrou Oded Abramsky Haim Ovadia Tamir Ben-Hur Dimitrios Karussis Adi Vaknin-Dembinsky Department of Neurology and Laboratory of Neuroimmunology, and the Agnes-Ginges Center for Neurogenetics, HadassahHebrew University Medical Center Jerusalem, Israel Arnon Karni Neuroimmunology Laboratory, Department of Neurology, Tel Aviv Sourasky Medical Center, and 2Sackler’s Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
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Multiple Sclerosis Journal antibodies against AQP4 in the immunopathogenic cascade of NMO is not clear: There are still patients with classical NMO disease whom are seronegative to AQP4.11,12 Recently, it was shown that KIR4.1 is a target of the autoantibody response in a subgroup of patients with MS.13 KIR4.1 (also known as KCNJ10) is a potassium channel subunit expressed by astrocytes and it is involved in spatial K(+) buffering, regulating neural activity.14–17 KIR4.1 channels are shown to co-localize with AQP4, the water channel expressed on astrocytes’ foot processes.18,19 Furthermore, a protein complex consisting of KIR4.1, AQP4 and several members of the dystrophin glycoprotein family was identified using an immunoprecipitation assay, supporting that there is a functional interaction between the two proteins.20
with OND (51 female and 38 male; average age 30.5 ± 8.8; ONDs were: three individuals with pseudo-tumor cereberi, eight with amyotrophic lateral sclerosis (ALS), four with stroke and one with migraine). We obtained written informed consent from all patients and controls. We measured anti-nuclear antibodies (ANA-Ab) using an indirect immunofluorescence assay (IFA-ANA), as previously described.21 Our MS and NMO patients had been diagnosed according to the 2010 McDonald criteria22 for MS and the NMO diagnostic criteria, respectively.23 The patients with CIS had at least one episode compatible with a relapse of MS, and two or more lesions on magnetic resonance imaging (MRI). The MS patient sera (n = 80) obtained at the Hadassah MS Center were negative for anti-AQP4 antibodies.
Srivastava et al.13 recently identified a specific serum IgG directed against KIR4.1 in 47% of patients with MS. The serum levels of anti-KIR4.1 antibodies significantly higher in the MS group than in patients with other neurological diseases (OND) or healthy controls (HCs). No correlation was established between anti-KIR4.1 antibodies and disease subtype, severity nor oligoclonal bands (OCBs) in the cerebrospinal fluid (CSF).
At the time of sampling, there were 43 patients with MS treated with interferon-beta (IFNβ), seven with glatiramer acetate, five with azathioprine, three with fingolimod and three with natalizumab; while 47 patients were not receiving immunomodulatory treatment and no treatment information was available for seven of our study patients. Out of the 45 NMO patients, seven were being treated with azathioprine and none received steroid treatment for the 2 months prior to testing.
To examine the possibility of using anti-KIR4.1 antibodies as a biomarker for MS, we measured their levels in patients with MS, clinically-isolated syndrome (CIS) and NMO, and compared those antibody levels during the relapse and remission phases of MS. We also assessed the clinical and laboratory characteristics of MS and NMO patients whom were positive for anti-KIR4.1 antibodies.
Serum samples obtained from 12 patients with MS were drawn at two time points that were 3–6 months apart, during their disease relapse and remission. The samples drawn during MS relapses were taken prior to receipt of intravenous (IV) steroid therapy. We used our samples to assess anti-KIR4.1 antibodies in an enzyme-linked immunosorbent assay (ELISA), as described below.
Methods
Anti-KIR4.1 antibody testing by ELISA and Western blot Serum anti-KIR4.1 antibody titers were measured by ELISA, using KIR4.1 peptide 83–120 as the capture antigen, as described by Srivastava et al.13 Based on those findings, which showed comparable results with the whole KIR4.1 protein and the KIR4.1 83– 120 peptide,13 KIR4.1 peptide 83–120 was established as the immunogenic KIR4.1 peptide. ELISA plate (Nunc Immunoplate, Thermo Scientific) wells were coated overnight with 6 µg/ml KIR4.1 83–120 peptide or Kcnq4 119–131 as the control antigen (potassium voltage-gated channel, KQT-like subfamily, Member 4, expressed in the human brain), in coating buffer (BioLegend, San Diego, CA, USA). As described by Srivastava et al.,13 an additional negative
Patients Our patient cohort included 115 patients with MS (83 female and 32 male; mean age 38 ± 12.5 years; disease duration 4.1 ± 4.1 years; expanded disability status scale (EDSS) 2 ± 1.8); 45 patients with NMO (34 female and 11 male; average age 39.4 ± 14 years; disease duration 5.3 ± 5 years); and 16 patients with CIS (12 female and 4 male; average age 38.2 ± 10.1 years), whom were seen at the Hadassah and Sourasky MS centers in Israel. We collected clinical data from the patient’s files, in order to investigate the correlation between clinical characteristics and the presence of KIR4.1 antibodies. An age- and sex-matched control group consisted of 73 HCs, plus a group of 16 patients
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L Brill, L Goldberg et al. solubilized by boiling in sample buffer for 10 min before loading onto 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels. Western blot was performed with eight serum samples from anti-KIR 4.1 seropositive and seronegative patients, diluted 1:500, or anti-KIR4.1 antibody as a positive control (#APC-035, Alomone Lab, Israel), anti-human IgG (H+L), APC conjugate antibody diluted 1:7000 or anti-rabbit IgG (H+L), APC conjugate antibody 1:30K (Promega). Figure 1. Anti-KIR4.1 antibody detection blocked with increasing concentrations of KIR4.1 peptide in a KIR4.1 peptide 83–120 competitive assay. Serum samples from seven individuals with anti-KIR4.1-positive antibodies were incubated with increasing concentrations of KIR 4.1 peptide (0, 6, 30, 60 and 120 µg/ml) prior to adding the samples to the ELISA plate. Results are displayed as mean OD for each concentration of the peptide. ELISA: Enzyme-linked immunosorbent assay; OD: optical density
control comprising plates that were coated with bovine serum albumin was included. Following a blocking step, serum samples, diluted 1:1000 in ELISA assay diluents (BioLegend), were added and incubated overnight at 4°C. Anti-human IgG antibody (Dako), diluted 1:5000, was added to each well for 2 hrs at room temperature. Plates were developed with 100 µl of substrate solution (TMB One Component, Southern Biotech), and 100 µl of sulfuric acid (2 M H2SO4) was added to terminate the reaction. We measured optical density (OD) at 450 nm. Serum was considered to be anti-KIR4.1 positive if the OD was more than 2 standard deviations (SDs) above the mean value for the normal control samples. To validate the specific binding of serum anti-KIR4.1 antibody to KIR4.1, we performed a competitive assay. Serum samples from seven individuals with anti-KIR4.1-positive titers were incubated with increasing concentrations of KIR4.1 peptide (0, 6, 30, 60 and 120 µg/ml) for 12 hrs prior to adding the samples to the ELISA plate. We measured the titer of serum anti-KIR4.1 antibody using ELISA, as described above. As shown in Figure 1, there was significant blocking by KIR4.1 peptide, confirming the specificity of the KIR4.1 ELISA assay. We further correlated the results obtained by the KIR4.1 peptide ELISA assay with serum reactivity to the whole KIR4.1 protein, using western blotting. Extracts of KIR4.1 that were over-expressed in Xenopus oocytes (as previously described)24 were
Validation Analyses were confirmed by a validation assay using 78 serum samples: 35 were from MS patients and 43 from HCs. Serum samples were obtained from the Sourasky MS Center in Israel and were delivered on dry ice to the neuroimmunology laboratory at the Hadassah Medical Center; the sample recipients were blinded as to the disease status of the patient providing samples. Statistical analysis Statistical significance was calculated using T-test, Pearson’s chi square test and Fisher’s exact test. Results Anti-KIR4.1 antibodies can be detected in a proportion of MS and CIS patients First, we analyzed serum specimens from 80 patients with MS, 16 CIS patients, 32 HCs and 16 patients with OND. In this initial analysis of patients from Hadassah MS Center, we detected antibodies to KIR4.1 in 21 of 80 patients with MS (26%); in 4 of 16 patients with CIS (25%); in 2 of 16 with OND (12.5%) and in 1 of the 32 HCs (3%) (Figure 2(a)). Noting that this proportion of patients with MS with antibodies to KIR4.1 was considerably lower than that described by Srivastava et al., we then analyzed an additional 78 serum samples from Sourasky MS Center. Only 5 out of 35 patients with MS (14%) and 3 out of 43 HCs (7%) had antibodies to KIR4.1 (Figure 2(b)). Combining the results from both MS centers, the total antibody titers against KIR4.1 were significantly higher in the serum samples from patients with MS than in those from HCs and patients with OND (p = 0.002), as seen in Figure 2. There was no significant difference between CIS patients and HCs in the prevalence of anti-KIR4.1 antibody seropositivity in this cohort (p = 0.13). The combination of CIS and MS
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Multiple Sclerosis Journal
Figure 2. (a) Anti KIR4.1 antibodies can be detected in a proportion of MS and CIS patients. Serum anti-KIR4.1 antibody levels were measured by ELISA using purified peptide KIR4.1 83–120 as the capture antigen. We found that 21 of 80 patients with MS (26%) were seropositive for anti-KIR4.1 antibodies; as well as 4 of 16 patients with CIS (25%), 2 of 16 with OND (12.5%) and 1 of the 32 HCs (3%). (b) Validation analysis. Validation analysis of 78 blinded serum samples drawn at a different MS center showed that 5 out of 35 (14%) patients with MS were seropositive for anti-KIR4.1 antibodies, as were 3 out of 43 HCs. Combining the results from both MS centers, the total antibody titers against KIR4.1 were significantly higher in the serum samples from patients with MS than in those from HCs and patients with OND (p = 0.002). (c) Anti-KIR4.1 antibodies cannot discriminate between patients with NMO and MS. We found that 10 out of 45 (22%) NMO patients were seropositive for anti-KIR4.1 antibodies (NMO versus HCs, p = 0.005; NMO versus MS, p = 0.6). In addition, 9 out of 37 (24%) patients who were seropositive for anti-AQP4 antibodies (NMO-SP) were also seropositive for anti-KIR4.1 antibodies; 1 out of 8 (12.5%) patients who were seronegative for anti-AQP4 antibodies (NMO-SN) was also seropositive for anti-KIR4.1 antibodies (p = 0.9). CIS: clinically-isolated syndrome; ELISA: enzyme-linked immunosorbent assay; HC: healthy control; MS: multiple sclerosis; NMO: neuromyelitis optica; OD: optical density; OND: other neurological disease; SN: seronegative; SP: seropositive
resulted in antibody titers against KIR4.1 that were significantly higher than that of healthy controls (26%, p = 0.005).
Anti-KIR4.1 antibodies do not define a specific disease subtype and cannot discriminate between patients with NMO and MS The majority of patients with NMO have serum anti-AQP4 antibodies.11 AQP4 co-localizes and might interact with KIR4.1.25 We found that 10 of 45 (22%) patients with NMO were seropositive for anti-KIR4.1 antibodies (Figure 2(c)). Serum levels of anti-KIR4.1 antibodies were significantly higher in the serum specimens from patients with NMO than in those from HCs and from patients with OND (p = 0.005). Anti-KIR4.1 antibody seropositivity was observed in patients whom were positive and negative for anti-AQP4 antibodies, with no significant difference between the groups. The prevalence of anti-KIR4.1 antibody seropositivity was not significantly different in patients with MS and NMO (p = 0.6). To investigate whether patients with anti-KIR4.1 antibody seropositivity have unique clinical or laboratory characteristics, we compared age, sex, EDSS, disease
duration, disease progression, CSF OCBs and ANA-Ab in our cohort. As seen in Table 1, no correlation was observed between KIR4.1 antibody positivity and age, sex, EDSS, OCBs, disease progression nor disease duration among patients with MS or NMO; however, there was a positive correlation between anti-KIR4.1 antibodies and detection of ANA-Ab in patients with NMO (p = 0.007). We analyzed whether receipt of MS therapy influenced anti-KIR4.1 antibody seropositivity. As is shown in Supplementary Figure 1 in the Supplementary Appendix, we found no difference between untreated MS patients and patients treated with IFNβ (p = 0.3). Of the 26 MS patients (from both MS centers) with positive antiKIR4.1 antibodies, 14 were untreated; 7 were treated with IFNβ; 2 with natalizumab; and 1 each with glatiramer acetate, fingolimod and azathioprine.
Increased anti-KIR 4.1 antibodies in MS relapse In 12 patients, we obtained blood samples and tested for anti-KIR4.1 antibodies during MS relapse and remission. In the majority of patients, the first blood test was obtained during the MS relapse (before
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L Brill, L Goldberg et al. Table 1. Clinical and laboratory characterization of patients with anti-KIR4.1 antibodies. KIR4.1 antibody positivity in patients with MS and NMO did not correlate with age, sex, disease duration, disease progression, EDSS, nor the occurrence of CSF OCBs. A positive correlation was found between anti-KIR4.1 antibodies and the detection of ANA-Ab, only in patients with NMO (p = 0.007). Data presented as mean ± SD.
Mean age Mean EDSS Mean disease duration (yrs) Mean disease progression (EDSS/disease duration) ANA-Ab OCB AQP4-Ab
MS KIR4.1(+) n = 26 (23%)
MS KIR4.1(-) n = 89 (77%)
p value
NMO KIR4.1(+) n = 10 (22%)
NMO KIR4.1(-) n = 35 (78%)
p value
10.6±37.5 2±2.2 4.2±5 0.45±0.3
13±38 1.7 ± 2 4±3.7 0.72 ±0.86
0.89 0.79 0.08 0.1
16±33.6 – 2.8±3.2 –
13.8±41 – 5.3±6 –
0.1 – 0.1 –
5.9% 31% ND
2.7% 44% ND
0.09 0.3 –
50% 20% 80%
8.7% 13% 84%
0.007 0.6 0.9
Ab: antibody; ANA-Ab: antinuclear antibodies; AQP: aquaporin; CSF: cerebrospinal fluid; EDSS: Expanded Disability Status Scale; MS: multiple sclerosis; ND: not determined; NMO: neuromyelitis optica; OCB: oligoclonal bands; yrs: years
therapy with intravenous (IV) steroids). As is shown in Figure 3, sera obtained during the MS relapses had significantly higher reactivity to KIR4.1, compared with sera obtained during remission (p = 0.04). Analysis of samples obtained from NMO patients 3–6 months apart revealed no correlation between antiKIR 4.1 levels, AQP4 positivity, disease relapse nor progression (data not shown). In order to further study patients with positive antiKIR4.1 antibodies, we examined the clinical outcome of CIS and patients with very high reactivity to antiKIR4.1 antibodies (OD > 1.2). Patients with CIS were followed for more than 2 years. The four CIS patients that were seropositive to KIR4.1 had no further clinical disease activity, whereas 2 of the 12 seronegative CIS patients had a second MS relapse, fulfilling the diagnosis of clinically definite MS (CDMS). Four patient blood samples exhibited very high reactivity to KIR4.1. Compared to those with lower reactivity and those seronegative to KIR4.1, no differences were observed with respect to age, sex, disease duration, disease progression nor severity. Accordingly, we saw no unique characteristics in the MS patients with high reactivity to KIR4.1 nor CIS patients with positive KIR4.1 reactivity, although the patient groups studied were small.
Discussion In the current study, we detected serum anti-KIR4.1 antibodies in a subgroup of MS patients, although the proportion was considerably lower than the 47% described by Srivastava et al.13 The proportion of MS and NMO patients with anti-KIR4.1 antibodies was comparable. Patients with serum anti-KIR4.1 antibodies were not characterized by unique clinical nor
Figure 3. Increased anti-KIR 4.1 antibodies in MS relapse. Blood samples of MS patients were obtained at two time points, 3–6 months apart: Once during MS relapse and once during remission. In the majority of patients, the first blood tests were drawn during a MS relapse (before therapy with IV steroids). Compared with serum drawn during remission, the serum obtained during a relapse had significantly higher reactivity to KIR4.1 (p = 0.04). IV: intravenous; MS: multiple sclerosis; OD: optical density
laboratory findings; however, reactivity to KIR4.1 was significantly higher during MS relapses. The lower proportion of seropositivity in our study, compared with that of Srivastava et al.13 could be attributed to different factors. Also recently, Watanabe et al.,26 Nerrant et al.27 and Brickshawana et al.28 found antiKIR4.1 antibody positivity rates of 3.9%, 7.5% and 1%, respectively, in their MS patients cohorts; which was lower than our observation (23%) and as described by Srivastava et al.13 (46.9%). The genetic and/or environmental background can affect the prevalence of serum autoantibodies; other autoimmune diseases tend to have a distinct prevalence, depending on the geographical
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Multiple Sclerosis Journal origin of the patients. For example, in patients with myasthenia gravis (MG) the frequency of anti-MuSK antibody-positivity varies according to latitude (low value is consistent with the northerly latitude).29,30 A second explanation for the lower prevalence of anti-KIR4.1 antibodies in our cohort could relate to the percentage of patients treated with disease-modifying therapy; however, in both our study and the Srivastava et al.13 study, the prevalence of anti-KIR 4.1 antibodies was equal in treated and untreated MS patients, and was the same as that detected in patients with CIS; consequently, this effect is likely to have been minor. Finally, the method used to detect antiKIR 4.1 antibodies might have affected the seropositivity. We used ELISA directed toward the 83–120 KIR4.1 peptide, whereas Srivastava et al.13 also used the whole KIR4.1 protein ELISA. In a recently published work from the same group, the authors mentioned that a whole protein ELISA might be more suitable, given the need for oligomerization and posttranslational modification of the KIR4.1 protein.31 However, Srivastava et al.13 demonstrated comparable results when using the 83–120 KIR4.1 peptide and the whole KIR4.1 protein, suggesting that this factor is unlikely to have had a major impact. In Figure 1 we demonstrated that increasing concentrations of 83–120 KIR4.1 peptide in the ELISA plate in our studies completely blocked detection of serum anti-KIR 4.1 antibodies, confirming that the antibody interaction is specific. Moreover, we validated the reactivity of representative sera from eight patients that were positive in the peptide ELISA assay, using Western blotting (data not shown). In the second part of our work, we measured anti-KIR 4.1 antibodies in patients with NMO. We demonstrated that the proportion of NMO patients with serum antiKIR4.1 antibodies was comparable to that in MS patients. The presence of a highly specific serum autoantibody, the NMO-IgG against the water channel AQP4, is the main biomarker that distinguishes NMO from MS.9,32 Autoantibodies that bind to AQP4 have been found in the majority (~80%) of NMO patients11,33 and are believed to play a role in the pathogenesis of the disease.34,35 Both AQP4 and KIR4.1 are channels expressed along the astroglial foot processes, and might mediate coupling between water homeostasis and potassium regulation in the CNS.25,36 It is therefore possible that in patients with NMO, astrocytic damage initiated by anti-AQP4 antibodies and followed by complement activation could mediate exposure of KIR4.1 protein to the immune system, thereby triggering production of specific anti-KIR4.1 antibodies. The
finding that anti-KIR4.1 antibody positivity is comparable in NMO and MS patients may support the hypothesis that different initial insults occur in CNS demyelinating diseases, causing the release or exposure of identical proteins. Therefore, anti-KIR4.1 antibodies may be present as a secondary event (epiphenomenon) following neuronal tissue and blood brain barrier destruction. We also observed increased ANA-Ab in NMO patients with anti-KIR4.1 antibody positivity. The detection of autoimmune antibodies in patients with an antibody-mediated disease such as NMO is not surprising. Patients with NMO are known to have increased serum anti-nuclear antibodies and Sjögren syndrome A (SSA) antibodies. 37 We and others have described increased comorbidity of NMO in patients with MG.38, 39 The presence of unique autoantibodies in patients with CNS demyelinating diseases is likely to provide a major contribution, both as a diagnostic tool, and in advancing our understanding of the pathogenesis of these diseases. In the last decades, considerable effort has been invested in the search for specific MS autoantibodies. Several autoantibodies have been detected, mainly against myelin antigens such as myelin associated glycoprotein (MBP), proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG)40,41; however, their roles are still controversial and most of the existing data represent indications of involvement, rather than proofs for primary and direct causation of anti-myelin antibodymediated tissue damage. It remains to be established whether anti-KIR4.1 antibodies can cause demyelination. This was partially achieved by the transfer of the antibodies to an animal model. Srivastava et al.13 injected KIR4.1 serum IgG into the cisternae magnae of mice, which led to a profound reduction of KIR4.1 expression, altered expression of glial fibrillary acidic protein (GFAP) in astrocytes, and activation of the complement cascade at sites of KIR4.1 expression. Comparing patients with anti-KIR4.1 antibody seropositivity and seronegativity revealed no differences in age, sex, disease duration, disease progression, EDSS, nor the occurrence of CSF OCBs. There was also no difference in anti-KIR4.1 antibody positivity between untreated and IFNβ-treated patients. The only positive laboratory correlation was a significantly increased ANA-Ab in NMO patients with positive anti-KIR4.1 antibodies. Recently, Kraus et al.31 found that children with MS, CIS or demyelinating encephalomyelitis (comprising acute disseminated and multiphasic
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L Brill, L Goldberg et al. disseminated encephalomyelitis) all have more than 50% positivity to KIR4.1. These findings support our data and suggest that anti-KIR 4.1 antibodies cannot be used as a discriminative biomarker for CNS demyelinating diseases. While we found no unique characteristics of MS patients with anti-KIR4.1 antibodies, we did observe increased KIR4.1 autoantibody levels during MS relapse. Further validation and exploration of these findings could expand our understanding of the antigenic triggers of NMO and MS, and might lead to the use of anti-KIR4.1 antibodies as a biomarker for MS relapse. Acknowledgements We want to thank Alomone Lab Israel for providing us with the human KIR4.1 protein (expressed in Xenopus oocytes) and antibody, as well as for their technical support. Conflict of interest The authors declare that there are no conflicts of interest. Funding This research received no specific grant from any funding agency in the public, commercial, or not-forprofit sectors.
References 1. Noseworthy JH, Lucchinetti C, Rodriguez M, et al. Multiple sclerosis. N Engl J Med 2000; 343: 938–952. 2. Compston A and Coles A. Multiple sclerosis. Lancet 2008; 372: 1502–1517. 3. McFarland HF and Martin R. Multiple sclerosis: A complicated picture of autoimmunity. Nat Immunol 2007; 8: 913–919. 4. Storch MK, Piddlesden S, Haltia M, et al. Multiple sclerosis: In situ evidence for antibody- and complement-mediated demyelination. Ann Neurol 1998; 43: 465–471. 5. Lassmann H, Bruck W and Lucchinetti CF. The immunopathology of multiple sclerosis: An overview. Brain Pathol 2007; 17: 210–218. 6. Breij EC, Brink BP, Veerhuis R, et al. Homogeneity of active demyelinating lesions in established multiple sclerosis. Ann Neurol 2008; 63: 16–25. 7. Hauser SL, Waubant E, Arnold DL, et al. B-cell depletion with rituximab in relapsing–remitting multiple sclerosis. N Engl J Med 2008; 358: 676–688.
8. Giacomini PS, Darlington PJ and Bar-Or A. Emerging multiple sclerosis disease-modifying therapies. Curr Opin Neurol 2009; 22: 226–232. 9. Lennon VA, Wingerchuk DM, Kryzer TJ, et al. A serum autoantibody marker of neuromyelitis optica: Distinction from multiple sclerosis. Lancet 2004; 364: 2106–2112. 10. Lennon VA, Kryzer TJ, Pittock SJ, et al. IgG marker of optic-spinal multiple sclerosis binds to the aquaporin-4 water channel. J Exp Med 2005; 202: 473–477. 11. Jiao Y, Fryer JP, Lennon VA, et al. Updated estimate of AQP4-IgG serostatus and disability outcome in neuromyelitis optica. Neurology 2013; 81: 1197–204. 12. Kitley J, Leite MI, Kuker W, et al. Longitudinally extensive transverse myelitis with and without aquaporin 4 antibodies. JAMA Neurol 2013; 70: 1375–1381. 13. Srivastava R, Aslam M, Kalluri SR, et al. Potassium channel KIR4.1 as an immune target in multiple sclerosis. N Engl J Med 2012; 367: 115–123. 14. Butt AM and Kalsi A. Inwardly rectifying potassium channels (Kir) in central nervous system glia: A special role for Kir4.1 in glial functions. J Cell Mol Med 2006; 10: 33–44. 15. Verkhratsky A and Steinhauser C. Ion channels in glial cells. Brain Res Brain Res Rev 2000; 32: 380–412. 16. Kofuji P and Newman EA. Potassium buffering in the central nervous system. Neuroscience 2004; 129: 1045–1056. 17. Newman EA. High potassium conductance in astrocyte endfeet. Science 1986; 233: 453–454. 18. Nagelhus EA, Horio Y, Inanobe A, et al. Immunogold evidence suggests that coupling of K+ siphoning and water transport in rat retinal Muller cells is mediated by a coenrichment of Kir4.1 and AQP4 in specific membrane domains. Glia 1999; 26: 47–54. 19. Tait MJ, Saadoun S, Bell BA, et al. Water movements in the brain: Role of aquaporins. Trends Neurosci 2008; 31: 37–43. 20. Connors NC, Adams ME, Froehner SC, et al. The potassium channel Kir4.1 associates with the dystrophin-glycoprotein complex via alphasyntrophin in glia. J Biolog Chem 2004; 279: 28387–28392. 21. Monce NM Jr, Bogusky RT and Cappel NN. An enzyme immunoassay screening test for the detection of total antinuclear antibodies. J Clin Lab Anal 1991; 5: 439–442.
http://msj.sagepub.com 7
Downloaded from msj.sagepub.com at Northeastern University on November 15, 2014
Multiple Sclerosis Journal 22. Polman CH, Reingold SC, Banwell B, et al. Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria. Ann Neurol 2011; 69: 292–302. 23. Wingerchuk DM, Lennon VA, Pittock SJ, et al. Revised diagnostic criteria for neuromyelitis optica. Neurology 2006; 66: 1485–1489. 24. Garcia-Caballero A, Dang Y, He H, et al. ENaC proteolytic regulation by channel-activating protease 2. J General Physiol 2008; 132: 521–535. 25. Masaki H, Wakayama Y, Hara H, et al. Immunocytochemical studies of aquaporin 4, Kir4.1 and alpha1-syntrophin in the astrocyte endfeet of mouse brain capillaries. Acta Histochem Cytochem 2010; 43: 99–105. 26. Watanabe M, Yamasaki R, Kawano Y, et al. Anti-KIR4.1 antibodies in Japanese patients with idiopathic central nervous system demyelinating diseases. Clin Exp Neuroimmunol 2013; 4: 241–242. 27. Nerrant E, Salsac C, Charif M, et al. Lack of confirmation of anti-inward rectifying potassium channel 4.1 antibodies as reliable markers of multiple sclerosis. Mult Scler 2014.
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32. Weinshenker BG. Neuromyelitis optica is distinct from multiple sclerosis. Arch Neurol 2007; 64: 899–901. 33. Jarius S, Probst C, Borowski K, et al. Standardized method for the detection of antibodies to aquaporin-4 based on a highly sensitive immunofluorescence assay employing recombinant target antigen. J Neurolog Sci 2010; 291: 52–56. 34. Cayrol R, Saikali P and Vincent T. Effector functions of antiaquaporin-4 autoantibodies in neuromyelitis optica. Ann NY Acad Sci 2009; 1173: 478–486. 35. Hinson SR, Roemer SF, Lucchinetti CF, et al. Aquaporin-4-binding autoantibodies in patients with neuromyelitis optica impair glutamate transport by down-regulating EAAT2. J Exp Med 2008; 205: 2473–2481. 36. Wen H, Nagelhus EA, Amiry-Moghaddam M, et al. Ontogeny of water transport in rat brain: Postnatal expression of the aquaporin-4 water channel. Eur J Neurosci 1999; 11: 935–945. 37. Pittock SJ, Lennon VA, De Seze J, et al. Neuromyelitis optica and non organ-specific autoimmunity. Arch Neurol 2008; 65: 78–83.
28. Brickshawana A, Hinson SR, Romero MF, et al. Investigation of the KIR4.1 potassium channel as a putative antigen in patients with multiple sclerosis: A comparative study. Lancet Neurol 2014; 13: 795–806.
38. Vaknin-Dembinsky A, Abramsky O, Petrou P, et al. Myasthenia gravis-associated neuromyelitis opticalike disease: An immunological link between the central nervous system and muscle? Arch Neurol 2011; 68: 1557–1561.
29. Niks EH, Kuks JB and Verschuuren JJ. Epidemiology of myasthenia gravis with anti-muscle-specific kinase antibodies in The Netherlands. J Neurol Neurosurg Psychiatry 2007; 78: 417–418.
39. Leite MI, Coutinho E, Lana-Peixoto M, et al. Myasthenia gravis and neuromyelitis optica spectrum disorder: A multicenter study of 16 patients. Neurology 78: 1601–1607.
30. Yeh JH, Chen WH, Chiu HC, et al. Low frequency of MuSK antibody in generalized seronegative myasthenia gravis among Chinese. Neurology 2004; 62: 2131–2132.
40. O’Connor KC, Lopez-Amaya C, Gagne D, et al. Antimyelin antibodies modulate clinical expression of childhood multiple sclerosis. J Neuroimmunol 2010; 223: 92–99.
31. Kraus V, Srivastava R, Kalluri SR, et al. Potassium channel KIR4.1-specific antibodies in children with acquired demyelinating CNS disease. Neurology 2014; 82: 470–473.
41. Tomassini V, De Giglio L, Reindl M, et al. Antimyelin antibodies predict the clinical outcome after a first episode suggestive of MS. Mult Scler 2007; 13: 1086–1094.
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