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Increased urinary free immunoglobulin light chain excretion in patients with multiple sclerosis
Journal of Neuroimmunology 220 (2010) 99–103
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Journal of Neuroimmunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n e u r o i m
Increased urinary free immunoglobulin light chain excretion in patients with multiple sclerosis Ruth Dobson a,⁎, R.F. Miller b, H.E. Palmer c,d, M. Feldmann e, E.J. Thompson f, A.J. Thompson f, D.H. Miller f, G. Giovannoni a,f a Neuroimmunology Unit, Queen Mary University of London, Blizard Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, 4 Newark Street, Whitechapel, London E1 2AT, United Kingdom b Research Department of Infection and Public Health, University College London, London WC1E 6JB, United Kingdom c Department of Ophthalmology, St Thomas' Hospital, London SW1 7EH, United Kingdom d University Hospitals, Birmingham B29 6JD, United Kingdom e Kennedy Institute of Rheumatology, London W6 8LH, United Kingdom f Institute of Neurology, University College London, Queen Square, London WC1N 3BG, United Kingdom
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Article history: Received 30 October 2009 Received in revised form 4 January 2010 Accepted 20 January 2010 Keywords: Urine Nitrate Nitrite Rheumatoid arthritis HIV-1 Posterior uveitis
a b s t r a c t Background: Plasma and B cells are implicated in multiple sclerosis (MS) and produce free light chains (FLC) that are excreted in urine. Objective: To confirm that demyelinating diseases (DD) cause increased urinary FLCs. Method: Urinary FLC in 50 patients with DD were compared to 20 patients with posterior uveitis (PU), 19 with AIDS, 34 with rheumatoid arthritis (RA) and 19 normal controls (NC). Result: Subjects with DD, PU, RA and AIDS have higher urinary FLCs than NC (p < 0.01). Urinary FLCs did not correlate with gadolinium-enhancing lesions on MRI. Conclusions: Urinary FLCs are raised in DD. Further studies are required to see if they correlate with disease activity.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction Multiple sclerosis (MS) is the commonest cause of neurological disability among young adults in the developed world. Plasma and B cells have been implicated in the pathogenesis of MS (Franciotta et al., 2008). The presence of intrathecal oligoclonal IgG (Freedman et al., 2005) and free light chains (FLCs) (Presslauer et al., 2008) is a very common diagnostic finding in MS. Recently Rituximab (anti-CD20) therapy that targets peripheral, and possibly intrathecal, B cells has been shown to be effective in MS (Hauser et al., 2008), (McFarland, 2008). Plasma and B cells produce excess light chains that are excreted in the urine. The level of urinary light chain excretion is therefore a putative marker of plasma and B cell activity in MS. It has been known for many years that people with MS have increased levels of free light chains (FLC) within their CSF when compared to healthy controls (Rudick et al., 1985). CSF κ free light chains have been shown to correlate with disability progression (Rinker et al., 2006). κ free light chains cannot reliably be detected in
⁎ Corresponding author. Tel.: +44 207 882 8813; fax: +44 207 377 7033. E-mail address: [email protected] (R. Dobson). 0165-5728/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2010.01.012
the serum of people with MS, although this may well be a dilutional effect (Mehta et al., 1991) or due to the rapid clearance of FLC by the kidneys (Tsai et al., 1992). Previous studies have established that the levels of both κ and λ free light chains are increased in the urine of people with MS when compared to normal controls and patients with other neurological diseases (Pezzoli and Pascali, 1987). Mehta et al. also established that a spot urine sample gave similar results to 24 h urine collection and that intercurrent urinary tract infection did not appear to affect the levels of free light chains in the urine. Whilst urinary free light chains have traditionally been used to monitor for B and plasma cell malignancies in the context of systemic rheumatological disease (Kelly et al., 1991), there is evidence that they are not only increased in the synovial fluid of those with rheumatoid arthritis, but that they are also excreted in the urine of these patients (Cooper and Bluestone, 1968). The presence of immunoglobulin within the iris and aqueous humor associated with uveitis was first demonstrated in post-mortem specimens in 1973 (Ghose et al., 1973), and is the result of local IgG synthesis (Murray et al., 1990). Free light chains are present in the CSF and in the serum of patients with Human Immunodeficiency Virus (HIV) infection (Elovaara et al., 1991), while the presence of urinary
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free light chains has only been documented in a small number of patients (Kahn, 1990), (Filomena, 1989). It is not known whether the immunoglobulin production in these two conditions causes an increase in the levels of urinary free light chains in a similar manner to that seen in MS and rheumatoid arthritis. The aim of this study was to confirm previous findings in patients with MS and rheumatoid arthritis, and to assess whether urinary free light chain excretion is raised in other conditions with locally increased immunoglobulin production. We also aim to establish whether subjects with clinically isolated syndromes (CIS) compatible with demyelination have elevated levels of urinary free light chain excretion. 2. Methods 2.1. Patients and subjects Forty patients with clinically or laboratory supported definite MS (Poser et al., 1983), 10 patients with clinically isolated syndromes compatible with demyelination (CIS), 20 patients with posterior uveitis (PU), 19 patients with AIDS, 34 patients with rheumatoid arthritis (RA) and 19 normal controls (NC) were studied. This was an exploratory study using banked samples. All participants provided informed consent and ethical approval had been granted. Patients with demyelinating diseases were recruited from the National Hospital for Neurology and Neurosurgery, London. Their disability was rated using Kurtzke's expanded disability status score (EDSS) (Kurtzke, 1983). Patients with CIS were not examined acutely, but within 3 months of their initial presentation. Patients with MS were classified as having relapsing remitting (RR), secondary progressive (SP), or primary progressive (PP) MS using established definitions (Lublin and Reingold, 1996). Patients with MS were studied at various times and only included 2 patients during a clinical relapse. The clinical relapse was defined using standard criteria (Poser et al., 1983). All patients with MS and CIS underwent a T1-weighted gadolinium (Gd)-enhanced MRI study of the brain, using a standardised protocol to assess disease activity (Miller et al., 1996). An experienced neuroradiologist, blinded to the clinical status, counted the number of Gd-enhancing lesions per MRI study. Patients with posterior uveitis were recruited from the ophthalmology outpatient department at St Thomas' Hospital, London. Active posterior uveitis was defined symptomatically or clinically using fundoscopy and/or fluorescein angiography. Patients with RA, fulfilling the American Rheumatism Association diagnostic criteria (Arnett et al., 1988), were recruited from Charing Cross Hospital, London. All patients with RA underwent a clinical examination and had their disease activity assessed using a joint count (Felson et al., 1993). AIDS patients were all inpatients at the University College London Hospitals' HIV/AIDS unit, and had a CD4+ T-cell count performed using standard methods; the clinical
details of these patients are described in detail in a previous publication (Giovannoni et al., 1999). NC were healthy volunteers, with no overt medical problems, and were recruited from the staff and the family and friends of the staff, working at the National Hospital for Neurology and Neurosurgery. 2.2. Urine assays Spot urine samples were collected at the time of the clinical examination of those with DD, RA, PU and AIDS. Samples were coded, frozen and stored at −20 °C. Samples were assayed without knowledge of the patients' disease status. Subjects with evidence of bacterial colonisation of the urinary tract, detected by the presence of excessive urinary nitrites (Giovannoni et al., 1997), were not included in the study. Urinary Ig-FLCurine was measured using two separate sandwich ELISAs, which were developed in-house. Polystyrene 96-well plates (Nunc, Maxisorb) were coated overnight at 4 °C with 100 μL of 1 in 500 dilution, in carbonate buffer (pH 9.0), of rabbit polyclonal anti-human κ (DAKO, A0100) and λ (DAKO, A0101) FLCs. The plates were then decanted and blocked for 1 h at room temperature with 200 μL of 2% bovine serum albumin in phosphate buffered saline (PBS) containing 0.1% TWEEN. Two sets of standards were made by pooling five κ and λ monoclonal Bence Jones proteins purified from the urine of patients with multiple myeloma (The Binding Site, Birmingham). Standard curves were constructed using doubling dilutions, in the blocking solution, from a concentration of 2 μg/mL downwards. Urine samples were tested in duplicate in a dilution of 1 in 200 in the blocking solution. Samples with light chain levels above the top standard were retested in dilutions of 1 in 1,000 or higher as appropriate. 100 μL of standard and samples was incubated on a plate shaker for 1 h at room temperature. The plate was then washed four times with a washing solution of PBS with 0.1% TWEEN and 0.2% BSA. 100 μL of a solution containing peroxidase conjugated rabbit polyclonal anti-human κ (DAKO, P0129) or λ (DAKO, P0130) light chains diluted 1:500 in blocking solution was then added for 1 h at room temperature. Plates were washed before the colour reagent was added; 100 μL/well (20 mg o-phenylenediamine (OPD) in 20 mL 0.02 M acetate buffer and 25 μL H2O2), and incubated in the dark for 10 min. The enzymatic reaction was stopped with 100 uL/well of 1 M HCl solution. The plate was read in an ELISA plate reader at 492 nm using 405 nm as a reference. The intra-assay coefficient of variation for the free κ light-chain assay was 4.1% (n = 10, range = 1.6%–9.8%) with an inter-assay coefficient of variation of 7.8% (n = 9). The intra-assay coefficient of variation for the free λ light-chain assay was 4.0% (n = 10, range= 0.8%–10.3%) with an inter-assay coefficient of variation of 12.9% (n = 9). To correct for variable degrees of proteinuria the concentration of Ig-FLCurine is expressed as ratio to urinary albumin which was measured using immunoelectrophoresis (Laurell technique).
Table 1 Demographic and urine Ig-FLC data by group.
No. of subjects Sex (M:F) Mean age yrs (SD) Urine kappa Ig-FLC:albumin median (25th–75th %tile) Urine lambda Ig-FLC:albumin median (25th–75th %tile) Urine total Ig-FLC:albumin median (25th–75th %tile) Urine Ig-FLC kappa:lambda mean (SD)
Normal controls (NC)
Demyelinating diseases (DD)
Posterior uveitis (PU)
Rheumatoid arthritis (RA)
Acquired Immunodeficiency syndrome (AIDS)
19 6:13 37.1 (10.0) 0.37 (0.01–0.72) 0.06 (0.00–0.09) 0.44 (0.01–0.80) 6.5 (4.0)
50 23:27 39.1 (9.4) 1.8 (0.55–4.12) 0.29 (0.10–0.65) 2.10 (0.65–4.77) 7.5 (4.5)
20 10:10 42.4 (12.1) 1.01 (0.47–2.22) 0.12 (0.04–0.20) 1.16 (0.53–2.40) 9.9 (4.7)
34 9:25 54.7 (11.7) 2.64 (1.02–5.12) 0.34 (0.12–0.64) 3.15 (1.10–5.59) 8.1 (3.8)
19 18:1 34.6 (7.7) 15.3 (6.00–28.2) 2.32 (0.86–5.42) 17.87 (6.86–36.42) 6.5 (2.4)
Post-hoc Tukey's honestly significant difference test. a RA > NC/DD/PU/AIDS, p < 0.001. b NC < DD/PU/RA/AIDS, p <0.006; AIDS > DD/PU/RA, p < 0.001. c NC < DD/RA/AIDS, p < 0.001; PU < DD/RA/AIDS, p < 0.04; AIDS > DD/PU/RA, p < 0.001. d NC < DD/RA/AIDS, p < 0.009; AIDS > DD/PU/RA, p < 0.001.
p-value
<0.001a <0.001a <0.006b <0.001c <0.001d n.s.
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2.3. Statistical methods Statistical analysis was performed using the PASW 18 statistics package. Variables were tested for normality using Q-Q plots and the Shapiro–Wilk test. Normally distributed continuous variables were compared using a one-way analysis of variance (ANOVA). If the ANOVA was significant individual groups were compared using a post-hoc Tukey's honestly significant difference test. Levene's test of homogeneity of variances was used to test if variables had equal variance. Variables which were not normally distributed were normalised using a simple natural logarithmic transformation. Sex ratios were compared using the Yates corrected Chi-squared test. A p-value of <0.05 was considered statistically significant. 3. Results The demographic and urinary Ig-FLC data are presented in Table 1. Significant differences were noted in the sex ratios and mean ages of the groups (Table 1). The majority of patients with AIDS was male compared to the other groups (p < 0.001). Patients with RA were significantly older compared to the subjects in the other groups (p < 0.001). The κ, λ and total Ig-FLC:alb.urine were significantly elevated in patients in all groups compared to NCs (p < 0.006 for κ Ig-FLC:alb.urine, p < 0.001 for λ and total Ig-FLC:alb.urine, Fig. 1a–c), with the AIDS group having significantly higher urine κ Ig-FLC:alb, λ Ig-FLC:alb and total Ig-FLC:alb ratio (p < 0.001 for all ratios, Table 1). The Ig-FLC κ:λ.urine did not differ between the groups. In patients with demyelinating disease, patients with CIS were significantly younger than patients with PP MS (p < 0.001). The κ, λ and total Ig-FLC:alb.urine of CIS patients were significantly higher than NC subjects (p < 0.004, Table 2 and Fig. 2a–c). Patients with RR and PP MS had significantly higher λ and total Ig-FLC:alb.urine than NC (p < 0.05, Table 2). There were too few patients in the SP MS group to reach statistical significance. The Ig-FLC κ:λ.urine did not differ between demyelinating disease sub-groups. After excluding patients with PP MS, who had minimal/no Gd-enhanced MRI activity (Ingle et al., 2005), no correlation was found between the Ig-FLC:alb.urine and the Gd-enhancing MRI activity in patients with demyelinating disease (data not shown). In the patients with PU, RA and AIDS, no correlation was found between active uveitis, joint count and the CD4+ T-cell count and Ig-FLC:alb.urine respectively (data not shown). 4. Discussion Our results show that the urinary excretion of κ and λ free light chains is increased in patients with MS. This is not a disease-specific phenomenon, as levels are also increased in patients with PU, RA and AIDS. Patients with established MS and CIS have higher levels of urinary free light chains than normal controls. In relation to HIV infection this observation has previously been documented in small numbers of patients (Kahn, 1990), (Filomena, 1989) but no conclusions were reached regarding causality or significance. The increased levels of urinary free light chains in patients with HIV compared to the other patient groups studied may be the result of non-specific polyclonal B cell activation, which has been well described in HIV infection (De Milito et al., 2004). In addition to this general phenomenon, most of the patients sampled for this study had clinical evidence of systemic infection in addition to HIV at the time of sampling, further contributing to their hypergammaglobulinaemia. The underlying reason for the presence of urinary free light chains in multiple sclerosis is yet to be determined. Free light chains can be detected in the CSF of those with multiple sclerosis but not in their plasma (Rudick et al., 1985), either due to rapid filtration by the kidneys (Tsai et al., 1992) or as a result of dilution (Mehta et al., 1991). There is currently no direct evidence that urinary free light chains are
Fig. 1. Combined scatter and box and whisker plots of (a) κIg-FLC:albuminurine, (b) λIg-FLC: albuminurine and (c) total urinary immunoglobulin free light chain to albumin ratios (Ig-FLC: alb.urine) in normal controls (○, n= 19), patients with demyelinating disease (□, n= 50), posterior uveitis (◊, n=20), rheumatoid arthritis (Δ, n= 34) and AIDS due to HIV-1 infection (+, n= 19). The box represents the 25th–75th percentile divided horizontally by the median, the whiskers the range, and the overlapping scatter plot the individual values from which the box and whiskers are derived.
Post-hoc Tukey's honestly significant difference test. a PP > CIS, p < 0.001. b NC < CIS, p = 0.003. c NC < CIS, p = 0.004. d NC < CIS, p < 0.001. e NC < RR, p = 0.19. f NC < RR, p = 0.05. g NC < RR, p = 0.04. h NC < DD, p < 0.001. i NC < DD, p < 0.001. j NC < DD, p = 0.003.
No. of subjects Sex (M:F) Mean age yrs (SD) Urine kappa Ig-FLC:albumin median (25th–75th %tile) Urine lambda Ig-FLC:albumin median (25th–75th %tile) Urine total Ig-FLC:albumin median (25th–75th %tile) Urine Ig-FLC kappa:lambda mean (SD)
19 6:13 37.1 (10.0) 0.37 (0.01–0.72) 0.06 (0.00–0.09) 0.44 (0.01–0.80) 6.5 (4.0)
Normal controls (NC)
Table 2 Demographic and urine Ig-FLC data by demyelinating disease subgroup. Clinically isolated syndrome (CIS) 10 6:4 30.2 (5.8) 0.56 (0.39–2.20)b 0.16 (0.09–0.33)c 0.66 (0.49–2.59)d 6.2 (2.8)
Relapsing remitting MS (RR) 19 5:14 38.3 (9.4) 1.99 (1.08–4.07)e 0.29 (0.14–0.71)f 2.12 (1.26–4.54)g 7.6 (4.3)
Secondary progressive MS (SP) 6 2:4 41.6 (7.8) 2.02 (0.28–9.18) 0.42 (0.03–1.08) 2.43 (0.31–10.25) 8.2 (2.5)
Primary progressive MS (PP) 15 10:5 45.2 (7.3) 3.20 (0.33–5.41) 0.46 (0.07–0.70) 3.33 (0.40–5.98) 7.9 (6.2)
n.s. <0.002a <0.001 h <0.001i = 0.003j n.s.
p-value
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Fig. 2. Combined scatter and box and whisker plots of (a) κIg-FLC:albuminurine, (b) λIg-FLC: albuminurine and (c) total urinary immunoglobulin free light chain to albumin ratios (Ig-FLC: alb.urine) in normal controls (○, n =19), patients with clinically isolated syndromes (□, n= 10), relapsing remitting MS (◊, n= 19), secondary progressive MS (Δ, n= 6) and primary progressive MS (+, n = 15). The box represents the 25th–75th percentile divided horizontally by the median, the whiskers the range, and the overlapping scatter plot the individual values from which the box and whiskers are derived.
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a direct marker for the presence of CSF oligoclonal bands, although this would seem a logical inference. Testing for urinary free light chains is now a commonplace procedure in many laboratories, and commercial assays are available (Freelite™, the Binding Site Ltd, Birmingham). Although our study did not show any correlation between the level of urinary free light chains and MRI activity (as measured by gadolinium enhancement), it is important to note that gadolinium enhancement is a surrogate marker of disease activity and does not necessarily correlate with immunological markers of disease progression (Giovannoni et al., 2000). Enhancing lesions have been associated with clinical relapses (Kappos et al., 1999), (Sormani et al., 2009) but not with the development of progressive disability (Kappos et al., 1999). It has also been shown that urinary neopterin:creatinine ratios correlate with new (<8 days old), gadolinium-enhancing lesions (Kappos et al., 1999), whereas gadolinium enhancement can persist for up to three months (Cotton et al., 2003). Currently MRI and the presence of oligoclonal bands unique to the CSF remain the most powerful predictors of the development of MS in those with CIS (Tintore et al., 2008), but these are costly and invasive investigations. B cell follicles have been described in the meninges of some patients with secondary progressive MS (Magliozzi et al., 2007), and those with B cell follicles present are described as having a younger age of onset and more severe disease course (Magliozzi et al., 2007). These outcome measures have not been correlated with the presence of CSF OCBs, but most patients have only a single lumbar puncture performed, limiting the evidence available. Urinary analysis has distinct advantages over blood or CSF monitoring — its noninvasive nature together with the ease of sample collection allow for the possibility of frequent monitoring. Further trials are needed to assess the relationship between urinary free light chains and CSF oligoclonal bands, in addition to identifying any relationship between the ratio of urine FLC:albumin and disease severity, progression and relapse rate. More work is needed in order to assess if among those with CIS the presence of urinary free light chains is predictive of progression to clinically definite MS and therefore could potentially be used as a biomarker in early disease.
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Journal of Neuroimmunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n e u r o i m
Increased urinary free immunoglobulin light chain excretion in patients with multiple sclerosis Ruth Dobson a,⁎, R.F. Miller b, H.E. Palmer c,d, M. Feldmann e, E.J. Thompson f, A.J. Thompson f, D.H. Miller f, G. Giovannoni a,f a Neuroimmunology Unit, Queen Mary University of London, Blizard Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, 4 Newark Street, Whitechapel, London E1 2AT, United Kingdom b Research Department of Infection and Public Health, University College London, London WC1E 6JB, United Kingdom c Department of Ophthalmology, St Thomas' Hospital, London SW1 7EH, United Kingdom d University Hospitals, Birmingham B29 6JD, United Kingdom e Kennedy Institute of Rheumatology, London W6 8LH, United Kingdom f Institute of Neurology, University College London, Queen Square, London WC1N 3BG, United Kingdom
a r t i c l e
i n f o
Article history: Received 30 October 2009 Received in revised form 4 January 2010 Accepted 20 January 2010 Keywords: Urine Nitrate Nitrite Rheumatoid arthritis HIV-1 Posterior uveitis
a b s t r a c t Background: Plasma and B cells are implicated in multiple sclerosis (MS) and produce free light chains (FLC) that are excreted in urine. Objective: To confirm that demyelinating diseases (DD) cause increased urinary FLCs. Method: Urinary FLC in 50 patients with DD were compared to 20 patients with posterior uveitis (PU), 19 with AIDS, 34 with rheumatoid arthritis (RA) and 19 normal controls (NC). Result: Subjects with DD, PU, RA and AIDS have higher urinary FLCs than NC (p < 0.01). Urinary FLCs did not correlate with gadolinium-enhancing lesions on MRI. Conclusions: Urinary FLCs are raised in DD. Further studies are required to see if they correlate with disease activity.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction Multiple sclerosis (MS) is the commonest cause of neurological disability among young adults in the developed world. Plasma and B cells have been implicated in the pathogenesis of MS (Franciotta et al., 2008). The presence of intrathecal oligoclonal IgG (Freedman et al., 2005) and free light chains (FLCs) (Presslauer et al., 2008) is a very common diagnostic finding in MS. Recently Rituximab (anti-CD20) therapy that targets peripheral, and possibly intrathecal, B cells has been shown to be effective in MS (Hauser et al., 2008), (McFarland, 2008). Plasma and B cells produce excess light chains that are excreted in the urine. The level of urinary light chain excretion is therefore a putative marker of plasma and B cell activity in MS. It has been known for many years that people with MS have increased levels of free light chains (FLC) within their CSF when compared to healthy controls (Rudick et al., 1985). CSF κ free light chains have been shown to correlate with disability progression (Rinker et al., 2006). κ free light chains cannot reliably be detected in
⁎ Corresponding author. Tel.: +44 207 882 8813; fax: +44 207 377 7033. E-mail address: [email protected] (R. Dobson). 0165-5728/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2010.01.012
the serum of people with MS, although this may well be a dilutional effect (Mehta et al., 1991) or due to the rapid clearance of FLC by the kidneys (Tsai et al., 1992). Previous studies have established that the levels of both κ and λ free light chains are increased in the urine of people with MS when compared to normal controls and patients with other neurological diseases (Pezzoli and Pascali, 1987). Mehta et al. also established that a spot urine sample gave similar results to 24 h urine collection and that intercurrent urinary tract infection did not appear to affect the levels of free light chains in the urine. Whilst urinary free light chains have traditionally been used to monitor for B and plasma cell malignancies in the context of systemic rheumatological disease (Kelly et al., 1991), there is evidence that they are not only increased in the synovial fluid of those with rheumatoid arthritis, but that they are also excreted in the urine of these patients (Cooper and Bluestone, 1968). The presence of immunoglobulin within the iris and aqueous humor associated with uveitis was first demonstrated in post-mortem specimens in 1973 (Ghose et al., 1973), and is the result of local IgG synthesis (Murray et al., 1990). Free light chains are present in the CSF and in the serum of patients with Human Immunodeficiency Virus (HIV) infection (Elovaara et al., 1991), while the presence of urinary
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free light chains has only been documented in a small number of patients (Kahn, 1990), (Filomena, 1989). It is not known whether the immunoglobulin production in these two conditions causes an increase in the levels of urinary free light chains in a similar manner to that seen in MS and rheumatoid arthritis. The aim of this study was to confirm previous findings in patients with MS and rheumatoid arthritis, and to assess whether urinary free light chain excretion is raised in other conditions with locally increased immunoglobulin production. We also aim to establish whether subjects with clinically isolated syndromes (CIS) compatible with demyelination have elevated levels of urinary free light chain excretion. 2. Methods 2.1. Patients and subjects Forty patients with clinically or laboratory supported definite MS (Poser et al., 1983), 10 patients with clinically isolated syndromes compatible with demyelination (CIS), 20 patients with posterior uveitis (PU), 19 patients with AIDS, 34 patients with rheumatoid arthritis (RA) and 19 normal controls (NC) were studied. This was an exploratory study using banked samples. All participants provided informed consent and ethical approval had been granted. Patients with demyelinating diseases were recruited from the National Hospital for Neurology and Neurosurgery, London. Their disability was rated using Kurtzke's expanded disability status score (EDSS) (Kurtzke, 1983). Patients with CIS were not examined acutely, but within 3 months of their initial presentation. Patients with MS were classified as having relapsing remitting (RR), secondary progressive (SP), or primary progressive (PP) MS using established definitions (Lublin and Reingold, 1996). Patients with MS were studied at various times and only included 2 patients during a clinical relapse. The clinical relapse was defined using standard criteria (Poser et al., 1983). All patients with MS and CIS underwent a T1-weighted gadolinium (Gd)-enhanced MRI study of the brain, using a standardised protocol to assess disease activity (Miller et al., 1996). An experienced neuroradiologist, blinded to the clinical status, counted the number of Gd-enhancing lesions per MRI study. Patients with posterior uveitis were recruited from the ophthalmology outpatient department at St Thomas' Hospital, London. Active posterior uveitis was defined symptomatically or clinically using fundoscopy and/or fluorescein angiography. Patients with RA, fulfilling the American Rheumatism Association diagnostic criteria (Arnett et al., 1988), were recruited from Charing Cross Hospital, London. All patients with RA underwent a clinical examination and had their disease activity assessed using a joint count (Felson et al., 1993). AIDS patients were all inpatients at the University College London Hospitals' HIV/AIDS unit, and had a CD4+ T-cell count performed using standard methods; the clinical
details of these patients are described in detail in a previous publication (Giovannoni et al., 1999). NC were healthy volunteers, with no overt medical problems, and were recruited from the staff and the family and friends of the staff, working at the National Hospital for Neurology and Neurosurgery. 2.2. Urine assays Spot urine samples were collected at the time of the clinical examination of those with DD, RA, PU and AIDS. Samples were coded, frozen and stored at −20 °C. Samples were assayed without knowledge of the patients' disease status. Subjects with evidence of bacterial colonisation of the urinary tract, detected by the presence of excessive urinary nitrites (Giovannoni et al., 1997), were not included in the study. Urinary Ig-FLCurine was measured using two separate sandwich ELISAs, which were developed in-house. Polystyrene 96-well plates (Nunc, Maxisorb) were coated overnight at 4 °C with 100 μL of 1 in 500 dilution, in carbonate buffer (pH 9.0), of rabbit polyclonal anti-human κ (DAKO, A0100) and λ (DAKO, A0101) FLCs. The plates were then decanted and blocked for 1 h at room temperature with 200 μL of 2% bovine serum albumin in phosphate buffered saline (PBS) containing 0.1% TWEEN. Two sets of standards were made by pooling five κ and λ monoclonal Bence Jones proteins purified from the urine of patients with multiple myeloma (The Binding Site, Birmingham). Standard curves were constructed using doubling dilutions, in the blocking solution, from a concentration of 2 μg/mL downwards. Urine samples were tested in duplicate in a dilution of 1 in 200 in the blocking solution. Samples with light chain levels above the top standard were retested in dilutions of 1 in 1,000 or higher as appropriate. 100 μL of standard and samples was incubated on a plate shaker for 1 h at room temperature. The plate was then washed four times with a washing solution of PBS with 0.1% TWEEN and 0.2% BSA. 100 μL of a solution containing peroxidase conjugated rabbit polyclonal anti-human κ (DAKO, P0129) or λ (DAKO, P0130) light chains diluted 1:500 in blocking solution was then added for 1 h at room temperature. Plates were washed before the colour reagent was added; 100 μL/well (20 mg o-phenylenediamine (OPD) in 20 mL 0.02 M acetate buffer and 25 μL H2O2), and incubated in the dark for 10 min. The enzymatic reaction was stopped with 100 uL/well of 1 M HCl solution. The plate was read in an ELISA plate reader at 492 nm using 405 nm as a reference. The intra-assay coefficient of variation for the free κ light-chain assay was 4.1% (n = 10, range = 1.6%–9.8%) with an inter-assay coefficient of variation of 7.8% (n = 9). The intra-assay coefficient of variation for the free λ light-chain assay was 4.0% (n = 10, range= 0.8%–10.3%) with an inter-assay coefficient of variation of 12.9% (n = 9). To correct for variable degrees of proteinuria the concentration of Ig-FLCurine is expressed as ratio to urinary albumin which was measured using immunoelectrophoresis (Laurell technique).
Table 1 Demographic and urine Ig-FLC data by group.
No. of subjects Sex (M:F) Mean age yrs (SD) Urine kappa Ig-FLC:albumin median (25th–75th %tile) Urine lambda Ig-FLC:albumin median (25th–75th %tile) Urine total Ig-FLC:albumin median (25th–75th %tile) Urine Ig-FLC kappa:lambda mean (SD)
Normal controls (NC)
Demyelinating diseases (DD)
Posterior uveitis (PU)
Rheumatoid arthritis (RA)
Acquired Immunodeficiency syndrome (AIDS)
19 6:13 37.1 (10.0) 0.37 (0.01–0.72) 0.06 (0.00–0.09) 0.44 (0.01–0.80) 6.5 (4.0)
50 23:27 39.1 (9.4) 1.8 (0.55–4.12) 0.29 (0.10–0.65) 2.10 (0.65–4.77) 7.5 (4.5)
20 10:10 42.4 (12.1) 1.01 (0.47–2.22) 0.12 (0.04–0.20) 1.16 (0.53–2.40) 9.9 (4.7)
34 9:25 54.7 (11.7) 2.64 (1.02–5.12) 0.34 (0.12–0.64) 3.15 (1.10–5.59) 8.1 (3.8)
19 18:1 34.6 (7.7) 15.3 (6.00–28.2) 2.32 (0.86–5.42) 17.87 (6.86–36.42) 6.5 (2.4)
Post-hoc Tukey's honestly significant difference test. a RA > NC/DD/PU/AIDS, p < 0.001. b NC < DD/PU/RA/AIDS, p <0.006; AIDS > DD/PU/RA, p < 0.001. c NC < DD/RA/AIDS, p < 0.001; PU < DD/RA/AIDS, p < 0.04; AIDS > DD/PU/RA, p < 0.001. d NC < DD/RA/AIDS, p < 0.009; AIDS > DD/PU/RA, p < 0.001.
p-value
<0.001a <0.001a <0.006b <0.001c <0.001d n.s.
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2.3. Statistical methods Statistical analysis was performed using the PASW 18 statistics package. Variables were tested for normality using Q-Q plots and the Shapiro–Wilk test. Normally distributed continuous variables were compared using a one-way analysis of variance (ANOVA). If the ANOVA was significant individual groups were compared using a post-hoc Tukey's honestly significant difference test. Levene's test of homogeneity of variances was used to test if variables had equal variance. Variables which were not normally distributed were normalised using a simple natural logarithmic transformation. Sex ratios were compared using the Yates corrected Chi-squared test. A p-value of <0.05 was considered statistically significant. 3. Results The demographic and urinary Ig-FLC data are presented in Table 1. Significant differences were noted in the sex ratios and mean ages of the groups (Table 1). The majority of patients with AIDS was male compared to the other groups (p < 0.001). Patients with RA were significantly older compared to the subjects in the other groups (p < 0.001). The κ, λ and total Ig-FLC:alb.urine were significantly elevated in patients in all groups compared to NCs (p < 0.006 for κ Ig-FLC:alb.urine, p < 0.001 for λ and total Ig-FLC:alb.urine, Fig. 1a–c), with the AIDS group having significantly higher urine κ Ig-FLC:alb, λ Ig-FLC:alb and total Ig-FLC:alb ratio (p < 0.001 for all ratios, Table 1). The Ig-FLC κ:λ.urine did not differ between the groups. In patients with demyelinating disease, patients with CIS were significantly younger than patients with PP MS (p < 0.001). The κ, λ and total Ig-FLC:alb.urine of CIS patients were significantly higher than NC subjects (p < 0.004, Table 2 and Fig. 2a–c). Patients with RR and PP MS had significantly higher λ and total Ig-FLC:alb.urine than NC (p < 0.05, Table 2). There were too few patients in the SP MS group to reach statistical significance. The Ig-FLC κ:λ.urine did not differ between demyelinating disease sub-groups. After excluding patients with PP MS, who had minimal/no Gd-enhanced MRI activity (Ingle et al., 2005), no correlation was found between the Ig-FLC:alb.urine and the Gd-enhancing MRI activity in patients with demyelinating disease (data not shown). In the patients with PU, RA and AIDS, no correlation was found between active uveitis, joint count and the CD4+ T-cell count and Ig-FLC:alb.urine respectively (data not shown). 4. Discussion Our results show that the urinary excretion of κ and λ free light chains is increased in patients with MS. This is not a disease-specific phenomenon, as levels are also increased in patients with PU, RA and AIDS. Patients with established MS and CIS have higher levels of urinary free light chains than normal controls. In relation to HIV infection this observation has previously been documented in small numbers of patients (Kahn, 1990), (Filomena, 1989) but no conclusions were reached regarding causality or significance. The increased levels of urinary free light chains in patients with HIV compared to the other patient groups studied may be the result of non-specific polyclonal B cell activation, which has been well described in HIV infection (De Milito et al., 2004). In addition to this general phenomenon, most of the patients sampled for this study had clinical evidence of systemic infection in addition to HIV at the time of sampling, further contributing to their hypergammaglobulinaemia. The underlying reason for the presence of urinary free light chains in multiple sclerosis is yet to be determined. Free light chains can be detected in the CSF of those with multiple sclerosis but not in their plasma (Rudick et al., 1985), either due to rapid filtration by the kidneys (Tsai et al., 1992) or as a result of dilution (Mehta et al., 1991). There is currently no direct evidence that urinary free light chains are
Fig. 1. Combined scatter and box and whisker plots of (a) κIg-FLC:albuminurine, (b) λIg-FLC: albuminurine and (c) total urinary immunoglobulin free light chain to albumin ratios (Ig-FLC: alb.urine) in normal controls (○, n= 19), patients with demyelinating disease (□, n= 50), posterior uveitis (◊, n=20), rheumatoid arthritis (Δ, n= 34) and AIDS due to HIV-1 infection (+, n= 19). The box represents the 25th–75th percentile divided horizontally by the median, the whiskers the range, and the overlapping scatter plot the individual values from which the box and whiskers are derived.
Post-hoc Tukey's honestly significant difference test. a PP > CIS, p < 0.001. b NC < CIS, p = 0.003. c NC < CIS, p = 0.004. d NC < CIS, p < 0.001. e NC < RR, p = 0.19. f NC < RR, p = 0.05. g NC < RR, p = 0.04. h NC < DD, p < 0.001. i NC < DD, p < 0.001. j NC < DD, p = 0.003.
No. of subjects Sex (M:F) Mean age yrs (SD) Urine kappa Ig-FLC:albumin median (25th–75th %tile) Urine lambda Ig-FLC:albumin median (25th–75th %tile) Urine total Ig-FLC:albumin median (25th–75th %tile) Urine Ig-FLC kappa:lambda mean (SD)
19 6:13 37.1 (10.0) 0.37 (0.01–0.72) 0.06 (0.00–0.09) 0.44 (0.01–0.80) 6.5 (4.0)
Normal controls (NC)
Table 2 Demographic and urine Ig-FLC data by demyelinating disease subgroup. Clinically isolated syndrome (CIS) 10 6:4 30.2 (5.8) 0.56 (0.39–2.20)b 0.16 (0.09–0.33)c 0.66 (0.49–2.59)d 6.2 (2.8)
Relapsing remitting MS (RR) 19 5:14 38.3 (9.4) 1.99 (1.08–4.07)e 0.29 (0.14–0.71)f 2.12 (1.26–4.54)g 7.6 (4.3)
Secondary progressive MS (SP) 6 2:4 41.6 (7.8) 2.02 (0.28–9.18) 0.42 (0.03–1.08) 2.43 (0.31–10.25) 8.2 (2.5)
Primary progressive MS (PP) 15 10:5 45.2 (7.3) 3.20 (0.33–5.41) 0.46 (0.07–0.70) 3.33 (0.40–5.98) 7.9 (6.2)
n.s. <0.002a <0.001 h <0.001i = 0.003j n.s.
p-value
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Fig. 2. Combined scatter and box and whisker plots of (a) κIg-FLC:albuminurine, (b) λIg-FLC: albuminurine and (c) total urinary immunoglobulin free light chain to albumin ratios (Ig-FLC: alb.urine) in normal controls (○, n =19), patients with clinically isolated syndromes (□, n= 10), relapsing remitting MS (◊, n= 19), secondary progressive MS (Δ, n= 6) and primary progressive MS (+, n = 15). The box represents the 25th–75th percentile divided horizontally by the median, the whiskers the range, and the overlapping scatter plot the individual values from which the box and whiskers are derived.
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a direct marker for the presence of CSF oligoclonal bands, although this would seem a logical inference. Testing for urinary free light chains is now a commonplace procedure in many laboratories, and commercial assays are available (Freelite™, the Binding Site Ltd, Birmingham). Although our study did not show any correlation between the level of urinary free light chains and MRI activity (as measured by gadolinium enhancement), it is important to note that gadolinium enhancement is a surrogate marker of disease activity and does not necessarily correlate with immunological markers of disease progression (Giovannoni et al., 2000). Enhancing lesions have been associated with clinical relapses (Kappos et al., 1999), (Sormani et al., 2009) but not with the development of progressive disability (Kappos et al., 1999). It has also been shown that urinary neopterin:creatinine ratios correlate with new (<8 days old), gadolinium-enhancing lesions (Kappos et al., 1999), whereas gadolinium enhancement can persist for up to three months (Cotton et al., 2003). Currently MRI and the presence of oligoclonal bands unique to the CSF remain the most powerful predictors of the development of MS in those with CIS (Tintore et al., 2008), but these are costly and invasive investigations. B cell follicles have been described in the meninges of some patients with secondary progressive MS (Magliozzi et al., 2007), and those with B cell follicles present are described as having a younger age of onset and more severe disease course (Magliozzi et al., 2007). These outcome measures have not been correlated with the presence of CSF OCBs, but most patients have only a single lumbar puncture performed, limiting the evidence available. Urinary analysis has distinct advantages over blood or CSF monitoring — its noninvasive nature together with the ease of sample collection allow for the possibility of frequent monitoring. Further trials are needed to assess the relationship between urinary free light chains and CSF oligoclonal bands, in addition to identifying any relationship between the ratio of urine FLC:albumin and disease severity, progression and relapse rate. More work is needed in order to assess if among those with CIS the presence of urinary free light chains is predictive of progression to clinically definite MS and therefore could potentially be used as a biomarker in early disease.
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