INTRODUCTION
Markers of inflammation such as erythrocyte sedimentation rate
(ESR) and acute phase reactants (APRs) such as C-reactive protein (CRP)
and serum amyloid-A (SAA) are widely used for laboratory and clinical
assessments of patients who have an inflammatory rheumatic diseases. The
ESR is a nonspecific measurement of increased serum proteins that occurs
during inflammation. APRs are produced by the liver under regulation of
circulating cytokines released by monocytes and macrophages. (1)
Immunoglobulins, produced by plasma cells and B lymphocytes, are
built of two identical heavy and light chains and these light chains
comprise one of two subtypes, known as kappa ([kappa]) and lambda
([lambda]). (2-4) When the light chains are attached to the heavy
chains, the light chains are referred to as bound light chains and when
they are not attached to heavy chains they are called as free light
chains. Normally, free light chains that are synthesized are quickly
filtered out and/or catabolized by the kidneys.
Increased levels of monoclonal free light chains (called
Bence-Jones proteins) have been measured as the consequence of clonal
expansion of plasma cells. Many autoimmune and inflammatory diseases are
accompanied with increased levels of oligoclonal free light chains in
different body fluids. Polyclonal B cell hyperactivity, hypersecretion
of antibodies and also increased local synthesis may be responsible for
the increase in free light chain levels in relation to disease activity
or relapses of disease. (5-11)
As an inflammatory disease, JIA is the most common form of
persistent arthritis in children that affects somewhere between 16-150
of every 100,000 children and is characterized with morning stiffness,
swelling of the affected joint(s), pain, limping and nonspecific
flu-like symptoms with respect to clinical type (12,13). In a study
including juvenile idiopathic arthtiris (JIA) patients, abnormalities in
the peripheral B cell pool, namely kappa light chain repertoire were
observed and the possibility of disturbance in B cell tolerance by
multiple pathogenic mechanisms in JIA was highlighted. (14) In Woulters
et al's study, all JIA patients had hypergammaglobulinemia
consistent with B cell hyperactivity. (15)
Familial mediterranean fever (FMF) is an autosomal recessively
inherited disorder characterized by recurrent, inflammatory self limited
episodes of fever and other symptoms. During the active stage of disease
and fever attacks, there is a substantial influx of polymorphonuclear
leukocytes into the affected tissues (16). Attack-free remission periods
are accompanied by the up-regulation of neutrophil and monocyte
phagocytic activity and oxidative burst. (16)
According to the recent literature, JIA and FMF have completely
different pathogenic mechanisms in which B cells play an important role
in the pathogenesis of JIA, but not in the pathogenesis of FMF. (17)
These facts led us to measure the concentrations of Ig kappa ([kappa])
and lambda ([lambda]) light chains in sera of patients with JIA (study
group) and FMF (disease control group) and in healthy children (healthy
control group) in order to compare them with other well-known markers of
inflammation to find out if they have any discriminating value in the
follow-up of these patients and if they correlate with classical markers
of inflammation.
PATIENTS and METHODS
In this prospective study of two years duration (20082009),
patients' sera were collected from the out-patient and in-patient
clinics of Ege University Faculty of Medicine, Department of Pediatric
Immunology, Izmir, Turkey. Parental informed consent was obtained and
the study was performed in concordance with the Declaration of Helsinki
ethical guidelines. A total of 117 patients (mean age: 11.3 [+ or -] 4.0
years, 54.7% were female and 45.3% were male) were included into the
study. Eighty-two of the patients had JIA and 35 patients had FMF.
Patients that followed up were included recurrently into the study which
some of them were at different clinical stages. A total of 174 sera were
collected from patients diagnosed with JIA (n=124, 71.2%) and FMF (n=50,
28.8%). FMF patients were taken as a "disease-control group".
JIA was defined as according to International League of Associations for
Rheumatology (ILAR) classification (18) and FMF patients were selected
according to Tel-Hashomer criteria. (19) A total of 30 age (11.6 [+ or
-] 3.26 years) and gender (16 male, 14 female) matched healthy children
were included as a "healthy control group". These children had
no chronic diseases and also no acute infections in the month prior to
blood collection.
JIA patients who did not have fever, arthralgia, arthritis, rash or
morning stiffness for at least three months were defined as "in
remission" according to disease-specific international criteria. If
the attack-free period was longer than three months in FMF patients,
they were thought of as being "in remission" stage.
Ig kappa ([kappa]) and lambda ([lambda]) light chain levels were
compared with other well-known markers of inflammation; namely ESR, SAA
and CRP. The ESR was determined by the Westergreen method. C-reactive
protein, serum amyloid A, [kappa] and [lambda] chain concentrations and
serum IgG, IgM levels were measured by nephelometry (with Dade Behring
BNII Nephelometer Analyzer and commercially available kits by Dade
Behring, Germany). The assay kit that was used, namely "antiserum
against human immunoglobulin light chains", recognized ([kappa])
and lambda ([lambda]) light chains both free and bound to heavy chains
in intact immunoglobulins. Reference intervals are 1.7-3.7 g/L for
[kappa], 0.9-2.1 g/L for [lambda] and 1.35-2.65 for [kappa/[lambda].
Reference curves were made up with multipoint calibration using N
protein standard SL (Siemens, Dade Behring, Germany).
Statistical analyses were performed using Pearson correlation,
Mann-Whitney U and Kruskal-Wallis test. A two-sided p-value less than
0.05 was considered to indicate statistical significance. Statistical
analyses were performed by using SPSS (Windows Version 17.0, SPSS Inc.,
Chicago, IL).
RESULTS
Immunoglobulin light chain levels, ESR and APRs both in active and
remission stages in JIA and FMF patients and in healthy controls were
measured and are listed in Tables 1 and 2.
In the JIA group, 55.7% (n=69) of the sera were from patients with
active JIA while 44.3% (n=55) of them were from patients in remission.
Not only ESR (p<0.0001), CRP (p<0.0001) and SAA (p<0.0001)
concentrations, but also [kappa] (p<0.0001) and [lambda]
(p<0.0001) chain levels showed statistically significant differences
between active and remission stages (Mann-Whitney U test). The
[kappa]/[lambda] (p=0.562) and [kappa]/IgG (p=0.391) ratios did not show
any significant difference compared to different disease stages, but the
[lambda]/IgG (p=0.025) ratio was significantly higher in the JIA with
active disease group as compared to the JIA patients in remission.
In the FMF group, 22.0% (n=11) of the sera were from patients with
active FMF while 78.0% (n=39) of them were from patients in remission
(Table-1). Although a significant increase was found in ESR (p=0.012),
CRP (p<0.0001) and SAA (p<0.0001) levels in patients in the active
disease group, there was no significant difference in [kappa] (p=0.981)
and [lambda] (p=0.535) chain levels, [kappa]/[lambda] ratio (p=0.419),
[kappa]/IgG (p=0.896) and [lambda]/IgG (p=0.896) between active and
remission periods of FMF patients (Mann-Whitney U test). In addition, in
the FMF group, [kappa] and [lambda] chain concentrations correlated
(p<0.0001, r=0.766) only with each other and showed no correlation
with ESR, APRs and immunoglobulin concentrations neither in entire FMF
patients nor with respect to clinical stages.
Correlations of Ig light chains and inflammatory markers in JIA
patients with respect to clinical stages are shown in Table-3 (Pearson
correlation). We found that [kappa] chain levels correlated very well
with SAA and ESR in both stages. On the other hand, [lambda] chain
levels correlated with SAA and ESR only in the remission period. No
correlation was observed between Ig light chain levels and CRP.
We also evaluated Ig light chain concentrations with respect to
immunoglobulin levels. There was no significant difference between three
immunoglobulin level (low, medium, high) group (Kruskal Wallis test).
Twelve patients had very high IgG levels (1893 [+ or -] 821 mg/dl) when
compared to age matched healthy children (1051 [+ or -] 228 mg/dl) and
[kappa] (p=0.031) and [lambda] (p=0.006) light chain concentrations
showed statistically significant relationship with high IgG
concentrations in sera of these patients. [kappa] levels positively
correlated with IgG (p=0.009, r=0.611) and IgM (p=0.041, r=0.500) in JIA
patients who were in the active stage of disease. In remission period,
there was no correlation between light chains and immunoglobulins
(pearson correlation).
Immunoglobulin light chains [kappa] and [lambda] as well as three
classical markers of inflammation were found to be significantly high in
JIA patients who were in the active stage of disease when compared to
data of healthy children and to JIA patients who were in remission
(Table 1 and 2). On the other hand, [kappa]/[lambda] ratio, [kappa]/IgG
and [lambda]/IgG did not show any significant difference between four
different patient subgroups and healthy controls (Table 1).
Active and remission stage specimens from the same JIA patients (27
samples from 13 patients) were examined and we observed that ESR
(p=0.001), SAA (p<0.0001) and CRP (p<0.0001) levels differed
significantly with respect to disease activity. There was a decrease in
[kappa] and [lambda] levels in remission period, but there was not
statistically significant difference (p=0.114 for [kappa], p=0.511 for
[lambda]) between both clinical stages which could be related to small
sample size.
DISCUSSION
Previously, relevance of increased Ig light chain concentrations in
the pathogenesis of some chronic inflammatory disorders such as systemic
lupus erythematosus or Sjogren's syndrome was previously reported.
(11,20) Low et al (21) assessed B cell activity by measuring the amount
of and [kappa]/[lambda] ratio in circulating immune complexes from the
sera of patients with JIA and they showed preferential selection of
lambda chains contributing to the formation of pathogenic circulating
immune complexes. In addition, they also concluded that a reversal of
the normal [kappa]/[lambda] ratio in JIA circulating immune complexes
may be used as a marker for increased B cell activity. (21) In the study
by Morbach et al (14) related with the role of light chains in JIA
pathogenesis, no clear evidence was found out.
Sensitive nephelometric assays for free [kappa] and [lambda] chains
are more effective in detecting conditions where only monoclonal light
chains are produced (light chain disease or amyloidosis) or where there
is minimal secretion of intact immunoglobulins (non-secretory or
oligosecretory multiple myeloma). Hypergammaglobulinemia and
musculoskeletal manifestations are common findings in both JIA and FMF
even though these diseases have different pathogenesis. Therefore, in
the light of above findings, we chose to measure the levels of free and
bound light chains together in addition to well-known APRs and serum
immunoglobulins.
Arthritis may be the initial or sole feature of FMF. Asymmetric
arthritis in larger joints of the lower extremity is most typical for
FMF and very similar to arthritis observed in JIA. However, the
diversity and the nonspecifity of the various clinical manifestations of
FMF can obscure the diagnosis. In the comparison of pathogenesis between
these two inflammatory disorders, neutrophils were found to play a more
important role than B cells in the pathogenesis of FMF. (22) Therefore,
light chain shedding is not expected in FMF patients although
hypergammaglobulinemia is often observed. In this study, [kappa] and
[lambda] chain concentrations correlated with each other but not with
other inflammatory markers in samples taken from FMF patients in acute
and remission stages. In addition, a significant increase was found in
ESR, CRP and SAA levels in the active FMF group as compared to the FMF
patients in remission, while there was no significant difference in
[kappa] and [lambda] chain levels between active and remission periods.
[kappa] and [lambda] chain levels of both active and remission stage FMF
patients also did not differ from levels measured in healthy controls.
Therefore, FMF patient samples served as a good disease control group
with no observation of increased light chain levels in relation to
clinical stage.
The ESR, CRP, SAA and Ig light chain levels showed a statistically
significant difference between JIA patients in active and remission
stages of disease. [kappa] levels showed better correlation with ESR and
SAA than [lambda] levels and it was thought that Ig light chain levels,
especially [kappa] chain, were good markers for detecting disease stage
in JIA patients.
In chronic inflammatory disorders, B cells can contribute to tissue
damage by autoantibody production and presentation of antigens to T
cells. Corcione et al23 demonstrated an expansion of activated switch
memory B cells and of IgG-secreting plasmablasts in the synovial fluid
from JIA patients. This finding is an evidence of long term immune
stimulation. Long-term stimulation of immune system leads an increase in
IgG levels as often observed in JIA. In our study, [kappa] and [lambda]
light chain concentrations were not correlated with whole IgG levels and
correlated only in a group of sera with very high IgG concentrations
suggesting to us that simply measuring total immunoglobulins is not
always helpful to determine disease stage.
Increased concentrations of immunoglobulin light chains might be an
indicator of B cell hyperactivity thereby reflecting disease activity
better than other inflammation parameters when B cell involvement is
suspected in the pathogenesis. As a result, Ig light chains especially
[kappa] chain concentrations are helpful to determine disease stage in
JIA patients but with our current data, they do not exhibit superiority
to any of the well-known tests for inflammation.
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Necil Kutukculer, M.D., Ege University, School of Medicine,
Division ofPediatric Immunology, Izmir, Turkey
Neslihan Edeer Karaca, M.D., Ege University, School of Medicine,
Division ofPediatric Immunology, Izmir, Turkey
Elif Azarstz, M.D., Ege University, School of Medicine, Division of
Pediatric Immunology, Izmir, Turkey
Guzide Aksu, M.D., Ege University, School of Medicine, Division of
Pediatric Immunology, Izmir, Turkey
Nesrin Gulez, M.D., Ege University, School of Medicine, Division of
Pediatric Immunology, Izmir, Turkey
Address for Correspondence: Dr. Necil Kutukculer, Faculty of
Medicine, Ege University, Department of Pediatrics, 35100 Bornova,
Izmir, Turkey, Phone number: 0090 232 3901016,
necil.kutukculer@ege.edu.tr
Table 1: Immunoglobulin light chain levels in patients with
juvenile idiopathic arthritis, familial mediterranean fever
and in healthy controls and their comparison with each other
(Data are given as median (range); p < 0.01, p < 0.05:
nonparametric tests).
JIA FMF
Active Remission Active Remission
(Group I) (Group II) (Group III) (Group IV)
n (sera) 69 55 11 39
Age 12 12 10 14
(2-19) (2-17) (2-17) (5-18)
[kappa] (g/L) 3.33 2.64 3.10 2.89
(1.70-6.56) (1.60-4.60) (1.77-4.58) (2.08-6.80)
[lambda] (g/L) 1.94 1.47 1.98 1.66
(0.4-10.7) (0.90-2.65) (0.9-4.02) (0.85-3.70)
[kappa]/[lambda] 1.76 1.77 1.68 1.74
(0.16-5.25) (1.08-2.67) (1.11-2.44) (1.17-3.13)
[kappa]/IgG 0.22 0.22 0.21 0.20
(0.14-0.43) (0.14-0.33) (0.16-0.39) (0.15-1.11)
[lambda]/IgG 0.14 0.12 0.14 0.11
(0.12-0.92) (0.08-0.20) (0.09-0.35) (0.09-0.60)
Healthy
Control *
(Group V) P < 0.05 P < 0.01
n (sera) 30
Age 11
(6-16) -- --
[kappa] (g/L) 2.39
(1.53-1.63) -- I-II, I-V
[lambda] (g/L) 1.40
(0.85-2.55) -- I-II, I-V
[kappa]/[lambda] 1.62
(1.27-1.97) -- --
[kappa]/IgG 0.22
(0.20-0.28) -- --
[lambda]/IgG 0.13
(0.11-0.16) I-II --
Table 2: Acute phase reactants in patients with juvenile idiopathic
arthritis, familial mediterranean fever and in healthy controls and
their comparison with each other. (Data are given as median (range);
p < 0.01, p < 0.05: nonparametric tests).
JIA FMF
Active Remission Active Remission
(Group I) (Group II) (Group III) (Group IV)
SAA 177 4.30 48.0 3.50
(mg/dL) (3.10-1966) (2.80-229) (3-616) (2.20-340)
CRP 3.42 0.30 3.60 0.30
(mg/dL) (0.30-134) (0.30-5.63) (0.30-13.7) (0.30-3)
ESR 42 12 46 12
(mm/hr) (12-140) (4-41) (30-100) (2-72)
Healthy
Control *
(Group V) P < 0.05 P < 0.01
SAA 5.80 I-II,
(mg/dL) (3.80-18.1) III-IV I-V
CRP 0.39 I-II,
(mg/dL) (0.33-0.50) -- III-IV,
I-V
ESR 10.5 I-II,
(mm/hr) (4-20) -- III-IV,
I-V,
III-V
Table 3: Correlation of Ig light chains and acute phase reactants
in juvenile idiopathic arthritis patient group with respect to
clinical stages.
SAA ESR CRP
[kappa] active p = 0.012 ** p = 0.006 * p = 0.682
r = 0.301 r = 0.336 r = 0.058
remission p = 0.019 ** p = 0.004 * p = 0.269
r = 0.315 r = 0.383 r = 0.161
[lambda] active r = 0.321 p = 0.174 p = 0.656
r = 0.140 r = 0.195 r = -0.064
remission p = 0.027 ** p = 0.002 * p = 0.264
r = 0.298 r = 0.403 r = 0.163
* p < 0.01, ** p < 0.05 (bivariate pearson correlation)