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Increase of L-chain proteins in the sera of patients with systemic lupus erythematosus and the synovial fluids of patients with peripheral rheumatoid arthritis.

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Increase of L-Chain Proteins in the Sera of Patients with
Systemic Lupus Erythematosus and the Synovial Fluids
of Patients with Peripheral Rheumatoid Arthritis
By WALLACE
V. EPSTEIN
AND MARGARET
TAN
B
specific for determinants of the light ( L ) polypeptide chains of the human immunoglobulins,
it has become possible to measure free
L-chains in the presence of normal or
increased quantities of intact immunoglohulins.1,2 The quantities of L-chain
proteins (Hence Jones proteins) found in
the serum and urine of patients with multiple myeloma and macroglobulinemia have
lieen established by both isolation technics3
and by direct immunologic methods2 The
present report deals with the presence of
increased quantities of L-chain proteins in
the synovial fluids of patients with rheumatoid arthritis and the sera of those having
systemic lupus erythematosus.
using 2 per cent agar in 0.075M barbital buffer,
pH 8.6. Serum was examined undiluted and synovial fluid was examined after the addition of
hyaluronidase. Plates were inspected for preeipitin bands after 24 hours in a moist chamber at
room temperature and again after staining. Concentration of the antigen was estimated by
doubling dilutions of the antigen using separate
pipettes at each dilution step.
Gel filtration chromatography of synovial fluid
was performed in 3.5 x 80 cm. columns of
medium grade Sephadex (2 parts G200 to 1 part
G100) in 0.1M NaCI, 0.01M PO,, pH 8 buffer.
Chromatography was performed at room temperature.
Y MEANS OF ANTISERUM
MATEHIALSAND METHODS
Antiserum to L-chain proteins was prepared by
iirimnnizatioii of rabbits with L-chains prepared
t)y the retliiction and alkylation of pooled human
y - ( : glol~ulins.?’he antiserum has specificity for Lchain determinants relatively unavailable in intact
g:imnia globulins. The antiserum was “absorbed”
11) the addition of y-G globulin until no precipitating ability for intact gamma globulin remained
when tested by precipitation in agar. The details
of these steps have been described previously.2
Antisrrnni specific for type K or type h deteriniiiants was prepared by absorption of this antiserinti with any type K or type h Bence Jones
protein. The minimum concentration of L-chain
protein that ciin be detected by these antisera is
approximatel) 0.02 mg./ml.
Precipitiii reactions in agar were performed
F r i m the Rkeurnutic. Disease Group, Departirierit ( i f Mediczrke, Uniuersity of California School
ot Mrdicitie, Sari Francisco, Calif.
Supportc.d by u grartt fTo?rL the National Instih t e s of Health ( U S P H S 0229), Bethesda, M d .
Presercted ‘n part at the Interim Meeting Of the
Rheurnatisrn Association, December,
Serum and Synovial Fluid
All sera were from patients with clinically active systemic lupus erythematosns or peripheral
rheumatoid arthritis. All of the former had positive LE cell preparations either at the time the
specimen was obtained or no longer than 6 months
earlier. All sera were examined for rheumatoid
factor by the FII tanned cell hemagglutination
procedure. Some serum specimens had been maintained in our serum reference collection at -20 C.
without preservatives, for as long as 2 years.
Synovial fluids were obtained by knee joint aspiration into syringes containing heparin, together
with simultaneous venous blood specimens. Tests
of synovial fluid for rheumatoid factor activity
were performed after the addition of hyaluronidase and without the application of heat for the
inactivation of complement.
Our attempts to classify the renal status of the
patients having systemic lupus erythematosus were
fraught with difficulty. In several instances. no
tests of renal function had been obtained at the
same time as the serum specimen was obtained. It
1964, Washington, D. C .
WALLACEv. EPSTEIN, M.D. : Associate Professor of Medicine; MARGARETTAN, B.A., M.S.: Research Biochemist; both of the Rheumatic Disease
Group, Department of Medicine, Unicersity of
California School of Medicine, Sun Francisco, Calif.
Arriericari
713
ARTHRITISAND RHEUMATISM,VOL. 9, No. 5 (OCTOBER),
1966
714
EPSTEIN AND TAN
Table 1.-Precipitin Test for Increased
L-Chain Protein in Sera of Patimts
with SLE
Number
Tested
With renal failure
Withont renal
failure
L-chain
Positive Negative
10
9
1
20
19
1
appeared most reliable to limit classification to
those with renal insufficiency at or within 3
months previous to the time the blood specimen
was obtained. The serum creatinine and the creatinine clearance were the determinations most
frequently found in the patients’ records and were
used with any other available tests to group patients into those with and those without gross
renal insufficiency.
RESULTS
except for one serum from a patient free of
renal failure, in which only type K light
chain protein was found. We have been
unable to demonstrate a direct relationship
between the amount of free L-chain protein in the serum and the presence or extent of renal insufficiency. In five cases
urine specimens of patients having positive
serum tests for increased free L-chain were
examined with serial determinations over a
2-month period. In all instances, the free Lchain concentration (0.04 to 0.16 mg. per
ml.) was equal or one doubling dilution
greater in urine than in that patient’s
serum. Again, no direct correlation was
noted between the daily total proteinuria
and the amount of free L-chain protein in
the serum or urine.
Systemic Lupus Erythematosus ( S L E )
Rheumatoid Arthritis
The sera of ten patients having definite
SLE associated with renal insufficiency and
20 without renal insufficiency were tested
tor increased quantities of free L-chain
protein. Tests of 47 normal sera using these
same reagents revealed that they cmtained
less than 0.02 mg./ml. of free L-chain proNine of ten patients with renal insufficiency due to SLE and 19 of 20 without renal insufficiency were found to conPain greater than 0.02 mg. per ml. (Table
1). Figure 1 shows the precipitin pattern
of six different sera from patients with SLE
arranged in a clockwise manner according
to intensity of precipitin band produced by
the whole serum. The center well contains
antiserum to human L-chains absorbed
with y-C: globulin. The double precipitin
band of serum 1 and 2 was often noted in
strongly positive sera.
Antiserum specific for type A or type K
light chain proteins was used to test the
serial dilution of all positive sera. It revealed that type h light chain was present
in amounts equal to or greater than type K
Some 40 sera of patients with definite
peripheral rheumatoid arthritis, having
positive serum tests for rheumatoid factor
and negative L. E. cell preparations, were
examined. Two of these formed a precipitin band under the same conditions used to
examine the SLE sera. Table 2 presents the
results obtained for those sera and their
corresponding synovial fluid specimens
when tested against L-chain antiserum.
The specimens are listed according to the
FII hemagglutination titer of the serum.
The quantities of free L-chain protein in
synovial fluid ranged between 0.02 to 0.64
mg. per ml. with no correlation noted between the concentration of L-chain protein
and the rheumatoid factor titer (Table 2).
The specimen listed under Osteoarthritis
that exhibits elevated rheumatoid factor
levels and increased L-chain concentration
in the synovial fluid had been obtained
from a patient cared for in our clinic, and
thought to have clinical osteoarthritis
despite the known positive test for rheumatoid factor. No attempt was made to cor-
LNCHEASE C F L-CHAIK P'HOTEINS IN SERA OF PATIENTS WITH SLE
715
Fig. 1.-Double diffusion in agar resulting in precipitin bands between six different
serd from patients with SLE in peripheral wells and antiserum to human L-chains
in
center well.
relate these results with drug therapy at the
time the specimens were obtained.
Fractionation of Synovial Fluids
Three specimens of- synovial fluid were
p'issed wparately through a column of a
mixture of Sephadex G200 and G100. The
protein distribution elution pattern as
5hown in Figure 2 is typical of the three
5pecimens. Rheumatoid factor activity was
regularly found concentrated in peak I.
The free L-chain activity was found only in
peak 111 protein as shown in Figure 3.
Specimens of Bence Jones proteins or of Lchain proteins of normal pooled 7-G globulin, when passed through these same columns, emerged in tubes corresponding to
the peak I11 distribution.
DrscussIoN
A series of s t ~ i d i e s ~
have
- ~ demonstrated
that proteins having characteristics of
Bence Tone5 proteins and the L-chains of
human immunoglobulins are found in the
serum and urine of normal individuals
716
EPSTEIN AND TAN
Table 2.-Precipitin Test for Increased L-Chain Protein in Serum and Synovial Fluid
Synovial Fluid
Serum
FII Titer
Rheumatoid
arthritis
FII Titer
L-chain (mg./ce.)
Type K
Type
0
0.08
0.08
0
0.08
0
0
0
0
0
0
0
0
0
0.02
0
0
0.02
224
1,280
112
28
7,000
14,000
7,000
56,000
3,584
14,000
112,000
7,000
14,000
0.08
0.08
0.08
0.16
0.08
0.32
0.08
0.02
0.08
0.02
0.02
0.02
0.08
0.08
0.16
0.64
0.16
<56
0.08
<56
0.02
0.04
<56
<56
<56
224
896
0
0
0
0
0
<56
<56
<58
56
224
0
0
0
0
0
0
0
0
0.02
0.02
<56
<56
<56
112
0
0
0
0
<56
<56
<56
<56
0
0
0
0
0
0
224
640
1,792
3.584
14,000
28,000
56,000
56,000
56,000
56,000
56,000
224,000
224,000
S.L.E.
Osteoarthritis
'I'ramriatit
arthriti5
L chain (mg./cc.)
O0
0
0
0
0
0
' 0 designates less than 0.02 mg./cc. The minimum concentration of L-chain protein to form precipitin line with the antiserum is 0.02 mg./cc.
Using the antisera described in the present
study, we have found that normal serum
contains 0.005-0.008 mg./cc. of free Lchain.2 Normal urine contains approximately 3 mg. per day.? The sera of normal
pregnant women contain approximately
0.016 mg./ml.*
Interpretation of our findings of greater
than normal quantities of L-chain proteins
in the sera of patients having SLE and in
the synovial fluids of patients having peripheral rheumatoid arthritis is made difficult by our lack of knowledge of the normal synthetic rate, metabolic fate, degradation and excretory pathways of L-chain
proteins. It must be assumed that the individual polypeptide chains of the immunoglobulins, like 7-C globulin itself, must be
synthesized and catabolized in connection
with mechanisms that regulate the total intravascular mass of the protein; the serum
concentration depends on the rate of synthesis, the plasma volume and the fractional catabolic rate. There may also be an
endogenous catabolism of L-chain protein
in the kidney itself3 as well as a normal
urinary excretion of the protein.
Our inability to correlate the magnitude
of the increased L-chain concentration in
the sera of patients having SLE with the
degree of renal insufficiency does not eliminate the possibility that this serum abnormality reflects renal involvement in this
disease in most, if not all, patients. Indeed,
in the cases in which we have performed
serial studies, L-chain proteinuria, but not
total proteinuria, parallels or precedes the
increase of the L-chain concentration in the
717
IlCCREASE OF L-CHAIN PROTEINS IN SERA OF PATIENTS WITH SLE
o.D.280
R.A. S Y N O V / A L FLU/D
1.0-
1.6 -
-
1.4 1.2
9
-
-
U
I
5
Fig. 2.-GeI
1
1
I
J
30 35 40 4 5 5 0 55 60 6 5 70 7 5 80
TUBE N U M B E R
filtration chromatography of synovial fluid obtained from a patient
10
15
20 2 5
having peripheral rheumatoid arthritis.
serum. Since decreased renal excretion alone
could not explain such findings, one might
postulate ;i decrease in the catabolic rate of
I,-chain protein. Solomon et aL3 have demonstrated a decrease in the Bence Jones
protein catabolic rates in patients with
hlood urea nitrogen levels above 30 mg.
per 100 ml. No catabolic or excretory organ
other than the kidney has been described.
It is also possible that the increased serum
concentration of L-chain protein reflects
the involvement of gamma globulins in the
disease process with accelerated synthesis
of the immunoglobulins and therefore of
the I,-chain protein. Andersen and Jensen!'
have found an increased rate of y-G globulin synthesis in 10 of 16 patients with collagen diseases and an increased fractional
catabolic rate in 11. Vaughan and Barnettlo
have recently reported an increase in serum
L ~ h a i n sin the course of a serum sickness
rewtion in it patient who had received
horse serum. The transient increase of
serum L-chains occurred at the time of the
maximum symptomatic reaction and may
have been related to both increased immunoglobulin synthesis and to peripheral
catabolism in tissues. Both of the major antigenic types of L-chain protein appear
represented in the serum increase found in
SLE. The equal or at times increased quantity of type h to type K protein appears at
variance with the reported normal excess of
type K to type h in intact immunoglobu1ins.ll Whether increased type h protein is
characteristic of the antibodies elaborated
in this disorder will await completion of a
wider ranging survey. It will be of interest
to examine the possibility that decrease or
loss of the catabolic role of the kidneys for
L-chain proteins is a feature of the renal
lesion of SLE.
It appeared likely that the sera of patients with peripheral rheumatoid arthritis
EPSTEIN AND TAN
Fig. 3.-Precipitin band formation in agar using fractions shown in Figure 2 in
concentrations of 1.0 mg./nil. Antiserum same as in Figure 1.
would constitute the most pertinent control
for the present observation of elevated Lchain in the serum of patients with SLE.
Wc applicd criteria which tend to eliminate
those cases in which one cannot easily decidc whether the patient has rheumatoid
arthritis or SLE. The presence of a strongly
positive test for rheumatoid factor and a
negative LE cell preparation were used as
criteria for the diagnosis of rheumatoid
arthritis. It appears that the sera of patients
with peripheral rheumatoid arthritis regularly lack increased concentrations of Lchain proteins. It was of considerable intercst to find increased concentration of Lchain protein in all but one of the synovial
fluid samples of the RA patients (Table 2).
One synovial fluid sample from a patient
diagnosed as having osteoarthritis also
showed detectable L-chain concentration. It
should be noted that no adjustment was
made for differences in the total protein
concentration of the RA synovial fluids
compared to those obtained from patients
with osteoarthritis or traumatic arthritis.
No correlation was found between the
amount of L-chain protein in synovial fluid
and its rheumatoid factor concentration. At
the present time, we have no information
bearing on whether L-chain is synthesized
de nouo in the synovial membrane resulting from an immunologic event within the
joint or is selectively concentrated from
serum into synovial fluid. The difference in
concentration between serum and synovial
fluid indicates the presence of a concentration gradient maintained by the synovial
membrane. The concentration of type A
light chain in the synovial fluid is in most
instances greater than that of type K . This
suggests either preferential involvement of
type A producing cells or preferential
breakdown of type A immunoglobulins.
ACKNOWLEDGMENTS
The authors wish to express their appreciation
to Mrs. Nancy IIelmers for her technical assistance.
INCREASE OF L-CHAIN PROTEINS IN SERA OF PATIENTS WITH SLE
719
SUMMARY
Greater than normal coricentrations of the free L-chains of human immuiioglobuliris
were fcnnd in the sera of 28 out of 30 patients having systemic lupus erythematosus.
No clear correlation between L-chain proteinemia a n d the presence or absence of
azotemin w a s noted. Only t w o of 40 sera of patients with peripheral rheumatoid
arthritis w a s found to have a n increased concentration of L-chain protein; 12 oiit of
1 :3 specinens of synovial fluids from patients with rheumatoid arthritis showed
iricreased concentrations of L-chain proteins in contrast t o one of nine from patients
with osteoarthritis or traumatic arthritis. The increased concentration of L-chain protein in t h e sera of patients with SLE, a n d the synovial fluid of patients with RA consisted of both of t h e two major antigenic types with type X in greater concentration
than type K in some cases.
SUMMARIO
IN INTERLINCUA
Plus-cii"-iiorniaI coriceiitrationes del libere catenas L d e immunoglobulinas human
esseva trovate in l e sero d e 28 de 30 patientes con disseminate lupus erythematose.
Nulle nette correlation esseva notate inter le proteinemia a catena L e le presentia
o absentia de azotemia. Un augmentate concentration de proteina a catena L esseva
constatate in solinente duo de 40 seros ah patientes con peripheric arthritis rheumatoide.
Dece-duo de 13 speciriiens de liquido synovial ab patientes con arthritis rheumatoide
moiistrava augmentate concentrationes de porteinas a catena L, sed iste mesme
constatation esseva facite in solmente u n de novem patientes con osteoarthritis o
arthritis traumatic. Le augmentate concentrationes de proteina a catena L in le sero
d e patientes con d:sseminate Iup~iserythematose e in le liquido synovial de patientes
con arthritis rheumatoide interessava a m b e major typos antigenic, sed in certe cases le
typo L esseva representate in n n plus alte concentration q u e le typo K.
REFERENCES
1. Epstein. W.; Tan, M.,and Gross, D. Blocked
antigenic sites on the L-chain of human
gamnra globillin. Nature 202: 1175, 1964.
9. Tan, M., and Epstein, W.: A direct immunologic assay of human sera for Bence
Jones proteins (L-chains). J. Lab. Clin. Med.
66:344, 1965.
' 3. Solomon, A,, Waldmann, T. A., Fahey, J. L.,
mcl McFarlane, A. S.: Metabolism of Bence
Jones proteins. J. Clin. Invest. 43:103, 1964.
4. Hcrgg'ird, I. On a y-globulin of low moleciilar weight in normal human plasma and
lirinr. Clin. Chim. Acta 6:545, 1962.
Fi. Webb, T.>Rose, B., and Sehon, A. I%.:Biocolloids in normal human urine. 11. Physicocheinical and iimnunochemical characteristics. Canad. J. Biochem. Physiol. 36:1167,
1958.
6. Hcrggard, I., and Edelman, G. M.: Normal
counterparts to Bence-Jones proteins: free L
polypeptide chains of human y-globulin.
Proc. Nat. Acad. Sci. 49:330, 1963.
7. Stevenson, G. T.: Further studies of the
8.
9.
10.
11.
gamma related proteins of normal urine. J.
Clin. Invest. 41:1190, 1962.
Epstein, W., Fong, S., and Tan, M.: Naturally
occurring macroglohulin antibody of fetal
origin in the normal human newborn. Immunology 10259, 1966.
Andersen, S. B., and Jensen, K. B.: Metabolism of yG-globulin in collagen disease. Clin.
Sci. 29 533, 1965.
Vaughan, J. H., ancl Barnett, E. V.: Increased
production of circulating L-chains in acute
serum sickness. Arthritis Rheum. 8:476,
1965. Presented at the Anniial Meeting of the
ARA, June 17-18, 1965, Philaclelphia, Pa.
Fahey, J. L.: Two types of 6.6s gamma
globulin, Beta ,,.,-globulins and 18s gamma,macroglobulins in normal sernm ancl gamma
niic.roglobulins in normal iirine. J . Innniinol.
91:438. 1963.
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