close

Вход

Забыли?

вход по аккаунту

?

Metabolism of the third component of complement C3 in patients with rheumatoid arthritis.

код для вставкиСкачать
Metabolism of the Third Component of Complement (C3) in
Patients with Rheumatoid Arthritis
Arthur Weinstein, Keith Peters, David Brown and Rodney Bluestone
The metabolism of radioiodinated purified C3 was studied in 8 patients with
classical seropositive rheumatoid arthritis, 1 patient with osteoarthritis, and 1
normal subject. An increased fractional catabolic rate and a decreased half-life
was found in 2 subjects. Both these patients showed evidence of rheumatoid
necrotizing vasculitis, and these results suggest an immune-mediated pathogenesis for this lesion. The high serum level of C3 found in some rheumatoid
patients is due to an increased rate of C3 synthesis.
Although the etiology of rheumatoid arthritis (KA) is obscure, there is much evidence implicating immune mechanisms in
the pathogenesis of rheumatoid synovitis
(1). Despite normal or elevated levels of
hemolytic complement in the serum of rheumatoid patients (Z), low levels have been
found consistently in rheumatoid synovial
fluids (3), and more recently, synovial fluid
degradation products of the third component of complement (C3) have been
From the Departments of Medicine and Haematology, Royal Postgraduate Medical School, Hammersmith Hospital, London, W.12.
This work was presented in part at the Fourth
Canadian Conference on Research in the Rheumatic
Diseases, Toronto, October 15, 1970 and at the
Medical Research Society, London, February 12,
1971.
ARTHUR WEINSTEIN,
MD, MRCP:
Department of
Medicine, Toronto General Hospital, Toronto 2.
Fellow of the Canadian Arthritis and Rheumatism
Society. RODNEY BLUEWONE, MB, MRCP: Wadsworth
\'A Hospital, Wilshire and Sawtelle Blvds., Los
Angela, Calif 90073. Recipient of an Arthritis and
Rheumatism Council of Great Britain research
grant. KEITH PETERS, MB, MRCP: Consultant Physician,
Hammersmith Hospital. DAVID BROWN, MB: Lecturer,
Hammersmith Hospital.
Reprint requests should he addressed to Dr.
Weinstein at the Toronto General Hospital.
Submitted for publication May 17, 1971; accepted
August 23, 1971.
demonstrated (4,5).Furthermore, rheumatoid factor, immunoglobulins and C3 have
been found in the rheumatoid synovial
membrane by immunofluorescence (6, 7).
These observations suggest continuing local
intra-articular complement consumption in
patients with RA.
T h e recent isolation and purification of
human complement components have permitted specific complement metabolic
studies in normal and disease states (8-10).
Since C3 has been isolated in a pure and,
more important, functionally active state
( l l ) , it has been the component most
studied. I n fact, hypercatabolism of C3, has
been found in a number of diseases in
which complement fixation and activation
are thought to play a role, such as systemic
lupus erythematosus and autoimmune hemolytic anemia, occasionally in the presence
of normal serum complement and C3 levels
(8,9).I t is therefore possible that increased
complement consumption could be occurring in RA despite the normal serum complement levels in this disease.
We have therefore studied the metabolism of radioiodine labeled C3 in patients
with seropositive rheumatoid arthritis in
order to detect evidence of a continuing
Arthritis and Rheumatism, Vol. 15, No. 1 (JanuayFebruaty 1972)
49
WEINSTEIN El A1
immunological process involving increased
CS consumption.
METHODS
Preparation and labeling of C3. C3 was isolated
from Australia antigen-free serum by a modification
of a previously established technic (11) The
euglobulin fraction of the serum was subjected to
ion exchange chromatography using DEAE cellulose, and pure C3 was obtained by subsequent
separation on a column of hydroxyapatite. T h e
C3-containing fractions were then concentrated by
vacuum dialysis against 0.065M borate buffer, pH
= 8.3, to a final concentration of 5-10 mg/ml i n
preparation for labeling. All procedures were done
at 4OC.
Purity was judged by Ouchterlony analysis and
immunoelectrophoresis against a monospecific rabbit anti-C3 antiserum (12) (Fig 1).
T h e purified protein was labeled with *%I or
'"I by the iodine monochloride method (13) to a
specific activity of 10-20 mCi/mg. T h e mean iodine
substitution ratio was 0.3 atoms of iodine per
molecule of C3.
Integrity of labeling was demonstrated by radioimmunoelectrophoresis and Sephadex G200 chromatography; 95-99y0 of the label could be precipitated with trichloroacetic acid. Before injection the
protein solutions were passed through 0 . 2 2 ~Milliporen filters.
.
Blofoglc acrivlty. A red cell-guinea pig complement intermediate, EA C142, was prepared (14).
EA C142, 1%. was suspended in guinea pig R4
reagent, diluted 1:200 in modified bartitone buffer
at O°C, and 0.1 ml of this suspension was added to
0.2 ml titrations of the test material (both labeled
and unlabeled C3 preparations were tested) . After
incubation at 37°C for 30 minutes, the tubes were
gently centrifuged and read for hemolysis by inspection. T h e titer of hemolytic activity was 1:SOOO and
was unchanged by the labeling procedure.
Each C3 preparation tested in this way was then
aliquoted and stored in liquid nitrogen until used.
Serologic rests. Total hemolytic complement
was measured by a standard assay (15). C3 was
quantitated by radial immunodiffusion (16) . Rheumatoid factor was measured by the sheep cell
agglutination test (17). ANF was done by a standard procedure using rat liver cells as substrate (18).
Parfenrs. Eight patients with
50
active classical
(19) seropositive rheumatoid arthritis, 1 patient
with osteoarthritis and 1 normal volunteer were
studied. T h e patients were selected a t random. T h e
patients and volunteer gave informed consent befoie the radioactive tracer was administered. Each
received sufficient potassium iodide for 3 days prior
to and during the study to block the uptake of free
radioiodine by the thyroid. Approximately 5-15
mCi of activity in 1-2 mg of protein was injected
per person, the exact amount being determined by
weighing the syringe before and after injection. A t
least two rheumatoid patients were studied simultaneously with the same batch preparation so that
abnormalities in metabolism due to denatured
material might be recognized.
Collection and treatment of samples. Blood
was collected in EDTA a t 10-15 minutes after
injection and then 2-4 times a day for 6 days. All
urine was collected throughout the study. T h e
radioactivity of 2.0-ml aliquots of plasma or urine
was counted on a PackardR gamma scintillation
counter. Protein-bound radioactivity for each sample was determined by precipitation with 10%
trichloroacetic acid. Using this method, no proteinbound radioactivity was detected in the urine.
Analysls of data. Plasma radioactivity curves
were analyzed by the Matthews' method to calculate the fractional catabolic rate (FCR), half-life
(Ty2)and extravascular/intravascularratios of the
protein (20).
Another measure of the catabolic rate was made
by analyzing the urinary excretion of free radioactivity (21).
Synthesis rates, assuming steady state conditions,
were calculated from the following formula:
Synthesis rate =
FCR X total intravascular C3
body weight (kg)
Normal ranges were derived from previously
published data (S-10) .
RESULTS
T h e clinical and serologic data of the
subjects are given in Tables 1 and 2. With
the exception of DB, the patients are listed
in the order studied. Most of the rheumatoid patients had evidence of active disease
as judged by symptomatology, clinical synovitis, and elevated sedimentation rate
(ESR) . All were seropositive and four had
Arthritis and Rheumatism, Vol. 15, No. 1 (January-February 1972)
Fig 1. lrnrnunoelectrophoresis demonstrating the
purity of the C3 preparation (above) and the monospecificity of the anti43 antiserum (below).
WEINSTEIN E l A 1
Table 1
Clinical Data
Duration
of
disease
ESR
(yr)
(mm/hr) Nodules
Extra-articular
features
Patient
Sex
Age
(yr)
AW
control
DB OA
WR RA
MG RA
M
26
0
-
0
0
F
M
M
65
47
73
3
4
7
21
39
59
+
0
0
0
0
EN
RA
M
70
20
60
+
FM
RA
M
53
9
65
+
WH RA
MM RA
M
F
57
55
4
5
40
113
+
MH
BH
M
F
53
43
17
13
30
50
Treatment
~~~
RA
OA
RA
RA
0
0
+
0
Episcleritis
Digital vasculitis,
ne uropathy,
pericarditis
0
Digital vasculitis,
Sjogren's
syndrome
Digital vasculitis
0
0
Aspirin
Aspirin
Aspirin
I ndomethacin
Aspirin
lndomethacin
Prednisone
Paracetamol
(acetaminophen)
Aspirin
Indomethacin
Aspirin
lndornethacin
Rheumatoid Arthritis
Osteoarthritis
- _- Not Done
0 = None
= Present
=
=
+
antinuclear antibodies. Assessment by urinalysis and blood urea showed renal function was normal in all patients. All the
subjects had normal or elevated serum hemolytic complement (CH,,) and C3 levels.
T h e metabolic results of the group are
shown in Table 3. T h e fractional catabolic
rate and half-life values were normal in the
control, the patient with osteoarthritis, and
6 of the patients with rheumatoid arthritis
(Fig 2). I n most instances, the metabolic
data obtained from analysis of the urinary
excretion of free radioiodine correlated well
with that obtained from analysis of the
plasma curves. I n some cases, however,
slightly higher FCR values were obtained
from the plasma data. I n one such case, this
52
was caused by incomplete urine collections.
I n the others, the presence of some denatured protein caused an excessive drop in
plasma protein-bound activity in the first
2 4 4 8 hours (reflected also in high urinary
excretion of free radioactivity during that
time) arid thereby falsely elevated the calculated FCR (see WH). I n all these cases after
48 hours, the final linear slope and therefore
TY2 was normal and the catabolic rate from
the urine data settled to a constant normal
level.
However, patients FM and MM had
grossly accelerated FCR and shortened T1/2
of C3 (Fig 3), whereas the same preparations were metabolized normally in other
rheumatoid subjects.
Arthritis and Rheumatism, Vol. 15, No. 1 (January-February 1972)
METABOLISM OF C3
IN RA
DISCUSSION
Our results have shown that 6 of 8 patients with rheumatoid arthritis had normal
catabolism of intravenously administered
Table 2
Serologic Data
Total
haemolytic
RheumaAnticomple- C3 (mg%)
toid
nuclear
ment
(N =
Patient
factor
factor
(CHa)
120-200)
AW
DB
WR
MG
EN
FM
WH
MM
MH
BH
-
-
0
1:512
1:32
1:32
1: 128
1:16
1:64
1:256
1:32
0
0
0
+
+
0
+
+
0
160
150
150
150
180
240
300
400
300
2 70
N
N
N
N
N
I
I
I
I
I
-- Not done
0 = None
+
= Present
N = Normal
I = Increased
Table 3
C3. These patients were seropositive, had
moderately severe disease and some had
extra-articular lesions such as nodules and
episcleritis. One patient (MH) with digital
lesions suggestive of vasculitis had minimal
synovitis, and he too showed a normal C3
turnover. T w o patients had clinical features
of necrotizing vasculitis (22) and severe
rheumatoid disease (FM and MM) and
showed accelerated C3 catabolism in the
face of high serum levels.
Unfortunately, the sensitivity of this type
of study is somewhat limited because of the
difficulty in preparing C3 completely free of
denatured material. The presence of this
denatured material may give falsely wide
ranges for the normal C3 catabolic rate and
thus slight deviations from normal could be
missed. However, the greatly accelerated C3
catabolism in both patients with necrotizing
vasculitis is striking, and this adds support
to the hypothesis of an immunologic pathogenesis of rheumatoid vasculitis.
It seems unlikely that rheumatoid vasculitis per se has a pathogenic basis distinct
from rheumatoid synovitis. Conceivably,
Metabolic Data
Patient
Fractional
catabolic
rate (FCR)
(%/ hr)
N=l-3%/hr*+
Half life
UM (hr)
N=40-70 hr*
C3 (mg%)
N=120-200 mg%
Extravascular/
intravascular ratio
N=0.3-1.5*
AW
DB
WR
MG
EN
FM
WH
MM
MH
BH
2.7
2.7
2.5
2.9
2.6
7.8
3.3
3.7
2.3
2.5
48
45
53
43
54
28
54
34
72
59
160
150
150
150
180
240
300
400
300
270
0.604
0.625
0.725
0.493
0.740
1.630
1.120
0.686
1.090
1.020
Synthesis rate
(mg/kg/ h r)
N=0.!3-2.0
mg/ kg/ hr*
1.43
1.95
1.30
1.62
1.79
11.40
7.30
7.00
2.80
3.20
* Normal range taken from other studies (8-10)
+
Derived from plasma curve analysis
Arthritis and Rheumatism, Vol. 15, No. 1 (January-February 1972)
53
WEINSTEIN E l A 1
FRACTION
0.1
OF
PROTEIN
BOUND
ACT1 VlTY
10
-
METABOLIC RATE
%/HOUR
(ulp)
5
0
7
-
I
"
"
I
'
I
I
.
1
1
1
'
Fig 2. C3 turnover in a patient with R/A. Upper pa& fraction of the protein-bound radioactivity remaining in the plasma with time (semilog). From this curve the FCR and T1/2 are calculated. lower part: fraction
of free radioactivity excreted in the urine over a period of time (u) divided by the mean plasma proteinbound activity over that time (p). This ratio expressed as a percentage is another measure of the FCR.
1.0
0.5
FRACTION
0.2
OF
PROTEIN 0.1
BOUND
ACTIVITY 0.05
0.02
0.01
FM
I
L
10
'20
30
40
50
60
70
80
TIME AFTER INJECTION
90
100
110
120
130
140
(Hours)
Fig 3. C3 turnovers in normal subject (AW), a patient with R/A (M6),and 2 patients with rheumatoid
vasculitis (MM, FM), showing hypercatabolism of C3 in the latter two.
54
Arthritis and Rheumatism, Vol. 15, No. 1 (January-February 1972)
METABOLISM OF C3
IN R4
turnover studies may be too insensitive to
detect intra-articular complement consumption. However, if immune-mediated tissue
damage becomes widespread, as in arteritis,
accelerated C3 consumption could then be
easily demonstrated.
Vaughn and his associates found that
when their rheumatoid patients were
grouped as seronegative, seropositive and
seropositive with vasculitis, a successive significant lowering of the mean serum complement levels could be seen. I n contrast,
our 2 patients with vasculitis had increased
C3 levels, presumably due to increased C3
synthesis. T h e reason for this difference is
unclear, but it is of interest that diminished
C3 synthesis has been reported in some
patients with hypocomplementemic progressive glomerulonephritis (8) and hypocomplementemic lupus nephritis (24).
N o direct measure of C3 synthesis was
made in our patients, but increased C3
synthesis is inferred in those patients with
elevated C3 levels and normal or increased
C3 catabolism (see formula in Analysis of
Data and Table 3). Thus, normal C3 levels
may offer little guide to the presence of
accelerated C3 turnover since the serum
levels of complement components depend
on synthesis rates as well as catabolic rates.
Finally, while the role of vasculitis in the
pathogenesis of rheumatoid arthritis is still
uncertain, our findings lend weight to the
concept of complement-mediated vascular
injury in rheumatoid vasculitis.
ACKNOWLEDGMENTS
We gratefully acknowledge the technical assistance of Miss Brenda Atkins and Mr. Nicholas Amos.
REFERENCES
1. ZvaiHer NJ: A speculation on tlie pathogenesis of joint inflammation in rheumatoid
arthritis.
Arthritis
Rheum
8~289-293, 1965
2. Schubart AF, Ewald RW, Scliroeder WC, et
al: Serum complement levels in rheumatoid arthritis. Ann Rheum Dis 24:439-450,
1965
3. Pekin TJ, Zvaifler NJ: Measurement of
liemolytic complement in synovial fluids.
Arthritis Kheum 5:314, 1%2
4. Zvaifler NJ: Breakdown products of C’3 in
human synovial fluids. J Clin Invest
48: 1532-1542, 1969
5. Hedberg H, Lundh B, Laurel1 A-W: Studies
of the third component of complement in
synovial Huid from arthritic patients. Clin
Exp Immunol 6:707-712, 1970
6. Rodman WS, Williams RC Jr, Bilka PJ, et
al: Immunofluorescent localization of the
third and fourth component of complement in synovial tissue from patients with
rheumatoid arthritis. J Lab Clin bled
69:141-150, 1967
7. Kinsella TD, Baum J, Ziff R I : Immunofluorescent demonstration of an IgC-p,,
complex in synovial lining cells of rheumatoid synovial membrane. Clin Exp Immuno1 4:265-271, 1969
8. Alper CA, Rosen FS: Studies of the in vivo
beliaviour of human C’3 in normal subjects and patients.
J
Clin Invest
46:2021-2034, 1967
9. Petz LD, Fink DJ. Letsky EA, et al: I n vivo
metabolism of complement. I. Metabolism
of tlie third component (C’3) in acquired
J
Clin
Invest
liemolytic
anemia.
47:2469-2484, 1968
10. Carpenter CB, Ruddy S, Shehadeli IH, et
al: Complement metabolism in man: hypercatabolism of the fouyth (C4) and third
(C3) components in patients with renal
allograft rejection and hereditary angioedema ( H A E ) . J Clin Invest 48:1495-1505,
I969
1 I . Nilsson UR, Muller-Eberhard HJ: Isolation of p,F-globulin from human serum
and its characterization as the fifth component of complement. J Exp bled
122:277-298, 1965
12. Mardiney MR Jr, Muller-Eberhard H J:
Mouse p,(,-globulin: production of an-
Arthritis and Rheumatism, Vol. 15, No. 1 (January-February 1972)
55
WEINSTEIN ET A 1
13.
14.
15.
16.
17.
18.
56
tiserum and characterization in the complement reaction. J Immunol 94:877-882,
1965
McFarlane AS: Efficient trace-labelling of
proteins with iodine. Nature (Lond)
182:53, 1958
Lachmann PJ, Liske K: T h e preparation
and properties of alexinated intermediates
that react with conglutinin. I. Guinea pig
complement.
Immunology
11:243-254,
1966
Kabat EA, Mayer MM: Experimental Immunochemistry.
Second
edition.
Springfield, Charles C Thomas, 1964, pp
149-153
Mancini G, Carbonara AO, Heremans JF:
Immunochemical quantitation of antigens
by single radial immunodiffusion. Int J
Immunochem 2:235-254, 1965
Bywaters EGL, Scott FET: Rheumatism
and connective tissue diseases, Recent Advances in Clinical Pathology (Series 111).
Edited by SC Dyke. London, Churchill,
1960, pp 278-302
Holborow EJ, Johnson GD: T h e immu-
19.
20.
21.
22.
23.
24.
nofluorescent test for serum antinuclear
factor. Association of Clinical PathologistsBroadsheet 65:l-5, 1969
Ropes MW, Bennett GA, Cobb S , et al:
1958 revision of diagnostic criteria for rheumatoid arthritis. Arthritis Rheum 2:16-20,
1959
Matthews CME: T h e theory of tracer experiments with 1~1I-labelledplasma proteins. Phys Med Biol 236-53, 1957
Campbell RM, Cuthbertson DP, Matthews
CM, et al: Behaviour of 14C- and 1311label!ed plasma proteins in the rat. Int J
Appl Radiat Isot 1:66-84, 1956
Schmid FR, Cooper NS, Ziff M, et al:
Arteritis in rheumatoid arthritis. Am J
Med 30:5683, 1961
Vaughan JH, Morgan ES, Jacox RF: Role
of gammaglobulin complexes in rheumatoid arthritis. Trans Assoc Am Physicians
81:231-239, 1968
Sliwinski AJ, Zvaifler NJ: Decreased synthesis of the third component of complement in hypocomplementemic systemic lupus. Arthritis Rheum 13:349, 1970
Arthritis and Rheumatism, Vol. 15, No. 1 (Januav-February 1972)
Документ
Категория
Без категории
Просмотров
2
Размер файла
394 Кб
Теги
complement, patients, metabolico, components, arthritis, third, rheumatoid
1/--страниц
Пожаловаться на содержимое документа