close

Вход

Забыли?

вход по аккаунту

?

The effect of a cobra venom factor on complement and adjuvant-induced disease in rats.

код для вставкиСкачать
The Effect of a Cobra Venom Factor on Complement and
Adjuvant-Induced Disease in Rats
Lygeri Kourounakis, R. A. Nelson, Jr and M. A. Kupusta
The parenteral administration of mycobacterial adjuvant into rats induces
an arthropathy which is generally believed to be due to cellular hypersensitivity. The role of complement in this phenomenon has not been
defined. The levels of individual complement components were measured
in sera of normal and adjuvant-treatedrats. The effect of decomplementation with cobra venom factor (CVF) was studied in adjuvant-induced
disease (AID). It was found that C9 is elevated in AID; and that CVF
temporarily depletes C3 and delays the onset of arthritis. This suggests
that certain complement components may play a role in this form of
hypersensitivity.
The parenteral administration of Freund’s
adjuvant into rats induces an arthropathy with
features which resemble rheumatoid arthritis
and .Reiter’s syndrome in man (1,2). While
there is no definite insight into the mechanisms
involved in adjuvant-induced disease (AID),
From the Lady Davis Institute for Medical Research of
the Jewish General Hospital, the Department of Medicine,
Subdivision of Rheumatology, Jewish General Hospital
and Department of Microbiology and Immunology, University of Montreal, Montreal, Quebec, Canada.
This work was supported by grants from the Canadian
Arthritis and Rheumatism Society and from The John A.
Hartford Foundation, Inc.
L. KOUROUNAKIS, BPH, PHD (Chem Microb): Research
Associate, Rheumatolog Department of the Lady Davis
Institute for Medical Research of the Jewish General Hospital, Montreal, Quebec, Canada. R.A. NELSON, JR. MD: Professor of Microbiology and Immunology, University of
Montreal, Montreal, Quebec, Canada, and Senior Investigator, Lady Davis Institute for Medical Research of the
Jewish General Hospital, Montreal, Quebec, Canada. M.A.
KAPUSTA, MD, CM, FRCP ( c ) , FACP: Lecturer in Medicine,
McGill University, Montreal, Quebec, Canada, and Project
Director, Rheumatology Department a1.d of the Lady Davis
Institute for Medical Research of the Jewish General
Hospital, Montreal, Quebec,Canada.
Reprint requests should be addressed to: Dr. Kapusta,
Project Director, Lady Davis Institute for Medical Research of the Jewish General Hospital, 3755 Cote St,
Catherine Road, Montreal 249, Canada.
Submitted for publication July 11, 1972; accepted Sept
18,1972.
there is evidence that it represents a form of cellular hypersensitivity. This conclusion is based
mainly on the fact that the disease can be transferred between highly inbred rats by means of
intact lymphoid cells (3, 4).
At the present time the role of complement in
cellular hypersensitivity is unclear. One of the
authors (RAN) has suggested that some examples of cellular hypersensitivity may involve the
fixation to antigenic determinants of lymphoid
cell membrane of specific, but cross reacting antibody and certain complement components.
Data were presented which indicated that the
activation of the complement sequence by this
antibody on lymphoid cells and subsequent
cell-cell interactions could produce either
phagocytosis or direct cytotoxic reactions
(5-7). In addition, Perlmann et a1 (8,9)
have demonstrated that complement may facilitate in vitro cytotoxic effects of normal lymphocytes and monocytes on certain target cells. In
contrast, several other authors (10-12) have
suggested that complement may play no role in
cellular hypersensitivity.
A model system whereby the third component of complement (C3) may be depleted in
vitro and in vivo was developed by Nelson and
collaborators (13-1 5). Definite evidence was
Arthritis and Rheumatism, Vol. 16, No. 1(January-February 1973)
71
KOUROUNAKlS El AL
Table 1. Hemolytic Reactivity of Serum Complement Components in Normal and Adjuvant-Treated Rats*
Complement components (approximate CH,
Sera
Normal rat
7 days
postadjuvant
14 days
postadjuvant
titers)
c1
c2
c3
c4
c5
C6
c7
C8
c9
4.000
320
3.000
1.200
800
12.000
2.000
370.000
20.000
4.000
320
3.000
1.200
800
16.000
2,000
250.000
80,000
4,000
320
3,000
1.600
800
12.000
2,000
250,000 80,000
*These are representative results of two to three independent experiments. The variation never
exceeded 1 serial dilution in either direction. In the case of the C9 studies, three independent
experiments gave identical results.
venom factor (CVF) on the development and intensity of arthritis.
obtained that this component of complement
was activated and destroyed by the action of a
purified, nontoxic, 6.6s protein from cobra
venom acting in conjunction with a labile 5s
globulin of normal serum called the cofactor.
The present experiments were designed t'
employ AID in rats as a model of cellular hYpersensitivity and to study the effect of cobra
MATERIALS AND METHODS
Rats
Highly inbred male Fisher rats (Simonsen Lab) weighing between 180 to 220 g were housed in groups of 4 or 5
in laree caees with wire mesh floors and received a diet of
commercial food pellets and water ad libitum.
V
"
ml of CVF
( 0.155 mg protein/ml )
-
c
320
P
0-0
m-¤
.-.
6-A
0-0
9-9
1
2
3
4
5
6
7
8
9
0
0.05
0.12
0.25
0.50
1 .oo
10
-
Days Post CVF Injection
Fig. 1 Time and dose response of CVF in vivo. Each point represents the mean titer of 2 rats.
72
Arthritis and Rheumatism,Vol. 16, No. 1 (January-February 1973)
COBRA VENOM FACTOR
MEAN ARTHRITIC SCORE IN ADJUVANT-TREATED RATS
-
4.4 10.3
1.8 5.2
1.2 3.5
m
0
P
the tail was not scored because it was the site of adjuvant
injection.
control
16.6 0-0
14.3
12.5 m--.
CVF on Day 0
CVF on Day +9
Cobra venom Factor (CVF)
2
Q
This material was purified according to the method of
Nelson (14). In brief, crude venom from Nuju nuju (Miami
Serpentarium) was dissolved in 0.005 M phosphate buffer, p H 7.5 and then applied to DEAE-cellulose, p H 7.5,
0.02 M saline. Two major toxins passed into the effluent.
A gradient of increasing saline concentration to 0.30 M
then was applied. T h e well-known venom phospholipase
eluted first at about 0.12 M. The anticomplementary
factor appeared when the gradient reached an ionic
strength of about 0.16 p . The fractions containing the
anticomplementary factor also contained a small amount
of an antibody-like material which reacted with several
different species of erythrocytes. Further purification was
achieved by chromatography, first on CM-cellulose, at
pH 5.0 as previously described (14), and then by passage
through Sephadex G-200. The protein obtained showed
a single band on disc electrophoresis and a sedimentation
coefficient of 6.6s. The final preparation contained about
155 fig protein/ml determined with the Fohn phenol
reagent (15). Varying amounts of the CVF were diluted
up to 2 ml with saline and injected intraperitoneally into
rats with a 26-gauge needle.
n
m
n
0
3
12
13
14
15
16
17
18
Days Postadjuvant Induction
Fig 2. The effect of single dose of CVF (78 r g
protein) on the onset of arthritis of AID in rats;
8 rats per experimental group.
Adjuvant Injection
Desiccated Myobacterzum butyricum (Difco Lab) was
suspended in light mineral oil (Fisher) to give a suspension
of 6 mg/ml of M butyricum and autoclaved. Normal saline was added in a ratio of 1 :50 v/v and the suspension
was emulsified for 10 minutes in a Sorvall Omni-mixer at
maximum speed. A 21-gauge needle was used to inject 0.1
ml of this adjuvant into the skin of the distal third of the
tail.
Collection of Rat Serum and Measurement of Hemolytic Complement
Quantification of Arthritis
Blood was obtained from the tail vein. The serum was
separated after centrifugation of the clotted blood at 700g
for 30 minutes and stored in ice for testing the same day or
quick-frozen and kept at -75" C.
A single point was assigned for each red and swollen
wrist or ankle area, and an additional point was given for
each similarly involved phalangeal joint. Inflammation of
MEAN ARTHRITIC SCORE IN ADJUVANT-TREATED RATS
-
.--.
9.3 11.2 13.5 12.3 10.6
9.1
3.5
6.6
8.0
7.7
7.0
3.1
3.9
5.5
7.3
8.4
7.8 &-A
14
15
16
17
I8
0
2.2
0
0-0
control
CVF on Dayr-l.+2and+5
CVF on Days-1 and+2
a
n
c
'?
-t
(
I
(L
0
.-"
L
x
Fig 3. The effect of multiple
doses of CVF on the onset of
arthritis of AID in rats; 10 rats
per experimental group. (Each
CVF injection contained 78 pg
protein.)
2
+2
Days
Portadjuvant Induction
Arthritis and Rheumatism, Vol. 16, No. 1(January-February 1973)
73
KOUROUNAKIS ET AL
Complement. The nine components of rat complement were determined in the rat serum by the microtiter
plate method of Nelson et a1 (16) and Vroon et a1 (17).
Briefly, sheep erythrocytes (E) were sensitized with purified
rabbit IgG antibody (A). These cells were mixed with
guinea pig C1 to produce EACl, and then with human or
guinea pig C4 to produce EAC1,4. The complex EACl-7
was generated by mixing EAC1,4 with about 50 units each
of purified human C2, C3, C5, C6 and C7, which were isolated as described by Vroon el a1 (17). Sera from rats were
tested along with normal human and guinea pig as references. In certain experiments, sera from normal or treated
rats were surveyed for component reactivities of C2 through
C9 by testing serial serum dilutions against EAC1.4 complex. Since any decrease of activity due to venom factor was
shown to be due to the destruction of C3 (10, 13, 14), this
was a convenient method to obtain significant results without using large amounts of the costly reagents necessary for
precise titrations of C3.
The destruction of C3 by CVF depends upon
the concentration of the normal serum cofactor.
Sufficient normal rat serum cofactor was
present to cause destruction of C3 even at a dilution of 1:64. This is interpreted to mean that
the cofactor is present at relatively high concentration in normal rat serum.
The Eflect of CVF In Vivo on Serum
Levels of C?. Single doses of CVF were given
intraperitoneally to normal rats in order to find
an effective in vivo dose (Figure 1). Cobra
venom factor, 0.5 ml, containing 78 pg protein
(ie, four times the minimum effective dose) was
used in the following experiments. This dose of
CVF was found to be as effective in depleting
C 3 in adjuvant-treated rats as in normal controls.
RESULTS
The Componentsof Complement in Rats
and the Effect of CVF In Vitro
Pooled normal rat sera were found to contain
all nine components of complement as was the
serum of adjuvant-treated rats taken at various
intervals from 5 to 19 days postadjuvant. The
ninth component of complement of adjuvanttreated rats was found to be four times higher
than the normal (Table 1). A marked elevation
of serum C9 has also been reported (18) in
some rheumatic diseases.
14
.
g
12.
.
10.
::
.2
The Eflect of CVF on AID. Cobra venom
factor was administered to groups of 8 rats as a
single intraperitoneal injection either on the
day of adjuvant administration or on the fifth,
ninth or fourteenth day postadjuvant. The effect
of multiple doses of CVF during the first week
postadjuvant was also studied in groups of 10
rats. The results reported here are representative of three complete experiments done during
the course of 1 year. It may be seen in Figures 2
f
a
8 .
C
a
i
6 .
4.
2.
0 1 1
12
1
1
I
I
I
I
I
I
I
l
l
1
14
16
18
20
22
24
26
28
30
32
34
36
Days Postadiuvant Induction
74
Fig 4. Clinical course of AID
in rats; 8 rats per experimental
group. (control, o-o; rats treated
with CVF on fourteenth day
postadjuvant. 0-0).
A single
dose of CVF (78 pg protein) was
injected in each rat.
Arthritis and Rheumatism, Vol. 16, No. 1(January-February1973)
COBRA VENOM FACTOR
and 3 that single or multiple doses of CVF administered during the first 9 days postadjuvant
delayed the onset of arthritis for approximately
3 days. Figure 4 shows that CVF may temporarily suppress inflammation if administered
on Day 14-ie, just prior to the peak of arthritic inflammation.
DISCUSSION
T h e administration of small doses to rats-eg,
78 pg of highly purified CVF, produced a maximal depression of C3 for 3 days. T h e onset of
arthritis also was delayed for approximately 3
days if CVF was administered within the first 9
days after injection of adjuvant. An antiinflammatory effect of a few days duration was
noted if CVF was given on the fourteenth day
postadjuvant-ie, only 2 to 4 days prior to the
usual peak of maximal arthritis in untreated
controls.
These results would seem to indicate a role
for the third component of complement in adjuvant-induced arthritis.
The antiinflammatory effect of depletion of
total hemolytic complement on various forms of
experimental acute inflammation has been
demonstrated by Willoughby et a1 (19).
The antiinflammatory effect of CVF administered at the onset of clinical arthritis probably
represents a nonspecific antiinflammatory effect
due to the depletion of C3. This would not explain the delay in onset of inflammation due to
CVF administered more than 2 weeks prior to
the onset of arthritis.
Cobra venom factor delayed the onset of
maximal arthritis in rats when it was administered at any time during the first 9 days after
the injection of adjuvant. The first 4 days postadjuvant comprise the stage of sensitization and
the sixth to tenth days represent the phase of
cellular production in AID (20). Since AID can
be transferred between highly inbred rats 8 to
10 days after the administration of adjuvant,
cellular sensitization and proliferation must be
relatively complete at that time. Since CVF is
effective on Day 9, this suggests that certain
specific proteins of the complement system may
be involved in the expression of some forms of
cellular hypersensitivity. This may be due to a
mechanism involving complement fixing antibody-like protein on the surface of the sensitized
cells. Although this hypothesis is under study by
the authors, it is recognized that there may be
other as yet undefined complement dependent
processes in AID.
REFERENCES
1. Pearson CM, Waksman BH, Sharp JT: Studies
of arthritis and other lesions induced in rats by
the injection of mycobacterial adjuvant. V.
Changes affecting the skin and mucous membranes. Comparison of the experimental process
with human disease. J Exp Med 113:485-510,
1961
2. Kourounakis L, Kourounakis P, Kapusta MA:
Experimental arthritis (Greek). Acta Microbiol
Hellenica 16:215-228,1971
3. Waksman BH, Wennersten C: Passive transfer
of adjuvant arthritis in rats with living lymphoid
cells of sensitized donors. Int Arch Allerg Appl
Immunol23:129-139,1963
4. Pearson CM, Wood FD: Passive transfer of adjuvant arthritis by lymph node or spleen cells. J
Exp Med 120:547-560,1964
5. Nelson RA Jr: Immunologic mechanisms for
homograft and heterograft rejections, Second International Symposium on Immunopathology
1961. Basel, Beno Schwabe & Co, Publisher,
1962, pp 223-232
6. Nelson RA Jr: The role of complement in immune phenomena, T h e Inflammatory Process.
Chapter 25. Edited by BW Zweifach, R T
McCluskey, LH Grant. New York, Academic
Press, 1965, pp 819-869
7. Fujii G , Nelson RA Jr: The cross-reactivity and
transfer of antibody in transplantation immunity. J Exp Med 118:1037-1058,1963
8. Perlmann P, Perlmann H, Muller-Eberhard
HJ, et al: Cytotoxic effects of leukocytes triggered by complement bound to target cells. Science 163:937-939,1969
9. Perlmann P, Holm G : Cytotoxic effects of
lymphoid cells in vitro. Adv Immunol 11:117193,1969
10. Cochrane CG, Miiller-Eberhard HJ, Aikin
Arthritis and Rheumatism,Vol. 16, No. 1(January-February1973)
75
KOUROUNAKIS ET AL
BS: Depletion of plasma complement in vivo by
a protein of cobra venom: its effect on various
immunologic reactions. J Immunol 105:55-69,
1970
11. Maillard JL, Zarco RM: Decomplementation
par un facteur extrait du venin de cobra. Effet
sur plusieurs reactions immunes du cobaye et du
rat. Ann Inst Pasteur (Paris) 114:756-774,1968
12. Schwartz HJ, Naff GB: The effect of complement depletion by cobra venom factor on delayed
hypersensitivity reactions. Proc Soc Exp Biol
Med 138:1041-1043,1071
13. Nelson RA Jr: Isolation of complement fractions
presents a new approach to immunosuppression.
JAMA 191:30, 1965
14. Nelson RA Jr: A new concept of immunosuppression in hypersensitivity reactions and in
transplantation immunity. Survey of Ophthalmology 11:498-505, 1966
15. Lowry OH, Rosebrough NT, Farr AL, et al:
Protein measurement with the Folin phenol rea-
76
gent. J Biol Chem 193:265-275,1951
16. Nelson RA Jr, Jensen J, Gigli I, et al: Methods
for the separation, purification and measurement of nine components of hemolytic complement in guinea pig serum. Immunochemistry
3:111-135,1966
17. Vroon D, Schultz D, Zarco RM: The separation
of nine components and two inactivators of components of complement in human serum. Immunochemistry 7:43-61, 1970
18. Ruddy S, Everson LK, Schur PH, et al: Hemolytic assay of the ninth complement component: elevation and depletion in rheumatic diseases. J Exp Med 134:2593-275S, 1971
19. Willoughby DA, Coote E, Turk JL: Complement in acute inflammation. J Pathol 77:295305,1969
20. Walz DT, Dimartino MJ, Misher A: Adjuvant
induced arthritis in rats. 11. Drug effects on
physiologic, biochemical and immunologic parameters. J Pharmacol Exp Ther 178:223-231,
1971
Arthritis and Rheumatism, Vol. 16,No. 1 (January-February1973)
Документ
Категория
Без категории
Просмотров
2
Размер файла
337 Кб
Теги
adjuvant, factors, effect, cobra, complement, induced, disease, rats, venok
1/--страниц
Пожаловаться на содержимое документа