Naunyn-Schmiedeberg's
Naunyn-Schmiedeberg's Arch. Pharmacol. 295, 237-241 (1976)
Archivesof Pharmacology 9 by Springer-Verlag1976
Effect of Hog Anaphylatoxin (C 5 a) on Vascular Permeability and Leukocyte Emigration in vivo B. D A M E R A U and W. V O G T Max-Planck-Institut fiir Experimentelle Medizin, Abteilung Biochemische Pharmakologie, Hermann-Rein-Strasse 3, D-3400 G6ttingen, Federal Republic of Germany
Summary. The effects of highly purified hog anaphylatoxin (C 5 a) on leukocyte emigration were investigated in guinea pigs in vivo using two experimental models: 1. Subcutaneous infusions. Sterile solutions of C 5 a in saline infused at a rate of 1.8 gg C 5 a / h (0.2 ml/h) for 10 h induced a dense accumulation of leukocytes, mainly neutrophils but also some eosinophils at the site of application. In control infusions with saline alone comparatively few leukocytes were found. 2. Single injections into the pleural cavity. 10 or 20 i.tg C 5 a (dissolved in 2 ml saline) caused a dosedependent increase in leukocyte number and volume of pleural exudate. Bradykinin in a dose of 18 gg produced similar fluid exudation as 20 lag C5 a but had no significant effect on leukocyte accumulation. Intrapleural injections of C 5 a further caused the appearance of i.v. injected Evans blue in the pleural cavity. This effect, indicating an increase in vascular permeability lasted for about 3 h. Since at least one of the two models used - subcutaneous i n f u s i o n - s i m u l a t e s natural c o n d i t i o n s continuous local generation of C 5 a in small amounts at the site of an inflammatory l e s i o n - t h e results indicate that C 5 a once formed by complement activation in natural defense reactions m a y contribute to local increase in vascular permeability and leukocyte infiltration. Key words: Anaphylatoxin (C 5 a) - Chemotaxis Leukocytes - Vascular permeability - Evans blue.
INTRODUCTION In vitro, in the,Boyden chamber anaphylatoxin (C 5 a) causes positive chemotaxis of leukocytes from various
Send offprint requests to: B. Damerau at the above address
species, and this effect has been well analyzed by various authors (Shin et al., 1968; Jensen et al., 1969; W a r d and Newman, 1969). In vivo a chemotactic effect of partially purified anaphylatoxin has been observed after intradermal injections of single doses in rabbits (Jensen et al., 1969). It can be expected that under natural conditions complement-derived products such as C 5 a would be continuously released in small amounts at the site of an immunological reaction where complement is activated, and they would accumulate in body cavities when an inflammation is going on there (e.g. in a joint cavity). In order to obtain information whether under such conditions C 5 a would also cause inflammatory reactions including leukocyte infiltration we studied its effects in two models. 1. C 5 a was infused at a slow rate and low concentration subcutaneously, and 2. it was injected into the pleural cavity of guinea pigs.
MATERIALS AND METHODS a) Purification of Anaphylatoxin ( C S a ). Anaphylatoxin was generated by activation of hog serum and with yeast; it was purified as
described earlier (Lieflfinder et al., 1972). The product appeared pure in acid disc electrophoresis and developed only a single band in immunoelectrophoresis, against anti-serum raised in rabbits with impure anaphylatoxin and containing antibodies against impurities.
b) In vivo-Infusions of Anaphylatoxin. Guinea pigs weighing about 300 g were anaesthetized with hexobarbital (40 mg/kg) and pretreated with 150000 IU penicillin G (i.p,) per kg body weight to prevent bacterial infections. After shaving and desinfection of the skin with iodine solution one steel cannula each (outside diameter 0.9 mm) was inserted s.c. in both sides of the back, fastened with plaster and connected with an infusion pump by thin polyethylene tubes. The whole infusion system had been sterilized before by autoclaving, the infusion fluids by membrane filtration. When the anesthesia had ceased both sides were infused for 10 h at a rate of 0.2 ml/h. One side received 0,9~ NaC1 solution as control, the other side anaphylatoxin dissolved in saline. After infusion the animals were killed by a blow on the neck and exsanguinated. The infused skin area was cut out and prepared for histological inspection.
238
c) Histological Preparation of Skin Samples. Skin samples were fixed in 3.6~ formaldehyde for 48 h and dehydrated in 80, 90, 95 ~, and absolute ethanol for 4 - 6 h each. After 2 x 1 h in toluene the samples were embedded in paraffin. Starting from the center of the infused area serial sections of 15 gm were cut and stained with hematoxylin and eosin. For the number of sections evaluated in each experiment see legend of Table 1. The extent of leukocyte emigration was estimated semiquantitatively: An even infiltration of the infused area (about 2 0 - 30 of the area consisting of leukocytes) was scored one point, denser infiltrations (about 5 0 - 70 ~ of the area) and zones in which the leukocytes covered the whole area were scored two and three points, respectively. If there were only few leukocytes detectable the score was 1/4 to 1/2 point. Examples of the evaluation are given in Figure 1.
d) IntrapleuralInjections. Volumes of 2 ml sterile isotonic NaC1 as control or C 5 a dissolved in saline in different concentrations were injected into the pleural cavity of guinea pigs weighing about 300 g. After 220 rain the animals were killed, by a blow on the neck and exsanguinated. The chest was opened and the fluid volume was measured and the number of leukocytes therein was counted in phase-contrast microscopy. Sanguineous exudates were not used.
Naunyn-Schmiedeberg's Arch. Pharmacol. 295 (1976) Table 1. Effect of anaphylatoxin (18 gg infused s.c. in the course of 10 h) and of control infusions with saline only, on local leukocyte accumulation in the guinea-pig skin. Control and anaphylatoxin infusions were given simultaneously into one side each of the back. Each value is the average score ofalI evaluated sections (4- 5 sections per slide, number of slides in brackets) of one individual animal Animal
Score of leukocyte accumulation Controls
Anaphylatoxin infusions
1. 2. 3. 4. 5. 6. 7. 8.
0.02 0.18 0.50 0.33 0.50 0.95 " "
0.60 1.88 1.31 1.55 2.26 1.95 1.45 3.00
Average • S.D.
0.4 • 0.3
(15) (16) (15) (13) (13) (14)
(20) (25) (18) (12) (17) (10) (10) (11)
1.8 • 0.7
e) Injections with Evans Blue. For estimating changes in vascular permeability Evans blue (1 ml of a 5 ~ solution per kg body weight) was injected into the V. saphena 20 min after intrapleural injections of C 5 a or control fluid. After 220 min the animals were killed, the chest was opened and the pleural fluid was removed and measured. After centrifugation the concentration of Evans blue in the supernatant was estimated photometrically at 623 nm. In kinetic experiments Evans blue was injected 30, 120, 180 or 240 rain after intrapleural injection of C5a or saline control. These animals were killed 60 rain after the injection of Evans blue.
RESULTS
a) Subcutaneous Infusions of Anaphylatoxin. T a b l e 1 a n d F i g u r e I s h o w the l e u k o c y t e a c c u m u l a t i o n in skin areas infused with 18 lag C 5 a in 2 ml saline, a n d in c o n t r o l a r e a s infused with saline only. I n a r e a s o f c o n t r o l infusion o n l y a few l e u k o c y t e s were d e t e c t e d except in a n i m a l 6) in w h i c h a l e u k o c y t e infiltration c o r r e s p o n d i n g to score 0.95 o c c u r r e d . A f t e r infusions o f a n a p h y l a t o x i n dense a c c u m u l a t i o n s o f l e u k o c y t e s were visible with their m a x i m u m n e a r the tip o f the c a n n u l a . I n the center o f the inflamed a r e a s n e u t r o p h i l l e u k o c y t e s p r e d o m i n a t e d b u t quite a n u m b e r o f e o s i n o phils were also f o u n d . I n the m a r g i n a l p a r t s the p r o p o r tion of monocytes increased gradually. The blood vessels o f the i n f l a m e d a r e a s were d i l a t e d a n d densely p a c k e d with red a n d white b l o o d cells.
b) Intrapleural Injections of Anaphylatoxin. T e n o r 20 lag a n a p h y l a t o x i n in 2 ml saline, o r saline a l o n e were injected into the p l e u r a l c a v i t y o f g u i n e a pigs. A f t e r 240 m i n the r e m a i n i n g fluid a n d the n u m b e r o f l e u k o c y t e s c o n t a i n e d in it were e s t i m a t e d . In c o n t r o l a n i m a l s a n a v e r a g e o f o n l y 0.47 m l fluid were r e c o v e r e d (Fig. 2). A f t e r injection o f a n a p h y l a t o x i n the r e m a i n i n g v o l u m e was c o n s i d e r a b l y higher, 1.2 m l after injection
In these experiments the cannula on the control side slepped out during the infusion
o f 10 gg a n d 1.4 ml after 20 gg. A n a p h y l a t o x i n thus led to a n increase in e x u d a t e v o l u m e , relative to the controls. 18 lag b r a d y k i n i n led to the s a m e relative increase in v o l u m e as 20 lag a n a p h y l a t o x i n . T h e t o t a l n u m b e r o f l e u k o c y t e s in c o n t r o l exudates was 1.5 x 106 cells ( 8 0 - 9 0 ~ m o n o n u c l e a r cells). A n a p h y l a t o x i n c a u s e d a m u c h h i g h e r a c c u m u l a t i o n o f leukocytes in the p l e u r a l fluid, 9.5 a n d 1 9 x 106 cells p e r t o t a l e x u d a t e after injection o f 10 a n d 20 lag a n a p h y l a t o x i n respectively (Fig. 2). G r a n u l o c y t e s p r e d o m i n a t e d (between 6 0 - 8 0 ~ o ) . I n c o n t r a s t , b r a d y k i n i n t h o u g h h a v i n g the s a m e effect o n e x u d a t e v o l u m e d i d only slightly increase the e m i g r a t i o n o f l e u k o c y t e s into the p l e u r a l c a v i t y (2.3 x 10 6 cells p e r exudate) (Fig. 2).
c) Accumulation of Evans Blue in the Pleural Cavity after Injections of Anaphylatoxin. A f t e r i n t r a p l e u r a l c o n t r o l injections o f saline 2.8 x 10 -5 g E v a n s blue were f o u n d in the p l e u r a l c a v i t y p e r ml fluid. A n a p h y l a t o x i n in doses o f 10 a n d 20 pg (dissolved in 2 ml) led to the p e r m e a t i o n o f 5.2 a n d 8.4 x 10 -5 g E v a n s b l u e / m l e x u d a t e (Fig. 3). Since a n a p h y l a t o x i n increased the e x u d a t e v o l u m e the t o t a l a m o u n t o f E v a n s blue w h i c h h a d diffused into the p l e u r a l c a v i t y was m u c h higher t h a n in the c o n t r o l s : 4 times as m u c h after 10 lag a n d 7.5 times after 20 gg a n a p h y l a t o x i n .
d) Time-Course of Evans Blue Accumulation in the Pleural Cavity. I n o r d e r to e s t i m a t e the d u r a t i o n o f the p e r m e a b i l i t y - i n c r e a s i n g effect caused b y i n t r a p l e u r a l injections o f a n a p h y l a t o x i n E v a n s blue was given i.v.
B. Damerau and W, Vogt: Effects of CSa on Permeability andCbemotaxis
239
Fig. l A and B. Histological appearance (magnification about 25 x) of cutaneous and subcutaneous tissue from guinea pigs after s.c. infusions of anaphylatoxin (18 gg in 10 h) ( = B) and saline as control ( = A). (A) Scattered leukocytes (score: approx. 0.5); (B) dense accumulation of leukocytes at the site of infusion (score: approx. 2)
at different times after the intrapleural application of 10 gg anaphylatoxin. The animals were killed 60 min after injection of the dye and the total amount of Evans blue in the pleural cavity was determined, Control animals received saline instead of anaphylatoxin. In controls almost identical amounts, 0.5-0.75 x 10 .5 g Evans blue, on an average, were detected in the pleural exudate regardless of the time intervals between intrapleural application of saline and intravenous injection of the dye (Fig. 4). Evans blue given 30 min after injection of anaphylatoxin accumulated in the cavity to an amount about 6 times higher (3.0 x 10 -~ g Evans blue) than in controls. Almost the same amount of dye, 2.7 x 10 .5 g, was found when the interval between injection of anaphylatoxin and Evans blue was 120 rain. At intervals of 180 and 240 min there were no longer significant differences between Evans blue accumulation in anaphylatoxin- and saline-treated animals (0.7 and 0.76x 10-Sg Evans blue). It is concluded from these results that the permeability-increasing effect of 10 gg anaphylatoxin injected into the pleural cavity lasted for about 3 h. In agreement with this anaphylatoxin activity (measured by
volume (ml ) FI.5 wbc 20-
I
JI I
-LO
10-0.5
C
I O P 9 A T 20,ugAT I8,ugBra~'
Fig.2. Number of leukocytes (wbc x 106, scale on left ordinate) and fluid volume (ml, scale on right ordinate) in the pleural cavity of guinea pigs taken 240 min after intrapleural injection (2 ml) of 10 and 20 pg anaphylatoxin (AT), 18 pg bradykinin (Brad) or saline as control (C). Hatched columns = number of leukocytes per exudate; open columns = exudate volume; vertical bars = standard deviation, Number of experiments = 3 - 5
240
Naunyn-Schmiedeberg's Arch. Pharmacol. 295 (1976)
ornount / m l
total
amount
E. bl. E. bL
15-
3-
i I
I0-
5-
~
1
I
1
.
I f f f
I
C
ii1
tO/Jg 20,ug AT
f f f f f
C
lOJJg 20JJ9 AT
30
120
180
240
rnio
Fig. 3. Accumulation of i.v. injected Evans blue (E.bl., ordinate) in the pleural cavity of guinea pigs after intrapleural injections of 10 or 20 lag anaphylatoxin (AT), or saline as control (C). Column 1 - 3: concentration of Evans blue (x 10 -5 g/ml exudate), column 4 - 6 : total amount of Evans blue in exudate (x i0 -s g). Number of experiments: 6 - 8
Fig. 4. Total content of Evans blue ( x 10-5 g, ordinate) recovered from the pleural cavity of guinea pigs 60 rain after i.v. injection. The injection was made at different times (indic~ited on the abscissa) after intrapleural application of 10 lag anaphylatoxin (open columns) or saline as control (hatched columns). Vertical bars: Standard deviation. Number of experiments : 3 - 5
bioassay on the isolated guinea-pig ileum) was regularly detected in pleural fluid taken 2 h after injection (6 experiments), but only in one out of 6 experiments 4 h after injection.
In both guinea-pig in vivo models used anaphylatoxin caused leukocyte accumulation. Subcutaneous infusion of very low amounts (1.8 gg/h), intended to mimick a natural continuous local release, led to dense accumulation of neutrophils, but also quite a number of eosinophils, at the site of application. Single intrapleural injections of anaphylatoxin were made with the intention to mimick an inflammatory complement-involving lesion in a body cavity. These injections not only caused massive leukocyte accumulation in the pleural fluid but in addition an increase in permeability of the adjacent vessels (evident from extravasation of Evans blue) and increase in exudate volume. Although the fluid volume recovered from the pleural cavity of anaphylatoxin-treated animals was never absolutely larger than the one injected, its larger size as compared to the control animals is very likely due to increased exudation, not decreased absorption. This is suggested by the concomitant extravasation of i.v. applied Evans blue, and the well-known general consequence of increased vascular permeability, oedema formation. The leukocyte accumulation in the pleural cavity is not simply the result of permeability increase; rather
DISCUSSION The preparation used in this study is classical anaphylatoxin, i.e. the principle which is generated in whole serum by complement activation and can be recovered from such serum without specific precautions. It should be noted that this peptide is already a secondary cleavage product. Its precursor obtained primarily from C5 by hydrolytic activation of the latter is a highly spasmogenic but very labile peptide, C5a. In serum, it is rapidly attacked by carboxypeptidase N losing its carboxy-terminal amino acid and thereby more or less of its spasmogenic activity, depending on the species of the serum (Vallota and Mtiller-Eberhard, 1973). Because of its stability the secondary peptide is of particular interest, biologically, as far as it has preserved (or even acquired?) pharmacological activities.
B. Damerau and W. Vogt: Effects of C 5 a on Permeability and Chemotaxis it is the c o n s e q u e n c e o f a specific c h e m o t a c t i c effect o f a n a p h y l a t o x i n . B r a d y k i n i n , in doses l e a d i n g to the s a m e increase in v a s c u l a r p e r m e a b i l i t y , p r o v e d to be ineffective as a l e u k o c y t e a t t r a c t a n t . T h e s e results f u r t h e r s t r e n g t h e n the role o f a n a p h y l a t o x i n as one i m p o r t a n t m e d i a t o r o f certain i n f l a m m a t o r y reactions. A s has been s h o w n earlier c h e m o t a c t i c f r a g m e n t s o f C 5 d o o c c u r at sites o f i n f l a m m a t i o n , e.g. in s y n o v i a l fluids f r o m p a t i e n t s w i t h r h e u m a t o i d a r t h r i t i s ( W a r d a n d Zvaifler, 1971) a n d in tissue s a m p l e s f r o m a n i m a l s w i t h reversed passive A r t h u s r e a c t i o n s ( W a r d a n d Hill, 1972).
Acknowledgements. The skillful technical assistance of Mrs. G. Werle is gratefully acknowledged.
REFERENCES Jensen, J. A., Snyderman, R., Mergenhagen, S. E.: Chemotactic activity, a property of guinea pig C'5-anaphylatoxin. In: Cellular and humoral mechanisms in anaphylaxis and allergy (H. Z. Movat, ed.), pp. 265-278. Basel: S. Karger 1969
241
Liefl/inder, M., Dielenberg, D., Schmidt, G., Vogt, W. : Structural elements of anaphylatoxin obtained by contact activation of hog serum. Hoppe-Seyler's Z. physiol. Chem. 353, 385-392 (1972) Shin, H. S., Snyderman, R., Friedman, E., Mellors, A., Mayer, M. M. : Chemotactic and anaphylatoxic fragment cleaved from the fifth component of guinea pig complement. Science 162, 361 - 363 (1968) Vallota, E. H., Mfiller-Eberhard, H.J.: Formation of C3a and C5a anaphylatoxins in whole human serum after inhibition of the anaphylatoxin inactivator. J. exp. Med. 13"7, 1109-1123 (1973) Ward, P. A., Hill, J. H. : Biological role of complement products. Complement-derived leukotactic activity extractable from lesions of immunologic vasculitis. J. Immunol. 108, 1137--1145 (1972) Ward, P. A., Newman, L. J. : A neutrophil chemotactic factor from human C5. J. Immunol. 102, 93-99 (1969) Ward, P. A., Zvaifler, N. J. : Complement-derived leukotactic factors in inflammatory synovial fluids of humans. J. clin. Invest. 50, 606- 616 (1971)
Received July 22/Accepted September 13, 1976
Naunyn-Schmiedeberg's Arch. Pharmacol. 295, 237-241 (1976)
Archivesof Pharmacology 9 by Springer-Verlag1976
Effect of Hog Anaphylatoxin (C 5 a) on Vascular Permeability and Leukocyte Emigration in vivo B. D A M E R A U and W. V O G T Max-Planck-Institut fiir Experimentelle Medizin, Abteilung Biochemische Pharmakologie, Hermann-Rein-Strasse 3, D-3400 G6ttingen, Federal Republic of Germany
Summary. The effects of highly purified hog anaphylatoxin (C 5 a) on leukocyte emigration were investigated in guinea pigs in vivo using two experimental models: 1. Subcutaneous infusions. Sterile solutions of C 5 a in saline infused at a rate of 1.8 gg C 5 a / h (0.2 ml/h) for 10 h induced a dense accumulation of leukocytes, mainly neutrophils but also some eosinophils at the site of application. In control infusions with saline alone comparatively few leukocytes were found. 2. Single injections into the pleural cavity. 10 or 20 i.tg C 5 a (dissolved in 2 ml saline) caused a dosedependent increase in leukocyte number and volume of pleural exudate. Bradykinin in a dose of 18 gg produced similar fluid exudation as 20 lag C5 a but had no significant effect on leukocyte accumulation. Intrapleural injections of C 5 a further caused the appearance of i.v. injected Evans blue in the pleural cavity. This effect, indicating an increase in vascular permeability lasted for about 3 h. Since at least one of the two models used - subcutaneous i n f u s i o n - s i m u l a t e s natural c o n d i t i o n s continuous local generation of C 5 a in small amounts at the site of an inflammatory l e s i o n - t h e results indicate that C 5 a once formed by complement activation in natural defense reactions m a y contribute to local increase in vascular permeability and leukocyte infiltration. Key words: Anaphylatoxin (C 5 a) - Chemotaxis Leukocytes - Vascular permeability - Evans blue.
INTRODUCTION In vitro, in the,Boyden chamber anaphylatoxin (C 5 a) causes positive chemotaxis of leukocytes from various
Send offprint requests to: B. Damerau at the above address
species, and this effect has been well analyzed by various authors (Shin et al., 1968; Jensen et al., 1969; W a r d and Newman, 1969). In vivo a chemotactic effect of partially purified anaphylatoxin has been observed after intradermal injections of single doses in rabbits (Jensen et al., 1969). It can be expected that under natural conditions complement-derived products such as C 5 a would be continuously released in small amounts at the site of an immunological reaction where complement is activated, and they would accumulate in body cavities when an inflammation is going on there (e.g. in a joint cavity). In order to obtain information whether under such conditions C 5 a would also cause inflammatory reactions including leukocyte infiltration we studied its effects in two models. 1. C 5 a was infused at a slow rate and low concentration subcutaneously, and 2. it was injected into the pleural cavity of guinea pigs.
MATERIALS AND METHODS a) Purification of Anaphylatoxin ( C S a ). Anaphylatoxin was generated by activation of hog serum and with yeast; it was purified as
described earlier (Lieflfinder et al., 1972). The product appeared pure in acid disc electrophoresis and developed only a single band in immunoelectrophoresis, against anti-serum raised in rabbits with impure anaphylatoxin and containing antibodies against impurities.
b) In vivo-Infusions of Anaphylatoxin. Guinea pigs weighing about 300 g were anaesthetized with hexobarbital (40 mg/kg) and pretreated with 150000 IU penicillin G (i.p,) per kg body weight to prevent bacterial infections. After shaving and desinfection of the skin with iodine solution one steel cannula each (outside diameter 0.9 mm) was inserted s.c. in both sides of the back, fastened with plaster and connected with an infusion pump by thin polyethylene tubes. The whole infusion system had been sterilized before by autoclaving, the infusion fluids by membrane filtration. When the anesthesia had ceased both sides were infused for 10 h at a rate of 0.2 ml/h. One side received 0,9~ NaC1 solution as control, the other side anaphylatoxin dissolved in saline. After infusion the animals were killed by a blow on the neck and exsanguinated. The infused skin area was cut out and prepared for histological inspection.
238
c) Histological Preparation of Skin Samples. Skin samples were fixed in 3.6~ formaldehyde for 48 h and dehydrated in 80, 90, 95 ~, and absolute ethanol for 4 - 6 h each. After 2 x 1 h in toluene the samples were embedded in paraffin. Starting from the center of the infused area serial sections of 15 gm were cut and stained with hematoxylin and eosin. For the number of sections evaluated in each experiment see legend of Table 1. The extent of leukocyte emigration was estimated semiquantitatively: An even infiltration of the infused area (about 2 0 - 30 of the area consisting of leukocytes) was scored one point, denser infiltrations (about 5 0 - 70 ~ of the area) and zones in which the leukocytes covered the whole area were scored two and three points, respectively. If there were only few leukocytes detectable the score was 1/4 to 1/2 point. Examples of the evaluation are given in Figure 1.
d) IntrapleuralInjections. Volumes of 2 ml sterile isotonic NaC1 as control or C 5 a dissolved in saline in different concentrations were injected into the pleural cavity of guinea pigs weighing about 300 g. After 220 rain the animals were killed, by a blow on the neck and exsanguinated. The chest was opened and the fluid volume was measured and the number of leukocytes therein was counted in phase-contrast microscopy. Sanguineous exudates were not used.
Naunyn-Schmiedeberg's Arch. Pharmacol. 295 (1976) Table 1. Effect of anaphylatoxin (18 gg infused s.c. in the course of 10 h) and of control infusions with saline only, on local leukocyte accumulation in the guinea-pig skin. Control and anaphylatoxin infusions were given simultaneously into one side each of the back. Each value is the average score ofalI evaluated sections (4- 5 sections per slide, number of slides in brackets) of one individual animal Animal
Score of leukocyte accumulation Controls
Anaphylatoxin infusions
1. 2. 3. 4. 5. 6. 7. 8.
0.02 0.18 0.50 0.33 0.50 0.95 " "
0.60 1.88 1.31 1.55 2.26 1.95 1.45 3.00
Average • S.D.
0.4 • 0.3
(15) (16) (15) (13) (13) (14)
(20) (25) (18) (12) (17) (10) (10) (11)
1.8 • 0.7
e) Injections with Evans Blue. For estimating changes in vascular permeability Evans blue (1 ml of a 5 ~ solution per kg body weight) was injected into the V. saphena 20 min after intrapleural injections of C 5 a or control fluid. After 220 min the animals were killed, the chest was opened and the pleural fluid was removed and measured. After centrifugation the concentration of Evans blue in the supernatant was estimated photometrically at 623 nm. In kinetic experiments Evans blue was injected 30, 120, 180 or 240 rain after intrapleural injection of C5a or saline control. These animals were killed 60 rain after the injection of Evans blue.
RESULTS
a) Subcutaneous Infusions of Anaphylatoxin. T a b l e 1 a n d F i g u r e I s h o w the l e u k o c y t e a c c u m u l a t i o n in skin areas infused with 18 lag C 5 a in 2 ml saline, a n d in c o n t r o l a r e a s infused with saline only. I n a r e a s o f c o n t r o l infusion o n l y a few l e u k o c y t e s were d e t e c t e d except in a n i m a l 6) in w h i c h a l e u k o c y t e infiltration c o r r e s p o n d i n g to score 0.95 o c c u r r e d . A f t e r infusions o f a n a p h y l a t o x i n dense a c c u m u l a t i o n s o f l e u k o c y t e s were visible with their m a x i m u m n e a r the tip o f the c a n n u l a . I n the center o f the inflamed a r e a s n e u t r o p h i l l e u k o c y t e s p r e d o m i n a t e d b u t quite a n u m b e r o f e o s i n o phils were also f o u n d . I n the m a r g i n a l p a r t s the p r o p o r tion of monocytes increased gradually. The blood vessels o f the i n f l a m e d a r e a s were d i l a t e d a n d densely p a c k e d with red a n d white b l o o d cells.
b) Intrapleural Injections of Anaphylatoxin. T e n o r 20 lag a n a p h y l a t o x i n in 2 ml saline, o r saline a l o n e were injected into the p l e u r a l c a v i t y o f g u i n e a pigs. A f t e r 240 m i n the r e m a i n i n g fluid a n d the n u m b e r o f l e u k o c y t e s c o n t a i n e d in it were e s t i m a t e d . In c o n t r o l a n i m a l s a n a v e r a g e o f o n l y 0.47 m l fluid were r e c o v e r e d (Fig. 2). A f t e r injection o f a n a p h y l a t o x i n the r e m a i n i n g v o l u m e was c o n s i d e r a b l y higher, 1.2 m l after injection
In these experiments the cannula on the control side slepped out during the infusion
o f 10 gg a n d 1.4 ml after 20 gg. A n a p h y l a t o x i n thus led to a n increase in e x u d a t e v o l u m e , relative to the controls. 18 lag b r a d y k i n i n led to the s a m e relative increase in v o l u m e as 20 lag a n a p h y l a t o x i n . T h e t o t a l n u m b e r o f l e u k o c y t e s in c o n t r o l exudates was 1.5 x 106 cells ( 8 0 - 9 0 ~ m o n o n u c l e a r cells). A n a p h y l a t o x i n c a u s e d a m u c h h i g h e r a c c u m u l a t i o n o f leukocytes in the p l e u r a l fluid, 9.5 a n d 1 9 x 106 cells p e r t o t a l e x u d a t e after injection o f 10 a n d 20 lag a n a p h y l a t o x i n respectively (Fig. 2). G r a n u l o c y t e s p r e d o m i n a t e d (between 6 0 - 8 0 ~ o ) . I n c o n t r a s t , b r a d y k i n i n t h o u g h h a v i n g the s a m e effect o n e x u d a t e v o l u m e d i d only slightly increase the e m i g r a t i o n o f l e u k o c y t e s into the p l e u r a l c a v i t y (2.3 x 10 6 cells p e r exudate) (Fig. 2).
c) Accumulation of Evans Blue in the Pleural Cavity after Injections of Anaphylatoxin. A f t e r i n t r a p l e u r a l c o n t r o l injections o f saline 2.8 x 10 -5 g E v a n s blue were f o u n d in the p l e u r a l c a v i t y p e r ml fluid. A n a p h y l a t o x i n in doses o f 10 a n d 20 pg (dissolved in 2 ml) led to the p e r m e a t i o n o f 5.2 a n d 8.4 x 10 -5 g E v a n s b l u e / m l e x u d a t e (Fig. 3). Since a n a p h y l a t o x i n increased the e x u d a t e v o l u m e the t o t a l a m o u n t o f E v a n s blue w h i c h h a d diffused into the p l e u r a l c a v i t y was m u c h higher t h a n in the c o n t r o l s : 4 times as m u c h after 10 lag a n d 7.5 times after 20 gg a n a p h y l a t o x i n .
d) Time-Course of Evans Blue Accumulation in the Pleural Cavity. I n o r d e r to e s t i m a t e the d u r a t i o n o f the p e r m e a b i l i t y - i n c r e a s i n g effect caused b y i n t r a p l e u r a l injections o f a n a p h y l a t o x i n E v a n s blue was given i.v.
B. Damerau and W, Vogt: Effects of CSa on Permeability andCbemotaxis
239
Fig. l A and B. Histological appearance (magnification about 25 x) of cutaneous and subcutaneous tissue from guinea pigs after s.c. infusions of anaphylatoxin (18 gg in 10 h) ( = B) and saline as control ( = A). (A) Scattered leukocytes (score: approx. 0.5); (B) dense accumulation of leukocytes at the site of infusion (score: approx. 2)
at different times after the intrapleural application of 10 gg anaphylatoxin. The animals were killed 60 min after injection of the dye and the total amount of Evans blue in the pleural cavity was determined, Control animals received saline instead of anaphylatoxin. In controls almost identical amounts, 0.5-0.75 x 10 .5 g Evans blue, on an average, were detected in the pleural exudate regardless of the time intervals between intrapleural application of saline and intravenous injection of the dye (Fig. 4). Evans blue given 30 min after injection of anaphylatoxin accumulated in the cavity to an amount about 6 times higher (3.0 x 10 -~ g Evans blue) than in controls. Almost the same amount of dye, 2.7 x 10 .5 g, was found when the interval between injection of anaphylatoxin and Evans blue was 120 rain. At intervals of 180 and 240 min there were no longer significant differences between Evans blue accumulation in anaphylatoxin- and saline-treated animals (0.7 and 0.76x 10-Sg Evans blue). It is concluded from these results that the permeability-increasing effect of 10 gg anaphylatoxin injected into the pleural cavity lasted for about 3 h. In agreement with this anaphylatoxin activity (measured by
volume (ml ) FI.5 wbc 20-
I
JI I
-LO
10-0.5
C
I O P 9 A T 20,ugAT I8,ugBra~'
Fig.2. Number of leukocytes (wbc x 106, scale on left ordinate) and fluid volume (ml, scale on right ordinate) in the pleural cavity of guinea pigs taken 240 min after intrapleural injection (2 ml) of 10 and 20 pg anaphylatoxin (AT), 18 pg bradykinin (Brad) or saline as control (C). Hatched columns = number of leukocytes per exudate; open columns = exudate volume; vertical bars = standard deviation, Number of experiments = 3 - 5
240
Naunyn-Schmiedeberg's Arch. Pharmacol. 295 (1976)
ornount / m l
total
amount
E. bl. E. bL
15-
3-
i I
I0-
5-
~
1
I
1
.
I f f f
I
C
ii1
tO/Jg 20,ug AT
f f f f f
C
lOJJg 20JJ9 AT
30
120
180
240
rnio
Fig. 3. Accumulation of i.v. injected Evans blue (E.bl., ordinate) in the pleural cavity of guinea pigs after intrapleural injections of 10 or 20 lag anaphylatoxin (AT), or saline as control (C). Column 1 - 3: concentration of Evans blue (x 10 -5 g/ml exudate), column 4 - 6 : total amount of Evans blue in exudate (x i0 -s g). Number of experiments: 6 - 8
Fig. 4. Total content of Evans blue ( x 10-5 g, ordinate) recovered from the pleural cavity of guinea pigs 60 rain after i.v. injection. The injection was made at different times (indic~ited on the abscissa) after intrapleural application of 10 lag anaphylatoxin (open columns) or saline as control (hatched columns). Vertical bars: Standard deviation. Number of experiments : 3 - 5
bioassay on the isolated guinea-pig ileum) was regularly detected in pleural fluid taken 2 h after injection (6 experiments), but only in one out of 6 experiments 4 h after injection.
In both guinea-pig in vivo models used anaphylatoxin caused leukocyte accumulation. Subcutaneous infusion of very low amounts (1.8 gg/h), intended to mimick a natural continuous local release, led to dense accumulation of neutrophils, but also quite a number of eosinophils, at the site of application. Single intrapleural injections of anaphylatoxin were made with the intention to mimick an inflammatory complement-involving lesion in a body cavity. These injections not only caused massive leukocyte accumulation in the pleural fluid but in addition an increase in permeability of the adjacent vessels (evident from extravasation of Evans blue) and increase in exudate volume. Although the fluid volume recovered from the pleural cavity of anaphylatoxin-treated animals was never absolutely larger than the one injected, its larger size as compared to the control animals is very likely due to increased exudation, not decreased absorption. This is suggested by the concomitant extravasation of i.v. applied Evans blue, and the well-known general consequence of increased vascular permeability, oedema formation. The leukocyte accumulation in the pleural cavity is not simply the result of permeability increase; rather
DISCUSSION The preparation used in this study is classical anaphylatoxin, i.e. the principle which is generated in whole serum by complement activation and can be recovered from such serum without specific precautions. It should be noted that this peptide is already a secondary cleavage product. Its precursor obtained primarily from C5 by hydrolytic activation of the latter is a highly spasmogenic but very labile peptide, C5a. In serum, it is rapidly attacked by carboxypeptidase N losing its carboxy-terminal amino acid and thereby more or less of its spasmogenic activity, depending on the species of the serum (Vallota and Mtiller-Eberhard, 1973). Because of its stability the secondary peptide is of particular interest, biologically, as far as it has preserved (or even acquired?) pharmacological activities.
B. Damerau and W. Vogt: Effects of C 5 a on Permeability and Chemotaxis it is the c o n s e q u e n c e o f a specific c h e m o t a c t i c effect o f a n a p h y l a t o x i n . B r a d y k i n i n , in doses l e a d i n g to the s a m e increase in v a s c u l a r p e r m e a b i l i t y , p r o v e d to be ineffective as a l e u k o c y t e a t t r a c t a n t . T h e s e results f u r t h e r s t r e n g t h e n the role o f a n a p h y l a t o x i n as one i m p o r t a n t m e d i a t o r o f certain i n f l a m m a t o r y reactions. A s has been s h o w n earlier c h e m o t a c t i c f r a g m e n t s o f C 5 d o o c c u r at sites o f i n f l a m m a t i o n , e.g. in s y n o v i a l fluids f r o m p a t i e n t s w i t h r h e u m a t o i d a r t h r i t i s ( W a r d a n d Zvaifler, 1971) a n d in tissue s a m p l e s f r o m a n i m a l s w i t h reversed passive A r t h u s r e a c t i o n s ( W a r d a n d Hill, 1972).
Acknowledgements. The skillful technical assistance of Mrs. G. Werle is gratefully acknowledged.
REFERENCES Jensen, J. A., Snyderman, R., Mergenhagen, S. E.: Chemotactic activity, a property of guinea pig C'5-anaphylatoxin. In: Cellular and humoral mechanisms in anaphylaxis and allergy (H. Z. Movat, ed.), pp. 265-278. Basel: S. Karger 1969
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Liefl/inder, M., Dielenberg, D., Schmidt, G., Vogt, W. : Structural elements of anaphylatoxin obtained by contact activation of hog serum. Hoppe-Seyler's Z. physiol. Chem. 353, 385-392 (1972) Shin, H. S., Snyderman, R., Friedman, E., Mellors, A., Mayer, M. M. : Chemotactic and anaphylatoxic fragment cleaved from the fifth component of guinea pig complement. Science 162, 361 - 363 (1968) Vallota, E. H., Mfiller-Eberhard, H.J.: Formation of C3a and C5a anaphylatoxins in whole human serum after inhibition of the anaphylatoxin inactivator. J. exp. Med. 13"7, 1109-1123 (1973) Ward, P. A., Hill, J. H. : Biological role of complement products. Complement-derived leukotactic activity extractable from lesions of immunologic vasculitis. J. Immunol. 108, 1137--1145 (1972) Ward, P. A., Newman, L. J. : A neutrophil chemotactic factor from human C5. J. Immunol. 102, 93-99 (1969) Ward, P. A., Zvaifler, N. J. : Complement-derived leukotactic factors in inflammatory synovial fluids of humans. J. clin. Invest. 50, 606- 616 (1971)
Received July 22/Accepted September 13, 1976
