COMPARISON O F T H E E F F E C T O F VARIOUS ANTISERA A N D COBRA VENOM FACTOR O N INFLAMMATORY REACTIONS IN GUINEA-PIG S K I N 11. THEARTHUSR E A C T I O N A N D T H E LOCAL SHWARTZMAN R E A C T I O N ELIZABETH LEWISAND J. L. TURK Department of Pathology, Royal College of Surgeons of England, Lincoln's Inn Fields, London WC2A 3PN PLATES
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INthe previous communication a comparison was made of the effect of cobra venom factor (CVF) and various antisera known to affect non-specific inflammation to turpentine in guinea-pig skin. It was suggested that serum complement, including the '' alternate pathway " and a normal level of platelets, were involved in the production of such a reaction. In experiments on the Arthus reaction in guinea-pigs Turk and Polak (1969) had found that the haemorrhagic component could be blocked by antisera prepared against guinea-pig lymphocytes, membrane-free extracts of lymph nodes and y globulin. They suggested that this effect might be produced by lowering of the serum complement levels following the antigen-antibody reaction in v i v a However, Polak and Turk (1969), found that the haemorrhagic component of the local Shwartzman reaction in guinea-pigs could only be blocked by anti-complement serum, and not by the other antisera used. The present investigation was designed to continue the study of the effect of anti-complement serum, and other antisera, on these two reactions, and to compare their effects with that of CVF which specifically depletes the terminal components, C3 to C9, of the complement system. In the previous experiments the anti-complement serum used was anti/ ~ , C / & A globulin (zymosan), that had been prepared against the C3-zymosan complex formed after incubation of zymosan with fresh guinea-pig serum at 37°C. Such an antiserum had been found to block the reversed passive Arthus reaction (Ward and Cochrane, 1965) and the Shwartzman reaction (Polak and Turk, 1969). This antiserum could have contained contaminating antibodies to " alternate pathway " complement components as well as demonstrable antibodies to zymosan. It also caused a marked fall in platelet numbers as well as serum complement levels when injected into guinea-pigs (Lewis and Turk, 1974). It was because of this variety of effects that other antisera were used in the present investigation. Anti-C3 was prepared against chemically extracted /3,c/j31~ globulin (Steinbuch, Quentin and Pejendier, 1963) (anti-/3lc/plA globulin (rivanol)) so as not to contain the same contaminating antibodies. An antiserum thought to contain antibodies against properdin (" anti-properReceived 16 March 1974; accepted 23 March 1974. 1. PATH.-VOL.
115 (1975)
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H
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ELIZABETH LEWIS A N D J. L. TURK
din ”) was prepared against zymosan and “ alternate pathway ” complement components which fixed to the zymosan at 0°C. Cobra venom factor was also used as an agent which would specifically deplete the serum complement (C3 to C9) levels and methysergide to cause depletion of histamine and serotonin. Using these agents it has been possible to correlate the serum complement levels and the platelet numbers with the development of the lesions of the active and reversed passive Arthus reaction, and the local Shwartzman reaction. MATERIALS AND METHODS Production of the active and reversedpassive Arthus reactions and the local Shwartzman reaction Hartley strain male guinea-pigs weighing between 400 and 500 g were used. All animals had a diet of RGP pellets supplemented by hay and fresh cabbage. To immunise animals for the active Arthus reaction the method described by Turk and Polak (1969) was used with alum precipitated ovalbumin as the antigen. Following the intradermal challenge with 1 mg ovalbumin in 0.1 ml saline the reactions were followed for 6 hr by which time they had reached maximal intensity. The diameter of the haemorrhage and the increase in skin thickness at the site of the lesion were measured. The reversed passive Arthus reaction was elicited as described by Bloch, Kourilsky, Ovary and Benacerraf (1963), using serum from guinea-pigs actively immunised as above. For the local Shwartzman reaction purified, lyophilised lipopolysaccharide (LPS) from E. coli B6.026 (Difco, USA) was used as described by Polak and Turk (1969). Guinea-pigs were injected intradermally with 1 mg of LPS. Following the challenging intravenous injection of 1 mg LPS 18 hr later, the skin sites were observed for 4 hr, during which time the haemorrhage appeared and was graded in the following way : 3 =confluent haemorrhage, 2=semi-confluent haemorrhage, 1=scattered haemorrhages, 0.5 =petechial, O=absence of haemorrhage. Histology Skin sections were taken from the active Arthus sites at 6 hr, or from the reversed passive Arthus site at 4 hr, fixed in formol-saline and stained with haematoxylin and eosin. Skin sections from the local Shwartzman site were taken at 4 hr and similarly treated. They were also stained for fibrin with phosphotungstic acid haematoxylin (PTAH). Measurement of serum complement levels and haematologicalparameters The number of total haemolytic complement units (CH50) per ml, the immune-adherence titre and the measurement of /3lC//3lA globulin by single radial immuno-diffusion, were all performed as described previously (Lewis and Turk, 1974). Total white cell counts, differentials and platelet counts were performed by standard methods. Experimental protocol The antisera given to the animals were prepared in the same way and administered in the same dosage as previously described (Lewis and Turk, 1974). For experiments on the active and reversed, passive Arthus reactions the antisera were injected 2 hr before the intradermal challenge with soluble ovalbumin. For experiments on the local Shwartzman reaction the antisera were administered 2 hr before the intravenous challenge with LPS. Zymosan was injected as described by Ward and Cochrane (1965), and methysergide as described by Crunkhorn and Meacock (1971). Animals were complement depleted with cobra venom factor 48 hr before challenge with antigen in the active and reversed passive Arthus reactions (Cordis Co.). The schedule differed from that in the previous experiments in the case of the administration of CVF to animals which had received intradermal LPS as preparation for the local Shwartzman reaction. In this case, to ensure survival of the animals for the duration of the experiment, the CVF was given 4 hr before the intravenous challenge.
EFFECT OF ANTISERA AND COBRA VENOM ON INFLAMMATION. II 113 Detection of antibodies to E. coli B6.026 LPS A specific antiserum to E. coli B6.026 LPS was obtained from Wellcome Laboratories which served as a positive control for the detection of antibody. The LPS was absorbed on to normal, washed sheep red blood cells (SRBC) after it had been treated by heating in a boiling water bath for 1 hr (Neter, Westphal, Luderitz, Gorzynski and Eichenberger, 1956). These antigen coated SRBC were then used for the detection of immunological cross-reactivity between the rabbit anti-zymosan serum and the LPS using the following techniques: 1. passive haemagglutination at 4°C and 37°C; 2. haemolysis in the presence of complement (Neter, 1956); 3. absorption studies in which anti-zymosan sera was absorbed with LPS-coated SRBC and then checked for the presence of agglutinating antibodies to zymosan; 4. the indirect anti-globulin test in which incomplete antibodies attached to the LPS-coated SRBC would be detected by agglutination of the cells following the addition of goat anti-rabbit y globulin.
Characterisation of the anti-ovalbumin sera causing the Arthus reaction
Sera were collected from a minimum of five guinea-pigs immediately before the challenging injection of ovalbumin for the following tests : 1. Precipitating antibody was detected by the Ouchterlony technique of double diffusion in gel. To determine the electrophoretic mobility of the anti-ovalbumin antibody, i.e., to try to distinguish between the faster moving y l , 7s immunoglobulin and the slower moving yz immunoglobulin described by Benacerraf, Ovary and Bloch and Franklin (1963), a modification of the immuno-electrophoretic technique was used. The guinea-pig serum was placed in the central well which was cut larger than usual (2 mm diameter). The quantity of antiserum placed in this well was now sufficient to ensure that after migration in the electric current, precipitation occurred with 0.5 mg/ml solution of ovalbumin placed in one trough. To show the migration of all the serum proteins anti-whole guinea-pig serum was diluted to 1 : 2 (to prevent antibody excess in the y globulin region) and placed in the opposite trough 1 cm distance from the well. In this way the relative position of the anti-ovalbumin antibody to the other serum proteins could be ascertained. 2. Haemagglutinating antibodies to ovalbumin were detected using 2.5 per cent. suspension of tanned sheep red blood cells coated with ovalbumin (Herbert, 1967) and haemolytic antibodies were detected by the addition of guinea-pig complement to the same system (Bloch, Kourilsky, Ovary and Benacerraf, 1963). 3. Skin sensitising antibodies responsible for passive cutaneous anaphylaxis (PCA) in guinea-pig skin were detected as described by Ovary, Benacerraf and Bloch (1963). Guineapigs weighing less than 300 g were injected with several dilutions of 0-1 ml aliquots of the test serum in normal saline. Four hr later a 0.1 per cent. solution of ovalbumin in 2 per cent. Evan’s Blue, both in physiological saline, were injected intravenously. Half an hour later the diameter of the skin reactions were measured. Each serum was tested in two animals. A positive control of rabbit anti-ovalbumin known to contain a high titre of PCA antibody was included in each animal.
RESULTS 1 . The active Arthus reaction (jigs. 1 and 2) During the active Arthus reaction the serum complement levels changed slightly. There was an average fall of 15 per cent. as measured by the total haemolytic complement, immune-adherence and j?lC/j?lA globulin levels. There were no changes in the platelet, total white blood cell or differential cell counts.
ELIZABETH LEWIS AND J. L. TURK
114
(a) Anti-/3tc//31Aglobulin (zyrnosan), anti-zymosun, anti-" properdin " and anti+ 1c/P 1~ globulin (rivunol) The effect of these agents on the serum complement levels have been described in the previous communication. The effect on the haematological parameters has also been described (Lewis and Turk, 1974). Briefly, it was found that the two anti-C3 sera, anti-/31c/PlA globulin (zymosan) prepared against the C3zymosan complex, and anti-/3lc//31A globulin (rivanol), prepared against chemically extracted /3lC//31A globulin, both caused a 50 to 60 per cent. fall in serum complement levels. They both had a profound effect on platelet numbers,
-
140
0
120 c
C
O
u
100
.+0
80
0,
a 60 rd a. .
= L o Q,
V
Q,
20
a
0
FIG.1 .-The active Arthus reaction. The effect of different treatments in skin thickness at 6 hr after intradermal challenge.
reducing them to around lOO,OOO/cu mm. " Anti-properdin ",which may have contained antibodies to " alternate pathway '' complement components, other than C3, which fixed to zymosan at O'C, did have some effect on complement levels, reducing them by one-third. However, this serum only caused a 40 per cent. reduction in platelet numbers. Anti-zymosan, prepared against the yeast particle alone, had none of these effects. When these agents were administered to animals undergoing an active Arthus reaction, it was found that the two antiC3 sera caused a reduction in the degree of induration (fig. 1). The reduction in the diameter and intensity of the haemorrhage was less marked. Both antisera caused a delay in the appearance of the haemorrhage when compared with the control animals. This delay was the only effect seen following " antiproperdin '' (fig. 2).
EFFECT OF ANTISERA AND COBRA VENOM ON INFLAMMATION. II 115
(b) Zymosan injected directly into the animals (Jigs. 1 and 2) As previously described, zymosan injected intravenously and intraperitoneally 2 hr before the intradermal challenge, again at the time of challenge and 2 hr later, caused a 50 per cent. fall in both complement and platelet numbers. In spite of this relatively modest effect, this procedure caused a 85 per cent. reduction of the induration and a delay of 3 hr in the appearance of the haemorrhage, which was more than that achieved by any other agent.
'r 3
1
s
FIG.2.--The active Arthus reaction. The effect of different treatments on the time of appearance of the haemorrhage after intradermal challenge.
(c) Anti-y globulin (Jigs. 1 and 2) Anti-y globulin was as effective as anti-C3 serum in lowering serum complement levels but it had less effect in reducing platelet numbers. The latter were reduced to one-half their normal level. It had similar effects on the Arthus reaction, causing a 40 per cent. reduction in induration, having little effect on the intensity or diameter of the haemorrhagic lesion, but causing considerable delay in its appearance. (d) Cobra venom factor and methysergide (jigs. 1 and 2) Cobra venom factor was administered to the animals 48 hr before the challenging injection. It lowered the serum complement levels by 80 to 90 per cent.
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UIZABETH LEWIS AND J. L. TURK
throughout this period, and the period of the reaction, while having no effect on platelet numbers. It caused a considerable polymorphonuclear leucocytosis. The only effect it had on the Arthus lesion was a delay in the appearance of the haemorrhage. Once it had developed, the diameter and intensity were the same as the controls, and the development of the induration was identical. Methysergide had no effect on serum complement levels or on platelet numbers. It caused some reduction in induration and delay in the appearance of the haemorrhage, but no reduction in the intensity of the latter.
His tology Histological sections taken from the Arthus sites 6 hr after challenge showed similar changes to those shown in figures of the reversed passive Arthus reaction, The control reaction showed extensive vascular damage, chiefly of the arteries, with necrosis of the walls and haemorrhage. Polymorphonuclear cells were seen within the vessel, lining the endothelium, passing through the vessel walls and infiltrating the dermus extensively. Sections taken from animals which had been treated with the two anti-C3 sera, anti-y globulin and zymosan, and which showed a reduction in development of induration at the site, all showed very much reduced changes histologically. Vessels were usually intact, minimal margination of leucocytes was occasionally seen and the intensity of the polymorphonuclear cell infiltration of the dermis was much reduced. Histological sections from the animals treated with CVF were similar to those in the untreated Arthus reaction. The vascular damage and cellular infiltration of the tissues was gross and indistinguishable from the sections taken from the animals eliciting a reaction without any other treatment.
2. The reversed passive Arthus reaction The macroscopic appearance of the control lesion was of an area of increased skin thickness at the site of antibody injection. Haemorrhage was only very occasionally visible on the underside of the skin. Histologically however, all the characteristic features of an Arthus reaction could be seen clearly (fig. 3). Zymosan was injected intravenously and intraperitoneally into animals 2 hr before challenging for the reversed passive Arthus reaction and at the time of challenge. There was marked reduction in the induration of the lesion, measured at 4 hr, and histologically there was less vascular destruction and less perivascular polymorphonuclear cell infiltration than the control animals which had received saline (fig. 4). This was in marked contrast to a second group of animals treated 48 hr earlier with cobra venom factor. In this group, despite 80 to 90 per cent. reduction in the complement levels for the duration of the experiment, there was no effect at all on the induration or on the histological appearance of the lesion (fig. 5). Anti-j3lc/j31A globulin (zymosan) was also given 2 hr before challenge and was found to reduce the induration and to cause a considerable reduction in the vascular damage and polymorphonuclear cell infiltration seen histologically (fig. 6).
EFFECT OF ANTISERA AND COBRA VENOM ON INFLAMMATION. 11 117
Characterisation of the anti-ovalbumin antibodies A precipitating antibody against ovalbumin developed in the serum during the immunisation schedule that was used to prepare the guinea-pigs for active Arthus sensitivity. The serum, which was also used for the passive Arthus experiments, contained specific antibody able to cause haemagglutination of tanned red cells coated with ovalbumin to a titre of 1 : 4000, and was able to cause lysis of these cells to a titre of 1/64 in the presence of complement. It also contained antibodies that could be detected to a titre of 1/400 to 1/800 by PCA. On immuno-electrophoresis only the y2 immuno-globulin fraction could be seen with the system used in this study, a faster moving y1 immuno-globulin which might have been responsible for the PCA activity was not visualised. 3. The local Shwartzman reaction (fig.7, table I) During the local Shwartzman reaction changes occurred in the serum complement levels and in the haematological parameters which are shown in table I. Blood collected 18 to 24 hr after the preparatory intradermal injection of LPS showed a 24 per cent. fall in complement titre. The platelet count was reduced by 30 per cent. and there was an increase in circulating polymorphonuclear leucocytes to an average of 10,600 per cu. mm. Blood taken 2 hr after the intravenous challenge with LPS showed an even greater fall in complement titre to 50 to 60 per cent. of normal. The platelet count showed no further significant change but leucocytes disappeared from the circulation leaving only 2000 to 3000 per cu. mm. Differential cell counts showed this fall to be due almost entirely to the disappearance of polymorphs. The histological appearance with haematoxylin and eosin staining showed a n intense polymorphonuclear cell infiltration of the dermis and the blood vessels were plugged with leucocyte-platelet thrombi. PTAH staining also showed fibrin deposition within the vessels. Since the decomplementing antisera and cobra venom factor were given to animals which were already 24 per cent. depleted of serum complement, the level of circulating complement at the time of challenge was considerably less than had been achieved by the same agent in normal animals, as is seen in table I.
(a) Anti-PlcllSlA globulin (zymosan), anti-“ properdin ”, anti-zymosan and anti-/3lc/p1Aglobulin (rivanol) (jig. 7, table I ) All three antisera which had been prepared using zymosan particles for the immunisation caused a 50 per cent. reduction in the haemorrhagic lesion compared with animals injected with normal rabbit serum. Anti-/31c//l1A globulin (rivanol) which was prepared against soluble /31c//31~ globulin in Freund’s Incomplete Adjuvent (FIA) also had this effect. The blocking activity was analysed statistically using Students’ t-test for ungrouped data. For the group receiving anti-/3lc//31Aglobulin (zymosan)p=
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INthe previous communication a comparison was made of the effect of cobra venom factor (CVF) and various antisera known to affect non-specific inflammation to turpentine in guinea-pig skin. It was suggested that serum complement, including the '' alternate pathway " and a normal level of platelets, were involved in the production of such a reaction. In experiments on the Arthus reaction in guinea-pigs Turk and Polak (1969) had found that the haemorrhagic component could be blocked by antisera prepared against guinea-pig lymphocytes, membrane-free extracts of lymph nodes and y globulin. They suggested that this effect might be produced by lowering of the serum complement levels following the antigen-antibody reaction in v i v a However, Polak and Turk (1969), found that the haemorrhagic component of the local Shwartzman reaction in guinea-pigs could only be blocked by anti-complement serum, and not by the other antisera used. The present investigation was designed to continue the study of the effect of anti-complement serum, and other antisera, on these two reactions, and to compare their effects with that of CVF which specifically depletes the terminal components, C3 to C9, of the complement system. In the previous experiments the anti-complement serum used was anti/ ~ , C / & A globulin (zymosan), that had been prepared against the C3-zymosan complex formed after incubation of zymosan with fresh guinea-pig serum at 37°C. Such an antiserum had been found to block the reversed passive Arthus reaction (Ward and Cochrane, 1965) and the Shwartzman reaction (Polak and Turk, 1969). This antiserum could have contained contaminating antibodies to " alternate pathway " complement components as well as demonstrable antibodies to zymosan. It also caused a marked fall in platelet numbers as well as serum complement levels when injected into guinea-pigs (Lewis and Turk, 1974). It was because of this variety of effects that other antisera were used in the present investigation. Anti-C3 was prepared against chemically extracted /3,c/j31~ globulin (Steinbuch, Quentin and Pejendier, 1963) (anti-/3lc/plA globulin (rivanol)) so as not to contain the same contaminating antibodies. An antiserum thought to contain antibodies against properdin (" anti-properReceived 16 March 1974; accepted 23 March 1974. 1. PATH.-VOL.
115 (1975)
111
H
112
ELIZABETH LEWIS A N D J. L. TURK
din ”) was prepared against zymosan and “ alternate pathway ” complement components which fixed to the zymosan at 0°C. Cobra venom factor was also used as an agent which would specifically deplete the serum complement (C3 to C9) levels and methysergide to cause depletion of histamine and serotonin. Using these agents it has been possible to correlate the serum complement levels and the platelet numbers with the development of the lesions of the active and reversed passive Arthus reaction, and the local Shwartzman reaction. MATERIALS AND METHODS Production of the active and reversedpassive Arthus reactions and the local Shwartzman reaction Hartley strain male guinea-pigs weighing between 400 and 500 g were used. All animals had a diet of RGP pellets supplemented by hay and fresh cabbage. To immunise animals for the active Arthus reaction the method described by Turk and Polak (1969) was used with alum precipitated ovalbumin as the antigen. Following the intradermal challenge with 1 mg ovalbumin in 0.1 ml saline the reactions were followed for 6 hr by which time they had reached maximal intensity. The diameter of the haemorrhage and the increase in skin thickness at the site of the lesion were measured. The reversed passive Arthus reaction was elicited as described by Bloch, Kourilsky, Ovary and Benacerraf (1963), using serum from guinea-pigs actively immunised as above. For the local Shwartzman reaction purified, lyophilised lipopolysaccharide (LPS) from E. coli B6.026 (Difco, USA) was used as described by Polak and Turk (1969). Guinea-pigs were injected intradermally with 1 mg of LPS. Following the challenging intravenous injection of 1 mg LPS 18 hr later, the skin sites were observed for 4 hr, during which time the haemorrhage appeared and was graded in the following way : 3 =confluent haemorrhage, 2=semi-confluent haemorrhage, 1=scattered haemorrhages, 0.5 =petechial, O=absence of haemorrhage. Histology Skin sections were taken from the active Arthus sites at 6 hr, or from the reversed passive Arthus site at 4 hr, fixed in formol-saline and stained with haematoxylin and eosin. Skin sections from the local Shwartzman site were taken at 4 hr and similarly treated. They were also stained for fibrin with phosphotungstic acid haematoxylin (PTAH). Measurement of serum complement levels and haematologicalparameters The number of total haemolytic complement units (CH50) per ml, the immune-adherence titre and the measurement of /3lC//3lA globulin by single radial immuno-diffusion, were all performed as described previously (Lewis and Turk, 1974). Total white cell counts, differentials and platelet counts were performed by standard methods. Experimental protocol The antisera given to the animals were prepared in the same way and administered in the same dosage as previously described (Lewis and Turk, 1974). For experiments on the active and reversed, passive Arthus reactions the antisera were injected 2 hr before the intradermal challenge with soluble ovalbumin. For experiments on the local Shwartzman reaction the antisera were administered 2 hr before the intravenous challenge with LPS. Zymosan was injected as described by Ward and Cochrane (1965), and methysergide as described by Crunkhorn and Meacock (1971). Animals were complement depleted with cobra venom factor 48 hr before challenge with antigen in the active and reversed passive Arthus reactions (Cordis Co.). The schedule differed from that in the previous experiments in the case of the administration of CVF to animals which had received intradermal LPS as preparation for the local Shwartzman reaction. In this case, to ensure survival of the animals for the duration of the experiment, the CVF was given 4 hr before the intravenous challenge.
EFFECT OF ANTISERA AND COBRA VENOM ON INFLAMMATION. II 113 Detection of antibodies to E. coli B6.026 LPS A specific antiserum to E. coli B6.026 LPS was obtained from Wellcome Laboratories which served as a positive control for the detection of antibody. The LPS was absorbed on to normal, washed sheep red blood cells (SRBC) after it had been treated by heating in a boiling water bath for 1 hr (Neter, Westphal, Luderitz, Gorzynski and Eichenberger, 1956). These antigen coated SRBC were then used for the detection of immunological cross-reactivity between the rabbit anti-zymosan serum and the LPS using the following techniques: 1. passive haemagglutination at 4°C and 37°C; 2. haemolysis in the presence of complement (Neter, 1956); 3. absorption studies in which anti-zymosan sera was absorbed with LPS-coated SRBC and then checked for the presence of agglutinating antibodies to zymosan; 4. the indirect anti-globulin test in which incomplete antibodies attached to the LPS-coated SRBC would be detected by agglutination of the cells following the addition of goat anti-rabbit y globulin.
Characterisation of the anti-ovalbumin sera causing the Arthus reaction
Sera were collected from a minimum of five guinea-pigs immediately before the challenging injection of ovalbumin for the following tests : 1. Precipitating antibody was detected by the Ouchterlony technique of double diffusion in gel. To determine the electrophoretic mobility of the anti-ovalbumin antibody, i.e., to try to distinguish between the faster moving y l , 7s immunoglobulin and the slower moving yz immunoglobulin described by Benacerraf, Ovary and Bloch and Franklin (1963), a modification of the immuno-electrophoretic technique was used. The guinea-pig serum was placed in the central well which was cut larger than usual (2 mm diameter). The quantity of antiserum placed in this well was now sufficient to ensure that after migration in the electric current, precipitation occurred with 0.5 mg/ml solution of ovalbumin placed in one trough. To show the migration of all the serum proteins anti-whole guinea-pig serum was diluted to 1 : 2 (to prevent antibody excess in the y globulin region) and placed in the opposite trough 1 cm distance from the well. In this way the relative position of the anti-ovalbumin antibody to the other serum proteins could be ascertained. 2. Haemagglutinating antibodies to ovalbumin were detected using 2.5 per cent. suspension of tanned sheep red blood cells coated with ovalbumin (Herbert, 1967) and haemolytic antibodies were detected by the addition of guinea-pig complement to the same system (Bloch, Kourilsky, Ovary and Benacerraf, 1963). 3. Skin sensitising antibodies responsible for passive cutaneous anaphylaxis (PCA) in guinea-pig skin were detected as described by Ovary, Benacerraf and Bloch (1963). Guineapigs weighing less than 300 g were injected with several dilutions of 0-1 ml aliquots of the test serum in normal saline. Four hr later a 0.1 per cent. solution of ovalbumin in 2 per cent. Evan’s Blue, both in physiological saline, were injected intravenously. Half an hour later the diameter of the skin reactions were measured. Each serum was tested in two animals. A positive control of rabbit anti-ovalbumin known to contain a high titre of PCA antibody was included in each animal.
RESULTS 1 . The active Arthus reaction (jigs. 1 and 2) During the active Arthus reaction the serum complement levels changed slightly. There was an average fall of 15 per cent. as measured by the total haemolytic complement, immune-adherence and j?lC/j?lA globulin levels. There were no changes in the platelet, total white blood cell or differential cell counts.
ELIZABETH LEWIS AND J. L. TURK
114
(a) Anti-/3tc//31Aglobulin (zyrnosan), anti-zymosun, anti-" properdin " and anti+ 1c/P 1~ globulin (rivunol) The effect of these agents on the serum complement levels have been described in the previous communication. The effect on the haematological parameters has also been described (Lewis and Turk, 1974). Briefly, it was found that the two anti-C3 sera, anti-/31c/PlA globulin (zymosan) prepared against the C3zymosan complex, and anti-/3lc//31A globulin (rivanol), prepared against chemically extracted /3lC//31A globulin, both caused a 50 to 60 per cent. fall in serum complement levels. They both had a profound effect on platelet numbers,
-
140
0
120 c
C
O
u
100
.+0
80
0,
a 60 rd a. .
= L o Q,
V
Q,
20
a
0
FIG.1 .-The active Arthus reaction. The effect of different treatments in skin thickness at 6 hr after intradermal challenge.
reducing them to around lOO,OOO/cu mm. " Anti-properdin ",which may have contained antibodies to " alternate pathway '' complement components, other than C3, which fixed to zymosan at O'C, did have some effect on complement levels, reducing them by one-third. However, this serum only caused a 40 per cent. reduction in platelet numbers. Anti-zymosan, prepared against the yeast particle alone, had none of these effects. When these agents were administered to animals undergoing an active Arthus reaction, it was found that the two antiC3 sera caused a reduction in the degree of induration (fig. 1). The reduction in the diameter and intensity of the haemorrhage was less marked. Both antisera caused a delay in the appearance of the haemorrhage when compared with the control animals. This delay was the only effect seen following " antiproperdin '' (fig. 2).
EFFECT OF ANTISERA AND COBRA VENOM ON INFLAMMATION. II 115
(b) Zymosan injected directly into the animals (Jigs. 1 and 2) As previously described, zymosan injected intravenously and intraperitoneally 2 hr before the intradermal challenge, again at the time of challenge and 2 hr later, caused a 50 per cent. fall in both complement and platelet numbers. In spite of this relatively modest effect, this procedure caused a 85 per cent. reduction of the induration and a delay of 3 hr in the appearance of the haemorrhage, which was more than that achieved by any other agent.
'r 3
1
s
FIG.2.--The active Arthus reaction. The effect of different treatments on the time of appearance of the haemorrhage after intradermal challenge.
(c) Anti-y globulin (Jigs. 1 and 2) Anti-y globulin was as effective as anti-C3 serum in lowering serum complement levels but it had less effect in reducing platelet numbers. The latter were reduced to one-half their normal level. It had similar effects on the Arthus reaction, causing a 40 per cent. reduction in induration, having little effect on the intensity or diameter of the haemorrhagic lesion, but causing considerable delay in its appearance. (d) Cobra venom factor and methysergide (jigs. 1 and 2) Cobra venom factor was administered to the animals 48 hr before the challenging injection. It lowered the serum complement levels by 80 to 90 per cent.
116
UIZABETH LEWIS AND J. L. TURK
throughout this period, and the period of the reaction, while having no effect on platelet numbers. It caused a considerable polymorphonuclear leucocytosis. The only effect it had on the Arthus lesion was a delay in the appearance of the haemorrhage. Once it had developed, the diameter and intensity were the same as the controls, and the development of the induration was identical. Methysergide had no effect on serum complement levels or on platelet numbers. It caused some reduction in induration and delay in the appearance of the haemorrhage, but no reduction in the intensity of the latter.
His tology Histological sections taken from the Arthus sites 6 hr after challenge showed similar changes to those shown in figures of the reversed passive Arthus reaction, The control reaction showed extensive vascular damage, chiefly of the arteries, with necrosis of the walls and haemorrhage. Polymorphonuclear cells were seen within the vessel, lining the endothelium, passing through the vessel walls and infiltrating the dermus extensively. Sections taken from animals which had been treated with the two anti-C3 sera, anti-y globulin and zymosan, and which showed a reduction in development of induration at the site, all showed very much reduced changes histologically. Vessels were usually intact, minimal margination of leucocytes was occasionally seen and the intensity of the polymorphonuclear cell infiltration of the dermis was much reduced. Histological sections from the animals treated with CVF were similar to those in the untreated Arthus reaction. The vascular damage and cellular infiltration of the tissues was gross and indistinguishable from the sections taken from the animals eliciting a reaction without any other treatment.
2. The reversed passive Arthus reaction The macroscopic appearance of the control lesion was of an area of increased skin thickness at the site of antibody injection. Haemorrhage was only very occasionally visible on the underside of the skin. Histologically however, all the characteristic features of an Arthus reaction could be seen clearly (fig. 3). Zymosan was injected intravenously and intraperitoneally into animals 2 hr before challenging for the reversed passive Arthus reaction and at the time of challenge. There was marked reduction in the induration of the lesion, measured at 4 hr, and histologically there was less vascular destruction and less perivascular polymorphonuclear cell infiltration than the control animals which had received saline (fig. 4). This was in marked contrast to a second group of animals treated 48 hr earlier with cobra venom factor. In this group, despite 80 to 90 per cent. reduction in the complement levels for the duration of the experiment, there was no effect at all on the induration or on the histological appearance of the lesion (fig. 5). Anti-j3lc/j31A globulin (zymosan) was also given 2 hr before challenge and was found to reduce the induration and to cause a considerable reduction in the vascular damage and polymorphonuclear cell infiltration seen histologically (fig. 6).
EFFECT OF ANTISERA AND COBRA VENOM ON INFLAMMATION. 11 117
Characterisation of the anti-ovalbumin antibodies A precipitating antibody against ovalbumin developed in the serum during the immunisation schedule that was used to prepare the guinea-pigs for active Arthus sensitivity. The serum, which was also used for the passive Arthus experiments, contained specific antibody able to cause haemagglutination of tanned red cells coated with ovalbumin to a titre of 1 : 4000, and was able to cause lysis of these cells to a titre of 1/64 in the presence of complement. It also contained antibodies that could be detected to a titre of 1/400 to 1/800 by PCA. On immuno-electrophoresis only the y2 immuno-globulin fraction could be seen with the system used in this study, a faster moving y1 immuno-globulin which might have been responsible for the PCA activity was not visualised. 3. The local Shwartzman reaction (fig.7, table I) During the local Shwartzman reaction changes occurred in the serum complement levels and in the haematological parameters which are shown in table I. Blood collected 18 to 24 hr after the preparatory intradermal injection of LPS showed a 24 per cent. fall in complement titre. The platelet count was reduced by 30 per cent. and there was an increase in circulating polymorphonuclear leucocytes to an average of 10,600 per cu. mm. Blood taken 2 hr after the intravenous challenge with LPS showed an even greater fall in complement titre to 50 to 60 per cent. of normal. The platelet count showed no further significant change but leucocytes disappeared from the circulation leaving only 2000 to 3000 per cu. mm. Differential cell counts showed this fall to be due almost entirely to the disappearance of polymorphs. The histological appearance with haematoxylin and eosin staining showed a n intense polymorphonuclear cell infiltration of the dermis and the blood vessels were plugged with leucocyte-platelet thrombi. PTAH staining also showed fibrin deposition within the vessels. Since the decomplementing antisera and cobra venom factor were given to animals which were already 24 per cent. depleted of serum complement, the level of circulating complement at the time of challenge was considerably less than had been achieved by the same agent in normal animals, as is seen in table I.
(a) Anti-PlcllSlA globulin (zymosan), anti-“ properdin ”, anti-zymosan and anti-/3lc/p1Aglobulin (rivanol) (jig. 7, table I ) All three antisera which had been prepared using zymosan particles for the immunisation caused a 50 per cent. reduction in the haemorrhagic lesion compared with animals injected with normal rabbit serum. Anti-/31c//l1A globulin (rivanol) which was prepared against soluble /31c//31~ globulin in Freund’s Incomplete Adjuvent (FIA) also had this effect. The blocking activity was analysed statistically using Students’ t-test for ungrouped data. For the group receiving anti-/3lc//31Aglobulin (zymosan)p=
