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The alternative complement pathway in chickens
DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol. 7, pp. 785-786, 1983. 0145-305X/83 $3.00 + .00 Printed in the USA. Copyright (c) 1983 Pergamon Press Ltd. All rights reserved.
THE ALTERNATIVE COMPLEMENT PATHWAY IN CHICKENS.
Claus Koch,
Leif Kongerslev and Lisbeth Bjerring Jensen.
Institute for Experimental Immunology, 71, DK-2100 Copenhagen 0, Denmark.
University
of Copenhagen,
Notre Alle
In a previous communication we have presented data on complement mediated lysis in chickens (1). We demonstrated a functional alternative complement pathway (ACP) which was activated when mammalian erythrocytes were injected intravenously into chickens or when chicken serum was incubated in vitro with mammalian erythrocytes. Antibodies did not seem to play any role in this reaction, and various observations lead us to doubt the functional existence in this lytic reaction of the classical complement pathway. Physico-chemical purification (PEG-precipitation, ion-exchange chromatography on CM 52 cellulose, g e l f i l t r a t i o n on Sephadex G 200, elution from a Con A column and finally ion-exchange chromatography) lead to a highly purified chicken factor B preparation wich had the capacity to re-establish the lytic activity of heat inactivated chicken serum. Immunization of rabbits with this factor B preparation gave rise to a monospecific antiserum which could be shown by various criteria to detect chicken factor B. Chicken factor B turned out to be a glycoprotein with a molecular weight of about 105.000. Upon activation of the ACP factor B is split into two fragments, Ba which has alfa-mobility and has a molecular weight around 34.000, and Bb with g a m m a - m o b i l i t y and a molecular weight around 58.000. Molecular weights of Ba and Bb were determined after SDS-PAGE of ACP-activated chicken serum and subsequent immunoblotting and staining with the monospecific antibody and enzyme-linked secondary antibody. In order to be able to dissect classical complement activity and ACP sctivity we then wanted to produce a monoclonal antibody to chicken factor B. After fusion of spleen cells from mice, immunized with the purified chicken factor B, with myeloma cells, we obtained 4 different clones which produced antibodies with reactivity against factor B. Three antibodies reacted with Ba and one with Bb. The monoclonal antibody with reactivity against Bb was coupled to cyanogenbromide activated Sepharose, and an immunosorbent column with the capacity to specifically remove factor B from chicken serum was made. 785
786
COMPLEMENT
IN CHICKENS
Vol.
7, No. 4
Fig. 1 shows normal chicken serum before and after passage through this column ( la and ib) and the eluted antigen (ic). The column turned out to have the capacity to deplete completely i00 ml of chicken serum of factor B.
Fig.
i.
Normal chicken serum before (la) and after (ib) passage through an immunosorbent column with monoclonal antibodies to chicken factor B. ic shows the eluted protein from the column. All three samples are tested with a rabbit antiserum to normal chicken serum in the second dimension.
We were now able to test show complement activation of normal serum.
this factor B-depleted serum for the ability to and to compare with the results from activation
C3 conversion was analysed in crossed immuno-electrophoresis against a rabbit antiserum to chicken C3. All activators used (Cobra venom factor, Lipopolysaccharide, Inulin and mammalian erythrocytes) gave rise to a clear C 3 - c o n v e r s i o n when incubated with normal serum, whereas no C3-conversion at all was seen when factor B-depleted serum was analysed. Normal chicken serum had a strong lyric activity when incubated with human or sheep red cells. This lyric activity was completely abolished when factor B was removed from the serum. The subsequent addition of factor B (either p h y s i c o - c h e m i c a l purified factor B or factor B eluted from the immunosorbent column) gave a complete restoration of the haemolytic activity. Haemagglutination-enhancing factor protein (HEF) is a factor in normal chicken serum which greatly enhances the a g g l u t i n a t i n g capacity of chicken IgG antibodies (2). When HEF activity was assayed in normal and factor B-depleted serum, they showed identical activity. Also C l - a c t i v i t y as assayed in a mixed complement assay with chicken serum as the source of Clq and guinea pig serum as the source of the rest of the classical complement components, indicated identical levels of Clq in normal and factor B-depleted serum. In conclusion, our results confirm earlier observations, that chicken serum has a powerful alternative complement system, whereas classical, antibody d e p e n d e n t complement function could not be demonstrated. References 1.
Koch, C., Josephsen, J., Nicolaisen, E.M., and Simonsen, M. Complement mediated lysis in chickens. Devel. Comp. Immunol. 6, 141, 1982. Nicolaisen, E.M. and Koch, C. H a e m a g g l u t i n a t i o n - e n h a n c i n g tein in chicken serum. Scand.J.Immunol. 13, ll, 1981.
factor pro-
THE ALTERNATIVE COMPLEMENT PATHWAY IN CHICKENS.
Claus Koch,
Leif Kongerslev and Lisbeth Bjerring Jensen.
Institute for Experimental Immunology, 71, DK-2100 Copenhagen 0, Denmark.
University
of Copenhagen,
Notre Alle
In a previous communication we have presented data on complement mediated lysis in chickens (1). We demonstrated a functional alternative complement pathway (ACP) which was activated when mammalian erythrocytes were injected intravenously into chickens or when chicken serum was incubated in vitro with mammalian erythrocytes. Antibodies did not seem to play any role in this reaction, and various observations lead us to doubt the functional existence in this lytic reaction of the classical complement pathway. Physico-chemical purification (PEG-precipitation, ion-exchange chromatography on CM 52 cellulose, g e l f i l t r a t i o n on Sephadex G 200, elution from a Con A column and finally ion-exchange chromatography) lead to a highly purified chicken factor B preparation wich had the capacity to re-establish the lytic activity of heat inactivated chicken serum. Immunization of rabbits with this factor B preparation gave rise to a monospecific antiserum which could be shown by various criteria to detect chicken factor B. Chicken factor B turned out to be a glycoprotein with a molecular weight of about 105.000. Upon activation of the ACP factor B is split into two fragments, Ba which has alfa-mobility and has a molecular weight around 34.000, and Bb with g a m m a - m o b i l i t y and a molecular weight around 58.000. Molecular weights of Ba and Bb were determined after SDS-PAGE of ACP-activated chicken serum and subsequent immunoblotting and staining with the monospecific antibody and enzyme-linked secondary antibody. In order to be able to dissect classical complement activity and ACP sctivity we then wanted to produce a monoclonal antibody to chicken factor B. After fusion of spleen cells from mice, immunized with the purified chicken factor B, with myeloma cells, we obtained 4 different clones which produced antibodies with reactivity against factor B. Three antibodies reacted with Ba and one with Bb. The monoclonal antibody with reactivity against Bb was coupled to cyanogenbromide activated Sepharose, and an immunosorbent column with the capacity to specifically remove factor B from chicken serum was made. 785
786
COMPLEMENT
IN CHICKENS
Vol.
7, No. 4
Fig. 1 shows normal chicken serum before and after passage through this column ( la and ib) and the eluted antigen (ic). The column turned out to have the capacity to deplete completely i00 ml of chicken serum of factor B.
Fig.
i.
Normal chicken serum before (la) and after (ib) passage through an immunosorbent column with monoclonal antibodies to chicken factor B. ic shows the eluted protein from the column. All three samples are tested with a rabbit antiserum to normal chicken serum in the second dimension.
We were now able to test show complement activation of normal serum.
this factor B-depleted serum for the ability to and to compare with the results from activation
C3 conversion was analysed in crossed immuno-electrophoresis against a rabbit antiserum to chicken C3. All activators used (Cobra venom factor, Lipopolysaccharide, Inulin and mammalian erythrocytes) gave rise to a clear C 3 - c o n v e r s i o n when incubated with normal serum, whereas no C3-conversion at all was seen when factor B-depleted serum was analysed. Normal chicken serum had a strong lyric activity when incubated with human or sheep red cells. This lyric activity was completely abolished when factor B was removed from the serum. The subsequent addition of factor B (either p h y s i c o - c h e m i c a l purified factor B or factor B eluted from the immunosorbent column) gave a complete restoration of the haemolytic activity. Haemagglutination-enhancing factor protein (HEF) is a factor in normal chicken serum which greatly enhances the a g g l u t i n a t i n g capacity of chicken IgG antibodies (2). When HEF activity was assayed in normal and factor B-depleted serum, they showed identical activity. Also C l - a c t i v i t y as assayed in a mixed complement assay with chicken serum as the source of Clq and guinea pig serum as the source of the rest of the classical complement components, indicated identical levels of Clq in normal and factor B-depleted serum. In conclusion, our results confirm earlier observations, that chicken serum has a powerful alternative complement system, whereas classical, antibody d e p e n d e n t complement function could not be demonstrated. References 1.
Koch, C., Josephsen, J., Nicolaisen, E.M., and Simonsen, M. Complement mediated lysis in chickens. Devel. Comp. Immunol. 6, 141, 1982. Nicolaisen, E.M. and Koch, C. H a e m a g g l u t i n a t i o n - e n h a n c i n g tein in chicken serum. Scand.J.Immunol. 13, ll, 1981.
factor pro-