Aust. N.Z. J. Med. (1978), 8, pp. 620-622
Alternative Pathway of Complement Activation by Candida Albicans Y. H. Thong" and A. Ferrantet From the Department of Paediatrics, University of Adelaide, The Adelaide Children's Hospital
SUmmary: Alternative pathway of complement activation by Candida albicans. Y H Thong and A. Ferrante, Aust N 2 J Med. 1978, 8, pp 620-622, Activation of the alternative pathway of complement by Candida albicans was examined using a chemotactic assay. Two serologically defined strains and eight clinical isolates of Candida albicans were used in these experiments. The results showed that all ten strains of Candida albicans were capable of alternative complement pathway activation. These findings may provide an insight into host resistance to this infection.
Candida albicans is an important fungal pathogen of man. Host resistance to this organism is complex and incompletely understood. Both cell-mediated immunity' and neutrophil polymorphs', have been shown to play a role in resistance to this infection. Chemotaxis, or the mechanism by which neutrophils are attracted to the site of infection, may determine the outcome of infection. Neutrophils in large numbers have been found in biopsies of Candida lesions in man and animal^.^. Denning and Davies6 described the generation of a chemotactic stimulus for neutrophils from the interaction of C. albicans and heat-labile serum factors, but only one study' has been done to examine complement activation by C. albicans. We have, therefore, studied the effects of C. albicans on serum complement. Activation of serum complement results in the production of the subcomponents C3a and CSa, which are chemotactic for neutrophils' and can be measured using the agarose technique.'-1° Materials and Methods Neutrophils were separated from heparinized blood obtained from healthy donors by hypaque-ficoll centrifugation." The cells were washed twice, resuspended in medium 199 *Senior Lecturer tscientific Officer Correspondence Dr Y H Thong, Department of Paediatrics, The Adelaide Children's Hospital, North Adelaide, SA 5006, Australia Accepted for publication 13 July, 1978
supplemented with 10% heat-inactivated foetal calf serum, and adjusted to a concentration of 4 x 10' cells mi-'. Two serologically defined strains and eight clinical isolates of C. albicans used in these experiments were obtained from Mrs. G. Kaminski of the Mycobgy Section, Adelaide Children's Hospital. They were maintained in agar slopes and cultured on Sabouraud agar plates at 37 for 48 hours for use in these experiments. The chemotactic stimulus was generated by incubating 1 x lo9 yeast cells of C. albicans in 0.5 ml pooled human serum for 30 min at 37°C. To determine the mechanism o f complement activation, the same pooled human serum was heated to 50°C for 20 min to inactivate properdin factor B of the alternative pathway; such treatment has no effect on the classical pathway.* In other experiments, we compared the chemotactic activity of pooled human serum with serum obtained fr6m a patient with hereditary deficiency of the second component of complement", essential for activation of complement via the classical pathway. This C2-deficient serum was tested in the laboratory of H. J. Muller-Eberhard using immunochemical techniques.13 In another set of experiments, pooled human serum was treated with 0.01 M EGTA in order to chelate calcium and interfere with the function of the calcium-dependent first component of complement, as previously de~cribed.'~ The chemotactic assay was performed using the agarose technique?, lo Briefly, 3 ml of 2% agarose solution was mixed with 3 ml medium 199containing 10% heat-inactivated foetal calf serum and allowed to set in 60 x 15 mm tissue culture dishes. Three holes, 2 . 3 mm diameter in size were punched 2 . 3 mm apart, measured edge-to-edge, with the help of a template. Neutrophils (2 x lo5 in 5 p l ) were delivered by micropipette to the centre well. The inner well received 5 p/of medium 199, while the outer well received 5 pl of the chemotactic stimulus. The culture dishes were placed in a humidified 5% C0,-air atmosphere at 37°C for two hours. The distance moved by the cells was measured with the aid of an eye-piece grid on a Nikon inverted microscope. The difference in migration distance towards the outer well and the inner well was taken as the index of chemotaxis, and expressed at mm/2 hr & SD of quadruplicate experiments. O
Results
It was found that human serum genetically deficient in C2 was able to promote a similar degree of chemotactic activity compared to pooled human serum (Table 1) for all ten strains of C. albicans tested. Since C2 is an essential complement component for classical pathway activation, the results provide indirect evidence of alternative pathway activation by C. albicans. Further confirmation is provided by heatinactivation of pooled human serum (50 "C for 20 min) to destroy factor B of the properdin
DECEMBER.
1978
COMPLEMENT ACTIVATION BY CANDIDA ALBICANS
TABLE 1 Generation of chemotactic activity by C. albicans from C2 deficient serum
C. albicans
Neutrophil chemotaxis (mm/2 hr &SD) C2 deficient Pooled serum serum -~
Serotype A Serotype B Clinical isolates 26565 26627 26588 26072 2608 1 26030 25555 25550
1 33+0 10 1 2 6 + 0 08
1 3 5 5 0 12 1 2 3 5 0 07
1.32k0.17 1.33k0.14 1.40+0.21 1,20+0.09 1.37k0.12 1.44k0.28 1.25k0.08 1.37k0.11
1.3350.14 1.2850.13 1.35k0.11 1.33t0.13 1.3050.06 1.31+0.12 1.30+0.09 1.2850.11
system. This resulted in almost total abolition of chemotactic activity (Table 2). A C1-bypass mechanism has been described by May and Frank14, in which activation of C1 by specific antibody can proceed along the direction of alternative pathway components, thereby by-passing C4 and C2. In order to exclude this possibility, pooled human serum was treated with EGTA which preferentially chelates calcium to a greater extent than magTABLE 2 Inability to generate chemotactic activity by C albicans from heat-inactivated (50'C for 20 min) serum
C albicans
Neutrophil chemotaxis (mm/2 h r f S D ) serum dilutions 1.4 Neat
Pooled human Serum
~
Serotype A Serotype B Clinical isolates 26565
26627 26588 26072 2608 1 26030 25555 25550
control heated control heated control heated control heated control heated control heated control heated control heated control heated control heated
0.15 i0.05 1.30f 0 . 0 6
l.12k0.05 0 0.42f0.10
0
0
1.401t0.12 0 1.28k0.06 0.13 kO.05 1.23f 0.07 0.27 f0.05 1 .32 f 0 . 0 5 0.13fO.05 1.35 f0.11
0 .8 8 f 0 .0 5
0 1 .37 i 0 . 0 7
0
1.45+0.10
0 1.27k0.05 0 1.30 f0.07 0
0
0.75 f0.05 0 0.73 f 0 . 0 5 0 0.67f0.05 0 0 .6 5 f 0 .0 5 0.67 0.05 0 0.26 f 0 . 0 3 0 0.55f0.05 0
62 1
nesium. This would interfere with the function of C1, which is calcium-dependent but not the alternative pathway, which requires magnesium. The results show that EGTA treatment did not affect the capacity of pooled human serum to generate chemotactic factors: neutrophil chemotaxis was 1.21 + O . 11 mm/2 hr (mean+SD of 7 experiments) in EGTA-treated serum compared to 1 '30 f0.29 mm/2 hr in untreated serum ( P > 0.1). This experiment provides further evidence for the direct activation of the alternative pathway by C. albicans. Discussion
The complement system consists of a group of 19 serum proteins with important functions in inflammati~n.'~They interact in an orderly sequential manner analogous to the cascade mechanism of blood coagulation. The activation of complement can occur by either the classical pathway involving the components C 142, or via the alternative pathway or properdin system. Neutrophils constitute an important mechanism of host resistance to i n f e ~ t i o n . ' ~ Serum antibody and complement potentiate neutrophil function by means of opsonic and chemotactic factors. Activation of the classical complement pathway occurs from the interaction of microorganisms with specific antibody. However, in the absence of specific antibody, the alternative pathway assumes a prominent role in defence. This role for the alternative complement pathway has been shown for some bacteria (PneumoCOCCUS'~) and other fungi (Cryptococcus neo-
form an^'^). Ray and Wuepper7 provided evidence for alternative pathway activation by C. albicans, using an immunoelectrophoretic technique to measure conversion of factor B of the alternative pathway during interaction of C. albicans with serum. In the present studies, we have used a neutrophil chemotactic assay to measure the generation of chemotactic factors by C. albicans. The findings support the contention that C. albicans is an efficient activator of the alternative complement pathway. The generation of chemotactic factors serves the important function of attracting neutrophils rapidly to the site of infection. Neutrophils possess excellent candidicidal mechanisms2. 3 , and activation of
622
THONG AND FERRANTE
complement would lead to more efficient phagocytosis and killing of fungus.'* Since the alternative complement pathway can function on the absence of specific antibody to C. albicans, this mechanism may play an important role in defence, especially in patients with low or absent serum antibody. References I . KIRKPATRICK, C H.. RICH. R R. and BENNETT,1 . E. (1971): Chronic rnucocutaneous candidiasis. model-building in cellular immunity. Ann. inrun M d . 74, 955 2. LCtmtm. R. I. and CLINE.M. J (1969): lnteraction ofCandida albicans with human leukocytes and serum, J . Burt. 89,996. 3 DAVIES.R. R. and DENNINC. T . J. V (1972). Candida albicans pnd the fungicidal activity of the blood, Sahouruudia 10, 301 4. MnlBArH, H. and KLIGMAN, A. (1962). The biology of experimental human cutaneous moniliasib (Candida albicans), Arch. Drrm 85, 233. 5. H U K L t Y . D. and FAUCI, A. (1975)- Disseminated candidiasis: 1 An experimental model in the guinea pig, J . infer!. DI., 131, 516. 6. D ~ ~ N N I T N.LI., V. and DAVIS, R R . (1973): Candida albicans and the chemotaxis of polymnrphonuclear neulrophils, Sahoumudia 11, 21 7. RAY, R. L. and WUFPPER,K D. (1976). Activation of the alternative (properdin) pathway of complement by Candida ahicans and related species. J invr\i Derm 67, 700.
Aust. N.Z. J. Med. (1978). 8.
VOL.
8,
NO.
6
8. MCI LER-EBFKHARU. H . J. (1976): Serum Complement. Texrhook oJ/mmunopatho1og.v (Meisher, P A. and Muller-Eberhard, H. J , eds.) pp. 45- 73. Grime & Strattoil, New York 9. NEtsoli, R. D , QUiE, P. G. and SIMMONS,K . (1975). Chemotaxia under agarose: a new and simple method for measuring chemotaxia and spontaneous migration of human polymorphonucledq leukocyte5 and monocytes, J . Immunol. 115, 1650. 10. JOHN,T . J . and SIEBER,0. F. (1976). Chemotactic migration of neutrophila under agarose, Lr/r Sci 18. 177. 11. BOYCM, A (1968): A one-stage procedure for isolation ofgranulocytes from human blood. General sedimentation of blood cells in an IG gravrty field. Scand J .
Alternative Pathway of Complement Activation by Candida Albicans Y. H. Thong" and A. Ferrantet From the Department of Paediatrics, University of Adelaide, The Adelaide Children's Hospital
SUmmary: Alternative pathway of complement activation by Candida albicans. Y H Thong and A. Ferrante, Aust N 2 J Med. 1978, 8, pp 620-622, Activation of the alternative pathway of complement by Candida albicans was examined using a chemotactic assay. Two serologically defined strains and eight clinical isolates of Candida albicans were used in these experiments. The results showed that all ten strains of Candida albicans were capable of alternative complement pathway activation. These findings may provide an insight into host resistance to this infection.
Candida albicans is an important fungal pathogen of man. Host resistance to this organism is complex and incompletely understood. Both cell-mediated immunity' and neutrophil polymorphs', have been shown to play a role in resistance to this infection. Chemotaxis, or the mechanism by which neutrophils are attracted to the site of infection, may determine the outcome of infection. Neutrophils in large numbers have been found in biopsies of Candida lesions in man and animal^.^. Denning and Davies6 described the generation of a chemotactic stimulus for neutrophils from the interaction of C. albicans and heat-labile serum factors, but only one study' has been done to examine complement activation by C. albicans. We have, therefore, studied the effects of C. albicans on serum complement. Activation of serum complement results in the production of the subcomponents C3a and CSa, which are chemotactic for neutrophils' and can be measured using the agarose technique.'-1° Materials and Methods Neutrophils were separated from heparinized blood obtained from healthy donors by hypaque-ficoll centrifugation." The cells were washed twice, resuspended in medium 199 *Senior Lecturer tscientific Officer Correspondence Dr Y H Thong, Department of Paediatrics, The Adelaide Children's Hospital, North Adelaide, SA 5006, Australia Accepted for publication 13 July, 1978
supplemented with 10% heat-inactivated foetal calf serum, and adjusted to a concentration of 4 x 10' cells mi-'. Two serologically defined strains and eight clinical isolates of C. albicans used in these experiments were obtained from Mrs. G. Kaminski of the Mycobgy Section, Adelaide Children's Hospital. They were maintained in agar slopes and cultured on Sabouraud agar plates at 37 for 48 hours for use in these experiments. The chemotactic stimulus was generated by incubating 1 x lo9 yeast cells of C. albicans in 0.5 ml pooled human serum for 30 min at 37°C. To determine the mechanism o f complement activation, the same pooled human serum was heated to 50°C for 20 min to inactivate properdin factor B of the alternative pathway; such treatment has no effect on the classical pathway.* In other experiments, we compared the chemotactic activity of pooled human serum with serum obtained fr6m a patient with hereditary deficiency of the second component of complement", essential for activation of complement via the classical pathway. This C2-deficient serum was tested in the laboratory of H. J. Muller-Eberhard using immunochemical techniques.13 In another set of experiments, pooled human serum was treated with 0.01 M EGTA in order to chelate calcium and interfere with the function of the calcium-dependent first component of complement, as previously de~cribed.'~ The chemotactic assay was performed using the agarose technique?, lo Briefly, 3 ml of 2% agarose solution was mixed with 3 ml medium 199containing 10% heat-inactivated foetal calf serum and allowed to set in 60 x 15 mm tissue culture dishes. Three holes, 2 . 3 mm diameter in size were punched 2 . 3 mm apart, measured edge-to-edge, with the help of a template. Neutrophils (2 x lo5 in 5 p l ) were delivered by micropipette to the centre well. The inner well received 5 p/of medium 199, while the outer well received 5 pl of the chemotactic stimulus. The culture dishes were placed in a humidified 5% C0,-air atmosphere at 37°C for two hours. The distance moved by the cells was measured with the aid of an eye-piece grid on a Nikon inverted microscope. The difference in migration distance towards the outer well and the inner well was taken as the index of chemotaxis, and expressed at mm/2 hr & SD of quadruplicate experiments. O
Results
It was found that human serum genetically deficient in C2 was able to promote a similar degree of chemotactic activity compared to pooled human serum (Table 1) for all ten strains of C. albicans tested. Since C2 is an essential complement component for classical pathway activation, the results provide indirect evidence of alternative pathway activation by C. albicans. Further confirmation is provided by heatinactivation of pooled human serum (50 "C for 20 min) to destroy factor B of the properdin
DECEMBER.
1978
COMPLEMENT ACTIVATION BY CANDIDA ALBICANS
TABLE 1 Generation of chemotactic activity by C. albicans from C2 deficient serum
C. albicans
Neutrophil chemotaxis (mm/2 hr &SD) C2 deficient Pooled serum serum -~
Serotype A Serotype B Clinical isolates 26565 26627 26588 26072 2608 1 26030 25555 25550
1 33+0 10 1 2 6 + 0 08
1 3 5 5 0 12 1 2 3 5 0 07
1.32k0.17 1.33k0.14 1.40+0.21 1,20+0.09 1.37k0.12 1.44k0.28 1.25k0.08 1.37k0.11
1.3350.14 1.2850.13 1.35k0.11 1.33t0.13 1.3050.06 1.31+0.12 1.30+0.09 1.2850.11
system. This resulted in almost total abolition of chemotactic activity (Table 2). A C1-bypass mechanism has been described by May and Frank14, in which activation of C1 by specific antibody can proceed along the direction of alternative pathway components, thereby by-passing C4 and C2. In order to exclude this possibility, pooled human serum was treated with EGTA which preferentially chelates calcium to a greater extent than magTABLE 2 Inability to generate chemotactic activity by C albicans from heat-inactivated (50'C for 20 min) serum
C albicans
Neutrophil chemotaxis (mm/2 h r f S D ) serum dilutions 1.4 Neat
Pooled human Serum
~
Serotype A Serotype B Clinical isolates 26565
26627 26588 26072 2608 1 26030 25555 25550
control heated control heated control heated control heated control heated control heated control heated control heated control heated control heated
0.15 i0.05 1.30f 0 . 0 6
l.12k0.05 0 0.42f0.10
0
0
1.401t0.12 0 1.28k0.06 0.13 kO.05 1.23f 0.07 0.27 f0.05 1 .32 f 0 . 0 5 0.13fO.05 1.35 f0.11
0 .8 8 f 0 .0 5
0 1 .37 i 0 . 0 7
0
1.45+0.10
0 1.27k0.05 0 1.30 f0.07 0
0
0.75 f0.05 0 0.73 f 0 . 0 5 0 0.67f0.05 0 0 .6 5 f 0 .0 5 0.67 0.05 0 0.26 f 0 . 0 3 0 0.55f0.05 0
62 1
nesium. This would interfere with the function of C1, which is calcium-dependent but not the alternative pathway, which requires magnesium. The results show that EGTA treatment did not affect the capacity of pooled human serum to generate chemotactic factors: neutrophil chemotaxis was 1.21 + O . 11 mm/2 hr (mean+SD of 7 experiments) in EGTA-treated serum compared to 1 '30 f0.29 mm/2 hr in untreated serum ( P > 0.1). This experiment provides further evidence for the direct activation of the alternative pathway by C. albicans. Discussion
The complement system consists of a group of 19 serum proteins with important functions in inflammati~n.'~They interact in an orderly sequential manner analogous to the cascade mechanism of blood coagulation. The activation of complement can occur by either the classical pathway involving the components C 142, or via the alternative pathway or properdin system. Neutrophils constitute an important mechanism of host resistance to i n f e ~ t i o n . ' ~ Serum antibody and complement potentiate neutrophil function by means of opsonic and chemotactic factors. Activation of the classical complement pathway occurs from the interaction of microorganisms with specific antibody. However, in the absence of specific antibody, the alternative pathway assumes a prominent role in defence. This role for the alternative complement pathway has been shown for some bacteria (PneumoCOCCUS'~) and other fungi (Cryptococcus neo-
form an^'^). Ray and Wuepper7 provided evidence for alternative pathway activation by C. albicans, using an immunoelectrophoretic technique to measure conversion of factor B of the alternative pathway during interaction of C. albicans with serum. In the present studies, we have used a neutrophil chemotactic assay to measure the generation of chemotactic factors by C. albicans. The findings support the contention that C. albicans is an efficient activator of the alternative complement pathway. The generation of chemotactic factors serves the important function of attracting neutrophils rapidly to the site of infection. Neutrophils possess excellent candidicidal mechanisms2. 3 , and activation of
622
THONG AND FERRANTE
complement would lead to more efficient phagocytosis and killing of fungus.'* Since the alternative complement pathway can function on the absence of specific antibody to C. albicans, this mechanism may play an important role in defence, especially in patients with low or absent serum antibody. References I . KIRKPATRICK, C H.. RICH. R R. and BENNETT,1 . E. (1971): Chronic rnucocutaneous candidiasis. model-building in cellular immunity. Ann. inrun M d . 74, 955 2. LCtmtm. R. I. and CLINE.M. J (1969): lnteraction ofCandida albicans with human leukocytes and serum, J . Burt. 89,996. 3 DAVIES.R. R. and DENNINC. T . J. V (1972). Candida albicans pnd the fungicidal activity of the blood, Sahouruudia 10, 301 4. MnlBArH, H. and KLIGMAN, A. (1962). The biology of experimental human cutaneous moniliasib (Candida albicans), Arch. Drrm 85, 233. 5. H U K L t Y . D. and FAUCI, A. (1975)- Disseminated candidiasis: 1 An experimental model in the guinea pig, J . infer!. DI., 131, 516. 6. D ~ ~ N N I T N.LI., V. and DAVIS, R R . (1973): Candida albicans and the chemotaxis of polymnrphonuclear neulrophils, Sahoumudia 11, 21 7. RAY, R. L. and WUFPPER,K D. (1976). Activation of the alternative (properdin) pathway of complement by Candida ahicans and related species. J invr\i Derm 67, 700.
Aust. N.Z. J. Med. (1978). 8.
VOL.
8,
NO.
6
8. MCI LER-EBFKHARU. H . J. (1976): Serum Complement. Texrhook oJ/mmunopatho1og.v (Meisher, P A. and Muller-Eberhard, H. J , eds.) pp. 45- 73. Grime & Strattoil, New York 9. NEtsoli, R. D , QUiE, P. G. and SIMMONS,K . (1975). Chemotaxia under agarose: a new and simple method for measuring chemotaxia and spontaneous migration of human polymorphonucledq leukocyte5 and monocytes, J . Immunol. 115, 1650. 10. JOHN,T . J . and SIEBER,0. F. (1976). Chemotactic migration of neutrophila under agarose, Lr/r Sci 18. 177. 11. BOYCM, A (1968): A one-stage procedure for isolation ofgranulocytes from human blood. General sedimentation of blood cells in an IG gravrty field. Scand J .
