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Electron microscopic demonstration of phagocytosis of Candida albicans by human eosinophilic leukocytes
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Electron microscopic demonstration of phagocytosis of Candida albicans by human eosinophilic leukocytes Takeru
Ishikawa,
M.D.,*
Mang
C. Yu, Ph.D., and Carl E. Arbesman,
M.D.
Buffalo, N. Y.
With the use of electron microscopy uw have shown that human eosiwphils can of Can&da organisms. By this method we of these organisms by eosinophils.
phagooytize antigen-antibody complexes have also shown the process of ingestion
Phagocytio activity of eosinophils to Can&da albicans-antibody complexes has been reported recently by using light microscopic and immunofluorescent techniques.l With the use of electron microscopy, it has been reported previously that eosinophils can phagocytize zymosan particles,2 Mycop1asma,3 and soluble ferritin-antif erritin complexes in vivo,4 and human eosinophils have been shown to phagocytize antigen-antibody complexes in vitro.5 In this report, we have confirmed the phagocytosis of Candida organisms by human eosinophils employing electron microscopy. In addition, the process of ingestion of these organisms by the eosinophil was observed. MATERIALS
AND
METHODS
Heparinized blood was obtained from patients with high eosinophilia (10 to 24 per cent), placed in plastic tubes, and allowed to stand for 30 minutes to 1 hour. Leukocytes in plasma were separated by centrifugation at 100 G for 10 minutes and washed 3 times with Tris (Tris ACM). The cell buffer containing human serum albumin, calcium, and magnesium count was adjusted to approximately 2 x 107 per milliliter. Can&da albioans, 1 x 109 spore3 per milliliter, was suspended in normal saline.1 Sera containing a high titer of IgG antibodies to Candida, estimated by indirect immunofluoreS(!ence,l were used for preparation of the antigen-antibody complexes. Candida
From the St.ate University of New York at Buffalo and the Allergy Research Laboratory and the Department of Neurosurgery of the Buffalo General Hospital. Supported in part by United States Public Health Service Research Grant 5-ROl-AI-01303 and in part by United States Public Health Service Training Grant 5-TOl-AI-00051. Received for publication March 17, 1972. Reprint request to: Dr. Carl E. Arbesman, 100 High St., Buffalo, N. Y. 14203. **Recipient of the Dr. Henry C. Buswell and Bertha H. Buswell Research Fellowship.
Vol. 50, No. 3, pp. 183-187
184
Ishikawa,
J. ALLERGY CLIN. IMMUNOL. SEPTEMBER 1972
Yu, and Arbesman
FIG. 1. Light microscopy of phagocytosis of Candida philic leukocyte; C, Candida organism. (Xl ,000.)
organisms
FIG. 2. A Candida organism is completely engulfed into A granule, arrow, appears to be in the process of being Candida organism. C, Candida organism; N, nucleus eosinophils; V, formed vacuole; X, electron-dense bar in x8,500.)
by eosinophils.
E, Eosino-
the cytoplasm of an eosinophil. released into the capsule of the of eosinophil; G, granules of granule. (Original magnification
suspension, 0.5 ml., wag mixed with 1.0 ml. of human sera containing antibodies and kept in the cold room (5” C.) for 2 hours. Leukocyte fraction, 1 ml., was suspended in the mixture and incubated at 37” C. for 15 minutes. After washing once with Tris ACM, the cells were spun down at 200 G for 20 minutes to obtain cell pellets in the bottom of the test tubes.
VOLUME 50 NUMBER 3
Phagocytosis
of Candida
albicans
185
FIG. 3. Two eosinophils become intermeshed as they both attempt to engulf the same Candida spore. C, Candida organism; N, nucleus of eosinophil; G, granules of eosinophils; X, electron-dense bar in granule. (Original magnification ~5,200.) The cell pellets were fixed in Millonig’s phosphate-buffered osmium tetroxide for 90 minutes.6 They were dehydrated in graded ethanol and embedded in Maraglas mixture. Thin sections were cut with a diamond knife, double-stained with uranyl acetate and lead citrate, and examined with a JEM T6S electron microscope.
RESULTS Phagocytosis of Candida organisms by eosinophilic leukocytes is clearly demonstrated in Figs. 1 to 4. Light microscopic demonstration of phagocytosis of Candida organisms, C, by 2 eosinophils, E, is shown in Fig. 1. Under the high resolution of the electron microscope, it is easy to distinguish eosinophils from other granulocytes, because of the characteristically electron-dense bars, X, in the granules of the eosinophils. In Fig. 2, a Candida organism is seen to be completely engulfed into the cytoplasm of the eosinophils. A part of this micrograph, indicated by an arrow, shows the initial contact of the granule components with the capsule of Candida spore. Figs. 3 and 4 show the competitive engulfment of a Candida spore by 2 eosinophi1.s. The eosinophil at the center of the micrograph has almost completely engulfed the Candida organism, which is also partly contacted by the cytoplasmic process of an adjacent eosinophil. The granules of the eosinophil in the micrograph at the top, right, can be seen attached to the capsule of the
186
Ishikawa,
Yu, and Arbesman
J. ALLERGY CLIN. IMMUNOL. SEPTEMBER 1972
FIG. 4. Serial section of the same cells as shown in Fig. 3. Arrow indicates the release of granules from an adjacent eosinophil. C, Candida organism; N, nucleus of eosinophils; X, electron-dense bar in granule. (Original magnification X7,800.)
organism (Fig, 3). In Fig. 4, the attached granule appears to be in the process of releasing its content. DISCUSSION Although there have been several reports on the phagocytic activity of eosinophils, there is at the present no unanimity of opinion. Some investigators7 *’ believe that phagocytic activity of eosinophils is rarely or never observed. On the other hand, the majority of studies indicate that eosinophils do have phagocytic function in regard to antigen-antibody complexes.1-5y g3lo We have demonstrated previously1 that Candida antibody complexes frequently were phagocytized by eosinophils. It was shown1 that the phagocytic activity of eosinophils parallels the serum anticandidal IgG titer but not titers of IgA or IgM antibodies. Anticandidal IgE activity was not detectable in any serum studied. At least the first 4 components of the complement system are also involved in the phagocytic process. The result of our present study demonstrated clearly the ability of the eosinophils to phagocytize Can&& dbicans. This process of phagocytosis consists of engulfing the organism, formation of a vacuole, and subsequent release of granules into the organism.
VOLUME 50 NUMBER 3
Phagocytosis
of Candida
albicans
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It is known that the major components in specific eosinophilic granules are peroxidase, ribonuclease, arylsulfatase, and cathepsin. The range of the hydrolytic enzymes of this granule is similar to that of neutrophilic leukocytes and rat liver lysosomes.ll LehreP recently demonstrated the ability of the eosinophils to kill Candida olbicans spores. The present electron micrographs show attachment of cell granules to the spores and also degranulation. This could represent an early stage of digestion of the spores. Actual destruction of the spores by eosinophils, however, was not seen in our experiments. Digestion of the spores by neutrophils was observed on many occasions. This would suggest that although eosinophils and neutrophils ingest the spores at a similar rate, the digestive activity of the neutrophil is greater. REFERENCES 1 Ishikawa, T., Dalton, A. C., and Arbesman, C. E.: Phagocytosis of Candida albicans by eosinophilic leukocytes, J. ALLERGYCLIN. IM&JNOL. 49: 3&i972. 2 Zucker-Franklin, D., and Hirsch, J. G.: Electron microscope studies on the degranulation of rabbit peritoneal leukocytes during phagocytosis, J. Exp. Med. 120: 569, 1964. 3 Zucker-Franklin, D., Davidson, M., and Thomas, L.: The interaction of Mycoplasmas with mammalian cells. I. Hela cells. neutroahils and eosinoahils. J. EXD. Med. 124: 521. 1966. 4 Sabesin, S. E.: A function of the eoiinophil: phagocitosis of antigen-antibody complexes, Proe. Sot. Exp. Biol. Med. 112: 667, 1963. 5 Ishikawa, T., Evans, R., Wither, K., and Arbesman, C. E.: In Vitro and in vivo adhesion of antigen-antibody complexes to eosinophils, Fed. Proc. 30: 2694, 1971. (Abst.) 6 Millonig, G.: Advantages of a phosphate buffer for 0~0, solutions in fixation, J. Appl. Physics 32: 1637, 1961. 7 Ross, R., and Klebanoff, S. J.: The eosinophilic leukocyte, J. Exp. Med. 124: 653, 1966. 8 Opie, E.: An experimental study of the relation of cells with eosinophil granulation to infection with an animal parasite (Trich. spir.), Am. J. Med. Sei. 127: 477, 1964. 9 Litt, M.: Eosinophils and antigen-antibody reactions, Ann. N. Y. Acad. Sci. 116: 964, 1964. 10 Archer, G. T., Nelson, M., and Johnston, J.: Eosinophils granule lysis in vitro induced by soluble antigen-antibody complexes, Immunology 17: 177, 1969. 11 Bloom, W., and Fawcett, D. W.: A textbook of histology, Philadelphia, 1969, W. B. Saunders Co., p. 150. 12 Lehrer, R. I.: Measurement of Candidacidal activity of specific leukocyte types in mixed cell populations. II. Normal and chronic granulomatous disease eosinophils, Infect. Immunol. 3: 800,197l.
Electron microscopic demonstration of phagocytosis of Candida albicans by human eosinophilic leukocytes Takeru
Ishikawa,
M.D.,*
Mang
C. Yu, Ph.D., and Carl E. Arbesman,
M.D.
Buffalo, N. Y.
With the use of electron microscopy uw have shown that human eosiwphils can of Can&da organisms. By this method we of these organisms by eosinophils.
phagooytize antigen-antibody complexes have also shown the process of ingestion
Phagocytio activity of eosinophils to Can&da albicans-antibody complexes has been reported recently by using light microscopic and immunofluorescent techniques.l With the use of electron microscopy, it has been reported previously that eosinophils can phagocytize zymosan particles,2 Mycop1asma,3 and soluble ferritin-antif erritin complexes in vivo,4 and human eosinophils have been shown to phagocytize antigen-antibody complexes in vitro.5 In this report, we have confirmed the phagocytosis of Candida organisms by human eosinophils employing electron microscopy. In addition, the process of ingestion of these organisms by the eosinophil was observed. MATERIALS
AND
METHODS
Heparinized blood was obtained from patients with high eosinophilia (10 to 24 per cent), placed in plastic tubes, and allowed to stand for 30 minutes to 1 hour. Leukocytes in plasma were separated by centrifugation at 100 G for 10 minutes and washed 3 times with Tris (Tris ACM). The cell buffer containing human serum albumin, calcium, and magnesium count was adjusted to approximately 2 x 107 per milliliter. Can&da albioans, 1 x 109 spore3 per milliliter, was suspended in normal saline.1 Sera containing a high titer of IgG antibodies to Candida, estimated by indirect immunofluoreS(!ence,l were used for preparation of the antigen-antibody complexes. Candida
From the St.ate University of New York at Buffalo and the Allergy Research Laboratory and the Department of Neurosurgery of the Buffalo General Hospital. Supported in part by United States Public Health Service Research Grant 5-ROl-AI-01303 and in part by United States Public Health Service Training Grant 5-TOl-AI-00051. Received for publication March 17, 1972. Reprint request to: Dr. Carl E. Arbesman, 100 High St., Buffalo, N. Y. 14203. **Recipient of the Dr. Henry C. Buswell and Bertha H. Buswell Research Fellowship.
Vol. 50, No. 3, pp. 183-187
184
Ishikawa,
J. ALLERGY CLIN. IMMUNOL. SEPTEMBER 1972
Yu, and Arbesman
FIG. 1. Light microscopy of phagocytosis of Candida philic leukocyte; C, Candida organism. (Xl ,000.)
organisms
FIG. 2. A Candida organism is completely engulfed into A granule, arrow, appears to be in the process of being Candida organism. C, Candida organism; N, nucleus eosinophils; V, formed vacuole; X, electron-dense bar in x8,500.)
by eosinophils.
E, Eosino-
the cytoplasm of an eosinophil. released into the capsule of the of eosinophil; G, granules of granule. (Original magnification
suspension, 0.5 ml., wag mixed with 1.0 ml. of human sera containing antibodies and kept in the cold room (5” C.) for 2 hours. Leukocyte fraction, 1 ml., was suspended in the mixture and incubated at 37” C. for 15 minutes. After washing once with Tris ACM, the cells were spun down at 200 G for 20 minutes to obtain cell pellets in the bottom of the test tubes.
VOLUME 50 NUMBER 3
Phagocytosis
of Candida
albicans
185
FIG. 3. Two eosinophils become intermeshed as they both attempt to engulf the same Candida spore. C, Candida organism; N, nucleus of eosinophil; G, granules of eosinophils; X, electron-dense bar in granule. (Original magnification ~5,200.) The cell pellets were fixed in Millonig’s phosphate-buffered osmium tetroxide for 90 minutes.6 They were dehydrated in graded ethanol and embedded in Maraglas mixture. Thin sections were cut with a diamond knife, double-stained with uranyl acetate and lead citrate, and examined with a JEM T6S electron microscope.
RESULTS Phagocytosis of Candida organisms by eosinophilic leukocytes is clearly demonstrated in Figs. 1 to 4. Light microscopic demonstration of phagocytosis of Candida organisms, C, by 2 eosinophils, E, is shown in Fig. 1. Under the high resolution of the electron microscope, it is easy to distinguish eosinophils from other granulocytes, because of the characteristically electron-dense bars, X, in the granules of the eosinophils. In Fig. 2, a Candida organism is seen to be completely engulfed into the cytoplasm of the eosinophils. A part of this micrograph, indicated by an arrow, shows the initial contact of the granule components with the capsule of Candida spore. Figs. 3 and 4 show the competitive engulfment of a Candida spore by 2 eosinophi1.s. The eosinophil at the center of the micrograph has almost completely engulfed the Candida organism, which is also partly contacted by the cytoplasmic process of an adjacent eosinophil. The granules of the eosinophil in the micrograph at the top, right, can be seen attached to the capsule of the
186
Ishikawa,
Yu, and Arbesman
J. ALLERGY CLIN. IMMUNOL. SEPTEMBER 1972
FIG. 4. Serial section of the same cells as shown in Fig. 3. Arrow indicates the release of granules from an adjacent eosinophil. C, Candida organism; N, nucleus of eosinophils; X, electron-dense bar in granule. (Original magnification X7,800.)
organism (Fig, 3). In Fig. 4, the attached granule appears to be in the process of releasing its content. DISCUSSION Although there have been several reports on the phagocytic activity of eosinophils, there is at the present no unanimity of opinion. Some investigators7 *’ believe that phagocytic activity of eosinophils is rarely or never observed. On the other hand, the majority of studies indicate that eosinophils do have phagocytic function in regard to antigen-antibody complexes.1-5y g3lo We have demonstrated previously1 that Candida antibody complexes frequently were phagocytized by eosinophils. It was shown1 that the phagocytic activity of eosinophils parallels the serum anticandidal IgG titer but not titers of IgA or IgM antibodies. Anticandidal IgE activity was not detectable in any serum studied. At least the first 4 components of the complement system are also involved in the phagocytic process. The result of our present study demonstrated clearly the ability of the eosinophils to phagocytize Can&& dbicans. This process of phagocytosis consists of engulfing the organism, formation of a vacuole, and subsequent release of granules into the organism.
VOLUME 50 NUMBER 3
Phagocytosis
of Candida
albicans
187
It is known that the major components in specific eosinophilic granules are peroxidase, ribonuclease, arylsulfatase, and cathepsin. The range of the hydrolytic enzymes of this granule is similar to that of neutrophilic leukocytes and rat liver lysosomes.ll LehreP recently demonstrated the ability of the eosinophils to kill Candida olbicans spores. The present electron micrographs show attachment of cell granules to the spores and also degranulation. This could represent an early stage of digestion of the spores. Actual destruction of the spores by eosinophils, however, was not seen in our experiments. Digestion of the spores by neutrophils was observed on many occasions. This would suggest that although eosinophils and neutrophils ingest the spores at a similar rate, the digestive activity of the neutrophil is greater. REFERENCES 1 Ishikawa, T., Dalton, A. C., and Arbesman, C. E.: Phagocytosis of Candida albicans by eosinophilic leukocytes, J. ALLERGYCLIN. IM&JNOL. 49: 3&i972. 2 Zucker-Franklin, D., and Hirsch, J. G.: Electron microscope studies on the degranulation of rabbit peritoneal leukocytes during phagocytosis, J. Exp. Med. 120: 569, 1964. 3 Zucker-Franklin, D., Davidson, M., and Thomas, L.: The interaction of Mycoplasmas with mammalian cells. I. Hela cells. neutroahils and eosinoahils. J. EXD. Med. 124: 521. 1966. 4 Sabesin, S. E.: A function of the eoiinophil: phagocitosis of antigen-antibody complexes, Proe. Sot. Exp. Biol. Med. 112: 667, 1963. 5 Ishikawa, T., Evans, R., Wither, K., and Arbesman, C. E.: In Vitro and in vivo adhesion of antigen-antibody complexes to eosinophils, Fed. Proc. 30: 2694, 1971. (Abst.) 6 Millonig, G.: Advantages of a phosphate buffer for 0~0, solutions in fixation, J. Appl. Physics 32: 1637, 1961. 7 Ross, R., and Klebanoff, S. J.: The eosinophilic leukocyte, J. Exp. Med. 124: 653, 1966. 8 Opie, E.: An experimental study of the relation of cells with eosinophil granulation to infection with an animal parasite (Trich. spir.), Am. J. Med. Sei. 127: 477, 1964. 9 Litt, M.: Eosinophils and antigen-antibody reactions, Ann. N. Y. Acad. Sci. 116: 964, 1964. 10 Archer, G. T., Nelson, M., and Johnston, J.: Eosinophils granule lysis in vitro induced by soluble antigen-antibody complexes, Immunology 17: 177, 1969. 11 Bloom, W., and Fawcett, D. W.: A textbook of histology, Philadelphia, 1969, W. B. Saunders Co., p. 150. 12 Lehrer, R. I.: Measurement of Candidacidal activity of specific leukocyte types in mixed cell populations. II. Normal and chronic granulomatous disease eosinophils, Infect. Immunol. 3: 800,197l.