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Rev Odontol Univ São Paulo vol.13 n.3 São Paulo July/Sept. 1999
Macrophage phagocytosis of Candida albicans. An in vitro study
Estudo in vitro da fagocitose de Candida albicans por macrófagos peritoneais ativados
WEINFELD, I.; BIRMAN, E. G.; PAULA, C. R. Macrophages phagocytosis of Candida albicans. An in vitro study. Rev Odontol Univ São Paulo, v. 13, n. 3, p. 233-238, jul./set. 1999.
Considering the role of macrophages in relation to fungi and the various utilized methodologies, the authors established an in vitro model to evaluate macrophage phagocytosis of Candida albicans. Activated macrophages were obtained from the peritoneal cavity of isogenic mice (A/Sn). Two different strains of Candida albicans serotype A and serotype B with different levels of pathogenicity in vivo and other similar characteristics were utilized in the study. Several microscopic fields containing about 200 macrophages were counted. The percentage of macrophages phagocytizing at least one viable or nonviable yeast cell determined an average number of phagocytized yeasts. Neutral red and fluorescein diacetate plus ethidium bromide were used for staining. It is possible to conclude that this is an efficient model related to the used methodology. The average number of yeasts in both strains were similar when inside macrophages, and there was a higher percentage of C. albicans serotype A phagocytosis, which was not experimentally pathogenic in vivo.
UNITERMS: Candida albicans; Macrophages; Phagocytosis.
The role of neutrophils and other defense cells in face of microorganisms has been experimentally studied in different models. The importance of yeast infections called the attention to macrophages and their function in C. albicans phagocytosis, since candidosis is an important marker in many diseases. The morphological characteristics of C. albicans yeasts are a relevant aspect that can affect the interaction between macrophage and fungi, leaving many doubts about the effectiveness of these cells in destroying budding yeasts (blastoconidium) or hyphae2,5,6,9,10,16,17,18,21,22. These complex mechanisms could be better understood when analyzing simplified systems and adding more variables to the system. The objective of this experiment was to evaluate a new model to study the role of macrophages facing two serotypes of C. albicans.
MATERIAL AND METHODS
Strains. Two strains of C. albicans (ICB-12A and ICB-156B) were utilized after growing for three days in Fava Netto medium23,24 at 37ºC. The final solution reached 18 x 106 yeasts/mL of PBS. Both strains were similar in relation to chlamydoconidium, germ tube formation, proteinase and phospholipase production, as well as adherence to Hella cells. They were different in experimental pathogenicity in vivo, antigenicity and serotype. Strain ICB-12, serotype A, was negative in experimental pathogenicity in mice, while strain ICB-156, serotype B, presented experimental pathogenicity, killing in nine days all tested animals15.
These strains have been previously studied under other aspects, as adhesion capacity and invasion to pre-established cellular lineage.
Phagocytosis assay. Peritoneal macrophages were obtained from a pool of cellular suspension of isogenic mice (A/Sn) inoculated with 1.3-ß glucan solution (1.5 mg glucan/animal) three days before the experiment. Animals were sacrificed with sulphuric ether and the peritoneal cavity washed with 3 mL of glycolated PBS. Then, 2 mL of peritoneal fluid was collected from each animal and maintained in ice (plastic tubes) during a maximum time of 20 min until the assays were performed. To control, crystal violet solution was added to one of the tubes utilized as reference to adjust macrophages concentration approximately at 9 x 106 cells/mL of PBS. Volumes of 150 µL macrophage suspension previously washed with PBS was incubated for 20 min at 25°C and put into glass slides which were on each well of a polyestirene plate. Eagle medium (0.9 mL) was added and the material incubated for 30 minutes at 37°C. Finally, 0.1 mL opsonized yeast serum sheep previously obtained from antigens 12 and 156, specific to strains ICB-12 and ICB-156 was added and incubated for 45 minutes at 37°C, being stained by neutral red for 15 minutes at 37°C8. After washing all glass slides with PBS, they were fixed with glutaraldehyde for 10 minutes. The cellular material was examined under phase microscopy, counting at least 200 cells in different fields. From this procedure the viable cells were uncolored and the nonviable stained red. Based on the viable and nonviable counted cells. The percentage of macrophages phagocytizing at least one yeast cell (P) was determined as well as the average number of yeasts observed inside these cells (F). The phagocytic index (I) was obtained and identified as: I = P x F13.
Other slides of both strains were stained with fluorescein diacetate and ethidium bromide solution4, and examined under fluorescence microscopy for 15 minutes. All the experiments were repeated five times, using a minimum of three slides for each strain.
Cells of phagocytized C. albicans ICB-12 represented an average of 37% ± 7, while strain ICB-156 21% ± 6. These percentages included viable and nonviable cells (Tables 1 and 2). A higher number of nonviable cells phagocytized by the macrophages was observed in both strains.
Utilizing the Student test, a significant level of 5% was observed comparing phagocytosis of both strains ICB-12A and ICB-156B.
The phagocytosis of strictly viable cells was not statistically significant when comparing both strains. Phagocytosis of nonviable cells, as well as completely viable and nonviable cells presented a high significance level (5%).
Phagocytic index was higher for strain ICB-12, serotype A (75.0 ± 15) compared to strain ICB-156, serotype B (42 ± 12) being statistically significant (5%).
The average number of yeast cells in each macrophage was similar for both strains. Nonviable yeast cells were observed in a higher frequence inside the macrophages, intensively stained by neutral red under the phase microscope or with an uniform red-orange color at fluorescence microscopy.
When the viable cells of strain ICB-12A were compared to the nonviable cells, the statistical analysis presented a level of 5%, the same occuring with strain ICB-156B for nonviable cells.
DISCUSSION AND CONCLUSIONS
The diversity of techniques refered in literature and the results obtained in relation to yeasts and macrophages lead to the development of this model based on previous studies23,24, enabling more defined patterns to phagocytosis studies in relation to C. albicans strains.
In order to create the best conditions for the assay, A/Sn mice were chosen for their capacity of phagocytosis compared to other animals23,24 and 1.3-b glucan solution has been chosen due to its specificity in similar trials.
In our study, macrophages from A/Sn mice were still viable after an hour in contact with the yeasts generally presenting one or two blastoconidium inside the cells, although occasionally five to six phagocytized yeasts were also found. STANLEY; HURLEY20 (1969) presented similar results, suggesting the hypothesis that macrophages are ready to defend but can act as vectors too, when yeast cells are not destroyed, thus collaborating to the proliferation and dissemination of these microorganisms. The utilized strains have been maintained in culture for more than a year, a factor which could accidentally modify their virulence, probably influencing the macrophage activity.
The serum for opsonization may not be essential to the intracellular killing of yeasts, as MORELLI; ROSENBERG11 (1971) observed. Nevertheless, it is an important factor to optimize the macrophages activity, as considered by THOMPSON; WILTON21 (1992). When not opsonized C. albicans was utilized, the phagocytosis by monocytes and macrophages presented a lower activity degree.
MORRISON; CUTLER12 (1981) verified that pre-heated serum, although less effective, offered a higher macrophage activity which was different when the serum was not used, representing an important requirement in experiments like this.
Few papers on phagocytosis identified serotypes of C. albicans and did not evaluate more than one serotype6,14. AUGER et al.1 (1983) reported serotype A as more virulent and HASENCLEVER; MITCHELL7 (1964) did not distinguish differences between the serotypes. In this study it was observed that serotype B was more virulent. Response will be found when using a greater number of strains and different serotypes, although the individual role of serotypes is not so important.
When considering the viability of yeast cells, neutral red offered better conditions as BORG et al.3 (1984), KOLOTILA et al.8 (1987) and WEINFELD et. al.23 (1994) previously confirmed. The fluorescence method is more accurate but restricts the working time, consequently influencing the experiment.
It was also verified a higher degree of macrophage phagocytosis in C. albicans strain ICB-12, serotype A, in comparison to C. albicans strain ICB-156, serotype B. This means no quantitative difference between the average number of yeast cells in both phagocytized strains, but a difference in the number of phagocytizing macrophages.
So, in view of these results, it was possible to conclude that the strain of C. albicans ICB-156 is more virulent when compared to ICB-12, as previously described by PIRES15 (1993). It has also been demonstrated that the phagocytosis index of virulent strains in vivo (ICB-156) was lower than the less virulent ones (ICB-12). SOUZA19 (1991) using intraperitoneal macrophages obtained similar results with a different methodology related to the collecting time of cells, as well as activation and opsonization, while LOURIA; BRAYTON10 (1964) confirmed this fact in relation to leucocytes.
Besides the studies conducted with experimental models and detailed observations in the literature, little is known about the virulence factors of C. albicans. The different virulence factors could be responsible for strain pathogenicity, with an influence on phagocytosis. It should be considered the in vivo characteristics of each host which allow conditions for yeasts colonization without inducing disease.
Changes in the behaviour of host defense cells could be observed regarding different strains. The phagocytosis index was higher for one of these strains, although this did not kill more cells. This fact brings perspectives to future studies with the objective of better understanding the yeast/macro-phage relation.
WEINFELD, I.; BIRMAN, E. G.; PAULA, C. R. Estudo in vitro da fagocitose de Candida albicans por macrófagos peritoneais ativados. Rev Odontol Univ São Paulo, v. 13, n. 3, p. 233-238, jul./set. 1999.
Tendo em vista a indefinida literatura em relação aos macrófagos e à fagocitose de fungos, bem como os resultados conflitantes frente a diversas metodologias empregadas, propusemo-nos a estabelecer um modelo experimental para estudar a fagocitose de Candida albicans. Utilizamos macrófagos peritoneais ativados, obtidos de camundongos isogênicos da linhagem A/SN e duas cepas de Candida albicans, sendo uma sorotipo A e a outra B, apresentando diferentes níveis de patogenicidade in vivo. Avaliamos a fagocitose pela contagem de macrófagos contendo em seu interior células vivas e/ou mortas, determinando-se o índice de fagocitose pela multiplicação da porcentagem de macrófagos que fagocitaram e do número médio de leveduras por macrófagos. Valemo-nos de corantes vitais como vermelho neutro e fluorescentes (diacetato de fluoresceína e brometo de etídio), examinando o material à luz da microscopia de fase e fluorescência. Pudemos concluir que este é um modelo eficiente, em face da metodologia utilizada, onde verificamos um comportamento diferente dos macrófagos em relação às duas cepas estudadas; a porcentagem e o índice de fagocitose dos macrófagos foram maiores frente à cepa de Candida albicans sorotipo A (não patogênica in vivo), quando comparada à de sorotipo B, embora o número médio de leveduras por macrófagos fosse semelhante para ambas as cepas.
UNITERMOS: Candida albicans; Macrófagos; Fagocitose.
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Recebido para publicação em 16/05/99
Aceito para publicação em 03/09/99
* PhD - Department of Pathology, School of Dentistry - Santa Cecília - Santos - SP, ** Chair Professor - Department of Stomatology, *** PhD - Department of Biomedical Sciences - University of São Paulo - USP, Brazil.