Vol. 18, No. 3 Printed in U.S.A.
INFECTION AND IMMUNrIy, Dec. 1977, p. 568-573 Copyright © 1977 American Society for Microbiology
Chernotactic Factor Produced by Candida albicans JIM E. CUTLER Department ofMicrobiology, Montana State University, Bozeman, Montana 59715
Received for publication 29 July 1977
Candida albicans was found to produce a substance that was chemotactically active for guinea pig polymorphonuclear neutrophils. The chemotactic factor was detected in culture filtrates of organisms grown under aeration and incubated at 37°C for at least 12 h. Nutrients found to be essential for the production of chemotactic factor included glucose, yeast extract, and a mixture of amino acids. Several strains of C. albicans isolated from humans were tested, and varying degrees of chemotactic activity were found to be associated with the culture filtrates. Only one of the eight isolates did not produce a measurable amount of chemotactic activity. Culture filtrates remained chemotactically active after several cycles of freezing and thawing and after heating at 90°C for 10 min. Substantial evidence is presented that the chemotactic activity is not dependent upon activation of complement. Candidiasis is a general term describing a variety of clinical conditions, all of which are caused by members of the genus Candida and most by the species C. albicans. Although Tlymphocyte-dependent, cell-mediated immune responses appear to be of primary importance in resistance to certain forms of candidiasis, notably chronic mucocutaneous candidiasis (3, 11, 12, 19), evidence has accumulated which indicates that phagocytic cells such as polymorphonuclear neutrophils (PMN) are important in resistance to various other forms of candidiasis (2, 13-15, 25). Because PMN appear to be important in nonspecific resistance to candidiasis, it would be desirable to know how PMN become attracted to a site of candidal activity. In biopsies of candidal lesions from either humans or laboratory animals, an abundance of neutrophils and macrophages is often apparent (6-8, 16). In candidal meningitis, spinal fluids are often suppurative, giving an initial impression of bacterial meningitis (26). Recently, workers have produced evidence indicating that C. albicans activates the alternative pathway of complement activation (5, 20, 23), resulting in the release of complement-derived, chemotactically active substances. These workers went on to speculate that the acute inflammatory response associated with candidal lesions is the consequence of complement activation. Indeed, some workers have suggested that Candida sp. do not themselves produce substances that are chemotactically active (20). However, a report has recently appeared which showed that sonic extracts of C. albicans were slightly chemotactic for PMN; culture filtrates were not found to be active (24).
In 1974, we reported on the development of a simple method for an in vitro evaluation of chemotaxis of PMN (4), a method that has since been used in several laboratories (9, 17, 21). By this method, we are able to show that C. albicans is capable of producing a substance that is chemotactically active for guinea pig neutrophils. Furthermore, the factor is not dependent upon heat-labile serum factors for its activity. MATERLAS AND METHODS Organisms. C. albicans 9938 was isolated from a skin lesion of a patient at the Mycology Unit, Tulane University. The remaining strains of C. albicans were obtained as gifts from T. G. Mitchell, Duke University, and were originally isolated from patients with various forms of candidiasis. Two strains of Saccharyomyces cerevisiae and one strain of C. krusei were obtained from the culture collection at Montana State University. The isolates were maintained at room temperature on Sabouraud dextrose slants and were transferred monthly. Preparation of PMN suspension. PMN used in the chemotactic tests were obtained from outbred guinea pigs (Hartley) as previously described (4). Animals (500 to 700 g) were injected intraperitoneally with 25 to 30 ml of 0.5% glycogen (Nutritional Biochemicals Corp., Cleveland, Ohio) in 0.15 M NaCl. After 4 to 12 h, peritoneal exudate cells were collected by lavage with 25 to 30 ml of Hanks balanced salt solution (HBSS; Grand Island Biological Co., Grand Island, N.Y.) plus 0.002 M ethylenediaminetetraacetic acid (Calbiochem, Los Angeles, Calif.). The exudate cells were pelleted by centrifugation at about 100 x g for 10 min and suspended in an equal volume of HBSS plus 5% fetal calf serum. The peritoneal cell suspension was kept on ice and used on the day of harvest. The percent neutrophils in the peritoneal cell suspension was determined by differential counts of Giemsastained smears, and viability was assessed by trypan
568
VOL. 18, 1977
blue exclusion. The peritoneal exudate cells were used only if they consisted of at least 85% PMN and if 98% or more of the cells excluded trypan blue. By examining Giemsa-stained agarose plates as reported previously (4), we found that over 98% of the cells that migrated in the chemotactic plates were neutrophils. Chemotactic assay. The chemotactic test was performed according to previous reports from this and other laboratories (4, 17), with minor differences. Briefly, 2.5 ml of melted 0.75% agarose (MCI Biomedical, Rockland, Maine) in tissue culture medium with Earle unmodified salts (M199, Grand Island Biological Co.) plus 5% fetal calf serum (final pH 7.2) was added to each of a number of plastic culture dishes (35 by 10 mm; Falcon Plastics, Oxnard, Calif.). After the medium gelled, three wells were cut (2.3-mm diameter) in a straight line equidistant from each other (4), and 10 pl of the neutrophil suspension was added to the center well. Control medium (M199) was added to one of the outside wells, and either control medium or chemotactic factor was added to the other outside well. The plates were incubated aerobically at 37°C in a humidified incubator under 5% CO2 for 1.5 to 3 h. After incubation, the distance that the neutrophils migrated toward the test well was compared with the distance of migration toward the well containing the control (neutral) medium. Measurements were done at x40 with an ocular micrometer calibrated in millimeters, as reported earlier (4). The results are expressed in two ways: first, as the distance of migration toward the test well minus migration toward the neutral well; and, second, as: (distance of migration toward the test well/distance of migration toward the neutral material) - 1. Values obtained from the latter calculation take into account the magnitude of the migration distances. Index values are interpreted as follows: values near zero indicate that chemotactic stimulation did not occur, values greater than zero, that there was positive chemotactic attraction; and values less than zero, that the test material negatively affected neutrophil migration. Conditions for the production of chemotactic factor. C. albicans 9938 was grown in Sabouraud dextrose broth for 3 days at 37°C. During incubation, the cultures were aerated by rotating the flasks at 180 rpm. The cells were harvested by centrifugation and some were heat killed (60°C, 20 min). Both viable and heat-killed C. albicans were washed three times with sterile saline. The viable C. albicans was suspended in HBSS to a concentration of 2 x 108/ml, and the heat-killed cells were suspended in HBSS to a concentration of 1 x 109/ml. Ten microliters of each cell suspension was tested for chemotactic activity. Cell-free culture filtrates were also tested. After growth at room temperature on Sabouraud dextrose slants, C. albicans was harvested, washed three times with sterile saline, and suspended to 2 x 108 yeast cells per ml. One drop (0.05 ml) of this suspension was used to inoculate 3-ml portions of filter-sterilized medium containing one or more of the following: glucose, yeast extract (Difco Laboratories, Detroit, Mich.), peptone (Difco), vitamins (BME Vitamin Mixture 5Ox, Microbiological Associates, Inc., Bethesda, Md.), amino acids (BME Amino Acid Mixture 10Ox, Microbiological Associates), HBSS, or tissue culture
C. ALBICANS CHEMOTACTIC FACTOR
569
medium (TC199, Grand Island Biological Co.). The inoculated media, contained in 15-ml flasks, were incubated for various periods of time at 37°C in an incubator shaker (New Brunswick Scientific Co., New Brunswick, N.J.) and aerated by rotating the flasks at 160 rpm. After incubation, the cultures were checked for contamination by direct microscopic examination, by plating onto blood agar and incubating at 37°C, and by plating onto Sabouraud dextrose agar and incubating at room temperature. After determination of pH, the cultures were centrifuged at 460 x g for 10 min, the packed-cell volumes were recorded, and the supernatants were removed and filtered through a 0.45-ttm membrane filter (Millipore Corp., Bedford, Mass.). The cell-free culture filtrates were then tested for chemotactic activity. The known chemotactic factor used in these studies was derived from culture filtrates of Escherichia coli grown in tissue culture medium as described by Keller and Sorkin (10). Determination of some physical characteristics of the fungal chemotactic factor. Fungal culture filtrates known to be chemotactically active for guinea pig neutrophils were dialyzed against HBSS. Three different dialysis tubings were used (Arthur H. Thomas Co., Philadelphia, Pa.), each with a different exclusion limit, specifically, 3,500, 8,000, and 12,000 daltons. Chemotactically active filtrates were also tested for stability by repeated cycles of freezing and thawing, by long-term storage (30 days) at 8°C, and by heating at various temperatures. Comparison of 8everal C. albicans strains. C. albicans strains designated 9938, 2252, Sweenie, 550, 4918, 3153, E-136, and E-139 were grown in Sabouraud dextrose broth at 37°C and aerated by rotation at 160 rpm for 48 to 72 h. Cells of the various strains were harvested by centrifugation, washed three times with sterile saline, and suspended in HBSS. An inoculum of each was prepared, consisting of 2 x 107 yeast cells in 0.05 ml of HBSS, added to a 15-ml flask containing 3.0 ml of filter-sterilized (0.45-jam membrane filter) medium (1% glucose, 0.3% yeast extract, and 0.1 ml of 100X amino acid mixture). After 24 h of incubation, all cultures were tested for contamination as described above, and the amount of growth as volume of packed cells and the pH of each were recorded. Culture filtrates were prepared, the pH of each was brought to neutrality by dropwise addition of either 0.1 N NaOH or 0.1 N HCI, and they were then tested for chemotactic activity.
RESULTS Production of chemotactic factor. All cultures were routinely tested for bacterial and fungal contamination; those that had evidence of contamination were discarded. When viable C. albicans 9938 was placed in the chemotactic well, guinea pig neutrophils became attracted toward the yeast cells (Table 1). By 3 h of incubation, extensive germ tube formation was noted of the fungal cells in the well, indicating that these cells were actively growing. Heatkilled cells did not induce a chemotactic response (Table 1). Culture filtrates of C. albicans
INFECT. IMMIJN.
CUTLER
570
at 370C for 18 h in TC199 were not found to be chemotactically active, and culture filtrates
grown
from TC199 containing 5% fetal calf serum were only minimally active (Table 1). Other media were then used to determine whether culture filtrates would contain detectable amounts of chemotactic material. Culture filtrates of C. albicans grown in glucose-yeast extract-peptone broth were chemotactically active (Table 2). By using various combinations of nutrients, it was determined that peptone could be replaced by a defined amino acid mixture, glucose was essential for optimum production of chemotactic factor, and media containing a defined mixture of vitamins in place of yeast extract did not appear to support the production of chemotactic factor as well as did yeast extract-containing media (Table 2). These data also show that production of chemotactic factor is not necessarily proportional to growth. By growing C. albicans in the glucose-yeast extract-amino acid medium at 37°C and harvesting culture filtrates at specified times of
incubation, it was found that chemotactic activity could be detected by 12 h of incubation but not 6 h or less. Chemotactic activity remained detectable in culture filtrates throughout the remainder of the 72-h incubation period. Activity could be detected in the 12-, 24-, 36and 72-h culture filtrates when tested undiluted or after a 1:4 dilution with HBSS. Upon a 1:8 dilution of each of the filtrates, chemotactic attraction was induced only by the 24-h filtrate. Complement independence of the chemotactic factor. Experiments were done in an attempt to rule out the possibility that the fungal chemotactic factor was dependent upon the complement cascade. The fetal calf serum used in the assay was heat inactivated (56°C, 60 min) before use. This treatment is known to inactivate Cl of the classical pathway as well as precursors of the alternative pathway (for example, 18). Also, because it is believed that cell wall material of C. albicans can activate the alternative pathway of complement activation and thus generate chemotactic factors (20, 23), C. albicans treated
TABLE 1. Attraction of guinea pig neutrophils toward actively growing C. albicans in vitro Migration toward test well - migration toward neutral well (mm)
Test material
Index of migration
0.34 (0.13)b Viable C. albicans (9938)a. ........................ 0.06 (0.05) Heat-kiRed C. albicans ... Culture filtrate 0.11 (0.05) .... .... TC199 + fetal calf serum .. 0.03 (0.05) TC119 .. Neutral material 0.01 (0.05) TC199 + fetal calf serum ......... 0.44 (0.10) E. coli chemotactic factor .. .... a 107 cells in 0.01 ml were added to the test well. b Number in parentheses indicates standard deviation of the mean of data collected separate experiments performed in duplicate each time.
0.55 (0.27)
0.04 (0.05) 0.17 (0.08) 0.04 (0.07) 0.01 (0.04) 0.48 (0.13)
from at least five
TABLE 2. Nutritional requirements for the production of neutrophil chemotactic factor by C. albicans Chemotaxis assay Culture mediuma
vol after Packed-cel incubation (mld)
Migration toward test well - migration toward neutral well (mm)
Index of migration
0.61 (0.29) 0.47 (0.20)b 0.36 (0.05) 0.67 (0.05) 0.16 (0.14) 0.13 (0.08) 0.07 Glu-vit-aa ........................ 0.06 0.09 (0.05) Glu-HBSS-aa ....................... 0.12 (0.06) 0.06 0.23 (0.02) 0.29 (0.02) Glu-ye-HBSS ............ 0.02 0.09 (0.05) 0.12 (0.06) HBSS-ye-aa ........................ 0.04 (0.02) 0.05 (0.02) Glu-ye-pep (uninoculated) 0.05 (0.03) 0.02 (0.02) Glu-ye-aa (uninoculated) ......... a An inoculum of 2 x 107 yeast cells in either HBSS or saline was added to 3.0 ml of filter-sterilized medium consisting variously of 1% glucose (glu), 0.3% yeast extract (ye), 0.1 ml of 10Ox amino acid mixture (aa), 1% peptone (pep), or 0.1 ml of 10OX vitamin mixture (vit). Culture filtrates were harvested after 36 h of incubation and tested for chemotactic activity. b Number in parentheses indicates standard deviation of the mean of data from at least two separate experiments performed in duplicate each time.
Glu-ye-pep .
Glu-ye-aa
..................
0.10 0.08
VOL. 18, 1977
C. ALBICANS CHEMOTACTIC FACTOR
either by heat (600C, 20 min) or lyophilization was suspended in HBSS and placed in the test well. Both of these treatments resulted in death of the C. albicans, as evidenced by the absence of growth upon plating onto Sabouraud dextrose agar plates. These two very different methods of killing were used in an attempt to rule out the possibility that a component of the cell wall necessary for complement activation was being lost during the killing process. Finally, bovine serum albumin was used as a replacement for fetal calf serum in some of the tests. The data obtained from these experiments strongly suggest that the chemotactic factor is not dependent upon activation of complement (Table 3). Physical characteristics of the chemotactic factor. The C. albicans chemotactic factor was not destroyed by heating at 900C for 10 min, storage at 80C for at least 2 weeks, or by three cycles of freezing and thawing. Activity was lost, however, upon dialysis at 80C against HBSS when dialysis tubing with an exclusion limit of either 12,000 or 8,000 daltons was used. Partial activity (0.20 index of migration) was
571
retained after dialysis when tubing with an exclusion limit of 3,500 daltons was used. Production of chemotactic factor by other strains of C. albican Excepting strain 2252, all strains of C. albicans isolated from humans produced detectable chemotactic factor when grown for 24 h at 370C in the glucoseyeast extract-amino acid medium (Table 4). Some strains were found to produce large numbers of germ tubes and hyphae, whereas only yeast cells were apparent after incubation of other strains. Production of chemotactic factor did not appear to relate directly to the resulting pH after incubation or the production of germ tubes and hyphae. Two S. cerevisiae strains and one C. krusei strain did not grow under these conditions, and filtrates collected after incubation were not chemotactically active.
DISCUSSION It is apparent from these studies that many strains of C. albicans have the capability, when grown in vitro, of producing an extracellular
TABLE 3. Independence on complement activation for production of C. albicans chemotactic factor Chemotaxis assay Protein in agarose me-
Test material
diuma
Migration toward test
well - migration toward neutral well (mm)
Index of migration
Viable C. albicans ......................... Heat-killed C. albicans ......... ........... Lyophilized C. albicans ....................
FCS 0.34 (0.13)b 0.55 (0.27) FCS 0.06 (0.05) 0.04 (0.05) FCS 0.01 (0.02) 0.03 (0.05) Culture filtrate" . BSA 0.30 (0.04) 0.75 (0.08) Bovine serum albumin was not chemotactically active. FCS, Fetal calf serum; BSA, bovine serum albumin. b Number in parentheses indicates standard deviation of the mean of data from at least two separate experiments run in duplicate each time. c 24-h culture filtrate of C. albicans 9938 grown in glucose-yeast extract-amino acid medium. TABLE 4. Production of chemotactic factor by various human isolates of C. albicans Chemotaxis assay Strain of C. albicans
Packed-cell vol (ml)
pH after in- Evidence of Migration toward test cubation germinationa well - migration toward neutral well (mm) 1+ 6.8 0.44 (0.10)b 6.8 None 0.08 (0.05) 7.4 None 0.23 (0.03) 1+ 0.30 (0.05) 8.6 7.6 4+ 0.36 (0.02) 8.6 None 0.23 (0.05) 8.5 1+ 0.27 (0.04) None 8.6 0.27 (0.08)
Index of migration
0.65 (0.07) 0.10 (0.07) 0.32 (0.10) Sweenie 0.48 (0.19) 550 0.56 (0.13) 4918 0.33 (0.10) 3153 0.46 (0.23) E-136 0.43 (0.19) E-139 8.8 0.03 (0.02) 0.05 (0.03) Uninoculated medium a Approximated by wet-mount exam at X430. 1+, Less than 1 germ tube per field; 4+, more than 10 per field. b Number in parentheses indicates standard deviation of the mean of data from two separate experiments run in duplicate each time.
9338 2252
0.10 0.05 0.08 0.09 0.06 0.07 0.06 0.09
572
CUTLER
substance (or substances) that attracts guinea pig neutrophils. The chemotactic factor was found to be actively produced when the fungus was grown in a complex medium consisting of glucose, yeast extract, and peptone. Peptone could be replaced by an amino acid mixture, but glucose and yeast extract were required for optimum production. We have not yet successfully developed a chemically defined medium that consistently supports the production of optimum levels of chemotactic factor. It is of interest that, when viable yeast cells were placed in the test well of the agarose chemotactic assay, guinea pig neutrophils became attracted toward the actively growing cells. However, culture filtrates of C. albicans grown in the tissue culture medium used in the assay, but without agarose, yielded little if any evidence of chemotactic factor. Although data are lacking at this time, we believe that chemotactic factor is produced in low concentrations when the organism is grown in the tissue culture medium, but, because only 10 pl of the culture filtrate is added to the test well, the factor becomes too dilute by the time it reaches the neutrophils. Actively growing cells in the test well, on the other hand, would provide a constant supply of chemotactic factor that could be detected by the neutrophils. Chemotactically active culture filtrates were detected by 12 h of incubation at 37°C. This might reflect either that 12 h of incubation was required for sufficient cell density to provide enough factor for detection in the test system, or that chemotactic factor begins to be produced only at a certain time in the growth cycle of the organism. In support of the latter possibility is the observation that 24-h culture filtrates had a greater activity of chemotactic factor than either 12- or 72-h filtrates. The fungal chemotactic factor appears to be amenable to purification and characterization techniques. It was not inactivated by freezing and thawing, storage at 80C, or heating at 900C. It also appears to be of relatively low molecular weight, since activity was lost upon dialysis. Low-molecular-weight chemotactic factors have been also described from nonfungal microorganisms such as E. coli (22). Our data showing that C. albicans produces a substance that is itself chemotactically active are in contrast to findings by other workers (5, 20), who report that chemotactically active substances were produced by C. albicans only if the organism was incubated in the presence of fresh serum. Serum inactivated by heat at 560C did not support the production of chemotactic factors. Presumably the mechanism involved is due to the activation of complement via the classical pathway, if natural or induced antibody is present, or via the altemative pathway in much the same fashion
INFECT. IMMUN.
as activation induced by zymosan. Indeed, recent reports have suggested that C. albicans does activate the alternative pathway (20, 23), a finding that should not be surpnsing since cell walls of S. cerevisiae (zymosan) are known to activate the pathway (18). Workers in one laboratory have even suggested that complement activation is the only way that C. albicans could promote the production of chemotactic factor, and that the organism does not produce substances that are directly active (20). We have provided data which strongly suggest that the chemotactic activity reported here is not associated with complement activation. Not only was activity recorded when heat-inactivated serum was used in the test system, but culture filtrates also showed activity when serum was replaced by albumin in the assay system. Furthermore, the presence of dead cells in the test well did not cause measurable attraction of neutrophils, and one of the strains of C. albicans, although it grew well, did not produce detectable chemotactic activity. Therefore, our data give evidence for the first time of chemotactic activity associated with culture filtrates of C. albicans. A recent study has provided evidence that sonic extracts of C. albicans are slightly chemotactically active, but the workers on this study found that culture filtrates were not chemotactic (24). Perhaps the lack of activity associated with culture filtrates in the studies by Sohnle et al. (24) was due to the absence of yeast extract in the growth medium. The importance of candidal chemotactic factors in the pathogenesis of candidiasis is unknown at this time; our observations have been made with in vitro systems. At a speculative level, if these substances are produced in vivo and do relate to virulence, then it would seem that those isolates that produce the least amount of chemotactic activity would be the most virulent. It is noteworthy that of the strains tested in this study, there was some strain variation noted, and one strain (2252), which was isolated from a patient with disseminated candidiasis, did not produce measurable chemotactic activity. Some workers have noticed an inverse relation between virulency of isolates of C. albicans and the amount of suppuration induced upon injection into laboratory animals (1). ACKNOWLEDGMENTS This research was supported by a grant from the BrownHazen Fund of the Research Corporation. I thank N. Reed for critical comments and Jerrie Beyrodt for preparation of the manuscript. LITERATURE CITED 1. Albano, M., and J. Schmitt. 1973. Pathogenicity in mice of strains of Candida albicans (Robin) Berk. isolated from burn patients. Mycopathol. Mycol. Appl.
VOL. 18, 1977 49:283-288. 2. Belcher, R., J. Carney, and F. Monahan. 1973. An electron microscopic study of phagocytosis of Candida albicans by polymorphonuclear leukocytes. Lab. Invest. 29:620-627. 3. Chilgren, R. A., P. G. Quie, H. J. Meuwissen, and R. Hong. 1967. Chronic mucocutaneous candidiasis, deficiency of delayed hypersensitivity and selective local antibody defect. Lancet ii:688-693. 4. Cutler, J. E. 1974. A simple in vitro method for studies on chemotaxis. Proc. Soc. Exp. Biol. Med. 7:471-474. 5. Denning, T., and R. R. Davies. 1973. Candida albicans and the chemotaxis of polymorphonuclear neutrophils. Sabouraudia 11:210-221. 6. Drake, T. E., and H. I. Maibach. 1973. Candida and candidiasis. 2. Clinical manifestations and therapy of candidal disease. Postgrad. Med. 53:120-125. 7. Emmons, C. W., C. Binford, and J. P. Utz. 1970. Medical mycology, 2nd ed., p. 172. Lea and Febiger, Philadelphia. 8. Hurley, D., and A. Fauci. 1975. Disseminated candidiasia. I. An experimental model in the guinsa pig. J. Infect. Dis. 131:516-521. 9. John, T., and 0. Sieber. 1976. Chemotactic migration of neutrophils under agarose. Life Sci. 18:177-182. 10. Keller, H. U., and E. Sorkin. 1967. Studies on chemotaxis. V. On the chemotactic effect of bacteria. Int. Arch. Allergy Appl. Immunol. 31:505-517. 11. Kirkpatrick, C. H., R. R. Rich, and J. E. Bennett. 1971. Chronic mucocutaneous candidissia: model-building in cellular immunity. Ann. Intern. Med. 74:955-978. 12. Kroll, J. J., J. M. Einbinder, and W. G. Merz. 1973. Mucocutaneous candidiasis in a mother and son. Arch. Dermatol. 8:259-262. 13. Lehrer, R., and M. J. Cline. 1969. Leukocyte myeloperoxidase deficiency and disseminated candidiasis: the role of myeloperoxidase in resistance to Candida infection. J. Clin. Invest. 48:1478-1488. 14. Lehrer, R. I. 1971. Inhibition by sulfonamides of the candidacidal activity of human neutrophils. J. Clin. Invest. 50:2498-2505. 15. Lehrer, R. I., and M. J. Cline. 1969. Interaction of
C. ALBICANS CHEMOTACTIC FACTOR
16. 17.
18. 19.
20.
21.
22.
573
Candida albicans with human leukocytes and serum. J. Bacteriol. 98:996-1004. Maibach, H., and A. Kligman. 1962. The biology of experimental human cutaneous moniliasis (Candida albicans). Arch. Dermatol. 85:233-257. Nelson, R. D., P. G. Quie, and K. Simmons. 1975. Chemotaxis under agarose: a new and simple method for measuring chemotaxis and spontaneous migration of human polymorphonuclear leukocytes and monocytes. J. Immunol. 115:1650-1656. Osler, A. G. 1976. Complement-mechanisms and functions. Prentice-Hall, Inc., Englewood Cliffs, N.J. Provost, T. T., L Garrettson, R. Zeschke, N. Rose, and T. Tomasi. 1973. Combined immune deficiency, autoantibody formation and mucocutaneous candidiasis. Clin. Immunol. Immunopathol. 1:429-445. Ray, T. L, and K. D. Wuepper. 1976. Activation of the alternative (properdin) pathway of complement by Candida albicans and related species. J. Invest. Dermatol. 67:700-703. Repo, H. 1977. Leukocyte migration agarose test for the assessment of human neutrophil chemotaxis. I. Effects of environmental factors on neutrophil migration under agarose. Scand. J. Immunol. 6:203-209. Schiffmann, E., H. V. Showell, B. A. Corcoran, P. A. Ward, E. Smith, anmd E. L Becker. 1975. The isolation and partial characterization of neutrophil chemotactic factors from Escherichia coli. J. Immunol. 114:1831-1837.
23. Sohnle, P. G., M. M. Frank, and C. H. Kirkpatrick. 1976. Deposition of complement components in the cutaneous lesions of chronic mucocutaneous candidiasis. Clin. Immunol. Immunopathol. 5:340-350. 24. Sohnle, P. G., M. M. Frank, and C. H. Kirkpatrick. 1976. Mechanisms involved in elimination of organisms from experimental cutaneous Candida albicans infections in guinea pigs. J. Immunol. 117:523-530. 25. Warden, G., A. Mason, and B. Pruitt. 1974. Evaluation of leukocyte chemotaxis in vitro in thermally injured patients. J. Clin. Invest. 54:1001-1004. 26. White, B. E. 1972. Cerebral candidiasis. N. Engl. J. Med.
286:321.
INFECTION AND IMMUNrIy, Dec. 1977, p. 568-573 Copyright © 1977 American Society for Microbiology
Chernotactic Factor Produced by Candida albicans JIM E. CUTLER Department ofMicrobiology, Montana State University, Bozeman, Montana 59715
Received for publication 29 July 1977
Candida albicans was found to produce a substance that was chemotactically active for guinea pig polymorphonuclear neutrophils. The chemotactic factor was detected in culture filtrates of organisms grown under aeration and incubated at 37°C for at least 12 h. Nutrients found to be essential for the production of chemotactic factor included glucose, yeast extract, and a mixture of amino acids. Several strains of C. albicans isolated from humans were tested, and varying degrees of chemotactic activity were found to be associated with the culture filtrates. Only one of the eight isolates did not produce a measurable amount of chemotactic activity. Culture filtrates remained chemotactically active after several cycles of freezing and thawing and after heating at 90°C for 10 min. Substantial evidence is presented that the chemotactic activity is not dependent upon activation of complement. Candidiasis is a general term describing a variety of clinical conditions, all of which are caused by members of the genus Candida and most by the species C. albicans. Although Tlymphocyte-dependent, cell-mediated immune responses appear to be of primary importance in resistance to certain forms of candidiasis, notably chronic mucocutaneous candidiasis (3, 11, 12, 19), evidence has accumulated which indicates that phagocytic cells such as polymorphonuclear neutrophils (PMN) are important in resistance to various other forms of candidiasis (2, 13-15, 25). Because PMN appear to be important in nonspecific resistance to candidiasis, it would be desirable to know how PMN become attracted to a site of candidal activity. In biopsies of candidal lesions from either humans or laboratory animals, an abundance of neutrophils and macrophages is often apparent (6-8, 16). In candidal meningitis, spinal fluids are often suppurative, giving an initial impression of bacterial meningitis (26). Recently, workers have produced evidence indicating that C. albicans activates the alternative pathway of complement activation (5, 20, 23), resulting in the release of complement-derived, chemotactically active substances. These workers went on to speculate that the acute inflammatory response associated with candidal lesions is the consequence of complement activation. Indeed, some workers have suggested that Candida sp. do not themselves produce substances that are chemotactically active (20). However, a report has recently appeared which showed that sonic extracts of C. albicans were slightly chemotactic for PMN; culture filtrates were not found to be active (24).
In 1974, we reported on the development of a simple method for an in vitro evaluation of chemotaxis of PMN (4), a method that has since been used in several laboratories (9, 17, 21). By this method, we are able to show that C. albicans is capable of producing a substance that is chemotactically active for guinea pig neutrophils. Furthermore, the factor is not dependent upon heat-labile serum factors for its activity. MATERLAS AND METHODS Organisms. C. albicans 9938 was isolated from a skin lesion of a patient at the Mycology Unit, Tulane University. The remaining strains of C. albicans were obtained as gifts from T. G. Mitchell, Duke University, and were originally isolated from patients with various forms of candidiasis. Two strains of Saccharyomyces cerevisiae and one strain of C. krusei were obtained from the culture collection at Montana State University. The isolates were maintained at room temperature on Sabouraud dextrose slants and were transferred monthly. Preparation of PMN suspension. PMN used in the chemotactic tests were obtained from outbred guinea pigs (Hartley) as previously described (4). Animals (500 to 700 g) were injected intraperitoneally with 25 to 30 ml of 0.5% glycogen (Nutritional Biochemicals Corp., Cleveland, Ohio) in 0.15 M NaCl. After 4 to 12 h, peritoneal exudate cells were collected by lavage with 25 to 30 ml of Hanks balanced salt solution (HBSS; Grand Island Biological Co., Grand Island, N.Y.) plus 0.002 M ethylenediaminetetraacetic acid (Calbiochem, Los Angeles, Calif.). The exudate cells were pelleted by centrifugation at about 100 x g for 10 min and suspended in an equal volume of HBSS plus 5% fetal calf serum. The peritoneal cell suspension was kept on ice and used on the day of harvest. The percent neutrophils in the peritoneal cell suspension was determined by differential counts of Giemsastained smears, and viability was assessed by trypan
568
VOL. 18, 1977
blue exclusion. The peritoneal exudate cells were used only if they consisted of at least 85% PMN and if 98% or more of the cells excluded trypan blue. By examining Giemsa-stained agarose plates as reported previously (4), we found that over 98% of the cells that migrated in the chemotactic plates were neutrophils. Chemotactic assay. The chemotactic test was performed according to previous reports from this and other laboratories (4, 17), with minor differences. Briefly, 2.5 ml of melted 0.75% agarose (MCI Biomedical, Rockland, Maine) in tissue culture medium with Earle unmodified salts (M199, Grand Island Biological Co.) plus 5% fetal calf serum (final pH 7.2) was added to each of a number of plastic culture dishes (35 by 10 mm; Falcon Plastics, Oxnard, Calif.). After the medium gelled, three wells were cut (2.3-mm diameter) in a straight line equidistant from each other (4), and 10 pl of the neutrophil suspension was added to the center well. Control medium (M199) was added to one of the outside wells, and either control medium or chemotactic factor was added to the other outside well. The plates were incubated aerobically at 37°C in a humidified incubator under 5% CO2 for 1.5 to 3 h. After incubation, the distance that the neutrophils migrated toward the test well was compared with the distance of migration toward the well containing the control (neutral) medium. Measurements were done at x40 with an ocular micrometer calibrated in millimeters, as reported earlier (4). The results are expressed in two ways: first, as the distance of migration toward the test well minus migration toward the neutral well; and, second, as: (distance of migration toward the test well/distance of migration toward the neutral material) - 1. Values obtained from the latter calculation take into account the magnitude of the migration distances. Index values are interpreted as follows: values near zero indicate that chemotactic stimulation did not occur, values greater than zero, that there was positive chemotactic attraction; and values less than zero, that the test material negatively affected neutrophil migration. Conditions for the production of chemotactic factor. C. albicans 9938 was grown in Sabouraud dextrose broth for 3 days at 37°C. During incubation, the cultures were aerated by rotating the flasks at 180 rpm. The cells were harvested by centrifugation and some were heat killed (60°C, 20 min). Both viable and heat-killed C. albicans were washed three times with sterile saline. The viable C. albicans was suspended in HBSS to a concentration of 2 x 108/ml, and the heat-killed cells were suspended in HBSS to a concentration of 1 x 109/ml. Ten microliters of each cell suspension was tested for chemotactic activity. Cell-free culture filtrates were also tested. After growth at room temperature on Sabouraud dextrose slants, C. albicans was harvested, washed three times with sterile saline, and suspended to 2 x 108 yeast cells per ml. One drop (0.05 ml) of this suspension was used to inoculate 3-ml portions of filter-sterilized medium containing one or more of the following: glucose, yeast extract (Difco Laboratories, Detroit, Mich.), peptone (Difco), vitamins (BME Vitamin Mixture 5Ox, Microbiological Associates, Inc., Bethesda, Md.), amino acids (BME Amino Acid Mixture 10Ox, Microbiological Associates), HBSS, or tissue culture
C. ALBICANS CHEMOTACTIC FACTOR
569
medium (TC199, Grand Island Biological Co.). The inoculated media, contained in 15-ml flasks, were incubated for various periods of time at 37°C in an incubator shaker (New Brunswick Scientific Co., New Brunswick, N.J.) and aerated by rotating the flasks at 160 rpm. After incubation, the cultures were checked for contamination by direct microscopic examination, by plating onto blood agar and incubating at 37°C, and by plating onto Sabouraud dextrose agar and incubating at room temperature. After determination of pH, the cultures were centrifuged at 460 x g for 10 min, the packed-cell volumes were recorded, and the supernatants were removed and filtered through a 0.45-ttm membrane filter (Millipore Corp., Bedford, Mass.). The cell-free culture filtrates were then tested for chemotactic activity. The known chemotactic factor used in these studies was derived from culture filtrates of Escherichia coli grown in tissue culture medium as described by Keller and Sorkin (10). Determination of some physical characteristics of the fungal chemotactic factor. Fungal culture filtrates known to be chemotactically active for guinea pig neutrophils were dialyzed against HBSS. Three different dialysis tubings were used (Arthur H. Thomas Co., Philadelphia, Pa.), each with a different exclusion limit, specifically, 3,500, 8,000, and 12,000 daltons. Chemotactically active filtrates were also tested for stability by repeated cycles of freezing and thawing, by long-term storage (30 days) at 8°C, and by heating at various temperatures. Comparison of 8everal C. albicans strains. C. albicans strains designated 9938, 2252, Sweenie, 550, 4918, 3153, E-136, and E-139 were grown in Sabouraud dextrose broth at 37°C and aerated by rotation at 160 rpm for 48 to 72 h. Cells of the various strains were harvested by centrifugation, washed three times with sterile saline, and suspended in HBSS. An inoculum of each was prepared, consisting of 2 x 107 yeast cells in 0.05 ml of HBSS, added to a 15-ml flask containing 3.0 ml of filter-sterilized (0.45-jam membrane filter) medium (1% glucose, 0.3% yeast extract, and 0.1 ml of 100X amino acid mixture). After 24 h of incubation, all cultures were tested for contamination as described above, and the amount of growth as volume of packed cells and the pH of each were recorded. Culture filtrates were prepared, the pH of each was brought to neutrality by dropwise addition of either 0.1 N NaOH or 0.1 N HCI, and they were then tested for chemotactic activity.
RESULTS Production of chemotactic factor. All cultures were routinely tested for bacterial and fungal contamination; those that had evidence of contamination were discarded. When viable C. albicans 9938 was placed in the chemotactic well, guinea pig neutrophils became attracted toward the yeast cells (Table 1). By 3 h of incubation, extensive germ tube formation was noted of the fungal cells in the well, indicating that these cells were actively growing. Heatkilled cells did not induce a chemotactic response (Table 1). Culture filtrates of C. albicans
INFECT. IMMIJN.
CUTLER
570
at 370C for 18 h in TC199 were not found to be chemotactically active, and culture filtrates
grown
from TC199 containing 5% fetal calf serum were only minimally active (Table 1). Other media were then used to determine whether culture filtrates would contain detectable amounts of chemotactic material. Culture filtrates of C. albicans grown in glucose-yeast extract-peptone broth were chemotactically active (Table 2). By using various combinations of nutrients, it was determined that peptone could be replaced by a defined amino acid mixture, glucose was essential for optimum production of chemotactic factor, and media containing a defined mixture of vitamins in place of yeast extract did not appear to support the production of chemotactic factor as well as did yeast extract-containing media (Table 2). These data also show that production of chemotactic factor is not necessarily proportional to growth. By growing C. albicans in the glucose-yeast extract-amino acid medium at 37°C and harvesting culture filtrates at specified times of
incubation, it was found that chemotactic activity could be detected by 12 h of incubation but not 6 h or less. Chemotactic activity remained detectable in culture filtrates throughout the remainder of the 72-h incubation period. Activity could be detected in the 12-, 24-, 36and 72-h culture filtrates when tested undiluted or after a 1:4 dilution with HBSS. Upon a 1:8 dilution of each of the filtrates, chemotactic attraction was induced only by the 24-h filtrate. Complement independence of the chemotactic factor. Experiments were done in an attempt to rule out the possibility that the fungal chemotactic factor was dependent upon the complement cascade. The fetal calf serum used in the assay was heat inactivated (56°C, 60 min) before use. This treatment is known to inactivate Cl of the classical pathway as well as precursors of the alternative pathway (for example, 18). Also, because it is believed that cell wall material of C. albicans can activate the alternative pathway of complement activation and thus generate chemotactic factors (20, 23), C. albicans treated
TABLE 1. Attraction of guinea pig neutrophils toward actively growing C. albicans in vitro Migration toward test well - migration toward neutral well (mm)
Test material
Index of migration
0.34 (0.13)b Viable C. albicans (9938)a. ........................ 0.06 (0.05) Heat-kiRed C. albicans ... Culture filtrate 0.11 (0.05) .... .... TC199 + fetal calf serum .. 0.03 (0.05) TC119 .. Neutral material 0.01 (0.05) TC199 + fetal calf serum ......... 0.44 (0.10) E. coli chemotactic factor .. .... a 107 cells in 0.01 ml were added to the test well. b Number in parentheses indicates standard deviation of the mean of data collected separate experiments performed in duplicate each time.
0.55 (0.27)
0.04 (0.05) 0.17 (0.08) 0.04 (0.07) 0.01 (0.04) 0.48 (0.13)
from at least five
TABLE 2. Nutritional requirements for the production of neutrophil chemotactic factor by C. albicans Chemotaxis assay Culture mediuma
vol after Packed-cel incubation (mld)
Migration toward test well - migration toward neutral well (mm)
Index of migration
0.61 (0.29) 0.47 (0.20)b 0.36 (0.05) 0.67 (0.05) 0.16 (0.14) 0.13 (0.08) 0.07 Glu-vit-aa ........................ 0.06 0.09 (0.05) Glu-HBSS-aa ....................... 0.12 (0.06) 0.06 0.23 (0.02) 0.29 (0.02) Glu-ye-HBSS ............ 0.02 0.09 (0.05) 0.12 (0.06) HBSS-ye-aa ........................ 0.04 (0.02) 0.05 (0.02) Glu-ye-pep (uninoculated) 0.05 (0.03) 0.02 (0.02) Glu-ye-aa (uninoculated) ......... a An inoculum of 2 x 107 yeast cells in either HBSS or saline was added to 3.0 ml of filter-sterilized medium consisting variously of 1% glucose (glu), 0.3% yeast extract (ye), 0.1 ml of 10Ox amino acid mixture (aa), 1% peptone (pep), or 0.1 ml of 10OX vitamin mixture (vit). Culture filtrates were harvested after 36 h of incubation and tested for chemotactic activity. b Number in parentheses indicates standard deviation of the mean of data from at least two separate experiments performed in duplicate each time.
Glu-ye-pep .
Glu-ye-aa
..................
0.10 0.08
VOL. 18, 1977
C. ALBICANS CHEMOTACTIC FACTOR
either by heat (600C, 20 min) or lyophilization was suspended in HBSS and placed in the test well. Both of these treatments resulted in death of the C. albicans, as evidenced by the absence of growth upon plating onto Sabouraud dextrose agar plates. These two very different methods of killing were used in an attempt to rule out the possibility that a component of the cell wall necessary for complement activation was being lost during the killing process. Finally, bovine serum albumin was used as a replacement for fetal calf serum in some of the tests. The data obtained from these experiments strongly suggest that the chemotactic factor is not dependent upon activation of complement (Table 3). Physical characteristics of the chemotactic factor. The C. albicans chemotactic factor was not destroyed by heating at 900C for 10 min, storage at 80C for at least 2 weeks, or by three cycles of freezing and thawing. Activity was lost, however, upon dialysis at 80C against HBSS when dialysis tubing with an exclusion limit of either 12,000 or 8,000 daltons was used. Partial activity (0.20 index of migration) was
571
retained after dialysis when tubing with an exclusion limit of 3,500 daltons was used. Production of chemotactic factor by other strains of C. albican Excepting strain 2252, all strains of C. albicans isolated from humans produced detectable chemotactic factor when grown for 24 h at 370C in the glucoseyeast extract-amino acid medium (Table 4). Some strains were found to produce large numbers of germ tubes and hyphae, whereas only yeast cells were apparent after incubation of other strains. Production of chemotactic factor did not appear to relate directly to the resulting pH after incubation or the production of germ tubes and hyphae. Two S. cerevisiae strains and one C. krusei strain did not grow under these conditions, and filtrates collected after incubation were not chemotactically active.
DISCUSSION It is apparent from these studies that many strains of C. albicans have the capability, when grown in vitro, of producing an extracellular
TABLE 3. Independence on complement activation for production of C. albicans chemotactic factor Chemotaxis assay Protein in agarose me-
Test material
diuma
Migration toward test
well - migration toward neutral well (mm)
Index of migration
Viable C. albicans ......................... Heat-killed C. albicans ......... ........... Lyophilized C. albicans ....................
FCS 0.34 (0.13)b 0.55 (0.27) FCS 0.06 (0.05) 0.04 (0.05) FCS 0.01 (0.02) 0.03 (0.05) Culture filtrate" . BSA 0.30 (0.04) 0.75 (0.08) Bovine serum albumin was not chemotactically active. FCS, Fetal calf serum; BSA, bovine serum albumin. b Number in parentheses indicates standard deviation of the mean of data from at least two separate experiments run in duplicate each time. c 24-h culture filtrate of C. albicans 9938 grown in glucose-yeast extract-amino acid medium. TABLE 4. Production of chemotactic factor by various human isolates of C. albicans Chemotaxis assay Strain of C. albicans
Packed-cell vol (ml)
pH after in- Evidence of Migration toward test cubation germinationa well - migration toward neutral well (mm) 1+ 6.8 0.44 (0.10)b 6.8 None 0.08 (0.05) 7.4 None 0.23 (0.03) 1+ 0.30 (0.05) 8.6 7.6 4+ 0.36 (0.02) 8.6 None 0.23 (0.05) 8.5 1+ 0.27 (0.04) None 8.6 0.27 (0.08)
Index of migration
0.65 (0.07) 0.10 (0.07) 0.32 (0.10) Sweenie 0.48 (0.19) 550 0.56 (0.13) 4918 0.33 (0.10) 3153 0.46 (0.23) E-136 0.43 (0.19) E-139 8.8 0.03 (0.02) 0.05 (0.03) Uninoculated medium a Approximated by wet-mount exam at X430. 1+, Less than 1 germ tube per field; 4+, more than 10 per field. b Number in parentheses indicates standard deviation of the mean of data from two separate experiments run in duplicate each time.
9338 2252
0.10 0.05 0.08 0.09 0.06 0.07 0.06 0.09
572
CUTLER
substance (or substances) that attracts guinea pig neutrophils. The chemotactic factor was found to be actively produced when the fungus was grown in a complex medium consisting of glucose, yeast extract, and peptone. Peptone could be replaced by an amino acid mixture, but glucose and yeast extract were required for optimum production. We have not yet successfully developed a chemically defined medium that consistently supports the production of optimum levels of chemotactic factor. It is of interest that, when viable yeast cells were placed in the test well of the agarose chemotactic assay, guinea pig neutrophils became attracted toward the actively growing cells. However, culture filtrates of C. albicans grown in the tissue culture medium used in the assay, but without agarose, yielded little if any evidence of chemotactic factor. Although data are lacking at this time, we believe that chemotactic factor is produced in low concentrations when the organism is grown in the tissue culture medium, but, because only 10 pl of the culture filtrate is added to the test well, the factor becomes too dilute by the time it reaches the neutrophils. Actively growing cells in the test well, on the other hand, would provide a constant supply of chemotactic factor that could be detected by the neutrophils. Chemotactically active culture filtrates were detected by 12 h of incubation at 37°C. This might reflect either that 12 h of incubation was required for sufficient cell density to provide enough factor for detection in the test system, or that chemotactic factor begins to be produced only at a certain time in the growth cycle of the organism. In support of the latter possibility is the observation that 24-h culture filtrates had a greater activity of chemotactic factor than either 12- or 72-h filtrates. The fungal chemotactic factor appears to be amenable to purification and characterization techniques. It was not inactivated by freezing and thawing, storage at 80C, or heating at 900C. It also appears to be of relatively low molecular weight, since activity was lost upon dialysis. Low-molecular-weight chemotactic factors have been also described from nonfungal microorganisms such as E. coli (22). Our data showing that C. albicans produces a substance that is itself chemotactically active are in contrast to findings by other workers (5, 20), who report that chemotactically active substances were produced by C. albicans only if the organism was incubated in the presence of fresh serum. Serum inactivated by heat at 560C did not support the production of chemotactic factors. Presumably the mechanism involved is due to the activation of complement via the classical pathway, if natural or induced antibody is present, or via the altemative pathway in much the same fashion
INFECT. IMMUN.
as activation induced by zymosan. Indeed, recent reports have suggested that C. albicans does activate the alternative pathway (20, 23), a finding that should not be surpnsing since cell walls of S. cerevisiae (zymosan) are known to activate the pathway (18). Workers in one laboratory have even suggested that complement activation is the only way that C. albicans could promote the production of chemotactic factor, and that the organism does not produce substances that are directly active (20). We have provided data which strongly suggest that the chemotactic activity reported here is not associated with complement activation. Not only was activity recorded when heat-inactivated serum was used in the test system, but culture filtrates also showed activity when serum was replaced by albumin in the assay system. Furthermore, the presence of dead cells in the test well did not cause measurable attraction of neutrophils, and one of the strains of C. albicans, although it grew well, did not produce detectable chemotactic activity. Therefore, our data give evidence for the first time of chemotactic activity associated with culture filtrates of C. albicans. A recent study has provided evidence that sonic extracts of C. albicans are slightly chemotactically active, but the workers on this study found that culture filtrates were not chemotactic (24). Perhaps the lack of activity associated with culture filtrates in the studies by Sohnle et al. (24) was due to the absence of yeast extract in the growth medium. The importance of candidal chemotactic factors in the pathogenesis of candidiasis is unknown at this time; our observations have been made with in vitro systems. At a speculative level, if these substances are produced in vivo and do relate to virulence, then it would seem that those isolates that produce the least amount of chemotactic activity would be the most virulent. It is noteworthy that of the strains tested in this study, there was some strain variation noted, and one strain (2252), which was isolated from a patient with disseminated candidiasis, did not produce measurable chemotactic activity. Some workers have noticed an inverse relation between virulency of isolates of C. albicans and the amount of suppuration induced upon injection into laboratory animals (1). ACKNOWLEDGMENTS This research was supported by a grant from the BrownHazen Fund of the Research Corporation. I thank N. Reed for critical comments and Jerrie Beyrodt for preparation of the manuscript. LITERATURE CITED 1. Albano, M., and J. Schmitt. 1973. Pathogenicity in mice of strains of Candida albicans (Robin) Berk. isolated from burn patients. Mycopathol. Mycol. Appl.
VOL. 18, 1977 49:283-288. 2. Belcher, R., J. Carney, and F. Monahan. 1973. An electron microscopic study of phagocytosis of Candida albicans by polymorphonuclear leukocytes. Lab. Invest. 29:620-627. 3. Chilgren, R. A., P. G. Quie, H. J. Meuwissen, and R. Hong. 1967. Chronic mucocutaneous candidiasis, deficiency of delayed hypersensitivity and selective local antibody defect. Lancet ii:688-693. 4. Cutler, J. E. 1974. A simple in vitro method for studies on chemotaxis. Proc. Soc. Exp. Biol. Med. 7:471-474. 5. Denning, T., and R. R. Davies. 1973. Candida albicans and the chemotaxis of polymorphonuclear neutrophils. Sabouraudia 11:210-221. 6. Drake, T. E., and H. I. Maibach. 1973. Candida and candidiasis. 2. Clinical manifestations and therapy of candidal disease. Postgrad. Med. 53:120-125. 7. Emmons, C. W., C. Binford, and J. P. Utz. 1970. Medical mycology, 2nd ed., p. 172. Lea and Febiger, Philadelphia. 8. Hurley, D., and A. Fauci. 1975. Disseminated candidiasia. I. An experimental model in the guinsa pig. J. Infect. Dis. 131:516-521. 9. John, T., and 0. Sieber. 1976. Chemotactic migration of neutrophils under agarose. Life Sci. 18:177-182. 10. Keller, H. U., and E. Sorkin. 1967. Studies on chemotaxis. V. On the chemotactic effect of bacteria. Int. Arch. Allergy Appl. Immunol. 31:505-517. 11. Kirkpatrick, C. H., R. R. Rich, and J. E. Bennett. 1971. Chronic mucocutaneous candidissia: model-building in cellular immunity. Ann. Intern. Med. 74:955-978. 12. Kroll, J. J., J. M. Einbinder, and W. G. Merz. 1973. Mucocutaneous candidiasis in a mother and son. Arch. Dermatol. 8:259-262. 13. Lehrer, R., and M. J. Cline. 1969. Leukocyte myeloperoxidase deficiency and disseminated candidiasis: the role of myeloperoxidase in resistance to Candida infection. J. Clin. Invest. 48:1478-1488. 14. Lehrer, R. I. 1971. Inhibition by sulfonamides of the candidacidal activity of human neutrophils. J. Clin. Invest. 50:2498-2505. 15. Lehrer, R. I., and M. J. Cline. 1969. Interaction of
C. ALBICANS CHEMOTACTIC FACTOR
16. 17.
18. 19.
20.
21.
22.
573
Candida albicans with human leukocytes and serum. J. Bacteriol. 98:996-1004. Maibach, H., and A. Kligman. 1962. The biology of experimental human cutaneous moniliasis (Candida albicans). Arch. Dermatol. 85:233-257. Nelson, R. D., P. G. Quie, and K. Simmons. 1975. Chemotaxis under agarose: a new and simple method for measuring chemotaxis and spontaneous migration of human polymorphonuclear leukocytes and monocytes. J. Immunol. 115:1650-1656. Osler, A. G. 1976. Complement-mechanisms and functions. Prentice-Hall, Inc., Englewood Cliffs, N.J. Provost, T. T., L Garrettson, R. Zeschke, N. Rose, and T. Tomasi. 1973. Combined immune deficiency, autoantibody formation and mucocutaneous candidiasis. Clin. Immunol. Immunopathol. 1:429-445. Ray, T. L, and K. D. Wuepper. 1976. Activation of the alternative (properdin) pathway of complement by Candida albicans and related species. J. Invest. Dermatol. 67:700-703. Repo, H. 1977. Leukocyte migration agarose test for the assessment of human neutrophil chemotaxis. I. Effects of environmental factors on neutrophil migration under agarose. Scand. J. Immunol. 6:203-209. Schiffmann, E., H. V. Showell, B. A. Corcoran, P. A. Ward, E. Smith, anmd E. L Becker. 1975. The isolation and partial characterization of neutrophil chemotactic factors from Escherichia coli. J. Immunol. 114:1831-1837.
23. Sohnle, P. G., M. M. Frank, and C. H. Kirkpatrick. 1976. Deposition of complement components in the cutaneous lesions of chronic mucocutaneous candidiasis. Clin. Immunol. Immunopathol. 5:340-350. 24. Sohnle, P. G., M. M. Frank, and C. H. Kirkpatrick. 1976. Mechanisms involved in elimination of organisms from experimental cutaneous Candida albicans infections in guinea pigs. J. Immunol. 117:523-530. 25. Warden, G., A. Mason, and B. Pruitt. 1974. Evaluation of leukocyte chemotaxis in vitro in thermally injured patients. J. Clin. Invest. 54:1001-1004. 26. White, B. E. 1972. Cerebral candidiasis. N. Engl. J. Med.
286:321.
