Vol. 26, No. 2

INFECTION AND IMMUNITY, Nov. 1979, p. 435440 0019-9567/79/11-0435/06$02.00/0

Chemotaxigenesis and Activation of the Alternative Complement Pathway by Encapsulated and NonEncapsulated Cryptococcus neoformans KRISTIN A. LAXALT AND THOMAS R. KOZEL Department of Microbiology, School of Medical Sciences, University of Nevada, Reno, Nevada 89557

Received for publication 6 August 1979

In the presence of serum, whole cells of encapsulated and non-encapsulated Cryptococcus neoformans generated a chemotactic response by neutrophils. Heat inactivation of serum ablated all chemotactic activity. Cryptococcal polysaccharide was not chemotaxigenic. Assays for alternative complement pathway activation such as depletion of alternative complement pathway factor B or electrophoretic conversion of factor B closely paralleled chemotaxis assays. Cells of encapsulated and non-encapsulated C. neoformans activated the alternative complement pathway, whereas cryptococcal polysaccharide was inactive. Failure of the capsular material to activate the alternative pathway was not due to serotype specificity because polysaccharide of several serotypes failed to achieve activation. The results suggest that chemotaxigenesis and alternative complement pathway activation are functions of the yeast cell wall. The results support our proposal that the cryptococcal capsule does not prevent potential opsonins from reaching binding and activation sites at the yeast cell wall or the release of biologically active soluble cleavage products into the surrounding medium; however, cell wall-bound cleavage products remain bound to the cell wall beneath the capsule. Therefore, they are unable to participate as opsonins in phagocytosis.

Tissue responses to infection by the yeast Cryptococcus neoformans are usually minimal, particularly in cryptococcal brain lesions (2, 4, 9, 24). Neutrophilic responses occur in a minority of cases (2). Farmer and Komorowski (9) attributed this lack of a strong inflammatory response to presence of the yeast's polysaccharide capsule since mouse inoculation studies of large and small capsule isolates showed a minimal neutrophil response to both isolates and a strong chronic inflammatory response to the small but not the large isolate. Macher et al. (16) reported that cryptococcal patients who were fungemic have serum with depressed levels of C3 and alternative complement pathway (ACP) factor B, suggesting that the yeast is able to activate the ACP. This activation of the ACP by some component of C. neoformans increases the likelihood that some component of the yeast is able to generate chemotactically active complement fragments from normal serum. Thus, the histopathology of cryptococcal infection may be related to the ability of the yeast to activate the ACP and generate a chemotactic response. The capsular polysaccharide could play one of several possible roles in this response. The capsular polysaccharide could directly activate the ACP as is the case with

some bacterial polysaccharides (3, 27), thus contributing to the chemotactic response. Cryptococcal polysaccharide could be inert with regard to the ACP, as is the case with other bacterial polysaccharides (22, 27); in which case, the capsule would have no effect on chemotaxigenesis by the yeast. Finally, the capsule could present a physical barrier that blocks activation of the ACP by activators located within the capsule at the cell wall, thus blocking chemotaxigenesis. The present study was undertaken (i) to determine the chemotaxigenic and ACP activating properties of whole cryptococci and (ii) to determine what role cryptococcal polysaccharide might have in chemotaxigenesis and ACP activation. MATERIALS AND METHODS Yeast isolates and soluble polysaccharide. C.

neoformnans 602 is a stable non-encapsulated isolate. C. neoformans 613 is a virulent, moderately encapsulated strain of cryptococcal serotype D. The characteristics of these strains have been described elsewhere in detail (13, 14). Cultures of C. neoformans serotypes B, C, and D were obtained from John E. Bennett, National Institute of Allergy and Infectious Diseases, Bethesda, Md. Yeast cells used in all assays were Formalin killed (15) and used as a suspension in saline (0.15 M NaCl).

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The procedure for purification of cryptococcal polysaccharide has been described previously (14). Chemotaxis assays. Blood was drawn from a rabbit via heart puncture into a plastic syringe containing 20 fd of 10% ethylenediaminetetraacetic acid (EDTA) per ml of blood. The blood was mixed with 0.75 volumes of 6% dextran T 500 (Pharmacia Fine Chemicals, Uppsala, Sweden) in saline, and the leukocyte-rich upper layer was drawn off after the erythrocytes had sedimented for 30 min at room temperature. The leukocytes were sedimented by centrifugation, and any remaining erythrocytes were lysed by differential hypotonic lysis in sodium bicarbonate-buffered (pH 7.2) Hanks balanced salt solution (HBSS; GIBCO, Grand Island, New York) diluted one-fifth in distilled water. The leukocytes were washed once in HBSS and resuspended to 1.5 x 106 neutrophils per ml in sodium bicarbonate-buffered medium 199 (M199; GIBCO). Cell counts were determined microscopically in a hemacytometer. Attractant solutions contained (i) a test attractant in saline, (ii) enough guinea pig serum to provide a final concentration of 2.5%, and (iii) M199 to yield a final volume of 2.5 ml. Positive and negative controls were included in each assay. Positive controls used 1.25 mg of zymosan (Sigma Chemical Co., St. Louis, Mo.) as the test attractant. The negative control was serum and M199 alone. All attractant solutions were incubated in a 370C water bath for 30 min before use. Chemotaxis was determined by a modified Boyden technique in acrylic Boyden chambers (Ahlco Corp., Southington, Conn.) as described by Maderazo and Woronick (17). The chambers were fitted with 13-mm diameter, 5-Mm pore-size filters (Millipore Corp., Bedford, Mass.; lot. no. C8E56812 A). The chemotactic attractant was added to the lower compartment, and 0.1 ml of the leukocyte suspension was added to the upper chamber. The chambers were incubated at 37°C for 90 min, and the membranes were fixed in methanol and stained with hematoxylin (17). Chemotactic indexes were determined by a modification of the technique described by Maderazo and Woronick (17). The number of migrating cells per 312x magnification field was determined at every 20,m interval from the original starting surface as shown by the fine adjustment scale on a Leitz Dialux microscope. The number of cells at each level was multiplied by the distance from the starting surface to that level, and the products were then summed for all levels. This cell migration factor for an attractant solution was then divided by the cell migration factor for the serum control to yield a chemotactic index for that attractant solution. Thus, an activator that failed to induce a significant chemotaxis would produce a chemotactic index of 1.0, i.e., the migration toward the attractant solution was equal to the migration toward the nonactivated serum control. Two to four replications were done for each attractant, and at least three fields were examined for each membrane. Tests for statistical significance were done by analysis of variance. ACP activation. Depletion of factor B was used to assess ACP activation. Factor B was assayed with the hemolytic diffusion plate assay described by Martin et al. (18). Hemolysis was determined in plates that

INFECT. IMMUN. contained (i) 1.2% agarose (Sigma Chemical Co., St. Louis, Mo.) in phosphate-buffered saline (pH 7.2) containing 10 mM [ethylene glycol-bis(,B-aminoethyl ether)]-N,N-tetraacetic acid (EGTA) and 7 mM MgCl2, (ii) 0.75% (vol/vol) guinea pig erythrocytes, and (iii) 10% human serum that had been heated at 50°C for 15 min to inactivate all factor B. Wells 5 mm in diameter were cut in the hemolysis plates, and 23 ,l of serum to be assayed for factor B was added to the well. The plates were incubated overnight at 4°C and for 2 h at 37°C, and the areas of hemolysis were determined. Agents to be assayed by the hemolytic technique for ACP activation were suspended in phosphatebuffered saline containing 10 mM EGTA and 7 mM MgCl2, an equal volume of normal human serum was added, and the sample was incubated for 2 h at 37°C. The serum was clarified by centrifugation, and residual factor B was measured. A standard curve of various dilutions of normal serum versus the area of lysis was used to calculate the percent depletion of factor B by ACP activators. A plot of log percent serum versus the area of lysis was linear. Cleavage products of factor B were identified by immunoelectrophoresis (12). Electrophoresis was done in 1.0% agarose containing Veronal acetate buffer (ionic strength = 0.1, pH 8.6) with 10 mM EDTA. A potential gradient of 2 V/cm was maintained for 90 min at room temperature. The trough was filled with rabbit antiserum to human C3 activator and incubated overnight at room temperature. Samples to be assayed by immunoelectrophoresis for ACP activating ability were incubated with normal human serum for 2 h at 37°C and clarified by centrifugation. Normal human serum incubated 2 h at 37°C was the negative control. Zymosan-treated serum (0.5 mg of serum per ml for 2 h at 37°C) was the positive control. Serum. Blood was drawn by venipuncture from normal human volunteers after obtaining informed consent. Blood was drawn from guinea pigs by heart puncture. The blood was allowed to clot at room temperature, the serum was separated, and all sera were immediately frozen at -85°C. Rabbit antiserum to human C3 activator was obtained from Behring Diagnostics (Somerville, N.J.). Heat-labile complement components were inactivated by heating serum at 56°C for 30 min. Determination of cell size. Comparisons of cell size were done on a Coulter Counter (model ABI, Hialeah, Fla.) equipped with a Coulter Channelyzer.

RESULTS Chemotactic response to C. neoformans. An initial experiment was done to determine the chemotactic response of neutrophils to cells of encapsulated strain 613 and non-encapsulated strain 602. The results (Fig. 1) showed that both strains generated a chemotactic response. The number of yeast cells needed to produce chemotaxis was similar for both strains; although, strain 613 appeared to generate a maximal response (relative to zymosan) with fewer yeast

CHEMOTAXIGENESIS BY C. NEOFORMANS

VOL. 26, 1979 602

cells with human serum produced substantial depletion of factor B as measured in hemolytic assays (Fig. 2). Cells of strain 613 were slightly,

3.0

2.0 x w z

1.0

400 I-) 0

100

25

12.5

Z

613

TABLE 1. Chemotactic response to C. neoformans soluble polysaccharide Chemotactic index' stinmulua 1.1 ± 0.2 1,000 pg of polysaccharide ...... 0.8 ± 0.01 400 pg of polysaccharide ....... 0.9 ± 0.2 40 pg of polysaccharide ........ 1.0 ± 0.2 4 pg of polysaccharide ......... 3.6 ± 1.7 a Attractant was incubated with M199 containing 2.5% guinea pig serum. b Chemotactic index ± standard deviation.

Zymosan

3.0

U

2.0

TABLE 2. Role of heat-labile serum components in the chemotactic response to encapsulated C. neoformans Chemotactic indeXb

stinul1sa

1.0 400

25 100 12.5 YEAST CELLS (X 10)

Z

FIG. 1. Chemotactic response of neutrophils to C. neoformans 602 (0) and 613 (0). Yeast ceUs were incubated in the presence of 2.5% guinea pig serum. Data shown are mean chemotactic index ± standard deviation.

cells than were needed for a maximal response

1.0 ± 0.2

2.2 ± 0.3 serum ....................... a with or without incubated Cells (108) of strain 613 2.5% guinea pig serum in a total volume of 2.5 ml. standard deviation. b Mean chemotactic index 00)

and (ii) that presence of a capsule does

75-

not prevent chemotaxigenesis by the yeast.

The previous experiment suggested that cryptococcal polysaccharide was unable to generate a chemotactic response. This was confirmed when the chemotactic response to various amounts of purified polysaccharide from strain 613 was determined (Table 1). No significant (P > 0.05) chemotactic response was observed at any polysaccharide concentration. An experiment was done to determine whether cryptococci alone were chemotactic or whether the yeast cells required heat-labile serum components for attraction of neutrophils. Cells of strain 613 were incubated with M199 alone, M199 supplemented with heat-inactivated serum, or M199 supplemented with normal guinea pig serum, and the mixtures were assayed for chemotactic activity. The results (Table 2) showed that cryptococci were chemotaxigenic only in the presence of normal serum. Activation of the ACP by C. neoforman& Consumption of human factor B was used as a measure of ACP activation by cryptococcal polysaccharide and by whole cells of strains 602 and 613. Incubation of C. neoformans 613 and 602

0.9 ± 0.2

C. neoformans + M199 .......... C. neoformans + M199 + heat-inactivated serum ........ ...... C. neoformans + M199 + normal

to strain 602. These results showed (i) that a capsule is not required to produce a chemotactic response,

437

50

-

25

t °

0

3

12

50*

200

.YEAST CELLS (X 10O)

100i_

cx75 U I

our 25k_

0

* pg

10 100 POLYSACCHARIDE

1000

FIG. 2. Depletion of factor B from human serum by cryptococcalpolysaccharide (A), cells of strain 602 (0), and cells of strain 613 (0). Data are shown as the percentage of normal factor B remaining after incubation of serum and activators for 2 h at 37°C.

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but significantly (P < 0.05) more active than cells of strain 602. Polysaccharide from strain 613 was unable to consume significant (P > 0.05) amounts of factor B, even at polysaccharide concentrations as high as 1 mg/ml. Immunoelectrophoretic analysis showed that the factor B in untreated serum migrated as a beta globulin. Treatment of serum with zymosan and cells of strain 613 and 602 produced conversion to the gamma migrating factor B cleavage product. Incubation of serum with 613 cryptococcal polysaccharide produced no cleavage of factor B. Winkelstein et al. (27) reported that some pneumococcal polysaccharides activated the ACP, whereas polysaccharides of other pneumococcal serotypes did not. Strain 613 is cryptococcal serotype D. It is possible that cryptococcal polysaccharides of other serotypes might be able to activate the ACP. Accordingly, we examined ACP activation by cryptococcal polysaccharides from several isolates. The results (Table 3) showed that polysaccharides of serotypes B and C, as well as another isolate of serotype D, were unable to significantly (P > 0.05) deplete factor B from normal human serum. Denning and Davies (5) suggested that differences in cell surface area could account for differences in chemotaxigenic activity between mycelium and blastospores of Candida albicans. It is possible that similar differences in surface area per cell could account for differences in the chemotaxigenesis and ACP activities of strains 602 and 613. Analysis of the size distribution of the two yeast strains in a Coulter Channelizer (Fig. 3) showed that cells of strain 613 are indeed larger than cells of strain 602, thus providing a larger area per cell for ACP activation.

DISCUSSION Our results showed that both encapsulated and non-encapsulated cryptococci were chemotaxigenic and activated the ACP. Chemotaxis was dependent on activation of heat-labile seTABLE 3. Failure of cryptococcal polysaccharides to deplete factor B from normal human serum Activatora Activator'

Cryptococcal polysaccharide serotype B Cryptococcal polysaccharide serotype C Cryptococcal polysaccharide serotype D Strain 613 polysaccharide ............

fac~~~Residual tor B (%)

100 100 100 100

Zymosan 0........................... a Serum was preincubated with 400 ,ug of cryptococcal polysaccharide or 0.5 mg of zymosan for 2 h at 37°C before hemolytic assay for residual factor B.

60

602

40 z 0

20

613 0

20

40 CHANNEL NUMBER

60

70

FIG. 3. Size distribution of C. neoformans 602 and 613 as shown by a Coulter Channelyzer.

rum components by the yeast. Cryptococcal polysaccharide played no apparent role either as a potentiator or inhibitor of chemotaxigenesis or ACP activation. It could be argued that our concentrations of polysaccharide were inadequate for ACP activation; however, the concentrations used exceeded concentrations at which other ACP activators were active (3) and greatly exceeded serum concentrations found during active cryptococcal disease (6). Nevertheless, it is possible that greater concentrations of polysaccharide or different assay techniques might show ACP activation. Our results differ considerably from an earlier report by Gadebusch (10) that extremely low concentrations of cryptococcal polysaccharide were able to significantly deplete properdin from mouse serum. Unfortunately, the assay technique used by Gadebusch differed considerably from our own, so direct comparisons are not possible. Our results also differ from a report by Diamond et al. (7) that 1,000 ,yg of cryptococcal polysaccharide produced 36.2% fixation of late complement components (C3 to 9) in normal human serum and 44.9% fixation in C4-deficient serum. These differences may be attributable to differences in assay techniques or to differences in purity of the polysaccharide preparations. Our results are in good agreement with results reported by Macher et al. (16) that showed little or no depletion of total hemolytic complement by cryptococcal polysaccharide. Several fungi have previously been shown to be chemotaxigenic. Extracts from mycelium and spherules of Coccidioides immitis (11), as well as whole blastospores and mycelia of C. albicans (1, 5, 23, 25), utilize heat-labile components of serum in generation of a chemotactic response. Chemotactic stimulation by C. albicans is directly proportional to the surface area of the fungus (5), a phenomenon similar to our observations on ACP activation by C. neoformans. Denning and Davies (5) and Weeks et al. (25) further demonstrated that the cell wall mannan

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CHEMOTAXIGENESIS BY C. NEOFORMANS

is the active component in chemotaxigenesis by C. albicans. The component of encapsulated and non-encapsulated cryptococci that was responsible for ACP activation and chemotaxigenesis is not known; however, cell wall mannans are a likely possibility. Bacterial capsular polysaccharides fit into one of two categories with regard to ACP activation. Pneumococcal polysaccharide serotypes 1, 4, and 25 activate the ACP (27). Other capsular polysaccharides such as pneumococcal serotypes 2, 3, 14, and 19 (27) and the polyribosephosphate capsule of Haemophilus influenzae type B (22) are unable to activate the ACP. Nonactivating polysaccharides do not prevent ACP activation by activators located within the capsule because whole cells of bacteria having nonactivating polysaccharide capsules will activate the ACP (8, 22, 27). Our studies have clearly placed C. neoformans into the latter category. A common structural or biochemical feature that identifies ACP activators or nonactivators has yet to be identified. Both encapsulated and non-encapsulated cryptococci activated the ACP and were chemotaxigenic. Thus, the capsule does not present a barrier that blocks complement components from reaching cell wall activators or the release of soluble cleavage products from the cell wall activation site as shown by the release of electrophoretically identifiable factor B cleavage products or chemotactically active cleavage products. However, it is evident that the capsule does block or mask opsonic particle-bound cleavage products because encapsulated cryptococci opsonized with normal serum are quite resistant to phagocytosis by macrophages and neutrophils (7, 15, 20). A similar result has been reported by Peterson et al. (21), who found that cell wall components of encapsulated staphylococci are able to activate complement, yet the bacteria remain resistant to phagocytosis. Thus, the present study lends further weight to the argument put forth by ourselves (19) and others (26) that masking of cell wall-bound opsonins is one mechanism by which capsular polysaccharides are able to inhibit phagocytosis. ACKNOWLEDGMENT This investigation was supported by Public Health Service grants AI 14209 and RR 09035 from the National Institutes of Health. LITERATURE CITED 1. Allan, R. B., and P. C. Wilkinson. 1978. A visual analysis of chemotactic and chemokinetic locomotion of human neutrophil leucocytes. Use of a new chemotaxis assay with Candida albicans as gradient source. Exp. Cell. Res. 111:191-203. 2. Baker, R. D., and R. K. Haugen. 1955. Tissue changes

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