INFECTION AND IMMUNITY, Sept. 1978, p. 714-720 0019-9567/78/0021-0714$02.00/0 Copyright i 1978 American Society for Microbiology
Vol. 21, No. 3
Printed in U.S.A.
Phagocytosis of Cryptococcus neoformans by Normal and Thioglycolate-Activated Macrophages FRANK J. SWENSON AND THOMAS R. KOZEL* Department of Microbiology, School of Medical Sciences, University of Nevada, Reno, Nevada 89557 Received for publication 7 June 1978
Phagocytosis of Cryptococcus neoformans by normal and thioglycolate-activated mouse peritoneal macrophages was studied. Thioglycolate-activated macrophages exhibited a lower percent phagocytosis than did normal macrophages. Differences in phagocytosis could not be attributed to differences in macrophage viability, minor variations in the concentration of adherent macrophages, or a general depression in activated macrophage phagocytosis. Thioglycolate-activated macrophages required heat-labile opsonins for optimal phagocytosis of nonencapsulated cryptococci, whereas nonactivated macrophages did not require heat-labile opsonins for phagocytosis of the yeast. Both types of macrophages exhibited similar sensitivity to the phagocytosis-inhibiting properties of cryptococcal polysaccharide. The results show that depletion of heat-labile opsonins from serum or inactivation of yeast-bound, heat-labile opsonins by polysaccharide cannot account for the phagocytosis-inhibiting properties of cryptococcal polysaccharide. The mechanism by which cryptococcal polysaccharide inhibits phagocytosis of Cryptococcus neoformans is not known. Previous studies have shown that cryptococcal polysaccharide inhibits attachment of the yeast to macrophages (10). Further, inhibition of phagocytosis is due to the presence of capsular material at the yeast surface rather than an indirect effect by the polysaccharide on macrophages or a depletion by polysaccharide of essential opsonins such as complement from the incubation medium (8). Studies by Diamond et al. (4, 5) demonstrated that components of both the classical and alternate complement pathways are active in phagocytosis of cryptococcus by human polymorphonuclear neutrophils. Although cryptococcal polysaccharide does not appear to inhibit phagocytosis by depleting serum of essential complement components (8), the polysaccharide may alter or modify complement components bound to the yeast such that the opsonizing complement fragments are no longer recognized by their receptors on the leukocyte membrane. Alternatively, the high-molecular-weight polysaccharide could present a barrier that physically prevents direct interaction between particlebound complement fragments and their leukocyte receptors. These possibilities are suggested by the studies of Diamond et al. (4), who used immunofluorescence techniques to demonstrate that incubation of normal human serum with encapsulated cryptococci leads to deposition of
C3 on the cryptococcal capsule. Diamond et al. (4) further observed that addition of fresh normal serum to cryptococci led to an increase in the refractile properties of the capsule and the formation of an inner capsular ring. Since this phenomenon was abrogated by heating the serum at 560C for 30 min, this inner ring may be composed of complement components that are located within the capsule and thus spatially unavailable to act in phagocytosis. Results from two studies on phagocytosis of C. neoformans by macrophages suggest a means to determine whether inhibition of phagocytosis by cryptococcal polysaccharide is due to some action by the polysaccharide on heat-labile opsonins. Mitchell and Friedman (11) reported that glycogen-stimulated rat peritoneal macrophages require heat-labile serum factors for phagocytosis of cryptococcus. In a later study, Kozel and Mastroianni (10) found that normal mouse peritoneal macrophages had little or no requirement for heat-labile opsonins in phagocytosis of a nonencapsulated strain of C. neoformans. Bianco et al. (2) showed that there is a qualitative difference between the complement receptors of thioglycolate-activated and normal macrophages since activated, but not normal, macrophages are able to ingest complementcoated erythrocytes. Thus, the reported differences in requirements for heat-labile opsonins in phagocytosis of C. neoformans may simply be due to differences in macrophage activation. If 714
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activated and nonactivated macrophages differ in their requirements for heat-labile opsonins, these differences can be exploited to determine whether cryptococcal polysaccharide inhibits phagocytosis by interfering with the opsonizing action of heat-labile serum components. Our study was designed to determine whether normal and thioglycolate-activated macrophages differ (i) in their ability to engulf cryptococci, (ii) in their requirements for heat-labile opsonins in phagocytosis of C. neoformans, and (iii) in their susceptibility to inhibition of phagocytosis by cryptococcal polysaccharide.
yeasts, and the slides were air dried. The monolayers were fixed in methanol and stained by the Giemsa procedure. Slides were examined microscopically, and 200 macrophages per monolayer were observed for ingested yeasts. To determine phagocytosis, only individual unclumped macrophages were examined. The percentage of macrophages with ingested yeasts (percent phagocytosis) was determined. Results are presented as mean values of at least four replications. All data were analyzed for significance (P = 0.05) by using two-way analysis of variance and Student's t test. Macrophage activation. Mice were stimulated by intraperitoneal injection of 1 ml of thioglycolate broth (Cal-Labs, Hollywood, Calif.). Macrophages were harvested 4 days after injection of the stimulant, and the thioglycolate-activated macrophages were cultured in the same manner as normal macrophages. A 4-day interval between injection of the stimulant and harvesting of macrophages ensured negligible neutrophil contamination. About 60% of the cells obtained from stimulated mice were macrophages, whereas the unstimulated mice yielded 50% macrophages. Trypan blue dye exclusion showed that viability of newly harvested macrophages was greater than 90% in both thioglycolate-activated and normal macrophage preparations. After being cultured for 48 h in monolayers, the viability of adherent cells was greater than 95% for both cell types. Adherence of thioglycolate-activated and nonactivated macrophages to glass was assessed by counting the number of macrophages per high-dry microscopic field (x500) in Giemsa-stained preparations 60 min after addition of identical numbers of thioglycolateactivated and nonactivated macrophages to the tissue culture chamber slides. Spreading of macrophages on glass was determined by observing under phase-contrast microscopy the percentage of macrophages that were flattened after a 30-min culture. Assays for macrophage Fc and C3 receptors were done by using immunoglobulin G-coated sheep erythrocytes [E(IgG)] and complement-coated sheep erythrocytes [E(IgM)C], respectively. Phagocytic indexes were determined by the techniques of Bianco (1). E(IgG) were prepared by mixing 1 ml of 4% washed sheep erythrocytes with 1 ml of a 1/800 dilution of the IgG fraction of rabbit antiserum to sheep erythrocytes (Cordis Laboratories, Miami, Fla.). This dilution of antiserum produced minimal, but consistent, opsonization of sheep erythrocytes. The mixture was incubated for 30 min at 370C and 30 min at 4VC. The E(IgG) were washed three times by centrifugation and resuspended in HBSS at a final concentration of 107 cells per ml. E(IgM)C were prepared by mixing 1 ml of 4% sheep erythrocytes with 1 ml of 1/10 dilution of the IgM fraction of rabbit antiserum to sheep erythrocytes (Cordis Laboratories). The mixture was incubated for 30 min at 370C, washed once with Veronalbuffered saline with glucose (VBG) (1), and resuspended in 1 ml of VBG. One milliliter of normal mouse serum diluted 1/5 in VBG was added, the mixture was incubated for 10 min at 370C, and 10 ml of cold VBG was added. The cells were washed three times in the cold with HBSS, and resuspended in HBSS at a final concentration of 107 cells per ml. Assays for phagocy-
MATERIALS AND METHODS Yeast strains and soluble polysaccharide. C. neoformans 613 was the encapsulated strain of cryptococcus used throughout this study. C. neoformans
602 is a non-encapsulated isolate of the yeast that has surface receptors for the soluble polysaccharide. The characteristics of these strains have been described elsewhere in detail (8,9). Organisms used in phagocytic assays were Formalin killed (10) and used as a suspension in Hank balanced salt solution (HBSS; Grand Island Biological Co., Grand Island, N.Y.) containing antibiotics (100 U of penicillin and 100 pig of streptomycin per ml; Grand Island Biological Co.) and buffered with sodium bicarbonate to pH 7.2. The procedure for purification of cryptococcal soluble polysaccharide has been described previously (9). Polysaccharide was prepared for use as a saline solution. Phagocytosis assays. Peritoneal exudate cells were obtained from 6- to 8-week old Swiss mice (Microbiological Associates, Bethesda, Md.). The procedure for collection of unstimulated macrophages has been described previously (10). Monolayers were prepared in four-chamber tissue culture chamber slides (Lab-Tek Products, Div. Miles Laboratories Inc., Westmont, Ill.) and incubated for 48 h at 370C in 5% CO2 before use. Unless otherwise indicated, ca. 2.5 x 10 macrophages were added to each monolayer. For phagocytosis assays, the culture medium was poured off, and each monolayer was washed two times with warmed (370C) HBSS. The test yeast suspension was warmed for 2 min at 370C in a water bath, and 1 ml was immediately added to each chamber. The test yeast suspension consisted of: (i) 106 yeast cells; (ii) calf serum (Grand Island Biological Co., lot no. A360810) at a final concentration of 10%; (iii) when required by an experimental protocol, 0.25 ml of cryptococcal polysaccharide in saline; and (iv) enough HBSS to give a final volume of 1 ml. Calf serum was stored at -801C as 10-ml portions and thawed immediately before use. When required by an experimental protocol, serum was depleted of heat-labile opsonins by heating the serum at 560C for 30 min. This procedure eliminated all hemolytic complement activity. Unless otherwise indicated, monolayers were incubated with test yeast suspensions at 370C for 1 h. After incubation, the medium was poured off, the slides were washed two times in HBSS to remove nonadherent
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tosis of E(IgG) and E(IgM)C used 10ropsonized erythrocytes per monolayer, yielding ca. 40 erythrocytes per
Although both cell preparations were adjusted to add similar numbers of cells per chamber, we macrophage. consistently observed more adherent macrophages in the thioglycolate-activated macroRESULTS phage monolayers than in the normal macroPhagocytosis of strain 602. An initial ex- phage monolayers after the 2-day incubation periment was done to determine whether thio- period. Preparations of activated macrophages glycolate-activated and nonactivated macro- contained about 1.5 times more macrophages phages differ in their ability to engulf the non- per microscopic field than did monolayers of encapsulated strain 602. Yeast cells were added normal macrophages. The greater concentration to monolayers of activated and normal macro- of activated macrophages was presumably due phages and incubated for various time intervals. to the greater adherent properties of thioglycoThe slides were fixed and stained, and the per- late-activated macrophages. As a result, nonaccent phagocytosis was determined (Fig. 1). Non- tivated macrophages had more yeasts available activated macrophages exhibited a significantly per macrophage in the test solutions, possibly higher percent phagocytosis than thioglycolate- increasing the percent phagocytosis observed activated macrophages at all time intervals stud- with these cells. Consequently, an experiment ied. However, after 3 to 4 h of incubation, the was done to determine the effect of a constant activated macrophages began to approach the number of yeast cells and a varying macrophage percent phagocytosis seen with nonactivated concentration on observed percent phagocytosis. macrophages. This data suggested that differ- Monolayers were prepared with various numences in phagocytosis were due to different rates bers of macrophages per monolayer, and 106 of phagocytosis. yeast cells were added to each monolayer. PerSince many reports (3, 13, 17, 18) indicate cent phagocytosis was determined after a 60-min enhanced phagocytosis by activated macro- incubation (Table 1). Nonactivated macrophages, several possible errors in interpreting phages exhibited no significant difference in perthe data shown in Fig. 1 were considered. De- cent phagocytosis at all macrophage concentracreased phagocytosis by thioglycolate-activated tions studied. Similarly, no difference in percent macrophages could be due to decreased macro- phagocytosis was noted between the four lower phage viability; however, studies with trypan concentrations of thioglycolate-activated macblue dye exclusion showed no difference in via- rophages. The highest concentration of macrobility between adherent activated and nonacti- phages showed a slight, but significant, decline vated macrophages. Further, after extended in- in percent phagocytosis. Microscopic examinacubation, the thioglycolate-activated macro- tion of monolayers at this high macrophage conphages approached the percent phagocytosis ex- centration showed masses of macrophages preshibited by nonactivated macrophages; therefore, ent in clumps, and this may account for the differences in macrophage viability seemed an decline in phagocytosis at very high macrophage unlikely explanation for an initial difference in concentrations. Nevertheless, within the remaining test mixtures, the percent phagocytosis phagocytic activity. Another possible error might be the yeast-to- was independent ofthe macrophage-to-yeast cell macrophage ratio in the incubation medium. ratio over a multiplicity range of 2.8 to 8.3 yeast cells per macrophage. Since the ratio between the highest and lowest multiplicity extended well beyond the 1.5:1 difference in adherent cell concentration between thioglycolate-activated TABLE 1. Effect of macrophage-yeast cell ratios on phagocytosis by normal and thioglycolate-activated macrophages
TIME (hrs)
FIG. 1. Phagocytosis of strain 602 by thioglycolateactivated (0) and normal (0) macrophages. Results are presented as mean ± standard deviation.
% Phagocytosis by % Phagocytoe by Macrophage activated macronormal macroconcn per monophagesb phagesb layer 58.7 ± 1.8 81.7 ± 1.8 5.0 x 105 68.4 ± 2.9 83.4 ± 2.8 3.5 X 105 68.5 + 2.1 81.1 1.6 2.5 X lo, 68.4 ± 1.9 84.0 2.5 1.8 X 106 69.5 ± 3.1 81.1 2.7 1.2 X10
as mean percent ± standard deviation. aExpressed b Yeast cells (106) were added to each monolayer.
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and nonactivated macrophages, the decreased phagocytosis by thioglycolate-activated macrophages (Fig. 1) was considered to be due to an alteration in some property of the macrophage rather than differences in macrophage concentration. Decreased phagocytosis of strain 602 could be due to either a generalized depression in phagocytosis by thioglycolate-activated macrophages or to a specific defect in phagocytosis of cryptococci. Consequently, normal and thioglycolateactivated macrophages were examined for their ability to ingest E(IgG), a particle that previous studies (1, 2) have shown to be engulfed at a higher rate by activated macrophages. E(IgG) were prepared with a minimal amount of IgG to ensure that any increased phagocytosis of E(IgG) by thioglycolate-activated macrophages would be apparent. The results (mean ± standard deviation) after a 60-min incubation of E(IgG) with macrophages showed 26 ± 6% of the normal macrophages with ingested E(IgG) compared with 65 ± 6% of the stimulated macrophages. Thus, decreased phagocytosis of strain 602 could not be attributed to a generalized depression in thioglycolate-activated macrophage phagocytosis. Requirements for heat-labile opsonins. An experiment was done to determine whether thioglycolate-activated and normal macrophages had similar requirements for the heatlabile components of serum for optimal phagocytosis of strain 602. Monolayers of both types of macrophages were prepared and tested with (i) yeast suspensions containing normal calf serum and (ii) yeast suspensions containing heatinactivated calf serum. Phagocytosis was determined after various time intervals. The results (Fig. 2) showed that normal macrophages exhibited similar phagocytosis of the yeast when opsonized by normal or heat-inactivated serum. On the other hand, thioglycolate-activated macrophages showed a significant depression in phagocytosis when the yeast was opsonized by heat-inactivated serum.
PHAGOCYTOSIS OF C. NEOFORMANS
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C')
0 CD 0
0
I
a-
z
w 0
w
a-
n0 0 75 0 0 " 50 z
m 25 0 L&J
aI
2
3
TIME (nrs)
FIG. 2. Phagocytosis of strain 602 by thioglycolateactivated (E, U) and normal (0,0 ) macrophages in normal serum (I, 0) or serum heated at 56°C for 30 min (U, 0). Results are presented as mean ± standard deviation.
ferential sensitivity to inhibition of phagocytosis should occur regardless of whether the polysaccharide depletes the serum of heat-labile opsonins or the polysaccharide renders yeast-bound opsonins incapable of interacting with their macrophage receptors. An initial experiment was done to determine whether thioglycolate-activated and nonactivated macrophages differed in their ability to engulf the fully encapsulated strain 613. ActiInhibition of phagocytosis by capsular vated and nonactivated macrophages were inpolysaccharide. Since thioglycolate-activated cubated for 60 min with cells of either strain 602 and nonactivated macrophages differ in their or strain 613. The results (Table 2) showed that requirements for heat-labile opsonins, we spec- both thioglycolate-activated and nonactivated ulated that if cryptococcal polysaccharide in- macrophages exhibited only minimal phagocyhibits phagocytosis either solely or in part by tosis of strain 613. There was no significant inhibiting the opsonizing activity of heat-labile difference between activated and nonactivated opsonins, the heat-labile opsonin-dependent macrophages in their ingestion of strain 613; cells (thioglycolate-activated macrophages) however, as in previous experiments, the thioshould exhibit a greater sensitivity to the phag- glycolate-activated macrophages were less effecocytosis-inhibiting properties of cryptococcal tive than nonactivated macrophages in phagopolysaccharide than do heat-labile opsonin-in- cytosis of strain 602. The previous experiment indicated that fully dependent cells (normal macrophages). This dif-
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encapsulated yeasts were as resistant to phagocytosis by thioglycolate-activated macrophages as they were to phagocytosis by nonactivated macrophages; however, activated and nonactivated macrophages might exhibit different sensitivities to inhibition of phagocytosis by smaller amounts of cryptococcal polysaccharide. Accordingly, various amounts of purified cryptococcal polysaccharide were added to strain 602, and phagocytosis of the polysaccharide-treated yeast cells by thioglycolate-activated and nonactivated macrophages was determined. A yeast suspension containing cells of strain 602 with no added polysaccharide was used as a positive control, and a test suspension of strain 613 served as a negative control. The results (Fig. 3) showed that similar amounts of cryptococcal polysaccharide were required to inhibit phagocytosis of strain 602 by both thioglycolate-activated and nonactivated macrophages. Thus, within the limits of the experimental design, thioglycolate-activated and normal macrophages did not differ in their sensitivity to inhibition of phagocytosis by cryptococcal polysaccharide. Evidence for activation of thioglycolatestimulated macrophages. Since several pre-
vious studies have shown increased rather than decreased phagocytosis of microorganisms by activated macrophages, additional morphological and functional evidence for "activation" of thioglycolate-stimulated macrophages was sought. First, the thioglycolate-stimulated macrophages exhibited increased adherence to glass. One hour after addition of 2.5 x 105 macrophages to each monolayer, about 33% more adherent thioglycolate-activated macrophages than adherent normal macrophages were observed per microscopic field. Second, examination of 30-min cultures of normal and thioglycolate-activated macrophages showed that about 67% ofthe stimulated macrophages were flattened, whereas only 14% of the normal macrophages were in the spreading form. Third, normal and thioglycolate-activated macrophages were examined for their ability to ingest E(IgM)C, a particle that is engulfed by activated, but not normal, macrophages (1, 2, 12). The results (mean ± standard deviation) after a 60-min incubation of E(IgM)C with macrophages showed 16 ± 3% of the normal macrophages with ingested E(IgM)C compared with 66 ± 10% of the stimulated macrophages. Phagocytosis of E(IgM) was negligible for both cell types.
TABLE 2. Phagocytosis of encapsulated and nonencapsulated strains of C. neoformans by normal and thioglycolate-activated macrophages
DISCUSSION Macrophage "activation" has been shown to follow exposure of macrophages to supernatants of lymphocytes from animals exposed to various antigens, intraperitoneal injection of chemicals such as thioglycolate broth, or infection by bacteria such as Mycobacterium tuberculosis or Listeria monocytogenes. Depending upon the method of activation, several morphological, metabolic, and functional changes may be observed in activated macrophages. These include increased adherence to glass (13), increased spreading on glass (14), increased production of neutral proteinases such as elastase (19), increased glucose oxidation through the hexose monophosphate shunt (13, 17), enhanced macrophage bacteriostasis (17), increased phagocytosis of microorganisms (13, 17, 18), decreased phagocytosis of microorganisms (7, 15, 16, 20), complement receptor-mediated ingestion of E(IgM)C (1, 2, 12), and increased phagocytosis of E(IgG) (1, 2). Indeed, Hamburger (6) has expressed the need for investigators to specify both the experimental conditions producing activated macrophagesand the parameters chosen to identify macrophage activation. Clearly, specific criteria for macrophage activation have not been established; however, the thioglycolate-activated macrophages used in our study complied
C. neofornans
% Phagocytosis by % Phagocytosis by normal macroactivated macrophagee phageea 82.4 ± 3.3 65.1 ± 2.1 1.0 ± 0.8 0.2 ± 0.2
602 613 a Expressed as mean percent ± standard deviation.
(n 0
0
I
a. z
U
CRYPTOCOCCAL POLYSACCHARIDE Lug/mi)
FIG. 3. Phagocytosis of strain 602 by thioglycolateactivated (0) and normal (0) macrophages in the presence of various amounts of cryptococcal capsular polysaccharide. Results are presented as mean standard deviation.
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with each of four parameters that have previously been associated with macrophage activation, namely increased adherence to glass, increased spreading, increased phagocytosis of E(IgG), and ingestion of E(IgM)C. However, given the diverse effects reported to follow macrophage activation, thioglycolate-activated macrophages undoubtedly differ in one or more metabolic or functional respects from macrophages activated by other means. Compared with normal macrophages, thioglycolate-activated macrophages exhibited depressed phagocytosis of non-encapsulated C. neoformans. This effect was most evident at shorter incubation times. With extended incubation, both types of macrophages became saturated with yeast cells and approached maximum percent ingestion. Differences in observed percent ingestion could not be attributed to differences in macrophage viability or to minor variations in the concentration of adherent macrophages. Presumably, these differences represent a real loss of phagocytic ability by thioglycolate-activated macrophages. It could be argued that this decreased phagocytosis is due to an overall depression in phagocytosis by thioglycolate-activated macrophages; however, increased phagocytosis of E(IgG) by activated macrophages demonstrates that the depressed phagocytosis does not apply to all particles. Activated macrophages display enhanced phagocytosis of a variety of particles (3, 13, 17, 18); however, decreased phagocytosis of Trypanosoma cruzi (7, 20), Candida albicans (16), starch granules (15), and Formalinized L. monocytogenes (15) have also been reported to follow activation of macrophages. Since the phenomenon of decreased phagocytosis has been observed with macrophages activated by a variety of techniques, it is likely that the decreased phagocytosis is a function ofthe ingested particle rather than the method of activation. David and Remold (3) suggested that macrophage membranes are altered by activation leading either to increased or decreased phagocytosis depending upon the surface properties of the particle undergoing ingestion. The nature of these alterations is not known, but particles such as T. cruzi, C. albicans, and non-encapsulated C. neoformans may possess some similar intrinsic surface property or a similarity in the manner of their opsonization that lowers their affinity for the altered cytoplasmic membranes of activated macrophages. The data in Fig. 2 explain reported differences in requirements for heat-labile opsonins in phagocytosis of C. neoformans by macrophages. Mitchell and Friedman (11) reported that gly-
cogen-stimulated rat peritoneal macrophages required heat-labile opsonins for phagocytosis of C. neoformans. Previous reports from our laboratory showed that unstimulated mouse peritoneal macrophages have little or no requirement for heat-labile opsonins in phagocytosis of the yeast (10). The data in Fig. 2 suggest that these differences in requirements for heat-labile opsonins were due to the state of activation of the macrophages. The exact nature of this alteration is unknown. Normal macrophages may have a very efficient receptor for a serum opsonin other than complement that allows the normal macrophage to function without the need for opsonizing complement components. This receptor that is operative in normal macrophages may be altered or absent in the activated macrophage. As a consequence, the activated macrophage utilizes the complement receptor that is present on activated macrophages for phagocytosis of opsonized C. neoformans in a manner that is probably similar or identical to ingestion of E(IgM)C by activated macrophages. This study was undertaken to determine whether inhibition of heat-labile opsonins is the mechanism by which cryptococcal polysaccharide inhibits phagocytosis of the yeast. If inhibition, inactivation, or masking of either free or yeast-bound heat-labile opsonins is the mechanism, the activated macrophages should have exhibited a greatly increased sensitivity to the phagocytosis-inhibiting properties of cryptococcal polysaccharide due to the increased dependence of activated macrophages on heat-labile opsonins. This was not the case since the data (Fig. 3) showed that heat-labile, opsonin-dependent (activated) macrophages and heat-labile, opsonin-independent (normal) macrophages exhibited identical sensitivities to inhibition of phagocytosis by cryptococcal polysaccharide.
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ACKNOWLEDGMENTS This investigation was supported by a grant from the Reno Cancer Center and by Public Health Service research grant A114209 from the National Institute of Allergy and Infectious Diseases.
LITERATURE CMD 1. Bianco, C. 1976. Methods for the study of macrophage Fc and C3 receptors, p. 407-415. In B. R. Bloom and J. R. David (ed.), In vitro methods in cell-mediated and tumor immunity. Academic Press Inc., New York. 2. Bianco, C., F. ML Griffin, Jr., and S. C. Silaversteiu. 1975. Studies of the macrophage complement receptor. Alteration of receptor function upon macrophage activation. J. Exp. Med. 141:1278-1290. 3. David, J. R., and H. G. Remold. 1976. Macrophage activation by lymphocyte mediators and studies on the interaction of macrophage inhibitory factor (MIF) with its target cell, p. 401-423. In D. S. Nelson (ed.), Immu-
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nobiology of the macrophage. Academic Press Inc., New York. 4. Diamond, R. D., J. E. May, M. A. Kane, M. M. Frank, and J. E. Bennett. 1974. The role of the classical and alternate complement pathways in host defenses against Cryptococcus neoformans infection. J. Immunol. 112:2260-2270. 5. Diamond, R. D., R. K. Root, and J. E. Bennett. 1972. Factors influencing killing of Cryptococcus neoformans by human leukocytes in vitro. J. Infect. Dis. 125: 367-376. 6. Hamburger, J. 1977. Macrophage activation: imperfect terminology hiding imperfect knowledge. Ann. Immunol. (Paris) 128:731-732. 7. Hoff, R. 1975. Killing in vitro of Trypanosoma cruzi by macrophages from mice immunized with T. cruzi or BCG, and absence of cross-immunity on challenge in vivo. J. Ezp. Med. 142:299-311. 8. Kozel, T. RK 1977. Non-encapsulated variant of Cryptococcus neoformans. H. Surface receptors for cryptococcal polysaccharide and their role in inhibition of phagocytosis by polysaccharide. Infect. Immun. 16:99-106. 9. Kozel, T. R., and J. Cazin, Jr. 1971. Nonencapsulated variant of Cryptococcus neoformans. I. Virulence studies and characterization of soluble polysaccharide. Infect. Immun. 3:287-294. 10. Kozel, T. R., and R. P. Mastroianni. 1976. Inhibition of phagocytosis by cryptococcal polysaccharide: dissociation of the attachment and ingestion phases of phagocytosis. Infect. Immun. 14:62-67. 11. Mitchell, T. G., and L Friedman. 1972. In vitro phagocytosis and intracellular fate of variously encapsulated strains of Cryptococcus neoformans. Infect. Immun. 5:491-498.
INFECT. IMMUN. 12. Morland, B., and G. Kaplan. 1977. Macrophage activation in vivo and in vitro. Esp. Cell Res. 108:279-288. 13. Nathan, C. F., M L. Karnovsky, and J. R David. 1971. Alterations of macrophage functions by mediators from lymphocytes. J. Ezp. Med. 133:1356-1376. 14. Nathan, C. F., and W. D. Terry. 1975. Differential stimulation of murine lymphoma growth in vitro by normal and BCG-activated macrophages. J. Exp. Med. 142:887-902. 15. Nathan, C. F., and W. D. Terry. 1977. Decreased phagocytosis by peritoneal macrophages from BCG-treated mice. Induction of the phagocytic defect in normal macrophages with BCG in vitro. Cell. Immunol. 29:295-311. 16. Neta, R., and S. B. Salvin. 1971. Cellular immunity in vitro: migration inhibition and phagocytosis. Infect. Immun. 4:697-702. 17. Ratzan, K. R., D. M. Musher, G. T. Keuswh, and L. Weinstein. 1972. Correlation of increased metabolic activity, resistance to infection, enhanced phagocytosis, and inhibition of bacterial growth by macrophages from listeria- and BCG-infected mice. Infect. Immun. 5:499-504. 18. Stubbs, M., A. V. Kiihner, E. A. Glass, J. R. David, and M. L. Karnovsky. 1973. Metabolic and functional studies on activated mouse macrophages. J. Exp. Med. 137:537-542. 19. Werb, Z., and S. Gordon. 1975. Elastase secretion by stimulated macrophages. Characterization and regulation. J. Exp. Med. 142:361-377. 20. Williams, D. M., and J. S. Remington. 1977. Effects of human monocytes and macrophages on Trypanosoma cruzi. Immunology 32:19-23.
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Phagocytosis of Cryptococcus neoformans by Normal and Thioglycolate-Activated Macrophages FRANK J. SWENSON AND THOMAS R. KOZEL* Department of Microbiology, School of Medical Sciences, University of Nevada, Reno, Nevada 89557 Received for publication 7 June 1978
Phagocytosis of Cryptococcus neoformans by normal and thioglycolate-activated mouse peritoneal macrophages was studied. Thioglycolate-activated macrophages exhibited a lower percent phagocytosis than did normal macrophages. Differences in phagocytosis could not be attributed to differences in macrophage viability, minor variations in the concentration of adherent macrophages, or a general depression in activated macrophage phagocytosis. Thioglycolate-activated macrophages required heat-labile opsonins for optimal phagocytosis of nonencapsulated cryptococci, whereas nonactivated macrophages did not require heat-labile opsonins for phagocytosis of the yeast. Both types of macrophages exhibited similar sensitivity to the phagocytosis-inhibiting properties of cryptococcal polysaccharide. The results show that depletion of heat-labile opsonins from serum or inactivation of yeast-bound, heat-labile opsonins by polysaccharide cannot account for the phagocytosis-inhibiting properties of cryptococcal polysaccharide. The mechanism by which cryptococcal polysaccharide inhibits phagocytosis of Cryptococcus neoformans is not known. Previous studies have shown that cryptococcal polysaccharide inhibits attachment of the yeast to macrophages (10). Further, inhibition of phagocytosis is due to the presence of capsular material at the yeast surface rather than an indirect effect by the polysaccharide on macrophages or a depletion by polysaccharide of essential opsonins such as complement from the incubation medium (8). Studies by Diamond et al. (4, 5) demonstrated that components of both the classical and alternate complement pathways are active in phagocytosis of cryptococcus by human polymorphonuclear neutrophils. Although cryptococcal polysaccharide does not appear to inhibit phagocytosis by depleting serum of essential complement components (8), the polysaccharide may alter or modify complement components bound to the yeast such that the opsonizing complement fragments are no longer recognized by their receptors on the leukocyte membrane. Alternatively, the high-molecular-weight polysaccharide could present a barrier that physically prevents direct interaction between particlebound complement fragments and their leukocyte receptors. These possibilities are suggested by the studies of Diamond et al. (4), who used immunofluorescence techniques to demonstrate that incubation of normal human serum with encapsulated cryptococci leads to deposition of
C3 on the cryptococcal capsule. Diamond et al. (4) further observed that addition of fresh normal serum to cryptococci led to an increase in the refractile properties of the capsule and the formation of an inner capsular ring. Since this phenomenon was abrogated by heating the serum at 560C for 30 min, this inner ring may be composed of complement components that are located within the capsule and thus spatially unavailable to act in phagocytosis. Results from two studies on phagocytosis of C. neoformans by macrophages suggest a means to determine whether inhibition of phagocytosis by cryptococcal polysaccharide is due to some action by the polysaccharide on heat-labile opsonins. Mitchell and Friedman (11) reported that glycogen-stimulated rat peritoneal macrophages require heat-labile serum factors for phagocytosis of cryptococcus. In a later study, Kozel and Mastroianni (10) found that normal mouse peritoneal macrophages had little or no requirement for heat-labile opsonins in phagocytosis of a nonencapsulated strain of C. neoformans. Bianco et al. (2) showed that there is a qualitative difference between the complement receptors of thioglycolate-activated and normal macrophages since activated, but not normal, macrophages are able to ingest complementcoated erythrocytes. Thus, the reported differences in requirements for heat-labile opsonins in phagocytosis of C. neoformans may simply be due to differences in macrophage activation. If 714
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activated and nonactivated macrophages differ in their requirements for heat-labile opsonins, these differences can be exploited to determine whether cryptococcal polysaccharide inhibits phagocytosis by interfering with the opsonizing action of heat-labile serum components. Our study was designed to determine whether normal and thioglycolate-activated macrophages differ (i) in their ability to engulf cryptococci, (ii) in their requirements for heat-labile opsonins in phagocytosis of C. neoformans, and (iii) in their susceptibility to inhibition of phagocytosis by cryptococcal polysaccharide.
yeasts, and the slides were air dried. The monolayers were fixed in methanol and stained by the Giemsa procedure. Slides were examined microscopically, and 200 macrophages per monolayer were observed for ingested yeasts. To determine phagocytosis, only individual unclumped macrophages were examined. The percentage of macrophages with ingested yeasts (percent phagocytosis) was determined. Results are presented as mean values of at least four replications. All data were analyzed for significance (P = 0.05) by using two-way analysis of variance and Student's t test. Macrophage activation. Mice were stimulated by intraperitoneal injection of 1 ml of thioglycolate broth (Cal-Labs, Hollywood, Calif.). Macrophages were harvested 4 days after injection of the stimulant, and the thioglycolate-activated macrophages were cultured in the same manner as normal macrophages. A 4-day interval between injection of the stimulant and harvesting of macrophages ensured negligible neutrophil contamination. About 60% of the cells obtained from stimulated mice were macrophages, whereas the unstimulated mice yielded 50% macrophages. Trypan blue dye exclusion showed that viability of newly harvested macrophages was greater than 90% in both thioglycolate-activated and normal macrophage preparations. After being cultured for 48 h in monolayers, the viability of adherent cells was greater than 95% for both cell types. Adherence of thioglycolate-activated and nonactivated macrophages to glass was assessed by counting the number of macrophages per high-dry microscopic field (x500) in Giemsa-stained preparations 60 min after addition of identical numbers of thioglycolateactivated and nonactivated macrophages to the tissue culture chamber slides. Spreading of macrophages on glass was determined by observing under phase-contrast microscopy the percentage of macrophages that were flattened after a 30-min culture. Assays for macrophage Fc and C3 receptors were done by using immunoglobulin G-coated sheep erythrocytes [E(IgG)] and complement-coated sheep erythrocytes [E(IgM)C], respectively. Phagocytic indexes were determined by the techniques of Bianco (1). E(IgG) were prepared by mixing 1 ml of 4% washed sheep erythrocytes with 1 ml of a 1/800 dilution of the IgG fraction of rabbit antiserum to sheep erythrocytes (Cordis Laboratories, Miami, Fla.). This dilution of antiserum produced minimal, but consistent, opsonization of sheep erythrocytes. The mixture was incubated for 30 min at 370C and 30 min at 4VC. The E(IgG) were washed three times by centrifugation and resuspended in HBSS at a final concentration of 107 cells per ml. E(IgM)C were prepared by mixing 1 ml of 4% sheep erythrocytes with 1 ml of 1/10 dilution of the IgM fraction of rabbit antiserum to sheep erythrocytes (Cordis Laboratories). The mixture was incubated for 30 min at 370C, washed once with Veronalbuffered saline with glucose (VBG) (1), and resuspended in 1 ml of VBG. One milliliter of normal mouse serum diluted 1/5 in VBG was added, the mixture was incubated for 10 min at 370C, and 10 ml of cold VBG was added. The cells were washed three times in the cold with HBSS, and resuspended in HBSS at a final concentration of 107 cells per ml. Assays for phagocy-
MATERIALS AND METHODS Yeast strains and soluble polysaccharide. C. neoformans 613 was the encapsulated strain of cryptococcus used throughout this study. C. neoformans
602 is a non-encapsulated isolate of the yeast that has surface receptors for the soluble polysaccharide. The characteristics of these strains have been described elsewhere in detail (8,9). Organisms used in phagocytic assays were Formalin killed (10) and used as a suspension in Hank balanced salt solution (HBSS; Grand Island Biological Co., Grand Island, N.Y.) containing antibiotics (100 U of penicillin and 100 pig of streptomycin per ml; Grand Island Biological Co.) and buffered with sodium bicarbonate to pH 7.2. The procedure for purification of cryptococcal soluble polysaccharide has been described previously (9). Polysaccharide was prepared for use as a saline solution. Phagocytosis assays. Peritoneal exudate cells were obtained from 6- to 8-week old Swiss mice (Microbiological Associates, Bethesda, Md.). The procedure for collection of unstimulated macrophages has been described previously (10). Monolayers were prepared in four-chamber tissue culture chamber slides (Lab-Tek Products, Div. Miles Laboratories Inc., Westmont, Ill.) and incubated for 48 h at 370C in 5% CO2 before use. Unless otherwise indicated, ca. 2.5 x 10 macrophages were added to each monolayer. For phagocytosis assays, the culture medium was poured off, and each monolayer was washed two times with warmed (370C) HBSS. The test yeast suspension was warmed for 2 min at 370C in a water bath, and 1 ml was immediately added to each chamber. The test yeast suspension consisted of: (i) 106 yeast cells; (ii) calf serum (Grand Island Biological Co., lot no. A360810) at a final concentration of 10%; (iii) when required by an experimental protocol, 0.25 ml of cryptococcal polysaccharide in saline; and (iv) enough HBSS to give a final volume of 1 ml. Calf serum was stored at -801C as 10-ml portions and thawed immediately before use. When required by an experimental protocol, serum was depleted of heat-labile opsonins by heating the serum at 560C for 30 min. This procedure eliminated all hemolytic complement activity. Unless otherwise indicated, monolayers were incubated with test yeast suspensions at 370C for 1 h. After incubation, the medium was poured off, the slides were washed two times in HBSS to remove nonadherent
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tosis of E(IgG) and E(IgM)C used 10ropsonized erythrocytes per monolayer, yielding ca. 40 erythrocytes per
Although both cell preparations were adjusted to add similar numbers of cells per chamber, we macrophage. consistently observed more adherent macrophages in the thioglycolate-activated macroRESULTS phage monolayers than in the normal macroPhagocytosis of strain 602. An initial ex- phage monolayers after the 2-day incubation periment was done to determine whether thio- period. Preparations of activated macrophages glycolate-activated and nonactivated macro- contained about 1.5 times more macrophages phages differ in their ability to engulf the non- per microscopic field than did monolayers of encapsulated strain 602. Yeast cells were added normal macrophages. The greater concentration to monolayers of activated and normal macro- of activated macrophages was presumably due phages and incubated for various time intervals. to the greater adherent properties of thioglycoThe slides were fixed and stained, and the per- late-activated macrophages. As a result, nonaccent phagocytosis was determined (Fig. 1). Non- tivated macrophages had more yeasts available activated macrophages exhibited a significantly per macrophage in the test solutions, possibly higher percent phagocytosis than thioglycolate- increasing the percent phagocytosis observed activated macrophages at all time intervals stud- with these cells. Consequently, an experiment ied. However, after 3 to 4 h of incubation, the was done to determine the effect of a constant activated macrophages began to approach the number of yeast cells and a varying macrophage percent phagocytosis seen with nonactivated concentration on observed percent phagocytosis. macrophages. This data suggested that differ- Monolayers were prepared with various numences in phagocytosis were due to different rates bers of macrophages per monolayer, and 106 of phagocytosis. yeast cells were added to each monolayer. PerSince many reports (3, 13, 17, 18) indicate cent phagocytosis was determined after a 60-min enhanced phagocytosis by activated macro- incubation (Table 1). Nonactivated macrophages, several possible errors in interpreting phages exhibited no significant difference in perthe data shown in Fig. 1 were considered. De- cent phagocytosis at all macrophage concentracreased phagocytosis by thioglycolate-activated tions studied. Similarly, no difference in percent macrophages could be due to decreased macro- phagocytosis was noted between the four lower phage viability; however, studies with trypan concentrations of thioglycolate-activated macblue dye exclusion showed no difference in via- rophages. The highest concentration of macrobility between adherent activated and nonacti- phages showed a slight, but significant, decline vated macrophages. Further, after extended in- in percent phagocytosis. Microscopic examinacubation, the thioglycolate-activated macro- tion of monolayers at this high macrophage conphages approached the percent phagocytosis ex- centration showed masses of macrophages preshibited by nonactivated macrophages; therefore, ent in clumps, and this may account for the differences in macrophage viability seemed an decline in phagocytosis at very high macrophage unlikely explanation for an initial difference in concentrations. Nevertheless, within the remaining test mixtures, the percent phagocytosis phagocytic activity. Another possible error might be the yeast-to- was independent ofthe macrophage-to-yeast cell macrophage ratio in the incubation medium. ratio over a multiplicity range of 2.8 to 8.3 yeast cells per macrophage. Since the ratio between the highest and lowest multiplicity extended well beyond the 1.5:1 difference in adherent cell concentration between thioglycolate-activated TABLE 1. Effect of macrophage-yeast cell ratios on phagocytosis by normal and thioglycolate-activated macrophages
TIME (hrs)
FIG. 1. Phagocytosis of strain 602 by thioglycolateactivated (0) and normal (0) macrophages. Results are presented as mean ± standard deviation.
% Phagocytosis by % Phagocytoe by Macrophage activated macronormal macroconcn per monophagesb phagesb layer 58.7 ± 1.8 81.7 ± 1.8 5.0 x 105 68.4 ± 2.9 83.4 ± 2.8 3.5 X 105 68.5 + 2.1 81.1 1.6 2.5 X lo, 68.4 ± 1.9 84.0 2.5 1.8 X 106 69.5 ± 3.1 81.1 2.7 1.2 X10
as mean percent ± standard deviation. aExpressed b Yeast cells (106) were added to each monolayer.
VOL. 21, 1978
and nonactivated macrophages, the decreased phagocytosis by thioglycolate-activated macrophages (Fig. 1) was considered to be due to an alteration in some property of the macrophage rather than differences in macrophage concentration. Decreased phagocytosis of strain 602 could be due to either a generalized depression in phagocytosis by thioglycolate-activated macrophages or to a specific defect in phagocytosis of cryptococci. Consequently, normal and thioglycolateactivated macrophages were examined for their ability to ingest E(IgG), a particle that previous studies (1, 2) have shown to be engulfed at a higher rate by activated macrophages. E(IgG) were prepared with a minimal amount of IgG to ensure that any increased phagocytosis of E(IgG) by thioglycolate-activated macrophages would be apparent. The results (mean ± standard deviation) after a 60-min incubation of E(IgG) with macrophages showed 26 ± 6% of the normal macrophages with ingested E(IgG) compared with 65 ± 6% of the stimulated macrophages. Thus, decreased phagocytosis of strain 602 could not be attributed to a generalized depression in thioglycolate-activated macrophage phagocytosis. Requirements for heat-labile opsonins. An experiment was done to determine whether thioglycolate-activated and normal macrophages had similar requirements for the heatlabile components of serum for optimal phagocytosis of strain 602. Monolayers of both types of macrophages were prepared and tested with (i) yeast suspensions containing normal calf serum and (ii) yeast suspensions containing heatinactivated calf serum. Phagocytosis was determined after various time intervals. The results (Fig. 2) showed that normal macrophages exhibited similar phagocytosis of the yeast when opsonized by normal or heat-inactivated serum. On the other hand, thioglycolate-activated macrophages showed a significant depression in phagocytosis when the yeast was opsonized by heat-inactivated serum.
PHAGOCYTOSIS OF C. NEOFORMANS
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C')
0 CD 0
0
I
a-
z
w 0
w
a-
n0 0 75 0 0 " 50 z
m 25 0 L&J
aI
2
3
TIME (nrs)
FIG. 2. Phagocytosis of strain 602 by thioglycolateactivated (E, U) and normal (0,0 ) macrophages in normal serum (I, 0) or serum heated at 56°C for 30 min (U, 0). Results are presented as mean ± standard deviation.
ferential sensitivity to inhibition of phagocytosis should occur regardless of whether the polysaccharide depletes the serum of heat-labile opsonins or the polysaccharide renders yeast-bound opsonins incapable of interacting with their macrophage receptors. An initial experiment was done to determine whether thioglycolate-activated and nonactivated macrophages differed in their ability to engulf the fully encapsulated strain 613. ActiInhibition of phagocytosis by capsular vated and nonactivated macrophages were inpolysaccharide. Since thioglycolate-activated cubated for 60 min with cells of either strain 602 and nonactivated macrophages differ in their or strain 613. The results (Table 2) showed that requirements for heat-labile opsonins, we spec- both thioglycolate-activated and nonactivated ulated that if cryptococcal polysaccharide in- macrophages exhibited only minimal phagocyhibits phagocytosis either solely or in part by tosis of strain 613. There was no significant inhibiting the opsonizing activity of heat-labile difference between activated and nonactivated opsonins, the heat-labile opsonin-dependent macrophages in their ingestion of strain 613; cells (thioglycolate-activated macrophages) however, as in previous experiments, the thioshould exhibit a greater sensitivity to the phag- glycolate-activated macrophages were less effecocytosis-inhibiting properties of cryptococcal tive than nonactivated macrophages in phagopolysaccharide than do heat-labile opsonin-in- cytosis of strain 602. The previous experiment indicated that fully dependent cells (normal macrophages). This dif-
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encapsulated yeasts were as resistant to phagocytosis by thioglycolate-activated macrophages as they were to phagocytosis by nonactivated macrophages; however, activated and nonactivated macrophages might exhibit different sensitivities to inhibition of phagocytosis by smaller amounts of cryptococcal polysaccharide. Accordingly, various amounts of purified cryptococcal polysaccharide were added to strain 602, and phagocytosis of the polysaccharide-treated yeast cells by thioglycolate-activated and nonactivated macrophages was determined. A yeast suspension containing cells of strain 602 with no added polysaccharide was used as a positive control, and a test suspension of strain 613 served as a negative control. The results (Fig. 3) showed that similar amounts of cryptococcal polysaccharide were required to inhibit phagocytosis of strain 602 by both thioglycolate-activated and nonactivated macrophages. Thus, within the limits of the experimental design, thioglycolate-activated and normal macrophages did not differ in their sensitivity to inhibition of phagocytosis by cryptococcal polysaccharide. Evidence for activation of thioglycolatestimulated macrophages. Since several pre-
vious studies have shown increased rather than decreased phagocytosis of microorganisms by activated macrophages, additional morphological and functional evidence for "activation" of thioglycolate-stimulated macrophages was sought. First, the thioglycolate-stimulated macrophages exhibited increased adherence to glass. One hour after addition of 2.5 x 105 macrophages to each monolayer, about 33% more adherent thioglycolate-activated macrophages than adherent normal macrophages were observed per microscopic field. Second, examination of 30-min cultures of normal and thioglycolate-activated macrophages showed that about 67% ofthe stimulated macrophages were flattened, whereas only 14% of the normal macrophages were in the spreading form. Third, normal and thioglycolate-activated macrophages were examined for their ability to ingest E(IgM)C, a particle that is engulfed by activated, but not normal, macrophages (1, 2, 12). The results (mean ± standard deviation) after a 60-min incubation of E(IgM)C with macrophages showed 16 ± 3% of the normal macrophages with ingested E(IgM)C compared with 66 ± 10% of the stimulated macrophages. Phagocytosis of E(IgM) was negligible for both cell types.
TABLE 2. Phagocytosis of encapsulated and nonencapsulated strains of C. neoformans by normal and thioglycolate-activated macrophages
DISCUSSION Macrophage "activation" has been shown to follow exposure of macrophages to supernatants of lymphocytes from animals exposed to various antigens, intraperitoneal injection of chemicals such as thioglycolate broth, or infection by bacteria such as Mycobacterium tuberculosis or Listeria monocytogenes. Depending upon the method of activation, several morphological, metabolic, and functional changes may be observed in activated macrophages. These include increased adherence to glass (13), increased spreading on glass (14), increased production of neutral proteinases such as elastase (19), increased glucose oxidation through the hexose monophosphate shunt (13, 17), enhanced macrophage bacteriostasis (17), increased phagocytosis of microorganisms (13, 17, 18), decreased phagocytosis of microorganisms (7, 15, 16, 20), complement receptor-mediated ingestion of E(IgM)C (1, 2, 12), and increased phagocytosis of E(IgG) (1, 2). Indeed, Hamburger (6) has expressed the need for investigators to specify both the experimental conditions producing activated macrophagesand the parameters chosen to identify macrophage activation. Clearly, specific criteria for macrophage activation have not been established; however, the thioglycolate-activated macrophages used in our study complied
C. neofornans
% Phagocytosis by % Phagocytosis by normal macroactivated macrophagee phageea 82.4 ± 3.3 65.1 ± 2.1 1.0 ± 0.8 0.2 ± 0.2
602 613 a Expressed as mean percent ± standard deviation.
(n 0
0
I
a. z
U
CRYPTOCOCCAL POLYSACCHARIDE Lug/mi)
FIG. 3. Phagocytosis of strain 602 by thioglycolateactivated (0) and normal (0) macrophages in the presence of various amounts of cryptococcal capsular polysaccharide. Results are presented as mean standard deviation.
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PHAGOCYTOSIS OF C. NEOFORMANS
with each of four parameters that have previously been associated with macrophage activation, namely increased adherence to glass, increased spreading, increased phagocytosis of E(IgG), and ingestion of E(IgM)C. However, given the diverse effects reported to follow macrophage activation, thioglycolate-activated macrophages undoubtedly differ in one or more metabolic or functional respects from macrophages activated by other means. Compared with normal macrophages, thioglycolate-activated macrophages exhibited depressed phagocytosis of non-encapsulated C. neoformans. This effect was most evident at shorter incubation times. With extended incubation, both types of macrophages became saturated with yeast cells and approached maximum percent ingestion. Differences in observed percent ingestion could not be attributed to differences in macrophage viability or to minor variations in the concentration of adherent macrophages. Presumably, these differences represent a real loss of phagocytic ability by thioglycolate-activated macrophages. It could be argued that this decreased phagocytosis is due to an overall depression in phagocytosis by thioglycolate-activated macrophages; however, increased phagocytosis of E(IgG) by activated macrophages demonstrates that the depressed phagocytosis does not apply to all particles. Activated macrophages display enhanced phagocytosis of a variety of particles (3, 13, 17, 18); however, decreased phagocytosis of Trypanosoma cruzi (7, 20), Candida albicans (16), starch granules (15), and Formalinized L. monocytogenes (15) have also been reported to follow activation of macrophages. Since the phenomenon of decreased phagocytosis has been observed with macrophages activated by a variety of techniques, it is likely that the decreased phagocytosis is a function ofthe ingested particle rather than the method of activation. David and Remold (3) suggested that macrophage membranes are altered by activation leading either to increased or decreased phagocytosis depending upon the surface properties of the particle undergoing ingestion. The nature of these alterations is not known, but particles such as T. cruzi, C. albicans, and non-encapsulated C. neoformans may possess some similar intrinsic surface property or a similarity in the manner of their opsonization that lowers their affinity for the altered cytoplasmic membranes of activated macrophages. The data in Fig. 2 explain reported differences in requirements for heat-labile opsonins in phagocytosis of C. neoformans by macrophages. Mitchell and Friedman (11) reported that gly-
cogen-stimulated rat peritoneal macrophages required heat-labile opsonins for phagocytosis of C. neoformans. Previous reports from our laboratory showed that unstimulated mouse peritoneal macrophages have little or no requirement for heat-labile opsonins in phagocytosis of the yeast (10). The data in Fig. 2 suggest that these differences in requirements for heat-labile opsonins were due to the state of activation of the macrophages. The exact nature of this alteration is unknown. Normal macrophages may have a very efficient receptor for a serum opsonin other than complement that allows the normal macrophage to function without the need for opsonizing complement components. This receptor that is operative in normal macrophages may be altered or absent in the activated macrophage. As a consequence, the activated macrophage utilizes the complement receptor that is present on activated macrophages for phagocytosis of opsonized C. neoformans in a manner that is probably similar or identical to ingestion of E(IgM)C by activated macrophages. This study was undertaken to determine whether inhibition of heat-labile opsonins is the mechanism by which cryptococcal polysaccharide inhibits phagocytosis of the yeast. If inhibition, inactivation, or masking of either free or yeast-bound heat-labile opsonins is the mechanism, the activated macrophages should have exhibited a greatly increased sensitivity to the phagocytosis-inhibiting properties of cryptococcal polysaccharide due to the increased dependence of activated macrophages on heat-labile opsonins. This was not the case since the data (Fig. 3) showed that heat-labile, opsonin-dependent (activated) macrophages and heat-labile, opsonin-independent (normal) macrophages exhibited identical sensitivities to inhibition of phagocytosis by cryptococcal polysaccharide.
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ACKNOWLEDGMENTS This investigation was supported by a grant from the Reno Cancer Center and by Public Health Service research grant A114209 from the National Institute of Allergy and Infectious Diseases.
LITERATURE CMD 1. Bianco, C. 1976. Methods for the study of macrophage Fc and C3 receptors, p. 407-415. In B. R. Bloom and J. R. David (ed.), In vitro methods in cell-mediated and tumor immunity. Academic Press Inc., New York. 2. Bianco, C., F. ML Griffin, Jr., and S. C. Silaversteiu. 1975. Studies of the macrophage complement receptor. Alteration of receptor function upon macrophage activation. J. Exp. Med. 141:1278-1290. 3. David, J. R., and H. G. Remold. 1976. Macrophage activation by lymphocyte mediators and studies on the interaction of macrophage inhibitory factor (MIF) with its target cell, p. 401-423. In D. S. Nelson (ed.), Immu-
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nobiology of the macrophage. Academic Press Inc., New York. 4. Diamond, R. D., J. E. May, M. A. Kane, M. M. Frank, and J. E. Bennett. 1974. The role of the classical and alternate complement pathways in host defenses against Cryptococcus neoformans infection. J. Immunol. 112:2260-2270. 5. Diamond, R. D., R. K. Root, and J. E. Bennett. 1972. Factors influencing killing of Cryptococcus neoformans by human leukocytes in vitro. J. Infect. Dis. 125: 367-376. 6. Hamburger, J. 1977. Macrophage activation: imperfect terminology hiding imperfect knowledge. Ann. Immunol. (Paris) 128:731-732. 7. Hoff, R. 1975. Killing in vitro of Trypanosoma cruzi by macrophages from mice immunized with T. cruzi or BCG, and absence of cross-immunity on challenge in vivo. J. Ezp. Med. 142:299-311. 8. Kozel, T. RK 1977. Non-encapsulated variant of Cryptococcus neoformans. H. Surface receptors for cryptococcal polysaccharide and their role in inhibition of phagocytosis by polysaccharide. Infect. Immun. 16:99-106. 9. Kozel, T. R., and J. Cazin, Jr. 1971. Nonencapsulated variant of Cryptococcus neoformans. I. Virulence studies and characterization of soluble polysaccharide. Infect. Immun. 3:287-294. 10. Kozel, T. R., and R. P. Mastroianni. 1976. Inhibition of phagocytosis by cryptococcal polysaccharide: dissociation of the attachment and ingestion phases of phagocytosis. Infect. Immun. 14:62-67. 11. Mitchell, T. G., and L Friedman. 1972. In vitro phagocytosis and intracellular fate of variously encapsulated strains of Cryptococcus neoformans. Infect. Immun. 5:491-498.
INFECT. IMMUN. 12. Morland, B., and G. Kaplan. 1977. Macrophage activation in vivo and in vitro. Esp. Cell Res. 108:279-288. 13. Nathan, C. F., M L. Karnovsky, and J. R David. 1971. Alterations of macrophage functions by mediators from lymphocytes. J. Ezp. Med. 133:1356-1376. 14. Nathan, C. F., and W. D. Terry. 1975. Differential stimulation of murine lymphoma growth in vitro by normal and BCG-activated macrophages. J. Exp. Med. 142:887-902. 15. Nathan, C. F., and W. D. Terry. 1977. Decreased phagocytosis by peritoneal macrophages from BCG-treated mice. Induction of the phagocytic defect in normal macrophages with BCG in vitro. Cell. Immunol. 29:295-311. 16. Neta, R., and S. B. Salvin. 1971. Cellular immunity in vitro: migration inhibition and phagocytosis. Infect. Immun. 4:697-702. 17. Ratzan, K. R., D. M. Musher, G. T. Keuswh, and L. Weinstein. 1972. Correlation of increased metabolic activity, resistance to infection, enhanced phagocytosis, and inhibition of bacterial growth by macrophages from listeria- and BCG-infected mice. Infect. Immun. 5:499-504. 18. Stubbs, M., A. V. Kiihner, E. A. Glass, J. R. David, and M. L. Karnovsky. 1973. Metabolic and functional studies on activated mouse macrophages. J. Exp. Med. 137:537-542. 19. Werb, Z., and S. Gordon. 1975. Elastase secretion by stimulated macrophages. Characterization and regulation. J. Exp. Med. 142:361-377. 20. Williams, D. M., and J. S. Remington. 1977. Effects of human monocytes and macrophages on Trypanosoma cruzi. Immunology 32:19-23.
