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A new and simple method for studying the binding and ingestion steps in the phagocytosis of yeasts
Journal of Immunological Methods, 154 (1992) 185-193
185
© 1992 Elsevier Science Publishers B.V. All rights reserved 0022-1759/92/$05.00
JIM 06432
A new and simple method for studying the binding and ingestion steps in the phagocytosis of yeasts J. Giaimis, Y. L o m b a r d , M. M a k a y a - K u m b a , P. F o n t e n e a u a n d P. P o i n d r o n Unicersitd Louis Pasteur, Centre de RecherchesPharmaceatiques, D(partement d'lmmumdogic et Immunopharmatvdogie, lllkirch, France
(Received 23 October 1991, revised received4 March 1992,accepted .30April 1992)
Autoclaved yeasts are stained light pink by May-Griinwald Giemsa (MGG). if treated with tannic acid solution just before M G G staining, they display a deep violet color. It seemed possible that these properties could be used to discriminate between extra- and intracellular yeasts in a phagocytosis test, extracellular yeasts being violet and intraeellular yeasts being pink. To validate this protocol, quantitative studies of phagocytosis by MALU cells (a murine macrophage cell line) were performed in the presence or absence of drugs known to interfere with phagocytosis. After treatment of cells with cytochalasin B, the mean number of pink yeasts per cell decreased in a dose-dependent manner, the mean number of violet yeasts increased in a dose-dependent manner, whereas the total number of cell-associated yeasts remained almost unchanged whatever the dose used. After treatment with a - m a n n a n s or chloroquine, the mean numbers of both violet and pink yeasts decreased in a dose-dependent manner. These results confirmed that (i) violet yeasts are extracellular, (ii) autoclaved yeasts recognize lectin receptors, and (iii) unstained (pink) yeasts are intracellular. We show that this simple method can be used for quantitative light microscopic analysis of both the attachment and internalization steps in the phago%,i ~3sis of yeasts. Key words: Phagocytosis(quantitative study of); Yeast; a-Mannan; Cytochalasin B: Chloroquine
Introduction Phagocytosis is a fundamental function of mononuclear phagocytes (Silverstein et al., 1977).
Correspondence to: P. Poindron, Universit6Louis Pasteur, Centre de Recherches Pharmaeeutiques, D6partement d'lmmunologie et Immunopharmacologie,BP 24, F-67401 lllkirch Cedex, France. Tel.: 88 67 69 27; Fax: 88 66 01 90. Abbreciations: CAY, cell-associatedyeasts; FCS, fetal calf serum; MGG, May-GrilnwaldGiemsa; VY, violet yeasts; PY, pink yeasts; Y/C, yeast to cell ratio.
it is a two-step process in which large-sized particles such as microorganisms 'bacteria, yeasts, protozoa, etc.), cells or cell ~.Sr~s as well as inert substances (iron, carbon and silica particles, latex beads, etc.) bind to a cell before being internalized. Opsonophagocytosis is mediated by serum opsonins, namely C3b and C3bi, and antimicrobial antibodies (mainly lgG). The opsonins bind specifically to both surface receptors of the maerophage and microorganisms. The Fc receptors mediate phagocytosis of lgG-coated microorganisms (Mantovani et al., 1972), whereas the
186
so.called CR1 and CR3 receptors are involved in the internalization of C3b- and C3bi-coated microorganisms, respectively (Bianco et al., 1975; Brown, 1991). C3b and C3bi, which are covalently linked to the microbial surface, arise from C3 activated by the alternative pathway after its contact with some microbial surface components (Fearon and Austen, 1980). CR1 and CR3 also enhance the phagocytosis of IgG-coated microorganisms: the antigen-z,ntibody complexes activate complement, and C3b and C3bi are deposited onto the microbial surface. Under these conditions, microorganisms bind more firmly to macrophages than when coated with either IgG or complement fragment alone. Lectinophagocytosis of microorganisms occurs independently of opsonins, in non-immune hosts (Rollag, 1979), according to two modes (Ofek and Sharon, 1988). Microorganisms may express on their surface lectins that recognize specific sugars on the macrophage membrane (Bar-Shavit et al., 1980; Svanborg-Ed6n et al., 1984; Athamma and Ofek, 198~. Alternatively, sugars of the bacterial or yeast surfa,'e may specifically bind to lectin-like receptors on the macrophage membrane (Ogmundsdottir ano Weir, 1976; Freimer et al., 1978; Perry and Ofek, 1984; Sharon, 1984; Perry et al., 1985). Among the lectin-like receptors, the mannose receptor is probably the best known (Stahl et al., 1980; Gordon and Mokoena, 1989; Stahl, 1990). Various methods have been used to study and measure phagocytosis (Lehrer, 1981; Shaw and Griffin, 1981) but they all have their limitations. The microscopic and the radioisotopic techniques are the two methods most frequently used (Verhoef and Waldvogel, 1985) but more recently flow cytofluorometric methods have been introduced (Bassoe et al., 1980; Bassoe and Solberg, 1984; Wilson et al., 1985). Measurement of leukocyte-associated radioactivity (Verhoef et al., 1977) after ingestion of labeled microorganisms is easy to perform, but it does not provide any information about parameters such as the percentage of phagocytosing cells or the distribution of intracellular particles within individual cells. These parameters can be evaluated by either light microscopy or flow cytofluorometry. To our knowledge only sophisticated
flow cytofluorometric techniques are able to differentiate between ingested and adherent particles, by the use of quenching agents that suppress the fluorescence of the non-ingested particles (Hed, 1977; HOd et al., 1987). We describe here a new and simple light microscopic method for studying the binding and internalization steps during the phagocytosis of yeasts by adherent cells. In this method, non-ingested yeasts display properties unequivocally different from ingested yeasts.
Materials and methods
Cells The MALU macrophage cell line was used throughout this study. This cell line was established in our laboratory (Lombard et al., 1988) from C57BL/6 Ipr/Ipr mouse lung, using techniques previously described for establishing mouse resident peritoneal cell lines (Lombard et al., 1985). Cells were grown in RPMI 1640 medium (Gibco BRL, Cergy-Pontoise, France), containing 10% heat-inactivated (30 min, 56°C) fetal calf serum (FCS; Gibco), 2 × 105 U / I sodium benzylpenicillinateand 40 mg/I streptomycin sulfate. The absence of Mycoplasma contamination was assessed by electron microscopic examination. The culture medium contained less than 1 ng/ml endotoxin as assayed by the Limulus test. The cells were regularly passaged as described by Lombard et al. (1988). Similar lines have been shown to express surface mannose receptors (Muller et al., 1988).
Yeasts The straia of Saccharomyces cerevisiae used throughout this study was a local strain. It was grown in Sabouraud's broth for 48 h at 28°C using agitation. Yeasts were then used either alive or after they had been killed by a 45-min autoclaving at 120"C in the culture medium. After autoclaving, the suspension was washed three times in calcium- and magnesium-free phosphatebuffered saline (Ca2+-MgZ+-free PBS). The suspension was then aliquoted (1 mi) and stored at +4°C. Just before use, the suspension of autoclaved yeasts was gently sonicated (water bath
D A
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Fig. l. Appearance of yeasts and phagocytosing cells after staining with MGG. (A) Live yeasts. (B) Autoclaved yeasts (from pale pink to pink). (C) Autoclaved yeasts treated with tannic acid (from blue to violet). (D) MALU cells phagocytosing autoclaved yeasts (without tannic acid treatment). (E) Typical aspect of a MALU cell phagocytosiug autoclaved yeasts (with tannic acid treatment). Blue particles are outside the cells. (F) As in E. Note the blue/violet yeasts around the cell but also at the top of the cells. (G, H ) Cytochalasin B treatment (10 p.g/ml) of MALU cells phagocytosing autoclaved yeasts. Phagocytosis is totally inhibited, but yeastscan still adhere to the surface of the cells. (G) Without tannic acid treatment: it is impossible to determine the location of the particles. (H) With tannic acid treatment: most of the yeasts are blue/violet indicating that they are outside the cells. Bar represents 10 p+m.
188 TABLE I DETERMINATION OF THE OPTIMAL CONCENTRATION OF TANNIC ACID TO BE USED Tannic acid a (w/v)
I% 0.1% 0.01% 0.~1% 0.0001%
Exposuretime 5 min
I min
Db
py c
CAY d
Db
py c
CAY d
+++ ++ + ±
4.5±0.3 4.6±0.7 5.0±0.6 6.3±0.7 *
6.5±0.5 5.8±0.8 6.7±0.9 7.3±0.6 5.9±0.2
+++ ++ ++ +±
4.6±0.1 5.0±0.2 4.8±0.1 5.9±0.1 *
6.4±0.1 7.0±0.2 6.6±0.1 7.2±0.1 6.3±1.2
a Without tannic acid, the mean number of cell-associated yeasts was 5.5 +0.2. b Degree of discrimination between pink and violet yeasts. ¢ Mean number of pink yeasts per cell (except for 0.0001% (*): in this case, all the yeasts were pink). ,t Mean number of total (pink + violet) cell-associated yeasts. Y/C ratio = 20. a p p a r a t u s , Bransonic, OSI, Paris, F r a n c e ) to disr u p t t h e residual c l u m p s a n d it was t h e n diluted in R P M I 1640 m e d i u m w i t h o u t FCS a n d antibiotics.
Quantitation of phagocytosis M A L U cells were s e e d e d in multiwell plates (24 wells, N u n c l o n ; Poly Labo, Strasbourg, F r a n c e ) c o n t a i n i n g sterile glass coverslips ( d i a m e ter: 14 m m , C M L ; N e m o u r s , France). 1 ml o f cell s u s p e n s i o n (10 s cells) was a d d e d to e a c h well. 2 h after seeding, t h e culture m e d i u m was r e m o v e d , cells were w a s h e d once, a n d fresh R P M I 1640 m e d i u m w i t h o u t F C S a n d antibiotics w a s a d d e d (1 m l / w e l l ) . 2 h later, y e a s t s u s p e n s i o n (100 p,l) w a s a d d e d a n d t h e plates i n c u b a t e d for 1 h at 37°(2 in a C O 2 ( 5 % ) h u m i d i f i e d incubator. W h e n m o d u l a t i n g a g e n t s were u s e d , they were a d d e d 15
m i n before t h e yeasts. A f t e r incubation, t h e cells were gently w a s h e d twice with c u l t u r e m e d i u m to r e m o v e u n b o u n d yeasts a n d t a n n i c acid solution was a d d e d as d e s c r i b e d in t h e results section. T h e coverslips were w a s h e d a g a i n with m e d i u m a n d t h e n covered with a d r o p of h e a t - i n a c t i v a t e d F C S to avoid crystallization o f t h e stain d u r i n g t h e following steps. A f t e r drying, t h e coverslips were s t a i n e d with M a y - G r t i n w a l d G i e m s a ( M G G ) , inv e r t e d o n glass slides a n d o b s e r v e d u n d e r light microscopy at a m a g n i f i c a t i o n o f × 1000. W h e n c h l o r o q u i n e was u s e d as t h e pbagocytosis-modulating drug, t h e culture m e d i u m w a s s u p p l e m e n t e d with H E P E S (25 r a M ) a n d t h e p H adj u s t e d to 7.4. T h i s p r o c e d u r e did n o t significantly i n f l u e n c e phagocytosis. A f t e r c o u n t i n g 200 cells, t h e following w e r e d e t e r m i n e d : t h e p e r c e n t a g e o f cells having at
TABLE II DETERMINATION OF THE OPTIMAL Y/C RATIO Y/Crati~ %PY+a pyb VY c CAY d
1 ~.0±4 0.5±0.1 0.1 0.6±0.2
10 ~.5~7.5 3.4±0.5 1.2±0.1 4.6±0.6
~ ~.5±0.5 4.3±0.1 1.5±0.3 5.8±0.4
50 ~.0~0.5 5.4±0.4 2.0±0.3 7.4±0.1
1~ 95.0±0.5 8.8±0.7 4.3±1.2 13.1±0.5
200 94.5±1.5 11.2±0.8 6.9±0.9 18.1±0.1
In this typical example, the optimal Y/C ratio was 1(14):1 (three identical experiments were performed). Values are mean + SE of three successive determinations on a given coverslip. a Percentage of cells having at least one pink cell-associated yeast. b Mean number of pink yeasts per cell. c Mean number of violet yeasts per cell. d Mean number of total cell-associated yeasts.
189 least one cell-associated pink yeast (subsequently referred to as % P Y + ), the mean number of pink yeasts per cell (PY/cell), the mean number of violet yeasts per cell (VY/cell) and the mean number of total cell-associated yeasts (CAY).
Chemicals and reagents Cytochalasin B, a - m a n n a n s and chloroquine were purchased from Sigma (Sigma France, La Verpilli~re, France). Tannic acid (Merck, Darmstadt, Germany) stock solution (10% (w/v)) was prepared with freshly distilled water and sterilized by filtration using Millipore filters (pore size: 0.40 ~ m ; Dachstein, France).
TABLE 111 ~FFECT OF ~-MANNAN ON P H A ~ O S I S a-Mannans 0 i (mg/ml) %py+a 96.0+2 91.0±4 pyb 16.5±0.4 8.1+0.3 VYc 6.0+0.4 1.2±0.1 CAY d 22.5±0.2 9.3±0.4
5
10
72.0±5 57.0±! 3.1±0.3 2.0+0.3 1.0±0.1 !.i ±0.1 4.1±0.2 3.1±0.2
a Percentage of cells having at least one pink cell-associated yeast. b Mean number of pink yeasts per cell. c Mean number of violet yeasts per cell. d Mean number of total cell-associatedyeasts. Three identical experiments were performed. Results of a typical experiment are presented. Values are mean±SE of three successivedeterminations, on a givencoverslip.
Results
Staining properties of yeasts Living yeasts stained with M G G appeared deep blue or violet (Fig. 1A). By contrast, autoclaved yeasts appeared to be uniformly light pink (Fig. 1B). However, if treated with tannic acid just before M G G staining, autoclaved yeasts exhibited a deep violet color (Fig. 1C). We considered whether these staining properties could be used for distinguishing extra- and intracellular yeasts in a phagocytosis test. Only extracellular autoclaved yeasts would be accessible to tannic acid solution, and appear violet after M G G staining, while ingested yeasts should remain pink.
Determination of the optimal experimental conditions In order to optimize the method, we used different concentrations of tannic acid and differ-
ent times of contact (Table I). Even at the highest concentration of tannic acid, the pH of the solution remained close to neutrality, when mannose receptor activity would not be modified (Stephenson and Shepherd, 1987). Whatever the conditions, the number of FY was not affected by the treatment. Neither was the total number of CAY. This suggested that the treatment had no effect on the binding of yeasts to the cells. Because of the easier discrimination between the two classes of yeasts, we chose 1% tannic acid and 1 min exposure as standard conditions. In preliminary experiments we determined the optimal yeast/cell ratio ( Y / C ) to be used in the phagocytosis studies. At a ratio of 50:1, the percentage of cells with PY reached a plateau (about 90%) although the mean number of PY per cell was rather low (5.45) (Table 11). Ratios up to 200:1 did not increase the percentage of cells with PY but did enhance the number of VY and
TABLE IV EFFECT OF CHLOROQUINE ON PHAGOCYTOSIS ~ Chioroquine(/tM) %PY + PY VY CAY a See footnotes of Table 111.
1 93.0± 1 14.3±0.2 5.9±0.2 20.2±0.1
10 93.0± I 12.1±0.4 4.3±0.1 16.4_+0.3
20 68.64-3 4.4±0.5 1.0+ 0.1 5.4+0.6
50 38.0± 3 2.15=0.1 0.6_+0.2 2.7+0.4
100 26.5± I 0.8±0.1 1.1 ±0.l 1.9±0.1
190
3O
PY per cell. From these results we chose a Y / C ratio of 100:1.
• CAY
i
Validation of the method In order to validate the method, we decided to perform comparative studies in the presence or absence of drugs known to interfere with phagocyt¢~s of yeasts, namely a-mannans, chloroquine and cytochalasin B. a - M a n n a n s compete with yeasts for mannose receptors (Sung et al., 1983). W h e n M A L U cells were treated with increasing doses of a - m a n n a n s before being challenged with autoclaved yeasts, the mean number of CAY per cell was reduced in a dose-dependent manner from 22.5 (control) to 3.1 (10 m g / m l a-mannans) (Table IID, as did the mean number of PY per cell (from 16.5 to 2.0), and the percentage of cells with PY (from 96% to 57%). The most striking feature of these experiments was the diminution in the mean n u m b e r of VY per cell. This decrease was almost maximal at the lowest dose of a-ma~aan. W h e n yeasts were opsonised with FCS, the addition of a-mannan, even at the highest dose (10 m g / m l ) , did not influence phagocytosis (data not shown). Chloroquine downregulates the expression of mannose receptors, most probably by trapping the receptors within intracellular compartments (Gonzales-Noriega et al., 1980; Tietze et al., 1980). W h e n M A L U cells were treated with chloroquine (100 p m o l / m l ) , we observed that the percentage of cells with PY decreased from 93% to 26.5% (Table IV). This was accompanied by a dose-dependent decrease of the mean number of CAY per cell, of PY per cell and of VY per cell.
0
1
5
96 ±2 96 ±2 92 ±1 16.5±0.4 11.9±0.9 6.0±0.4 6.0± 0.4 7.3± 2.0 15.5± 0.9 22.5±0.2 19.2±3.0 21.5±0.5
a See footnotes of Table 111.
10
61 ±1 2.0±0.1 22.9± 0.8 24.9+0.9
•
vY
15
'g 0
It l
10
5
20
mnnans (mgml)
~1
_
• c.Y
I
: t_khR'I 211 h050 01 ._s I
TABLE V EFFECI" OF CYTOCHALASIN B ON PHAGOCYTOSIS a Ojtochalasin (pg/mi) %PY+ PY VY CAY
20
20
IIvY
| " 1
o~
B
0
1 5 l0 ¢lnoehaluin S (~gml)
20
3O
0
" 0
O,5
-5
C 1o
2o
mnnans (mgml) Fig. 2. Quantitative study of the binding and internalizationof yeasts by MALU cells treated with a-mannan, cytochalasinB, alone or in combination.(A) mean number of CAY, PY and VY as a function of the dose of a-mannan. (B) mean number of CAY, PY and VY as a function of the dose of cytochalasin B. (C) mean number of CAY, PY and VY as a function of the dose of a-mannan in combination with c~ochalasin B (5 /~g/ml). Cytochalasin B impairs phagocytosis by disorganizing the cytoskeleton (Spudich and Lin, 1972; Axline and Reaven, 1974). As shown in Table V, the mean number of CAY per cell did not change
191 in presence of increasing doses of cytochalasin B, but the mean number of PY per cell dropped from 16.5 (control) to 2.0 and the percentage of cells with PY decreased from 96% (control) to 61% in a dose-dependent manner. The mean number of VY was augmented from 6.0 (control) to 22.9 (10 itg/ml cytochalasin B). When MALU cells were simultaneously treated with cytochalasin B ( 5 / t g / m l ) and various doses of a-mannans (Fig. 2C), the mean number of CAY per cell dropped from 21.6 (no a-mannans) to 1.62 (5 m g / m l a-mannans). Even at low concentrations, a-mannans induced a marked decrease of VY without affecting the number of PY, suggesting that the adhesion of yeast particles to the cell surface was indeed specific.
Discussion
The present study shows that autoclaved yeasts can be used to study attachment a n d / o r internalization using tannic acid pretreatment before M G G staining. Autoclaved yeasts acquire new staining properties which permit two classes of CAY to be clearly distinguished. The first class consists of stainable yeasts (VY); the second, of unstained or poorly stained yeasts (PY). Modulating phagocytosis with drugs known to impair this function, results in the predicted modifications of the distribution of these two classes of yeasts. We concluded that VY were indeed extracellular. When treated with a-mannans (Fig. 2 A ) or chloroquine, MALU cells displayed fewer VY presumably because lectin-like receptors that bind yeasts were either occupied (a-mannans) or unable to recycle after being internalized (chloroquine). The intracellular location of PY was confirmed by the effect of cytochalasin B which diminished both the percentage of PY-positive cells and the mean number of PY per cell, whereas the mean number of VY per cell increased so that tile mean number of CAY per cell did not change. This is exactly as expected if cytochalasin B does not impede the attachment step but impedes engulfment (Fig. 3). However, the increase in the number of VY per cell after cytochalasin B treatment represents specific binding since it is inhib-
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~110..s: oss. o /
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oo
I
o
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I
g%o oo
1 L'oOso Z
x
. x
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Numberof violety~tsts Fig. 3. Individual analysis of MALU cells challenged with autoclaved yeasts, after treatment with cytochalasin B or a-mannan. The number of PY and VY is recorded for each cell.
ited by the addition of low doses of a-mannans (Fig. 2C). Even with high doses of a-mannans (i.e. 10-20 mg/ml), we always observed a small population of cells with PY. We believe that this persistent phagocytosis, under conditions where all mannose receptors should have been occupied, is mediated by another receptor or another mechanism. This method cannot be performed with living yeasts since they always exhibit a deep violet color, whether located inside or outside the cells. We observed also that autoclaved yeasts digested by trypsin lost their ability to be stained by M G G (data not shown). This suggests that the n a t u r e o f the proteins is critical in the process of tannic acid binding. This light microscopic method is reproducible, sensitive and permits extracellular yeasts to be distinguished from intracellular yeasts. It is a simple and accessible alternative to the use of flow cytometry or UV microscopy.
Acknowledgements The authors are grateful to Professor Pesson for his help with the light microscopy and microphotography, and to Drs. K. Takeda, C.D. Muller and S. Braun for their help in the preparation of the manuscript.
192
References Athamma, A. and Ofek, I. (1988) Enzyme-linked [mmunosorbent assay for quantitation of attachment and ingestion stages of bacterial phagocytosis. J. Clin. Miero~iol. 26, 476. Axline, S.G. and Reaven, E.P. (1974) Inhibition of phagocytosis and plasma membrane mobility of the cultivated macrophage by cytochalasin B. Role of subplasmalemmal microfilaments. J. Cell Biol. 63, 647. Bar-Shavit, Z., Goldman, R., Ofek, I., Sharon, N. and Hirelman, D. (1980) Mannose-binding activity of Escherichia coli: a determinant of attachment and ingestion. Infect. lmmun. 29, 417. Bassoe, C.F. and Solberg, C.O. (1984) Phagocytosis of Staphylococcus aureus by human leukocytes: quantitation by a flow cytometric and a microbiological method. Acta Pathol. Microbiol. Immunol. Scand. Sect. C 92, 43. Bass~e, C.F., Solsvik, J. and Laerun, O.D. (1980) Quantitation of single cell phagocytic capacity by flow cytometry. Flow Cytometry 4, 170. Bianco, C., Griffin, F.M. and Silverstein, S.C. (1975) Studies on the macrophage complement receptor function upon macrophage activation. J. Exp. Med. 141,1278. Brown, EJ. (1991) Complement receptors and phagocytosis. Curt. Opinion Immunol. 3, 75. Fearon, D.T. and Austen, K.F. (1980) The alternative pathway of complement: a system for host resistance to microbial infection. N. Eng. J. Med. 303, 259. Freimer, N.B., Ogmundsdottir, H.M., Blackwell, C.C., Sutherland, 1. W., Graham, L. and Weir, DiM. (1978) The role of cell wall carbohydrate in binding of microorganisms to mouse peritoneal exudate macrophages. Acta Pathol. Microbiol. Scand., Sect. B 86, 53. Gonzalez-Noriega, A., Grubb, J.H., Talkad, V. and Sly, W.S. (1980) Chloroquine inhibits lysosomal enzyme pinocytosis and enhances enzyme secretion by impairing receptor recycling. J. Cell Biol. 85, 839. Gordon, S. and Mokoena, T. (1989) Receptors for mannose structures on mononuclear phagocytes. In: M. Zembala and G.L Asherson (Eds.), Mononuclear Phagocytes. Academic Press, San Diego, CA, p. 141. HOd, J. (1977) The extinction of fluorescence by crystal violet and its use to differentiate between attached and ingested microorganisms in phagocytosis. FEMS Lett. 1, 357. HOd, J., Hallden, G., Johansson, S.G.O. and Larsson, P. (1987) The use of fluorescence quenching in flow cytometry to measure the attachment and ingestion phases in phagocytosis in peripheral blood without prior cell separation. J. Immunol. Methods 101,119. Lehrer, R.L (1981) Ingestion and destruction of Candida albicans, in: D.O. Adams, P.J. Edelson and H. Koren (Eds.), Methods for Studying Mononuclear Phagocytes. Academic Press, New York, p. 693. Lombard, Y.. Ulrich, B. and Poindron, P. (1985) In vitro multiplication and apparently indefinite subcultures of normal mouse peritoneal resident macrophages. Biol. Cell 53, 219.
Lombard, Y., Bartholeyns, J., Chokri, M., lllinger, D., Hartmann, D., Dumont, S., Kauffmann, S.H., Landmann, R., Loor, F. and Poindron P. (1988) Establishment and characterization of long-term cultured cell lines of routine resident macrophagcs. J. Leukocyte Biol. 44, 391. Mantovani, B., Rabinovitch, M. and Nussenzweig, V. (1972) Phagocytosis of immune complexes by macrophages. J. Exp. Med. 135, 780. Muller, C.D., Lombard, Y., Bartholeyns, J., Poindron, P. and Schuber, F. (1988) Characterization of surface markers of continuously growing murine resident macophages. J. Leukocyte Biol. 49,165. Ofek, I. and Sharon, N. (1988) Lectinopbagocytosis: a molecular mechanism of recognition between cell surface sugars and lectins in the pbagocytosis of bacteria. Infect. Immun. 56, 539. Ogmundsdottir, H.M. and Weir, D.M. (1976) The characteristics of binding of Corynebacterium parvum to glass-adherent mouse peritoneal exudate cells. Clin. Exp. Immunol. 26, 334. Perry, A. and Ofek, I. (1984) Inhibition of blood clearance and hepatic tissue binding of Escherichia coil by liver lectin-specific sugars and glycoproteins. Infect. Immun. 43, 257. Perry, A., Keisari, Y. and Ofek, 1. (1985) Liver cell and macrophage surface lectins as determinants of recognition in blood clearance and cellular attachment. FEMS Microbiol. Left. 27, 345. Rollag, H. (1979) Uptake of non-opsonized Escherichia coil by unstimulated mouse peritoneal macrophages. Acta Pathol. Microbiol. Scaod., Sect. C 87, 99. Sharon, N. (1984) Surface carbohydrates and surface lectins are recognition determinants in phagocytosis. Immunol. Today 5, 143. Shaw, D.R. and Griffin, F.M. (1981) Antibody*dependent and antibody-independent phagocytosis. In: D.O. Adams, P.J. Edelson and H. Koren (Eds.), Methods for Studying Mononuclear Phagocytes. Academic Press, New York, p. 511. Silverstein, S.C., Steinman, R.S. and Cohn, Z.A. (1977) Endocytosis. Annu. Rev. Biochem. 46, 699. Spudich, J.A. and Lin, S. (1972) Cytochalasin B, its interaction with actin and actomyosin from muscle. Proc. Natl. Acad. Sci. USA 69, 442. Stahl, P. (1990) The macrophage mannose receptors: current status. Am. J. Resp. Cell. Mol. Biol. 2, 317. Stabl, P., Schlesinger, P., Sigardson, E., Rodman, J. and Lee, Y.C. (1980) Receptor-mediated pinocytosis of mannoseglycoconjugates by macrophages: characterization and evidence for receptor recycling. Cell 19, 207. Stephenson, J.D. and Shepherd, V.L. (1987) Purification of the human alveolar macrophage mannose receptor. Biochem. Biophys. Res. Commun. 148, 883. Suns, S.SJ., Nelson, R.S. and Silverstein, S.C. (1983) Yeast mannans inhibit binding and phagocytosis of zymosan by mouse peritoneal macrophages. J. Cell Biol. 96,160. Svanborg-Ed6n, C., Bjursten, L.-M., Hull, R., Hull, S., Magnusson, K.E., Moldovano, Z. and Leffer, H. (1984) Influ-
ence of adhesins on the interaction of Escherichia coli with human phagocytes. Infect. lmmun. 44, 672. Tietze, C,, Schlesinger, P. and Stahl, P. (1980) Chloroquine and ammonium ion inhibit receptor-mediated endocytosis of mannose-glycoconjugates by macrophages: apparent inhibition of receptor recycling. Biochem. Biophys. Res. Commun. 93,1. Verhoef, J. and Waldvogel, F.A. (1985) Testing phagocy~:c cell function. Eur. J. Clin. Microbiol. 4, 379.
VerhoeL J., Peterson, P.K. and Quie, P.O. (1977) Kinetics of Staphylococcal opsonization, attachment, ingestion and killing by human morphonuclear leukocytes: a quantitative assay using H. tbymidine labeled bacteria. J. lm,,q.unoL Methods 14, 303. Wilson, R.M., Galvin, A.M., Robins, R.A. and Reeves, W.G. (1985) A flow cytometric method for the measurement of phagocytosis by polymorphonuclear leucocytes. J. lmmunol. Methods 76, 247.
185
© 1992 Elsevier Science Publishers B.V. All rights reserved 0022-1759/92/$05.00
JIM 06432
A new and simple method for studying the binding and ingestion steps in the phagocytosis of yeasts J. Giaimis, Y. L o m b a r d , M. M a k a y a - K u m b a , P. F o n t e n e a u a n d P. P o i n d r o n Unicersitd Louis Pasteur, Centre de RecherchesPharmaceatiques, D(partement d'lmmumdogic et Immunopharmatvdogie, lllkirch, France
(Received 23 October 1991, revised received4 March 1992,accepted .30April 1992)
Autoclaved yeasts are stained light pink by May-Griinwald Giemsa (MGG). if treated with tannic acid solution just before M G G staining, they display a deep violet color. It seemed possible that these properties could be used to discriminate between extra- and intracellular yeasts in a phagocytosis test, extracellular yeasts being violet and intraeellular yeasts being pink. To validate this protocol, quantitative studies of phagocytosis by MALU cells (a murine macrophage cell line) were performed in the presence or absence of drugs known to interfere with phagocytosis. After treatment of cells with cytochalasin B, the mean number of pink yeasts per cell decreased in a dose-dependent manner, the mean number of violet yeasts increased in a dose-dependent manner, whereas the total number of cell-associated yeasts remained almost unchanged whatever the dose used. After treatment with a - m a n n a n s or chloroquine, the mean numbers of both violet and pink yeasts decreased in a dose-dependent manner. These results confirmed that (i) violet yeasts are extracellular, (ii) autoclaved yeasts recognize lectin receptors, and (iii) unstained (pink) yeasts are intracellular. We show that this simple method can be used for quantitative light microscopic analysis of both the attachment and internalization steps in the phago%,i ~3sis of yeasts. Key words: Phagocytosis(quantitative study of); Yeast; a-Mannan; Cytochalasin B: Chloroquine
Introduction Phagocytosis is a fundamental function of mononuclear phagocytes (Silverstein et al., 1977).
Correspondence to: P. Poindron, Universit6Louis Pasteur, Centre de Recherches Pharmaeeutiques, D6partement d'lmmunologie et Immunopharmacologie,BP 24, F-67401 lllkirch Cedex, France. Tel.: 88 67 69 27; Fax: 88 66 01 90. Abbreciations: CAY, cell-associatedyeasts; FCS, fetal calf serum; MGG, May-GrilnwaldGiemsa; VY, violet yeasts; PY, pink yeasts; Y/C, yeast to cell ratio.
it is a two-step process in which large-sized particles such as microorganisms 'bacteria, yeasts, protozoa, etc.), cells or cell ~.Sr~s as well as inert substances (iron, carbon and silica particles, latex beads, etc.) bind to a cell before being internalized. Opsonophagocytosis is mediated by serum opsonins, namely C3b and C3bi, and antimicrobial antibodies (mainly lgG). The opsonins bind specifically to both surface receptors of the maerophage and microorganisms. The Fc receptors mediate phagocytosis of lgG-coated microorganisms (Mantovani et al., 1972), whereas the
186
so.called CR1 and CR3 receptors are involved in the internalization of C3b- and C3bi-coated microorganisms, respectively (Bianco et al., 1975; Brown, 1991). C3b and C3bi, which are covalently linked to the microbial surface, arise from C3 activated by the alternative pathway after its contact with some microbial surface components (Fearon and Austen, 1980). CR1 and CR3 also enhance the phagocytosis of IgG-coated microorganisms: the antigen-z,ntibody complexes activate complement, and C3b and C3bi are deposited onto the microbial surface. Under these conditions, microorganisms bind more firmly to macrophages than when coated with either IgG or complement fragment alone. Lectinophagocytosis of microorganisms occurs independently of opsonins, in non-immune hosts (Rollag, 1979), according to two modes (Ofek and Sharon, 1988). Microorganisms may express on their surface lectins that recognize specific sugars on the macrophage membrane (Bar-Shavit et al., 1980; Svanborg-Ed6n et al., 1984; Athamma and Ofek, 198~. Alternatively, sugars of the bacterial or yeast surfa,'e may specifically bind to lectin-like receptors on the macrophage membrane (Ogmundsdottir ano Weir, 1976; Freimer et al., 1978; Perry and Ofek, 1984; Sharon, 1984; Perry et al., 1985). Among the lectin-like receptors, the mannose receptor is probably the best known (Stahl et al., 1980; Gordon and Mokoena, 1989; Stahl, 1990). Various methods have been used to study and measure phagocytosis (Lehrer, 1981; Shaw and Griffin, 1981) but they all have their limitations. The microscopic and the radioisotopic techniques are the two methods most frequently used (Verhoef and Waldvogel, 1985) but more recently flow cytofluorometric methods have been introduced (Bassoe et al., 1980; Bassoe and Solberg, 1984; Wilson et al., 1985). Measurement of leukocyte-associated radioactivity (Verhoef et al., 1977) after ingestion of labeled microorganisms is easy to perform, but it does not provide any information about parameters such as the percentage of phagocytosing cells or the distribution of intracellular particles within individual cells. These parameters can be evaluated by either light microscopy or flow cytofluorometry. To our knowledge only sophisticated
flow cytofluorometric techniques are able to differentiate between ingested and adherent particles, by the use of quenching agents that suppress the fluorescence of the non-ingested particles (Hed, 1977; HOd et al., 1987). We describe here a new and simple light microscopic method for studying the binding and internalization steps during the phagocytosis of yeasts by adherent cells. In this method, non-ingested yeasts display properties unequivocally different from ingested yeasts.
Materials and methods
Cells The MALU macrophage cell line was used throughout this study. This cell line was established in our laboratory (Lombard et al., 1988) from C57BL/6 Ipr/Ipr mouse lung, using techniques previously described for establishing mouse resident peritoneal cell lines (Lombard et al., 1985). Cells were grown in RPMI 1640 medium (Gibco BRL, Cergy-Pontoise, France), containing 10% heat-inactivated (30 min, 56°C) fetal calf serum (FCS; Gibco), 2 × 105 U / I sodium benzylpenicillinateand 40 mg/I streptomycin sulfate. The absence of Mycoplasma contamination was assessed by electron microscopic examination. The culture medium contained less than 1 ng/ml endotoxin as assayed by the Limulus test. The cells were regularly passaged as described by Lombard et al. (1988). Similar lines have been shown to express surface mannose receptors (Muller et al., 1988).
Yeasts The straia of Saccharomyces cerevisiae used throughout this study was a local strain. It was grown in Sabouraud's broth for 48 h at 28°C using agitation. Yeasts were then used either alive or after they had been killed by a 45-min autoclaving at 120"C in the culture medium. After autoclaving, the suspension was washed three times in calcium- and magnesium-free phosphatebuffered saline (Ca2+-MgZ+-free PBS). The suspension was then aliquoted (1 mi) and stored at +4°C. Just before use, the suspension of autoclaved yeasts was gently sonicated (water bath
D A
L~
)c
t.
rll
Fig. l. Appearance of yeasts and phagocytosing cells after staining with MGG. (A) Live yeasts. (B) Autoclaved yeasts (from pale pink to pink). (C) Autoclaved yeasts treated with tannic acid (from blue to violet). (D) MALU cells phagocytosing autoclaved yeasts (without tannic acid treatment). (E) Typical aspect of a MALU cell phagocytosiug autoclaved yeasts (with tannic acid treatment). Blue particles are outside the cells. (F) As in E. Note the blue/violet yeasts around the cell but also at the top of the cells. (G, H ) Cytochalasin B treatment (10 p.g/ml) of MALU cells phagocytosing autoclaved yeasts. Phagocytosis is totally inhibited, but yeastscan still adhere to the surface of the cells. (G) Without tannic acid treatment: it is impossible to determine the location of the particles. (H) With tannic acid treatment: most of the yeasts are blue/violet indicating that they are outside the cells. Bar represents 10 p+m.
188 TABLE I DETERMINATION OF THE OPTIMAL CONCENTRATION OF TANNIC ACID TO BE USED Tannic acid a (w/v)
I% 0.1% 0.01% 0.~1% 0.0001%
Exposuretime 5 min
I min
Db
py c
CAY d
Db
py c
CAY d
+++ ++ + ±
4.5±0.3 4.6±0.7 5.0±0.6 6.3±0.7 *
6.5±0.5 5.8±0.8 6.7±0.9 7.3±0.6 5.9±0.2
+++ ++ ++ +±
4.6±0.1 5.0±0.2 4.8±0.1 5.9±0.1 *
6.4±0.1 7.0±0.2 6.6±0.1 7.2±0.1 6.3±1.2
a Without tannic acid, the mean number of cell-associated yeasts was 5.5 +0.2. b Degree of discrimination between pink and violet yeasts. ¢ Mean number of pink yeasts per cell (except for 0.0001% (*): in this case, all the yeasts were pink). ,t Mean number of total (pink + violet) cell-associated yeasts. Y/C ratio = 20. a p p a r a t u s , Bransonic, OSI, Paris, F r a n c e ) to disr u p t t h e residual c l u m p s a n d it was t h e n diluted in R P M I 1640 m e d i u m w i t h o u t FCS a n d antibiotics.
Quantitation of phagocytosis M A L U cells were s e e d e d in multiwell plates (24 wells, N u n c l o n ; Poly Labo, Strasbourg, F r a n c e ) c o n t a i n i n g sterile glass coverslips ( d i a m e ter: 14 m m , C M L ; N e m o u r s , France). 1 ml o f cell s u s p e n s i o n (10 s cells) was a d d e d to e a c h well. 2 h after seeding, t h e culture m e d i u m was r e m o v e d , cells were w a s h e d once, a n d fresh R P M I 1640 m e d i u m w i t h o u t F C S a n d antibiotics w a s a d d e d (1 m l / w e l l ) . 2 h later, y e a s t s u s p e n s i o n (100 p,l) w a s a d d e d a n d t h e plates i n c u b a t e d for 1 h at 37°(2 in a C O 2 ( 5 % ) h u m i d i f i e d incubator. W h e n m o d u l a t i n g a g e n t s were u s e d , they were a d d e d 15
m i n before t h e yeasts. A f t e r incubation, t h e cells were gently w a s h e d twice with c u l t u r e m e d i u m to r e m o v e u n b o u n d yeasts a n d t a n n i c acid solution was a d d e d as d e s c r i b e d in t h e results section. T h e coverslips were w a s h e d a g a i n with m e d i u m a n d t h e n covered with a d r o p of h e a t - i n a c t i v a t e d F C S to avoid crystallization o f t h e stain d u r i n g t h e following steps. A f t e r drying, t h e coverslips were s t a i n e d with M a y - G r t i n w a l d G i e m s a ( M G G ) , inv e r t e d o n glass slides a n d o b s e r v e d u n d e r light microscopy at a m a g n i f i c a t i o n o f × 1000. W h e n c h l o r o q u i n e was u s e d as t h e pbagocytosis-modulating drug, t h e culture m e d i u m w a s s u p p l e m e n t e d with H E P E S (25 r a M ) a n d t h e p H adj u s t e d to 7.4. T h i s p r o c e d u r e did n o t significantly i n f l u e n c e phagocytosis. A f t e r c o u n t i n g 200 cells, t h e following w e r e d e t e r m i n e d : t h e p e r c e n t a g e o f cells having at
TABLE II DETERMINATION OF THE OPTIMAL Y/C RATIO Y/Crati~ %PY+a pyb VY c CAY d
1 ~.0±4 0.5±0.1 0.1 0.6±0.2
10 ~.5~7.5 3.4±0.5 1.2±0.1 4.6±0.6
~ ~.5±0.5 4.3±0.1 1.5±0.3 5.8±0.4
50 ~.0~0.5 5.4±0.4 2.0±0.3 7.4±0.1
1~ 95.0±0.5 8.8±0.7 4.3±1.2 13.1±0.5
200 94.5±1.5 11.2±0.8 6.9±0.9 18.1±0.1
In this typical example, the optimal Y/C ratio was 1(14):1 (three identical experiments were performed). Values are mean + SE of three successive determinations on a given coverslip. a Percentage of cells having at least one pink cell-associated yeast. b Mean number of pink yeasts per cell. c Mean number of violet yeasts per cell. d Mean number of total cell-associated yeasts.
189 least one cell-associated pink yeast (subsequently referred to as % P Y + ), the mean number of pink yeasts per cell (PY/cell), the mean number of violet yeasts per cell (VY/cell) and the mean number of total cell-associated yeasts (CAY).
Chemicals and reagents Cytochalasin B, a - m a n n a n s and chloroquine were purchased from Sigma (Sigma France, La Verpilli~re, France). Tannic acid (Merck, Darmstadt, Germany) stock solution (10% (w/v)) was prepared with freshly distilled water and sterilized by filtration using Millipore filters (pore size: 0.40 ~ m ; Dachstein, France).
TABLE 111 ~FFECT OF ~-MANNAN ON P H A ~ O S I S a-Mannans 0 i (mg/ml) %py+a 96.0+2 91.0±4 pyb 16.5±0.4 8.1+0.3 VYc 6.0+0.4 1.2±0.1 CAY d 22.5±0.2 9.3±0.4
5
10
72.0±5 57.0±! 3.1±0.3 2.0+0.3 1.0±0.1 !.i ±0.1 4.1±0.2 3.1±0.2
a Percentage of cells having at least one pink cell-associated yeast. b Mean number of pink yeasts per cell. c Mean number of violet yeasts per cell. d Mean number of total cell-associatedyeasts. Three identical experiments were performed. Results of a typical experiment are presented. Values are mean±SE of three successivedeterminations, on a givencoverslip.
Results
Staining properties of yeasts Living yeasts stained with M G G appeared deep blue or violet (Fig. 1A). By contrast, autoclaved yeasts appeared to be uniformly light pink (Fig. 1B). However, if treated with tannic acid just before M G G staining, autoclaved yeasts exhibited a deep violet color (Fig. 1C). We considered whether these staining properties could be used for distinguishing extra- and intracellular yeasts in a phagocytosis test. Only extracellular autoclaved yeasts would be accessible to tannic acid solution, and appear violet after M G G staining, while ingested yeasts should remain pink.
Determination of the optimal experimental conditions In order to optimize the method, we used different concentrations of tannic acid and differ-
ent times of contact (Table I). Even at the highest concentration of tannic acid, the pH of the solution remained close to neutrality, when mannose receptor activity would not be modified (Stephenson and Shepherd, 1987). Whatever the conditions, the number of FY was not affected by the treatment. Neither was the total number of CAY. This suggested that the treatment had no effect on the binding of yeasts to the cells. Because of the easier discrimination between the two classes of yeasts, we chose 1% tannic acid and 1 min exposure as standard conditions. In preliminary experiments we determined the optimal yeast/cell ratio ( Y / C ) to be used in the phagocytosis studies. At a ratio of 50:1, the percentage of cells with PY reached a plateau (about 90%) although the mean number of PY per cell was rather low (5.45) (Table 11). Ratios up to 200:1 did not increase the percentage of cells with PY but did enhance the number of VY and
TABLE IV EFFECT OF CHLOROQUINE ON PHAGOCYTOSIS ~ Chioroquine(/tM) %PY + PY VY CAY a See footnotes of Table 111.
1 93.0± 1 14.3±0.2 5.9±0.2 20.2±0.1
10 93.0± I 12.1±0.4 4.3±0.1 16.4_+0.3
20 68.64-3 4.4±0.5 1.0+ 0.1 5.4+0.6
50 38.0± 3 2.15=0.1 0.6_+0.2 2.7+0.4
100 26.5± I 0.8±0.1 1.1 ±0.l 1.9±0.1
190
3O
PY per cell. From these results we chose a Y / C ratio of 100:1.
• CAY
i
Validation of the method In order to validate the method, we decided to perform comparative studies in the presence or absence of drugs known to interfere with phagocyt¢~s of yeasts, namely a-mannans, chloroquine and cytochalasin B. a - M a n n a n s compete with yeasts for mannose receptors (Sung et al., 1983). W h e n M A L U cells were treated with increasing doses of a - m a n n a n s before being challenged with autoclaved yeasts, the mean number of CAY per cell was reduced in a dose-dependent manner from 22.5 (control) to 3.1 (10 m g / m l a-mannans) (Table IID, as did the mean number of PY per cell (from 16.5 to 2.0), and the percentage of cells with PY (from 96% to 57%). The most striking feature of these experiments was the diminution in the mean n u m b e r of VY per cell. This decrease was almost maximal at the lowest dose of a-ma~aan. W h e n yeasts were opsonised with FCS, the addition of a-mannan, even at the highest dose (10 m g / m l ) , did not influence phagocytosis (data not shown). Chloroquine downregulates the expression of mannose receptors, most probably by trapping the receptors within intracellular compartments (Gonzales-Noriega et al., 1980; Tietze et al., 1980). W h e n M A L U cells were treated with chloroquine (100 p m o l / m l ) , we observed that the percentage of cells with PY decreased from 93% to 26.5% (Table IV). This was accompanied by a dose-dependent decrease of the mean number of CAY per cell, of PY per cell and of VY per cell.
0
1
5
96 ±2 96 ±2 92 ±1 16.5±0.4 11.9±0.9 6.0±0.4 6.0± 0.4 7.3± 2.0 15.5± 0.9 22.5±0.2 19.2±3.0 21.5±0.5
a See footnotes of Table 111.
10
61 ±1 2.0±0.1 22.9± 0.8 24.9+0.9
•
vY
15
'g 0
It l
10
5
20
mnnans (mgml)
~1
_
• c.Y
I
: t_khR'I 211 h050 01 ._s I
TABLE V EFFECI" OF CYTOCHALASIN B ON PHAGOCYTOSIS a Ojtochalasin (pg/mi) %PY+ PY VY CAY
20
20
IIvY
| " 1
o~
B
0
1 5 l0 ¢lnoehaluin S (~gml)
20
3O
0
" 0
O,5
-5
C 1o
2o
mnnans (mgml) Fig. 2. Quantitative study of the binding and internalizationof yeasts by MALU cells treated with a-mannan, cytochalasinB, alone or in combination.(A) mean number of CAY, PY and VY as a function of the dose of a-mannan. (B) mean number of CAY, PY and VY as a function of the dose of cytochalasin B. (C) mean number of CAY, PY and VY as a function of the dose of a-mannan in combination with c~ochalasin B (5 /~g/ml). Cytochalasin B impairs phagocytosis by disorganizing the cytoskeleton (Spudich and Lin, 1972; Axline and Reaven, 1974). As shown in Table V, the mean number of CAY per cell did not change
191 in presence of increasing doses of cytochalasin B, but the mean number of PY per cell dropped from 16.5 (control) to 2.0 and the percentage of cells with PY decreased from 96% (control) to 61% in a dose-dependent manner. The mean number of VY was augmented from 6.0 (control) to 22.9 (10 itg/ml cytochalasin B). When MALU cells were simultaneously treated with cytochalasin B ( 5 / t g / m l ) and various doses of a-mannans (Fig. 2C), the mean number of CAY per cell dropped from 21.6 (no a-mannans) to 1.62 (5 m g / m l a-mannans). Even at low concentrations, a-mannans induced a marked decrease of VY without affecting the number of PY, suggesting that the adhesion of yeast particles to the cell surface was indeed specific.
Discussion
The present study shows that autoclaved yeasts can be used to study attachment a n d / o r internalization using tannic acid pretreatment before M G G staining. Autoclaved yeasts acquire new staining properties which permit two classes of CAY to be clearly distinguished. The first class consists of stainable yeasts (VY); the second, of unstained or poorly stained yeasts (PY). Modulating phagocytosis with drugs known to impair this function, results in the predicted modifications of the distribution of these two classes of yeasts. We concluded that VY were indeed extracellular. When treated with a-mannans (Fig. 2 A ) or chloroquine, MALU cells displayed fewer VY presumably because lectin-like receptors that bind yeasts were either occupied (a-mannans) or unable to recycle after being internalized (chloroquine). The intracellular location of PY was confirmed by the effect of cytochalasin B which diminished both the percentage of PY-positive cells and the mean number of PY per cell, whereas the mean number of VY per cell increased so that tile mean number of CAY per cell did not change. This is exactly as expected if cytochalasin B does not impede the attachment step but impedes engulfment (Fig. 3). However, the increase in the number of VY per cell after cytochalasin B treatment represents specific binding since it is inhib-
30]0°°0°0 / ° o ooo
~' ....
I o i
S.o
•
s
~110..s: oss. o /
I
I x C~oclniiteB(lOlql) I
oo
I
o
i(lOmg/mi)
I
g%o oo
1 L'oOso Z
x
. x
xxX,,lllXX
0
l0
o x
x
x x o
IIx:~x
20
x:,~
:30
~
x
x
40
Numberof violety~tsts Fig. 3. Individual analysis of MALU cells challenged with autoclaved yeasts, after treatment with cytochalasin B or a-mannan. The number of PY and VY is recorded for each cell.
ited by the addition of low doses of a-mannans (Fig. 2C). Even with high doses of a-mannans (i.e. 10-20 mg/ml), we always observed a small population of cells with PY. We believe that this persistent phagocytosis, under conditions where all mannose receptors should have been occupied, is mediated by another receptor or another mechanism. This method cannot be performed with living yeasts since they always exhibit a deep violet color, whether located inside or outside the cells. We observed also that autoclaved yeasts digested by trypsin lost their ability to be stained by M G G (data not shown). This suggests that the n a t u r e o f the proteins is critical in the process of tannic acid binding. This light microscopic method is reproducible, sensitive and permits extracellular yeasts to be distinguished from intracellular yeasts. It is a simple and accessible alternative to the use of flow cytometry or UV microscopy.
Acknowledgements The authors are grateful to Professor Pesson for his help with the light microscopy and microphotography, and to Drs. K. Takeda, C.D. Muller and S. Braun for their help in the preparation of the manuscript.
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