[11] Assay of phagosome-lysosome fusion

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neutrophils from patients longitudinally or in one assay with those of other patients. Also, neutrophil cytoplasts can be frozen and serve as reference material or can be collected for studies that require large amounts o f material from one particular donor. [11] Assay of Phagosome-Lysosome By MARGARET

Fusion

KIELIAN

Introduction Particles ingested by cells are contained within a membrane-bounded vacuole or p h a g o s o m e . In many instances this vacuole undergoes fusion with lysosomes, resulting in exposure of the particle to both the acidic p H ~ and hydrolytic e n z y m e s 2 of the lysosome. The mechanisms which control the specificity, triggering, and kinetics of this and other intracellular fusion events are unclear. Also not understood is the mechanism by which a n u m b e r o f parasites inhibit fusion with lysosomes, in some cases resulting in their survival and multiplication within the host. 3,4 Better understanding of the effectors of the p h a g o s o m e - l y s o s o m e ( P - L ) 4a fusion reaction requires systems for its assay in cultured cells, and several types of assays have been described. Electron microscopy has been used to look for the appearance of lysosomal markers such as acid phosphatase or exogenously fed peroxidase, ferritin, colloidal gold, or thorium dioxide in the phagocytic vacuole. The degradation of radiolabeled particles by lysosomal hydrolases has been used as an indirect assay of P - L fusion. 5 Phagocytized particles of low density have been isolated by gradient centrifugation after ingestion, and the transfer of lysosomal enzymes into this " p h a g o s o m e f r a c t i o n " assayed. 6,7 An in v i t r o fusion system has also been described, in which light and electron microscopy were used to monitor the fusion of differentially labeled phagolysosome fractions. 8-1° l S. O h k u m a a n d B. P o o l e , Proc. Natl. Acad. Sci. U.S.A. 75, 3327 (1978).

2 C. deDuve, Science 189, 186 (1975). 3 M. B. Goren, Annu. Rev. Microbiol. 31, 507 (1977). 4 M. A. Horwitz, J. Exp. Med. 158, 2108 (1983). Abbreviations: AO, acridine orange; FCS, fetal calf serum; HRP, horseradish peroxidase; MEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline, pH 7.4; P-L fusion, phagosome-lysosome fusion. 5 Z. A. Cohn, J. Exp. Med. 117, 27 (1963). 6 T. P. Stossei, R. J. Mason, T. D. Pollard, and M. Vaughan, J. Clin. Invest. 51, 604 (1972). 7 E. L. Pesanti and S. Axline, J. Exp. Med. 142, 903 (1975). 8 p. j. Oates and O. Touster, J. Cell Biol. 68, 319 (1976). 9 p. j. Oates and O. Touster, J. Cell Biol. 79, 217 (1978). l0 p. j. Oates and O. Touster, J. Cell Biol. 85, 804 (1980). METHODS IN ENZYMOLOGY, VOL. 132

Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

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In order to evaluate factors affecting P - L fusion in intact cells, we adapted the assay first described by Hart and Young. 11 Lysosomes are labeled with a fluorescent marker, and fluorescence microscopy is used to follow transfer of the marker into phagocytic vacuoles. This assay is simple, rapid, enables detailed and reproducible rate studies, and makes possible the analysis of P - L fusion using intact and viable cells as a test system. Cultured cells are prelabeled with acridine orange (AO), a fluorescent vial dye which by its weakly basic nature becomes concentrated primarily in the low pH environment of lysosomes.12,13 AO is a metachromatic dye 14 which displays a brilliant orange fluorescence when concentrated and a green fluorescence when more dilute. After phagocytosis, the transfer of lysosomal fluorescence into the particle-containing vacuoles is evaluated by fluorescence microscopy, and orange-stained particles are counted as positive. Although it cannot be concluded that n o fusion has occurred in green-stained vacuoles, the relative amount is less and is easily distinguished visually from that of the orange-stained vacuoles. The cell used for these studies is the phagocytic macrophage, either primary cultures from mouse peritoneal cavity or a permanent macrophage-derived cell line. The choice of test particle is critical. A heatkilled, reduced, and alkylated yeast particle may be used. These particles are freely and uniformly permeable to AO (making the staining easy to evaluate), do not themselves concentrate the dye, and remain stably orange stained (the assay end point) for hours in viable cells. Other particles are less suitable for the fluorescence assay, including live yeast cells (which only show a "rim" of fluorescence and also are reported to inhibit fusionl~), antibody-coated red blood cells (which are rapidly digested), and polystyrene latex beads (which bind AO nonspecifically). However, for some purposes these and other particles may prove useful. For example, modifications of this assay have been used to monitor the viability and characterize the fusion of various intracellular parasites. 3,15 It is important to synchronize particle phagocytosis by the cells. The yeast particles are therefore opsonized with complement via the alternate pathway, see this volume [13], and this ligand is used to bind yeast to the macrophage complement receptor in the cold. Upon warming of the culture to 37°, rapid phagocytosis results. Under conditions of low particle/ cell ratio (-2), more than 90% of the particles are ingested after I0 min at i1 p. D. Hart and M. R. Young, Nature (London) 256, 47 (1975). 12 E. Robbins and P. I. Marcus, J. Cell Biol. 18, 237 (1963). 13 A. C. Allison and M. R. Young, Life Sci. 3, 1407 (1964). 14 M. E. L a m m and D. M. Neville, J. Phys. Chem. 69, 3872 (1965). t5 H. W. Murray and Z. A. Cohn, J. Exp. Med. 150, 938 (1979).

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37°. 16 Phagocytosis is thus essentially complete by the first time point of the fusion assay, and the rate of subsequent P - L fusion may be followed independent of the phagocytic rate. Methods

Cells and Cell Culture Resident peritoneal cells are prepared from female or male NelsonCollins or Swiss Webster mice by lavaging the peritoneal cavity with 3-5 ml of sterile PBS without C a 2+ o r Mg 2+.16a Cell suspensions are pooled, pelleted for 5 min at 1500 rpm, 4 °, and resuspended in an appropriate volume of MEM containing 15-20% FCS (heat inactivated, 56°), 100 U/ml penicillin, and 100 ~g/ml streptomycin. Cells are plated at densities of 610 × 106 peritoneal cells per 35-mm dish or 1.5-2 × 107 per 60-mm dish. One to two hours after plating, monolayers are washed with medium to remove nonadherent cells, resulting in a relatively pure primary macrophage culture. ~7 For coverslip cultures to be used in the fluorescence assay, cells are resuspended to 4 × 106 peritoneal cells/ml and 100/xl layered onto a 12-mm glass coverslip previously sterilized by flaming in 90% ethanol. Ten coverslips can thus be conveniently placed in a 60-mm culture dish, and the cells cultured by the addition of 5 ml of medium after the 1-hr adherence and washing step. Cells are given flesh medium at least every other day, and for most experiments are used within 4 days of explant. P388D1, a macrophage-like permanent cell line ~8(obtainable from the American Type Culture Collection), is maintained in spinner culture in 10% FCS/MEM. 19 These cells are allowed to adhere on glass coverslips as described above for the fluorescence assay.

Particle Preparation for Fusion Studies Preparation of Yeast Particles. Fresh baker's yeast is processed by the method of Lachman and Hobart. 2° Two packages of yeast (1/2 oz.) are resuspended in PBS and autoclaved for 30 min at 120°, washed with PBS until the supernatant is clear, then resuspended to 80 ml total volume in ~6M. C. Kielian and Z. A. Cohn, J. Cell Biol. 85, 754 (1980). ~6, This series, Vol. 108 [25]. t7 Z. A. Cohn and B. Benson, J. Exp. Med. 121, 153 (1965). 18 H. S. Koren, B. S. Handwerger, and J. R. Wunderlick, J. Immunol. 114, 894 (1975). 19 j. C. Unkeless and H. N. Eisen, J. Exp. Med. 142, 1520 (1975). 20 p. j. Lachman and M. J. Hobart, in "Handbook of Experimental Immunology" (D. M. Weir, ed.), Vol. 1, p. 5A.1. Blackwell, Oxford, 1978.

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0.1 M mercaptoethanol/PBS and stirred for 2 hr at 37 ° The yeast is then pelleted and washed once with PBS. It is next resuspended in 50 ml 0.85% NaC1 containing I0 m M phosphate buffer, pH 7.2, and 30 mM iodoacetamide, and stirred at room temperature for 2 hr, keeping the pH adjusted to 7.2 if necessary. It is then washed three times with PBS, autoclaved in PBS 30 min at 120°, washed in PBS until the supernatant is clear, and made to a 50% (v/v) sterile stock in PBS and 0.02% NAN3. This preparation can be stored indefinitely at 4 °. Opsonization of Yeast with Complement Components. Yeast is prepared as a 5% suspension in veronal buffer; 300/~1 is mixed with 300/zl of mouse serum (fresh or stored at - 7 0 °) and incubated 30 min at 37°. The mixture is brought to 10 ml with cold veronal buffer and pelleted 10 min, 4°. The wash is repeated once and the opsonized yeast stored as a 1% (v/v) suspension in veronal buffer, and used within 3 days. Veronal buffer: Made fresh daily; stocks are autoclaved separately and stored at 4°: 70 mM NaCI 2.5 mM Sodium 5,5-diethylbarbiturate, pH 7.35 2.5% Glucose 1 mM MgC12 0.15 m M CaCI2

Fluorescence Assay of Phagosome-Lysosome Fusion AO stock, 100 tzg/ml in PBS, is filter sterilized and stored in the dark at 4 °. It is made fresh monthly. It should be noted that AO binds to nucleic acids by intercalation and gloves should be worn when handling concentrated solutions. 1. The cells on coverslips are labeled by adding AO stock solution directly to the culture medium to give a final concentration of 5/xg/ml, and incubating for 20 min at 37 °. To label a 60-mm dish containing 10 coverslips, for example, 250 ~1 AO stock is added to the 5 ml complete culture medium in the dish. 2. Each coverslip is then transferred with forceps to a well of a 24-well culture plate (Costar, Data Packaging, Cambridge, MA) containing 1 ml of 37° MEM/5% FCS, and incubated in the absence of AO for 10 min at 37°. This step helps to concentrate any free AO into the lysosomal compartment, decreasing background fluorescence and photodamage. 3. The medium is aspirated and 1 ml of a dilute suspension of opsonized yeast (0.004%, v/v) in PBS, 4 °, is added to each well. The yeast suspension is centrifuged onto the cell monolayer by placing the culture dish in a centrifuge carrier designed for microtiter plates (Cooke Labora-

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tory Products, Alexandria, VA) and centrifuging in an International centrifuge for 2 min at 1200 rpm, 4 °. The opsonized yeast binds to the macrophage complement receptor; unbound particles are removed by washing each well with 1 ml of 4 ° MEM. 4. Each well is then given 1 ml 4 ° MEM/5% FCS and at time zero the entire plate is rapidly warmed to 37° by placing it in a 37 ° water bath inside a 37° incubator. Under these conditions, relatively synchronous phagocytosis of the bound yeast particles occurs. The average number of particles bound and ingested per cell is two. 5. Time points are taken by inverting coverslips over a drop of icecold PBS on a microscope slide, blotting, and rimming with nail polish. Slides are kept on ice and viewed immediately in a fluorescence microscope adjusted for fluorescein. A Zeiss photomicroscope III with a mercury lamp adjusted for epiilumination, a BG12 filter and fluorescein dichroic mirror for excitation, and a 53 barrier filter is appropriate for this purpose. 6. The intracellular yeast particles which are orange stained are counted as positive for P - L fusion; faintly green-stained particles are scored as negative. For each time point, at least l0 different microscope fields and duplicate coverslips should be examined, and a total of >200 particles counted. By using a diaphragm to control the intensity of the incident light, samples may be evaluated without extensive photodamage or cell death. A series of time points after the 10-min ingestion period enables the determination of the initial rate of P - L fusion as well as its final extent. 7. For the photography of the AO-labeled fusion samples the Zeiss photomicroscope III with an automatic exposure meter may be used. Kodak Tri-X film is used for black and white pictures and Kodak Ektachrome film, ASA 200, for color slides. Good, clear photographs require exposing the cells to the exciting light for as short a time as possible. Routinely, a field is located under reduced light, the picture is taken using the spot light meter of the microscope with a labeled cell centered in the spot, and the ASA setting of the camera is increased to 800 or even 1600 ASA. These factors contribute to decrease the exposure time of the film to less than 1 sec. Figure 1 is a fluorescence picture of typical AO fusion assays in l- and 4-day cultures. Punctate lysosomal staining occurs in the absence of extensive cytoplasmic or nuclear background. Both positive, brightly orange fluorescent yeast and negative, faintly green fluorescent yeast are observed. When counts of positive vs negative intracellular particles are made at varying times after ingestion, the results shown in Fig. 2 are typically obtained. The fusion rate is strongly affected by in vitro culture

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[1 1]

FIG. 1. Fluorescence micrographs of AO-fusion assays in 1- and 4-day-old cultures. (a) One-day cells 160 min after yeast ingestion. Both positively (arrow) and negatively (arrowhead) stained yeast are seen, as well as abundant punctate lysosomal staining, x 1500. (b) Four-day cells 40 min after ingestion. Abundant lysosomal staining is observed in cells containing several positively stained yeast, x 1598. From Kielian and Cohn. ~" Reproduced from The Journal of Cell Biology, by copyright permission of the Rockefeller University Press.

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263

I00

,.096 hours 8C

~ /

1

o

'

j

72 hours

~

48 hours

4G

2O ~

20

I 40

I I I 60 80 100 Time in minutes

I 120

I 140

FIc. 2. Rate and extent of P - L fusion in mouse macrophages cultured for varying periods of time. The percent of intracellular particles positively stained is plotted vs time after particle ingestion. Length of cell culture is shown in hours. Average results from three separate experiments. F r o m Kielian and C o h n J 6 Reproduced from The Journal of Cell Biology, by copyright permission of the Rockefeller University Press.

of mouse primary macrophages, being about 8-fold higher initially in 4day cells than in cells cultured for 5 hr.16 If l-day cells with intracellular particles are cultured overnight, 90% of the particles are positively stained, implying that P - L fusion continues at this slow rate. When P - L fusion is examined in the P388D~ macrophage line, the rate is similar to that of a 4-day primary culture.

Important Conditions for the Fluorescence Assay of P-L Fusion Besides the considerations of choice of test particle, opsonization, etc., described above, it is important to note that the fluorescence assay can only be used under certain conditions. Since AO uptake is dependent on low intralysosomal pH, conditions which modify lysosomal pH or quench AO fluorescence interfere with the assay. Lysosomotropic drugs such as ammonium chloride and chloroquine, or metabolic inhibitors such as 2-deoxyglucose plus sodium azide all act to increase lysosomal pH 1and also significantly decrease the lysosomal uptake of AO.16 When comparing P - L fusion in different cell populations in which the AO technique might be affected, it is important to quantitate the AO uptake by the cells to assure that comparable amounts of the fluorescent marker are concentrated. In addition, another P - L fusion assay should be used as a confirmatory assay when necessary. Electron microscopy, while less amenable to multiple time points, is unlikely to be affected by

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ISOLATION OF CELLS AND CELLULAR COMPONENTS

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[1 1]

PHAGOSOME--LYSOSOME FUSION ASSAY

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altered lysosomal pH. Lucifer yellow CH (Sigma Chemical Company), a recently described fluorescent marker which is taken up via pinocytosis, 21 might also be a useful control. Quantitation of Acridine Orange Uptake. Coverslip cultures are labeled with AO as described for the fluorescence assay, washed in PBS, and extracted in 2 ml 95% ethanol. The fluorescence is read on an MPF-44 fluorometer (Perkin-Elmer Corp., Norwalk, CT) using an excitation wavelength of 490 nm, and recording emission at 520 nm. A standard curve of known AO concentrations in the same solvent is used. This curve is not affected by the addition of unlabeled cell extracts. AO concentrations are normalized for cell protein as determined with the fluorescamine protein assay 22 on parallel coverslips. Typical uptake by mouse macrophages is - 5 0 pmol AO/~g cell protein.16 Electron Microscopic Assay of P-L Fusion. To mark secondary lysosomes, cells are pulsed with either HRP or colloidal thorium dioxide (thorotrast, Fellows Testagar Div., Fellows Mfg. Co., Inc.) in 35-mm culture dishes, and the transfer of marker to phagosomes evaluated by electron microscopy. 1. HRP specimens: To label, cultures are allowed to pinocytose 2 mg/ ml H R P in MEM for 2-3 hr at 37°, washed four times with MEM, and recultured in HRP-free medium for 20-60 min to allow transit of this fluidphase marker to lysosomes. Cells can then be given yeast or latex particles and fixed 1 hr. after ingestion. After fixation, but before osmication, HRP is visualized by staining for 10 min at room temperature with freshly made DAB reagent [5 mg diaminobenzidine (Sigma Chemical Company), 10 ml 0.1 M Tris, pH 7.6, and 3.3/~1 of 30% H202]. 23 Specimens are processed for electron microscopy by standard techniques, 16 and thin sections examined without uranyl acetate and lead citrate staining. Under these conditions, positive fusion is indicated by a "rim" of electron-dense HRP 21 D. K. Miller, E. Griffiths, J. Lenard, and R. A. Firestone, J. Cell Biol. 97, 1841 (1983). 22 p. Bohlen, S. Stein, W. Dairman, and S. Udenfriend, Arch. Biochem. Biophys. 155, 213 (1973). 23 R. C. Graham, Jr., and M. J. Karnovsky, J. Histochem. Cytochem. 14, 291 (1966). FIG. 3. Electron microscopic evaluation of P - L fusion using electron-dense lysosomal markers. (a) HRP assay in 2-day cultures given latex particles. Reaction product is seen in secondary lysosomes (Ly), and as a rim around latex vacoules. Much of the latex is dissolved by the propylene oxide used in specimen processing. ×4982. (b) Four-day cells prelabeled with thorotrast and given opsonized yeast. Thorotrast is seen in secondary lysosomes (Ly), and distributed linearly around an ingested yeast (Y). × 12,300. From Kielian and Cohn. ~6 Reproduced from The Journal of Cell Biology, by copyright permission of the Rockefeller University Press.

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MODULATORS OF P - L FUSION a

Fusion is unaffected by:

Number of particles phagocytosed Particle size Lysosome size Prior uptake of digestible or nondigestible solutes or particles ~ Enzyme pretreatment of the cell surface (trypsin, neuraminidase, pronase, chymotrypsin) Cell surface cross-linking (concanavalin A) Particle surface cross-linking (concanavalin A or antibody) Cytoskeletal drugs (colchicine, cytochalasin B or D) 2 Increased lysosomal pH (via NH4CI or chloroquine) 3

Fusion is enhanced by:

Increasing time of cell culture ~ In vivo macrophage activation 2 Phorbol myristate acetate pretreatment 4

Fusion is inhibited by:

Incubation at temperatures <37 ° (Q~0 = 2.5, no fusion seen below 15°) t Lysosomal uptake of polyanions [dextran sulfate, suramin, poly(D-glutamate), heparin, chondroitin sulfate] 3,5.6

" Key to references: (I) M. C. Kielian and Z. A. Cohn, J. Cell Biol. 85, 754 (1980); (2) M. C. Kielian and Z. A. Cohn, J. Exp. Med. 153, 1015 (1981); (3) M. C. Kielian, R. M. Steinman, and Z. A. Cohn, J. Cell Biol. 93, 866 (1982); (4) M. C. Kielian and Z. A. Cohn, J. Exp. Med. 154, 101 (1981); (5) P. D. Hart and M. R. Young, Nature (London) 256, 47 (1975); (6) P. D. Hart and M. R. Young, Exp. Cell Res. 118, 365 (1979).

reaction product around the test particle (see Fig. 3a). This marker is easy to visualize and assays can be scored on the electron microscope without photographing. However, HRP is degraded by lysosomes and thus labeling must be performed just prior to particle ingestion. 2. Thorotrast specimens: Thorotrast, or colloidal thorium dioxide, is a particulate, electron-dense tracer which is also taken up by fluid-phase pinocytosis. Thorotrast is not degraded, and thus cells can be labeled at equivalent times in culture when pinocytic uptake should be comparable, and then further cultured under various experimental conditions. To label, cell monolayers are washed 2 hr after explant, allowed to pinocytose thorotrast in medium (1/100, v/v) for 12 hr, washed four times with MEM, and recultured for 1-4 days. Again, fusion is evaluated 1 hr after particle ingestion. Thorotrast is present as discrete particles around the edge of the phagocytic vacuole, and within secondary lysosomes (Fig. 3b). The extent of fusion is determined by stereological analysis, to take the particulate nature of the marker into account. Micrographs at the same final magnification are projected through a 3× enlarger onto a grid of 1-cm squares. The number of times horizontal and vertical lines cross a phago-

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cytic vacuole membrane vs the number of times lines cross thorotrast in the vacuole are scored. For each determination, from 25-60 vacuoles are scored, and total line crossing of 600-1500 are obtained. Results are expressed as the percentage of total possible crossings which are thorotrast positive. Colloidal gold of small particle size should in similar fashion be useable as a marker. Conclusions Using these assays, a number of treatments of the cell are found to affect P - L fusion using yeast as a phagocytic particle. The conditions which we have tested are summarized in the table. It appears that alterations in vacuole size, number, pH, or luminal membrane surface do not affect P - L fusion. Culture time, in vivo macrophage activation, and phorbol myristate acetate treatment all dramatically increase P - L fusion, while incubation temperatures <15 ° and lysosomal accumulation of polyanions such as dextran sulfate block fusion. We found that our experimental results from the AO, HRP, and thorotrast assays were in relative agreement, in spite of the use of different markers, stereological analysis, and thin sections vs whole cell preparations. Acknowledgment The authorwouldlike to thank Dr. ZanvilCohnof the RockefellerUniversitywithwhom this work was performed.

[12] M e t h o d s for A s s e s s i n g E x o c y t o s i s b y Neutrophil Leukocytes

By BEATRICE DEWALD and MARCO BAGGIOLINI

Introduction

Exocytosis Exocytosis is the active release of preformed material, present in cytoplasmic storage organelles, into the extracellular space. It is a selective process which depends on the fusion of the membrane of the storage organelles with the plasma membrane, and which occurs without loss of METHODS IN ENZYMOLOGY, VOL. 132

Copyright © 1986by Academic Press, Inc. All rights of reproduction in any form reserved.