Journal of lmmunological Methods, 143 (1991) 103-109 © 1991 Elsevier Science Publishers B.V. All rights reserved 0022-1759/91/$03.50 ADONIS 002217599100296Q
103
JIM06072
Suspended mouse peritoneal macrophages Preparation and properties Marie-Th6r~se Chfiteau a,2, Herisoa R a b e s a n d r a t a n a 3 and Ren6 Caravano 1 11NSERM U65, D@artement de Biologie-Sant~ Universit£ de Montpellier II, CP 100, 34095 Montpellier Cedex 5, France, 2 Facult~ de Pharmacie, UniversitJ Montpellier I, Montpellier, France, and 3 Laboratoire d'Immunologie, H3pital LaPeyronie, Montpellier, France (Received 22 February 1991, revised received 24 April 1991, accepted 10 June 1991)
Since macrophages (MPH) are able to adhere firmly to solid surfaces, the recovery of viable and functional MPH has proven to be extremely difficult. We have developed a simple method using agarose coating for preparing MPH and culturing the cells in suspension. Their properties were tested over 72 h. The oxidative burst declined with time, but could be restored using the lymphokine rich supernatant of pokeweed-stimulated mouse spleen cells. In contrast, phagocytosis and Candida intra-cellular killing remained unchanged. Key words: Macrophage; Agarose-coating; Oxidative burst; Flow cytometry; Phagocytosis; Intracellular killing
Introduction
Mouse peritoneal macrophages provide a good model for studying the interaction of cells with drugs or microorganisms (Brett and Butler, 1988; Van den Broek, 1989). However, their use is restricted by the heterogeneity of the peritoneal exudate cell population (50-60% resident macrophages), and by the capacity of macrophages to adhere firmly to a variety of supports. We have developed a simple method which renders purified macrophages non-adherent. Their functional activity over time was assessed by assaying their capacity for phagocytosis, intracellular killing and their oxidative burst activity.
Correspondence to: M.-T. Chateau, INSERM U65, D6partement de Biologie-Sant& Universit6 de Montpellier II, CP 100, 34095 Montpellier Cedex 5, France (Tel.: 67-52-36-89).
Materials and methods
Preparation of coated vessels Tissue culture dishes were coated with serum as described by Kumagai et al. (1979). Briefly, 2 ml of heat-inactivated (56°C, 30 rain) foetal calf serum (FCS, Gibco BRL, France) were placed in 60 mm tissue culture dishes, which were incubated overnight at 4°C. The FCS was removed and the dishes were rinsed with sterile phosphate buffered saline (PBS). Polystyrene tubes (12 × 75 mm, Falcon, France) were coated by filling them with 0.8% agarose solution. The agarose solution was prepared by dissolving 0.16 g low-melting agarose (SeaPlaque, FMC, France) in 20 ml sterile distilled water, boiling, and cooling to approximately 50°C. The tubes were emptied, capped, and kept at 4° C for up to 2 weeks. They were rinsed with PBS before use.
104
Preparation of purified, non-adherent macrophages Resident peritoneal cells were obtained from 8-12-week-old Swiss mice ( I F F A - C R E D O , France) by peritoneal lavage with 5 ml Hanks' balanced salt solution (HBSS). Cells were centrifuged at 200 x g for 10 min, washed with cold HBSS, resuspended in 4 ml RPMI 1640 containing 10% FCS and transferred to serum-coated dishes. Macrophages were allowed to adhere for 20 min at 37°C in a 95% air - 5 % CO 2 atmosphere. The dishes were then thoroughly washed with warm RPMI to remove non-adherent cells, and incubated for 60 min at 4°C with 4 ml of PBS (Ca 2+ and Mg2+-free) containing 0.2% E D T A and 5% FCS. This solution was removed and saved. The cells were washed from the dish with jets of cold HBSS from a small bore, polyethylene pipette (CML, France). Medium and washing were collected and centrifuged in the cold at 200 x g for 10 min. Purified adherent cells were resuspended in 2 ml of a 1:1 mixture of fresh RPMI and conditioned RPMI (supernatant from a 3 - 4 days culture of L-929a cells), supplemented with 10% FCS and incubated in standing agarose-coated tubes at 37°C in a 95% air - 5% CO 2 atmosphere. Tests were performed after cultivation for 24, 48 or 72 h. The incubation medium of some tubes was supplemented, at 48 h, with 5% of the supernatant from a culture of mouse spleen cells (Brummer and Stevens, 1989) stimulated for 72 h with 5 0 / z g / m l of pokeweed mitogen (Sigma, France). The peritoneal cells in these tubes were used 24 h later and are subsequently referred to as 72 + SCS (spleen cell supernatant) cells. Cells were counted by flow cytometry (Epics profile analyzer, Coulter, Hialeah, FL) and cell viability was evaluated by trypan blue exclusion. The purity of the macrophages (MPH) obtained was estimated using cytocentrifuged preparations (Cytospin, Shandon, France) after staining for non-specific esterase.
Candidacidal activity Candidacidal activity was assessed by the method of Lehrer (1981). Candida albicans was incubated on Sabouraud agar slants for 18 h at 37 ° C. Opsonized yeasts were prepared by incubating a loopful of C. albicans with FCS for 60
min at 4°C. The suspension was centrifuged, the yeasts were washed and resuspended in phenol red-free HBSS to avoid further quenching of the dyes. C. albicans viability was checked by aqueous methylene blue exclusion (0.01%, final concentration) and the cells counted in hemocytometer. The macrophage suspension was washed in phenol red-free HBSS and adjusted to 105 cells/ml in agarose-coated tubes. Opsonized C. albicans were added to provide two living yeasts per macrophage. This suspension was supplemented with 10% FCS and incubated for 3 h at 37°C in a shaking water bath. 100 /xl of incubation mixture and 100/xl of phenol red-free HBSS containing 25% FCS were then spun in the cytocentrifuge, for 10 rain at 600 rpm. The slides were air dried, fixed in methanol, stained with 10% Giemsa (pH 7.4) for 20 min and examined under the microscope ( × 1000). Blue staining yeast cells were alive, unstained cells, which have lost their cytoplasmic affinity for the basic components of Giemsa after their RNA has undergone enzymatic hydrolysis, were dead. One hundred intracellular yeasts were counted in duplicate for each preparation. The candidacidal activity was expressed as the candidacidal index, i.e., number of intracellular dead yeasts x 100 total number of intracellular yeasts
Chemiluminescence assay Macrophage chemiluminescence elicited by phorbol myristate acetate (PMA) was assessed by a luminol-enhanced system (Allen and Loose, 1976; Thomas et al., 1988). Stock solutions of luminol (Boehringer Mannheim, France; 10 m g / m l , room temp.) and PMA (Sigma, France; 1 m g / m l , - 80°C) were prepared in dimethyisulfoxide (Sigma). In a semi-dark laboratory, macrophages were washed and resuspended in 0.5 ml PBS. The cell suspension was placed in 75 x 11 mm plastic tubes (Sarstedt, France) and 0.5 ml of luminol (stock solution diluted 1/1000 in PBS) was added. The tubes were shaken gently, placed in the luminometer chambers (at 37°C) and allowed to equilibrate. 100/xl of PMA (stock solution diluted 1/100 in PBS) were added and light emission was recorded with automatic back-
105
ground subtraction. The controls contained no PMA. Each test was performed in triplicate. Chemiluminescence readings were made at 5 s intervals over a 60 min period using a repeat mode. The amplitude of the response was computed and recorded as photons or counts per minute (cpm) per 105 cells. Phagocytosis assay Phagocytosis was assayed by the method of Dunn and Tyrer (1981) using Fluoresbrite microspheres. Macrophages were washed and resuspended in PBS at 105 cells/ml. Fresh frozen (-80°C) pooled human serum was added to a final concentration of 10%, plus 10 /zl of a suspension of 2/~m Fluoresbrite microspheres (Polysciences, Warrington, PA) diluted 1/20 in PBS. The tubes were incubated at 37°C in a shaking water bath for 60 min, placed on ice to stop phagocytosis, and the samples analysed in a flow cytofluorometer (Epics profile analyzer, Coulter, Hialeah, FL) equipped with a 15 mW argon laser (488 nm excitation filter, photomultiplier voltage: 800 V). Cytocentrifuged preparations were fixed in methanol, stained with Giemsa, examined by combined phase and fluorescence microscopy at 400 × . Trypan blue (0.5 mg/ml in 0.15 M NaC1) and crystal violet (1 mg/ml in 0.15 M NaC1) failed to quench the bright fluorescence of the latex beads this provided the basis of the method used to distinguish between intracellular and extracellular, adherent microspheres. Using the classic filter (450-490 nm excitation) no difference was observed but when the 340-380 nm excitation filter was used extracellular and adherent beads remained green while truly ingested beads appeared blue. Further corroboration was possible using xylene to dissolve extracellular
20000-
L
~ Controls [--] Assays
.L
5_ ~
10O00-
E
1000-
~
~ 24
~ 48
72
72+SCS
Duration (hr) of culture
Fig. 1. Chemiluminescence of peritoneal macrophages induced by PMA activation and expressed in cpm per 105 cells, after incubation for 24, 48, and 72 h (SCS = spleen cell supernatant).
beads (Van Furth and Diesselhoff-Den Dulk, 1980)
Results
The double-coating method provided highly purified murine peritoneal macrophages (85% non-specific esterase positive cells) with a good yield (70% of the MPH were recovered after the purification step). Flow cytometry also indicated a homogeneous population; the macrophage population was distinguished by the combination of low angle forward scattered and right angle side scattered laser light. The macrophages remained viable and non-adherent for at least 2 days (20% were lost on the third day) and candidacidal activity persisted during the 72 h incubation (Table I). The oxidative burst elicited by PMA declined during 72 h of cultivation, but was restored by the addition of SCS (Fig. 1). Before testing
TABLE I E V A L U A T I O N O F CELL PURITY, VIABILITY AND C A N D I D A C I D A L ACTIVITY O V E R TIME
Number a of viable cells/ml MPH (%) Viable MPH recovery (%) Candidacidal index (%) a Mean(+SD); n=5.
0h
24 h
48 h
72 h
1.04× 105 (0.30) 84.5 70 N.D.
1.06× 105 (0.26) 84.8 70.8 20.4
1.05 × 105 (0.19) 86.6 72.4 18.8
0.70× 105 (0.10) 92.2 50.9 21.2
106
A
B
1 2 3~t
:
:,;:i~
C
z INTERNAL STRUCTURE
LOG OF FLUORESCENCE INTENSITY
LOG OF FLUORESCENCE INTENSITY
Fig. 2. Visualization of microsphere population (A) based on size (forward scatter) and internal structure (light side scatter). The microspheres form clusters (B) of increasing size and fluorescence. Each peak corresponds to one or more microspheres (C).
phagocytosis, the fluorescent microspheres were first separately analysed in the flow cytofluorometer. A bitmap was drawn around the microsphere population on cytogram A (Fig. 2), and this determined the location of fluorescence on cytogram B and histogram C. The fluorescence indicated spontaneous aggregation of microspheres forming clusters of increasing size (Fig. 2B). The mean fluorescence per cluster was 10.7
for one microsphere, 21.0 for two, 32.0 for three, and 45.1 for four (Fig. 2C). Single cells showed no autofluorescence after the drawing of a new bitmap around the cell population. The cluster of ceils was readily distinguished from the population of microspheres. The number of cell-associated microspheres in the phagocytosis assay was estimated from the mean fluorescence per cell using the data given above: 9.1 U implied one
B
A
INTERNAL S T R U C T U R E
LOG O F F L U O R E S C E N C E INTENSITY LOG O F F L U O R E S C E N C E INTENSrrY
Fig. 3. Phagocytosis assay. Cytogram distribution (A) of fluorescent microsphere-associated peritoneal macrophages based on size and internal structure of cells. A bitmap is drawn around the analysed cell population. The cell population analysed shows clusters of cells phagocytosing 0, 1, 2, 3, 4, or more microspheres (B) when one considers the log of fluorescence intensity as a function of internal structure of cells. If the log of the fluorescent intensity is analysed as a function of the number of cells, each peak corresponds to a number of cell-associated microspheres (C).
TABLE II PHAGOCYTOSIS CAPACITY O V E R TIME Incubation time
Percentage of cells phagocytosing 1-4 microspheres 1 2 3
4
Total % of phagocytosis
24 48 72 72
5.60 6.12 6.50 8.37
0.I2 0.I0 0.10 0.22
7.25 8.0 8.75 11.2
a
h h h h + SCS a
Spleen cell supernatant.
1.10 1.35 1.67 2.07
0.42 0.40 0.42 0.55
107
.
l=i/
,","!
....
B
c ,.
L.X.,_ . . . . .
D
Fig. 4. Histogramof cell-associatedfluorescentmicrospheres over time, with and without lymphokines. 72+spleen cell supernatant (A), 72 (B), 48 (C), 24 (D) hours of incubation. microsphere, 20.0 two, 31.7 three, 45.7 four (Fig. 3). Since the microscopic control showed essentially ingested beads, the results were expressed as the percentage of cells phagocytosing 1, 2, 3, or 4 microspheres (Table II). The phagocytosis capacities after 24, 48, or 72 h of incubation were essentially the same, but were moderately increased by SCS (Fig. 4).
Discussion
Several of the sophisticated techniques such as flow cytofluorometry which are now used to study phagocyte functions (Bjerknes et al., 1989), require that the cells be in suspension. Until now, only teflon films (Van der Meer et al., 1981) have provided non-adherent macrophages. The double-coating method described here combines the technique described by Ku0aagai et al. (1979) for preparing highly purified peritoneal macrophages with a method of keeping macrophages non-adherent, without the need for continuous shaking. The ceils were purified using selective Ca2+-de pendent adherence to FCS-coated dishes. This technique was more convenient than that of Mantovani (1981) using peritoneal microexudate and gave better results, especially in term of cell purity, than the method of Hassan et al. (1986). Jones et al. (1989) recently reported that gelatine pre-coating followed by plasma coating provided better purity than did density gradient centrifugation or monoclonal antibodies specific for human monocytes. Since 70% of the macrophages was
recovered after the purification assay, the yield from this present method was greater than that obtained by density gradient centrifugation: 3060% of recovered cells (Stoll et al., 1986). In our experiments the macrophages were maintained in a non-adherent state on agarose. This is a low-cost, non-toxic, easily prepared support which is more likely than other solid surfaces to keep macrophages in a state akin to their physiologic peritoneal state. Indeed, Schumann et al. (1989) observed that plastic-cultured monocytes lost some of their surface markers. The functional capacities of macrophages were studied after they had been left overnight to recover from the separation procedures thereby avoiding possible transient activation of the cells. Investigations were then made on cells cultured for 24 h. Although Bjerknes et al. (1989) used crystal violet or trypan blue to quench the fluorescence of extracellular zymosan particles, bacteria or fungi, we found this technique to be inappropriate for fluorescent microspheres which were too bright. Xylene could not be used to dissolve adherent beads on cells in suspension since it induced cell clumping and affected the morphology of the cells (Van Furth and Diesselhoff-Den Dulk, 1980). This method has been used as a control for methanol fixed cytocentrifuged preparations but we found it was directly possible to distinguish between adherent (green) and ingested (blue) beads by a simple observation with a lower excitation filter. Since most of the cell-associated microspheres were ingested, it became possible to equate the blue beads with phagocytosis. This phagocytosis capacity was very poor, less than 10% of the cells being able to ingest latex beads. This low result could easily be explained by the assay conditions. We did not try to obtain optimal phagocytosis but rather to develop a rapid method to quantify the number of ingested beads per cell. Consequently we used a small quantity of microspheres (20 per cell) and we restricted the incubation time to 60 min. Phagocytosis is known to be limited by the number of beads accessible to the phagocytes. Ito et al. (1979) reported that phagocytic uptake was linear up to 6 h and in a later study Matsson et al. (1985) incubated cells and beads for 14-17 h. Nevertheless, Levitz et al. (1987) reported that only 10.6% of resident peri-
108
toneal macrophages internalized complementopsonized particles. In the present study phagocytosis and Candida intracellular killing remained unchanged over 3 days. It appears therefore that the agarose did not induce cell activation and the macrophages remained in their resident state. Agarose could provide a good support for further in vitro studies of cell activation and/or differentiation. '~ It was noted that the oxidative burst fell over time. This result agreed with the study of Murray et al. (1980) who demonstrated that endogenous scavengers of oxygen intermediates in macrophages increased during cell culture. However, in this earlier study the oxidative burst was restored with the supernatant of pokeweed-stimulated spleen cells, most likely by the endogenous lymphokine activity (Murray and Cohn, 1980). In the present study there was a lack of correlation between the oxidative burst and candidacidal activities in cells which had been cultured for 3 days. This seemed to prove that, at least up to this time, killing occurred by an oxygen-independent mechanism. Perhaps this mechanism becomes more important as the cells mature and replaces the oxygen dependent mechanism. It has been reported that oxygen radicals play an important role in monocyte candidacidal activity (Sasada et al., 1987). But, in macrophages, it is possible that such a strictly intracellular mechanism is oxygen-independent and not affected by oxidative burst variation. Such a mechanism could be important for killing organisms able to avoid or overcome the repiratory burst of the macrophage (Nathan, 1983); it might also permit the cell to kill some organisms during severely hypoxic conditions (Freedman et a1.,1984) such as exist in vivo at sites of low oxygen tension.
References Allen, R.C. and Loose, L.D. (1976) Phagocytic activation of a luminol-dependent chemiluminescence in rabbit alveolar and peritoneal macrophages. Biochem. Biophys. Res. Commun. 69, 245. Bjerknes, R., Bassoe, C-F., Sjursen, H., Laerum, O.D. and Solberg, C.O. (1989) Flow cytometry for the study of phagocyte functions. Rev. Infect. Dis. 11, 16.
Brett, S.J. and Butler, R. (1988) Interactions of Mycobacterium lepraemurium with resident peritoneal macrophages; phagocytosis and stimulation of the oxidative burst. Clin. Exp. Immunol. 71, 32. Brummer, E. and Stevens, D.A. (1989) Candidacidal mechanisms of peritoneal macrophages activated with lymphokines or y-interferon. J. Med. Microbiol. 28, 173. Dunn, P.A. and Tyrer, H.W. (1981) Quantitation of neutrophil phagocytosis, using fluorescent latex beads. Correlation of microscopy and flow cytometry. J. Lab. Clin. Med. 98, 374. Freedman, V.H., Gorrell, T.E., Nathan, C.F., Copeland, C.S. and Silverstein S.C. (1984) Bacillus Calmette-Gu~rinactivated murine macrophages kill syngeneic melanoma cells under strict anaerobic conditions. J. Exp. Med. 160, 94. Hassan, N.F., Campbell, D.E. and Douglas S.D. (1986) Purification of human monocytes on gelatin-coated surfaces. J. Immunol. Methods 95, 273. Ito, M., Ralph, P. and Moore, M.A.S. (1979) In vitro stimulation of phagocytosis in a macrophage cell line measured by a convenient radiolabeled latex beads assay. Cell. Immunol. 46, 48. Jones, B.M., Nicholson, J.K.A., Holman, R.C. and Hubbard, M. (1989) Comparison of monocyte separation methods using flow cytometric analysis. J. Immunol. Methods 125, 41. Kumagai, K., Itoh, K., Hinuma, S. and Tada, M. (1979) Pretreatment of plastic petri dishes with fetal calf serum. A simple method for macrophage isolation. J. Immunol. Methods 29, 17. Lehrer, R.I. (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. Levitz, S.M., DiBenedetto, D.J. and Diamond, R.D. (1987) A rapid fluorescent assay to distinguish attached from phagocytized yeast particles. J. Immunol. Methods 101, 37. Mantovani, A. (1981) Adherence to microexudate-coated plastic. In: H.B. Herscowitz, H.T. Holden, J.A. Bellanti and A. Ghaffar (Eds.), Manual of Macrophage Methodology: Collection, Characterization, and Function. M. Dekker, New York, p. 69. Matsson, P., Fossum, C. and Larsson, B. (1985) Evaluation of flow cytometry and fluorescence microscopy for the estimation of bovine mononuclear phagocytes. J. Immunol. Methods 78, 13. Murray, H.W. and Cohn, Z.A. (1980) Macrophage oxygendependent antimicrobial activity. III. Enhanced oxidative metabolism as an expression of macrophage activation. J. Exp. Med. 152, 1596. Murray, H.W., Nathan, C.F. and Cohn, Z.A. (1980) Macropl~age oxygen-dependent antimicrobial activity. IV. Role of endogenous scavengers of oxygen intermediates. J. Exp. Med. 152, 1610. Nathan, C.F. (1983) Mechanisms of macrophage antimicrobial activity. Trans. R. Soc. Trop. Med. Hyg. 77, 620.
109 Sasada, M., Kubo, A., Nishimura, T., Kakita, T., Moriguchi, T., Yamamoto, K. and Uchino, H. (1987) Candidacidal activity of monocyte-derived human macrophages: relationship between Candida killing and oxygen radical generation by human macrophages. J. Leuk. Biol. 41, 289. Schumann, R.R., Van der Bosch, J., Riiller, S., Ernst, M. and Schlaak, M. (1989) Monocyte long-term cultivation on microvascular endothelial cell monolayers: morphologic and phenotypic characterization and comparison with monocytes cultured on tissue culture plastic. Blood 73, 818. Stoll, H.P., Kr~imer, S. and Oberhausen, E. (1986) A new method of preparing monocytes suspensions from human whole blood. Blood Cells 11, 421.
Thomas, V.L., Sanford, B.A., Driscoll, M.S., Casto, D.T. and Ramamurthy, R.S. (1988) Luminol-dependent chemiluminescence microassay for phagocytic function. J. Immunol. Methods 111,227. Van den Broek, P.J. (1989) Antimicrobial drugs, microorganisms, and phagocytes. Rev. Infect. Dis. 11, 213. Van der Meet, J.W.M., Van de Gevel, J.S. and Van Furth, R. (1981) Teflon film as a substrate for the culture of mononuclear phagocytes. In: D.O. Adams, P.J. Edelson and H. Koren (Eds.), Methods for Studying Mononuclear Phagocytes. Academic Press, New York, p. 121. Van Furth, R. and Diesselhoff-Den Dulk, M.M.C. (1980) Method to prove ingestion of particles by macrophages with light microscopy. Scand. J. Immunol. 12, 265.
103
JIM06072
Suspended mouse peritoneal macrophages Preparation and properties Marie-Th6r~se Chfiteau a,2, Herisoa R a b e s a n d r a t a n a 3 and Ren6 Caravano 1 11NSERM U65, D@artement de Biologie-Sant~ Universit£ de Montpellier II, CP 100, 34095 Montpellier Cedex 5, France, 2 Facult~ de Pharmacie, UniversitJ Montpellier I, Montpellier, France, and 3 Laboratoire d'Immunologie, H3pital LaPeyronie, Montpellier, France (Received 22 February 1991, revised received 24 April 1991, accepted 10 June 1991)
Since macrophages (MPH) are able to adhere firmly to solid surfaces, the recovery of viable and functional MPH has proven to be extremely difficult. We have developed a simple method using agarose coating for preparing MPH and culturing the cells in suspension. Their properties were tested over 72 h. The oxidative burst declined with time, but could be restored using the lymphokine rich supernatant of pokeweed-stimulated mouse spleen cells. In contrast, phagocytosis and Candida intra-cellular killing remained unchanged. Key words: Macrophage; Agarose-coating; Oxidative burst; Flow cytometry; Phagocytosis; Intracellular killing
Introduction
Mouse peritoneal macrophages provide a good model for studying the interaction of cells with drugs or microorganisms (Brett and Butler, 1988; Van den Broek, 1989). However, their use is restricted by the heterogeneity of the peritoneal exudate cell population (50-60% resident macrophages), and by the capacity of macrophages to adhere firmly to a variety of supports. We have developed a simple method which renders purified macrophages non-adherent. Their functional activity over time was assessed by assaying their capacity for phagocytosis, intracellular killing and their oxidative burst activity.
Correspondence to: M.-T. Chateau, INSERM U65, D6partement de Biologie-Sant& Universit6 de Montpellier II, CP 100, 34095 Montpellier Cedex 5, France (Tel.: 67-52-36-89).
Materials and methods
Preparation of coated vessels Tissue culture dishes were coated with serum as described by Kumagai et al. (1979). Briefly, 2 ml of heat-inactivated (56°C, 30 rain) foetal calf serum (FCS, Gibco BRL, France) were placed in 60 mm tissue culture dishes, which were incubated overnight at 4°C. The FCS was removed and the dishes were rinsed with sterile phosphate buffered saline (PBS). Polystyrene tubes (12 × 75 mm, Falcon, France) were coated by filling them with 0.8% agarose solution. The agarose solution was prepared by dissolving 0.16 g low-melting agarose (SeaPlaque, FMC, France) in 20 ml sterile distilled water, boiling, and cooling to approximately 50°C. The tubes were emptied, capped, and kept at 4° C for up to 2 weeks. They were rinsed with PBS before use.
104
Preparation of purified, non-adherent macrophages Resident peritoneal cells were obtained from 8-12-week-old Swiss mice ( I F F A - C R E D O , France) by peritoneal lavage with 5 ml Hanks' balanced salt solution (HBSS). Cells were centrifuged at 200 x g for 10 min, washed with cold HBSS, resuspended in 4 ml RPMI 1640 containing 10% FCS and transferred to serum-coated dishes. Macrophages were allowed to adhere for 20 min at 37°C in a 95% air - 5 % CO 2 atmosphere. The dishes were then thoroughly washed with warm RPMI to remove non-adherent cells, and incubated for 60 min at 4°C with 4 ml of PBS (Ca 2+ and Mg2+-free) containing 0.2% E D T A and 5% FCS. This solution was removed and saved. The cells were washed from the dish with jets of cold HBSS from a small bore, polyethylene pipette (CML, France). Medium and washing were collected and centrifuged in the cold at 200 x g for 10 min. Purified adherent cells were resuspended in 2 ml of a 1:1 mixture of fresh RPMI and conditioned RPMI (supernatant from a 3 - 4 days culture of L-929a cells), supplemented with 10% FCS and incubated in standing agarose-coated tubes at 37°C in a 95% air - 5% CO 2 atmosphere. Tests were performed after cultivation for 24, 48 or 72 h. The incubation medium of some tubes was supplemented, at 48 h, with 5% of the supernatant from a culture of mouse spleen cells (Brummer and Stevens, 1989) stimulated for 72 h with 5 0 / z g / m l of pokeweed mitogen (Sigma, France). The peritoneal cells in these tubes were used 24 h later and are subsequently referred to as 72 + SCS (spleen cell supernatant) cells. Cells were counted by flow cytometry (Epics profile analyzer, Coulter, Hialeah, FL) and cell viability was evaluated by trypan blue exclusion. The purity of the macrophages (MPH) obtained was estimated using cytocentrifuged preparations (Cytospin, Shandon, France) after staining for non-specific esterase.
Candidacidal activity Candidacidal activity was assessed by the method of Lehrer (1981). Candida albicans was incubated on Sabouraud agar slants for 18 h at 37 ° C. Opsonized yeasts were prepared by incubating a loopful of C. albicans with FCS for 60
min at 4°C. The suspension was centrifuged, the yeasts were washed and resuspended in phenol red-free HBSS to avoid further quenching of the dyes. C. albicans viability was checked by aqueous methylene blue exclusion (0.01%, final concentration) and the cells counted in hemocytometer. The macrophage suspension was washed in phenol red-free HBSS and adjusted to 105 cells/ml in agarose-coated tubes. Opsonized C. albicans were added to provide two living yeasts per macrophage. This suspension was supplemented with 10% FCS and incubated for 3 h at 37°C in a shaking water bath. 100 /xl of incubation mixture and 100/xl of phenol red-free HBSS containing 25% FCS were then spun in the cytocentrifuge, for 10 rain at 600 rpm. The slides were air dried, fixed in methanol, stained with 10% Giemsa (pH 7.4) for 20 min and examined under the microscope ( × 1000). Blue staining yeast cells were alive, unstained cells, which have lost their cytoplasmic affinity for the basic components of Giemsa after their RNA has undergone enzymatic hydrolysis, were dead. One hundred intracellular yeasts were counted in duplicate for each preparation. The candidacidal activity was expressed as the candidacidal index, i.e., number of intracellular dead yeasts x 100 total number of intracellular yeasts
Chemiluminescence assay Macrophage chemiluminescence elicited by phorbol myristate acetate (PMA) was assessed by a luminol-enhanced system (Allen and Loose, 1976; Thomas et al., 1988). Stock solutions of luminol (Boehringer Mannheim, France; 10 m g / m l , room temp.) and PMA (Sigma, France; 1 m g / m l , - 80°C) were prepared in dimethyisulfoxide (Sigma). In a semi-dark laboratory, macrophages were washed and resuspended in 0.5 ml PBS. The cell suspension was placed in 75 x 11 mm plastic tubes (Sarstedt, France) and 0.5 ml of luminol (stock solution diluted 1/1000 in PBS) was added. The tubes were shaken gently, placed in the luminometer chambers (at 37°C) and allowed to equilibrate. 100/xl of PMA (stock solution diluted 1/100 in PBS) were added and light emission was recorded with automatic back-
105
ground subtraction. The controls contained no PMA. Each test was performed in triplicate. Chemiluminescence readings were made at 5 s intervals over a 60 min period using a repeat mode. The amplitude of the response was computed and recorded as photons or counts per minute (cpm) per 105 cells. Phagocytosis assay Phagocytosis was assayed by the method of Dunn and Tyrer (1981) using Fluoresbrite microspheres. Macrophages were washed and resuspended in PBS at 105 cells/ml. Fresh frozen (-80°C) pooled human serum was added to a final concentration of 10%, plus 10 /zl of a suspension of 2/~m Fluoresbrite microspheres (Polysciences, Warrington, PA) diluted 1/20 in PBS. The tubes were incubated at 37°C in a shaking water bath for 60 min, placed on ice to stop phagocytosis, and the samples analysed in a flow cytofluorometer (Epics profile analyzer, Coulter, Hialeah, FL) equipped with a 15 mW argon laser (488 nm excitation filter, photomultiplier voltage: 800 V). Cytocentrifuged preparations were fixed in methanol, stained with Giemsa, examined by combined phase and fluorescence microscopy at 400 × . Trypan blue (0.5 mg/ml in 0.15 M NaC1) and crystal violet (1 mg/ml in 0.15 M NaC1) failed to quench the bright fluorescence of the latex beads this provided the basis of the method used to distinguish between intracellular and extracellular, adherent microspheres. Using the classic filter (450-490 nm excitation) no difference was observed but when the 340-380 nm excitation filter was used extracellular and adherent beads remained green while truly ingested beads appeared blue. Further corroboration was possible using xylene to dissolve extracellular
20000-
L
~ Controls [--] Assays
.L
5_ ~
10O00-
E
1000-
~
~ 24
~ 48
72
72+SCS
Duration (hr) of culture
Fig. 1. Chemiluminescence of peritoneal macrophages induced by PMA activation and expressed in cpm per 105 cells, after incubation for 24, 48, and 72 h (SCS = spleen cell supernatant).
beads (Van Furth and Diesselhoff-Den Dulk, 1980)
Results
The double-coating method provided highly purified murine peritoneal macrophages (85% non-specific esterase positive cells) with a good yield (70% of the MPH were recovered after the purification step). Flow cytometry also indicated a homogeneous population; the macrophage population was distinguished by the combination of low angle forward scattered and right angle side scattered laser light. The macrophages remained viable and non-adherent for at least 2 days (20% were lost on the third day) and candidacidal activity persisted during the 72 h incubation (Table I). The oxidative burst elicited by PMA declined during 72 h of cultivation, but was restored by the addition of SCS (Fig. 1). Before testing
TABLE I E V A L U A T I O N O F CELL PURITY, VIABILITY AND C A N D I D A C I D A L ACTIVITY O V E R TIME
Number a of viable cells/ml MPH (%) Viable MPH recovery (%) Candidacidal index (%) a Mean(+SD); n=5.
0h
24 h
48 h
72 h
1.04× 105 (0.30) 84.5 70 N.D.
1.06× 105 (0.26) 84.8 70.8 20.4
1.05 × 105 (0.19) 86.6 72.4 18.8
0.70× 105 (0.10) 92.2 50.9 21.2
106
A
B
1 2 3~t
:
:,;:i~
C
z INTERNAL STRUCTURE
LOG OF FLUORESCENCE INTENSITY
LOG OF FLUORESCENCE INTENSITY
Fig. 2. Visualization of microsphere population (A) based on size (forward scatter) and internal structure (light side scatter). The microspheres form clusters (B) of increasing size and fluorescence. Each peak corresponds to one or more microspheres (C).
phagocytosis, the fluorescent microspheres were first separately analysed in the flow cytofluorometer. A bitmap was drawn around the microsphere population on cytogram A (Fig. 2), and this determined the location of fluorescence on cytogram B and histogram C. The fluorescence indicated spontaneous aggregation of microspheres forming clusters of increasing size (Fig. 2B). The mean fluorescence per cluster was 10.7
for one microsphere, 21.0 for two, 32.0 for three, and 45.1 for four (Fig. 2C). Single cells showed no autofluorescence after the drawing of a new bitmap around the cell population. The cluster of ceils was readily distinguished from the population of microspheres. The number of cell-associated microspheres in the phagocytosis assay was estimated from the mean fluorescence per cell using the data given above: 9.1 U implied one
B
A
INTERNAL S T R U C T U R E
LOG O F F L U O R E S C E N C E INTENSITY LOG O F F L U O R E S C E N C E INTENSrrY
Fig. 3. Phagocytosis assay. Cytogram distribution (A) of fluorescent microsphere-associated peritoneal macrophages based on size and internal structure of cells. A bitmap is drawn around the analysed cell population. The cell population analysed shows clusters of cells phagocytosing 0, 1, 2, 3, 4, or more microspheres (B) when one considers the log of fluorescence intensity as a function of internal structure of cells. If the log of the fluorescent intensity is analysed as a function of the number of cells, each peak corresponds to a number of cell-associated microspheres (C).
TABLE II PHAGOCYTOSIS CAPACITY O V E R TIME Incubation time
Percentage of cells phagocytosing 1-4 microspheres 1 2 3
4
Total % of phagocytosis
24 48 72 72
5.60 6.12 6.50 8.37
0.I2 0.I0 0.10 0.22
7.25 8.0 8.75 11.2
a
h h h h + SCS a
Spleen cell supernatant.
1.10 1.35 1.67 2.07
0.42 0.40 0.42 0.55
107
.
l=i/
,","!
....
B
c ,.
L.X.,_ . . . . .
D
Fig. 4. Histogramof cell-associatedfluorescentmicrospheres over time, with and without lymphokines. 72+spleen cell supernatant (A), 72 (B), 48 (C), 24 (D) hours of incubation. microsphere, 20.0 two, 31.7 three, 45.7 four (Fig. 3). Since the microscopic control showed essentially ingested beads, the results were expressed as the percentage of cells phagocytosing 1, 2, 3, or 4 microspheres (Table II). The phagocytosis capacities after 24, 48, or 72 h of incubation were essentially the same, but were moderately increased by SCS (Fig. 4).
Discussion
Several of the sophisticated techniques such as flow cytofluorometry which are now used to study phagocyte functions (Bjerknes et al., 1989), require that the cells be in suspension. Until now, only teflon films (Van der Meer et al., 1981) have provided non-adherent macrophages. The double-coating method described here combines the technique described by Ku0aagai et al. (1979) for preparing highly purified peritoneal macrophages with a method of keeping macrophages non-adherent, without the need for continuous shaking. The ceils were purified using selective Ca2+-de pendent adherence to FCS-coated dishes. This technique was more convenient than that of Mantovani (1981) using peritoneal microexudate and gave better results, especially in term of cell purity, than the method of Hassan et al. (1986). Jones et al. (1989) recently reported that gelatine pre-coating followed by plasma coating provided better purity than did density gradient centrifugation or monoclonal antibodies specific for human monocytes. Since 70% of the macrophages was
recovered after the purification assay, the yield from this present method was greater than that obtained by density gradient centrifugation: 3060% of recovered cells (Stoll et al., 1986). In our experiments the macrophages were maintained in a non-adherent state on agarose. This is a low-cost, non-toxic, easily prepared support which is more likely than other solid surfaces to keep macrophages in a state akin to their physiologic peritoneal state. Indeed, Schumann et al. (1989) observed that plastic-cultured monocytes lost some of their surface markers. The functional capacities of macrophages were studied after they had been left overnight to recover from the separation procedures thereby avoiding possible transient activation of the cells. Investigations were then made on cells cultured for 24 h. Although Bjerknes et al. (1989) used crystal violet or trypan blue to quench the fluorescence of extracellular zymosan particles, bacteria or fungi, we found this technique to be inappropriate for fluorescent microspheres which were too bright. Xylene could not be used to dissolve adherent beads on cells in suspension since it induced cell clumping and affected the morphology of the cells (Van Furth and Diesselhoff-Den Dulk, 1980). This method has been used as a control for methanol fixed cytocentrifuged preparations but we found it was directly possible to distinguish between adherent (green) and ingested (blue) beads by a simple observation with a lower excitation filter. Since most of the cell-associated microspheres were ingested, it became possible to equate the blue beads with phagocytosis. This phagocytosis capacity was very poor, less than 10% of the cells being able to ingest latex beads. This low result could easily be explained by the assay conditions. We did not try to obtain optimal phagocytosis but rather to develop a rapid method to quantify the number of ingested beads per cell. Consequently we used a small quantity of microspheres (20 per cell) and we restricted the incubation time to 60 min. Phagocytosis is known to be limited by the number of beads accessible to the phagocytes. Ito et al. (1979) reported that phagocytic uptake was linear up to 6 h and in a later study Matsson et al. (1985) incubated cells and beads for 14-17 h. Nevertheless, Levitz et al. (1987) reported that only 10.6% of resident peri-
108
toneal macrophages internalized complementopsonized particles. In the present study phagocytosis and Candida intracellular killing remained unchanged over 3 days. It appears therefore that the agarose did not induce cell activation and the macrophages remained in their resident state. Agarose could provide a good support for further in vitro studies of cell activation and/or differentiation. '~ It was noted that the oxidative burst fell over time. This result agreed with the study of Murray et al. (1980) who demonstrated that endogenous scavengers of oxygen intermediates in macrophages increased during cell culture. However, in this earlier study the oxidative burst was restored with the supernatant of pokeweed-stimulated spleen cells, most likely by the endogenous lymphokine activity (Murray and Cohn, 1980). In the present study there was a lack of correlation between the oxidative burst and candidacidal activities in cells which had been cultured for 3 days. This seemed to prove that, at least up to this time, killing occurred by an oxygen-independent mechanism. Perhaps this mechanism becomes more important as the cells mature and replaces the oxygen dependent mechanism. It has been reported that oxygen radicals play an important role in monocyte candidacidal activity (Sasada et al., 1987). But, in macrophages, it is possible that such a strictly intracellular mechanism is oxygen-independent and not affected by oxidative burst variation. Such a mechanism could be important for killing organisms able to avoid or overcome the repiratory burst of the macrophage (Nathan, 1983); it might also permit the cell to kill some organisms during severely hypoxic conditions (Freedman et a1.,1984) such as exist in vivo at sites of low oxygen tension.
References Allen, R.C. and Loose, L.D. (1976) Phagocytic activation of a luminol-dependent chemiluminescence in rabbit alveolar and peritoneal macrophages. Biochem. Biophys. Res. Commun. 69, 245. Bjerknes, R., Bassoe, C-F., Sjursen, H., Laerum, O.D. and Solberg, C.O. (1989) Flow cytometry for the study of phagocyte functions. Rev. Infect. Dis. 11, 16.
Brett, S.J. and Butler, R. (1988) Interactions of Mycobacterium lepraemurium with resident peritoneal macrophages; phagocytosis and stimulation of the oxidative burst. Clin. Exp. Immunol. 71, 32. Brummer, E. and Stevens, D.A. (1989) Candidacidal mechanisms of peritoneal macrophages activated with lymphokines or y-interferon. J. Med. Microbiol. 28, 173. Dunn, P.A. and Tyrer, H.W. (1981) Quantitation of neutrophil phagocytosis, using fluorescent latex beads. Correlation of microscopy and flow cytometry. J. Lab. Clin. Med. 98, 374. Freedman, V.H., Gorrell, T.E., Nathan, C.F., Copeland, C.S. and Silverstein S.C. (1984) Bacillus Calmette-Gu~rinactivated murine macrophages kill syngeneic melanoma cells under strict anaerobic conditions. J. Exp. Med. 160, 94. Hassan, N.F., Campbell, D.E. and Douglas S.D. (1986) Purification of human monocytes on gelatin-coated surfaces. J. Immunol. Methods 95, 273. Ito, M., Ralph, P. and Moore, M.A.S. (1979) In vitro stimulation of phagocytosis in a macrophage cell line measured by a convenient radiolabeled latex beads assay. Cell. Immunol. 46, 48. Jones, B.M., Nicholson, J.K.A., Holman, R.C. and Hubbard, M. (1989) Comparison of monocyte separation methods using flow cytometric analysis. J. Immunol. Methods 125, 41. Kumagai, K., Itoh, K., Hinuma, S. and Tada, M. (1979) Pretreatment of plastic petri dishes with fetal calf serum. A simple method for macrophage isolation. J. Immunol. Methods 29, 17. Lehrer, R.I. (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. Levitz, S.M., DiBenedetto, D.J. and Diamond, R.D. (1987) A rapid fluorescent assay to distinguish attached from phagocytized yeast particles. J. Immunol. Methods 101, 37. Mantovani, A. (1981) Adherence to microexudate-coated plastic. In: H.B. Herscowitz, H.T. Holden, J.A. Bellanti and A. Ghaffar (Eds.), Manual of Macrophage Methodology: Collection, Characterization, and Function. M. Dekker, New York, p. 69. Matsson, P., Fossum, C. and Larsson, B. (1985) Evaluation of flow cytometry and fluorescence microscopy for the estimation of bovine mononuclear phagocytes. J. Immunol. Methods 78, 13. Murray, H.W. and Cohn, Z.A. (1980) Macrophage oxygendependent antimicrobial activity. III. Enhanced oxidative metabolism as an expression of macrophage activation. J. Exp. Med. 152, 1596. Murray, H.W., Nathan, C.F. and Cohn, Z.A. (1980) Macropl~age oxygen-dependent antimicrobial activity. IV. Role of endogenous scavengers of oxygen intermediates. J. Exp. Med. 152, 1610. Nathan, C.F. (1983) Mechanisms of macrophage antimicrobial activity. Trans. R. Soc. Trop. Med. Hyg. 77, 620.
109 Sasada, M., Kubo, A., Nishimura, T., Kakita, T., Moriguchi, T., Yamamoto, K. and Uchino, H. (1987) Candidacidal activity of monocyte-derived human macrophages: relationship between Candida killing and oxygen radical generation by human macrophages. J. Leuk. Biol. 41, 289. Schumann, R.R., Van der Bosch, J., Riiller, S., Ernst, M. and Schlaak, M. (1989) Monocyte long-term cultivation on microvascular endothelial cell monolayers: morphologic and phenotypic characterization and comparison with monocytes cultured on tissue culture plastic. Blood 73, 818. Stoll, H.P., Kr~imer, S. and Oberhausen, E. (1986) A new method of preparing monocytes suspensions from human whole blood. Blood Cells 11, 421.
Thomas, V.L., Sanford, B.A., Driscoll, M.S., Casto, D.T. and Ramamurthy, R.S. (1988) Luminol-dependent chemiluminescence microassay for phagocytic function. J. Immunol. Methods 111,227. Van den Broek, P.J. (1989) Antimicrobial drugs, microorganisms, and phagocytes. Rev. Infect. Dis. 11, 213. Van der Meet, J.W.M., Van de Gevel, J.S. and Van Furth, R. (1981) Teflon film as a substrate for the culture of mononuclear phagocytes. In: D.O. Adams, P.J. Edelson and H. Koren (Eds.), Methods for Studying Mononuclear Phagocytes. Academic Press, New York, p. 121. Van Furth, R. and Diesselhoff-Den Dulk, M.M.C. (1980) Method to prove ingestion of particles by macrophages with light microscopy. Scand. J. Immunol. 12, 265.
