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An advantageous method for detection of Fc-receptors and for studying Fc-receptor-mediated phagocytosis
Immunology Letters, 7 (1983) 29 33 Elsevier
AN ADVANTAGEOUS METHOD FOR DETECTION FOR STUDYING Fc-RECEPTOR-MEDIATED
OF Fc-RECEPTORS PHAGOCYTOSIS
AND
L. FORNUSEK*, J. KOPE~EK** and V. VETVI(~KA* Czechoslovak Academy of Sciences, *Institute of Microbiology, CS-142 20 Prague 41l and **Institute of Macrornolecular Chemistry, CS-162 06 Prague 616, Czechoslovakia (Received 24 May 1983) (Accepted 6 June 1983)
1. Summary A method employing fluorescent 2-hydroxyethyl methacrylate copolymer particles with dinitrophenyl haptenic group and anti-haptenic murine monoclonal antibodies of different isotypes is described for tracing Fc-receptors and for studies of phagocytosis via these cell surface structures. The possibility of quenching the fluorescence of non-phagocytosed particles enables us to distinguish clearly and reliably between internalized and cell surface-bound particles.
specificity, were described during the sixties and seventies [3]. In an extensive literature greatly varying data on the proportions of Fc-receptor-bearing cells in numerous cell populations have been published. Different methods used for different subtypes of Fcreceptors may be one of the most relevant causes of these discrepancies. Additionally, in studies on phagocytosis the problem of reliable distinction between internalized and cell surface-bound prey appears. The principal objective of the current paper has been to develop an additional detection technique which could be useful for Fc-receptor studies (especially for that on phagocytosis) and which would provide some advantages over methods employed so far.
2. Introduction Phagocytosis, originally a primitive feeding mechanism, developed during phyUogenesis to an important mechanism of immune defence [1]. In vertebrates specialized cell surface structures exist which render this process specific; Fc-receptors rank among these structures [2]. Many subtypes of Fc-receptors, according to their immunoglobulin isotype
Key words: Fc-receptors - fluorescence 2-hydroxyethyl methacrylate copolymers - monoclonal antibodies - phagocytosis Abbreviations: DNP, dinitrophenyl; Fc-receptor, receptor for the Fc part of an immunoglobulin; F-DNP-HEMA particles, particles based on HEMA polymer bearing fluorescent label and dinitrophenyl haptenic group; FITC-AA, N-fluorescein-N'-allylthiourea; HEMA, 2-hydroxyethyl methacrylate; MA Gly Gly-Lys-DNP, N-methacryloylglycylglycyl-N~-dinit rophenyl-Llysine; TNP, trinitrophenyl. 0165-2478 / 83 / $03.00 © Elsevier Science Publishers B.V.
3. Material and methods 3.1. Cells An arbitrary cell population where Fc-receptorbearing cells were to be detected, or in which the process of phagocytosis was to be studied was collected, washed and adjusted to 1 to 10 X 106 ml i. For the experiments documented in this paper, murine peritoneal exudate cells, spleen cells, lymph node cells and thymocytes of the 3~-month-old females of the A/J strain were used. Cell viability was checked in cell samples by the trypan blue exclusion method. 3.2. Particles Particles based on 2-hydroxyethyl methacrylate (HEMA) copolymers were prepared by gamma-ir29
radiation-activated polymerization [4-6]. Monomers: HEMA (L6~iva, Prague) and methacrylic acid (Fluka, Buchs) were purified as described earlier [7]. Methylene-bis-acrylamide (Fluka, Buchs) was recrystallized from methanol. N-methacryloylglycylglycylN~-dinitrophenyl-L-lysine (MA-Gly-Gly-Lys-DNP) was prepared by the reaction of N-methacryloylglycyl-glycine p-nitrophenyl ester [8] and N'-DNP-Llysine hydrochloride (Sigma, St. Louis) in dimethylformamide in the presence of triethylamine. N-fluorescein-N'-allylthiourea (FITC-AA) was prepared from fluorescein isothiocyanate (Isomer I; Sigma, St. Louis) and allylamine (Fluka, Buchs). Typical procedure: By a reaction of 4 mg of fluorescein isothiocyanate with an excess of allylamine in dry dioxane at 50 °C, FITC-AA was prepared. Dioxan and excess of allylamine were distilled off under vacuum. To the oil obtained 0.4 ml of mixture of monomers was added (77.9 wt. parts HEMA, 0.1 wt.p. ethylenedimethacrylate, 2.0 wt.p. methylene-bis-acrylamide, 20.0 wt.p. methacrylic acid). After a homogeneous solution was formed, 5-20 mg of crude MA-Gly-Gly-Lys-DNP was added in order to obtain fluorescent particles bearing the DNP haptenic group (F-DNP-HEMA particles). The solution obtained was diluted with 19.6 ml of distilled water, spun down (2000 g, I0 min), purged with nitrogen and irradiated (6°Co, 8 kGy, 25-30 o C; Gamma-cell, AEC, Ottawa). The F-DNP-HEMA particle suspension thus obtained was then poured into a large excess of distilled water. Occasional particle aggregates or clumps were removed by sedimentation and/or by centrifugation (300 g, 3 min). 5-10-fold thorough washing (sedimentation) followed in order to remove all detectable monomers. This procedure yielded a suspension of highly durable single particles about 2.5 #m in size. It could be stored at 4 °C for many months.
3.3. Coating of particles with monoclonal antibodies F-DNP-HEMA particles were coated (22 °C, 1 h) with maximal subagglutinating doses of murine monoclonal anti-trinitrophenyl (anti-TNP) antibodies of IgGl, IgG2a, IgG2b, IgM, IgA and IgE isotypes. All these antibodies cross-reacted strongly with the DNP haptenic group. They were products of Hy 2.15, Hy 1.2, GK 14-1, Sp 6,51-9-17, and IGELa4 hybridomas, respectively. The hybridoma 30
cell lines were kindly provided by Dr. M. Wabl (Max-Planck-Gesellschaft, Ttibingen). Particles were then washed (600 g, 10 min), resuspended, stored in refrigerator and used until deterioration due to formation of particle aggregates appeared (3-5 days at most). 3.4. Fc-receptor detection (F-DNP-HEMA assay) Cells were mixed with antibody-coated F-DNPHEMA particles (ratio l:100) and incubated under occasional gentle agitation at 22 °C for 1 h. Samples were then observed under a fluorescent microscope with epi-illumination equipment (Leitz-Orthoplan). Filters KP490 (2 pieces), KP540, K480 and K515 were used. Cells labelled with 3 and more adhering particles were considered Fc-receptor-bearing cells. 3.5. Fc-receptor detection (rosette assay) "Classical" rosette assay with antibody-coated TNP-coupled sheep red blood cells [9] was used for comparison with the technique described above. Briefly, cells were mixed (ratio 1:100) with TNPcoupled sheep red blood cells coated with maximal subagglutinating doses of murine monoclonal antiTNP antibodies of different isotypes. After a short period of centrifugation (120 g, 5 min) 1 h incubation at 22 °C followed. Cells were then gently resuspended, fixed by adding glutaraldehyde to the final concentration of 0.6%. After adding the same volume of 0.002% acridine orange (Loba Chemic, Wien) samples were estimated under fluorescent microscope. Only rosettes with a fluorescent central cell were counted; cells with 3 and more bound red blood cells were considered a rosette. In all experiments, at least 500 cells were estimated, each experiment was performed 3-6 times. 3.6. Test for Fc-receptor-mediated phagoo'tosis Cells in RPMI 1640 medium (Flow, Irvine) without serum were mixed with F-DNP-HEMA particles as decribed above and incubated for various intervals (3-12 h) under gentle agitation at 37 °C. To the withdrawn drop sample, a drop of 0.25% glutaraldehyde in phosphate-buffered saline (pH =7.4-7.6) and after a few seconds a drop of 0.1-0.05% crystal violet (Lachema, Brno) in borate buffer (pH -- 8.0-8.5) were added in order to quench the green fluorescence of non-phagocytized particles
F-DNP-HEMA particles a Red blood cellsb F-DNP-HEMA particles a Red blood cells b F-DNP-HEMA particles a Red blood cellsb F-DNP-HEMA particles a Red blood cells b F-DNP-HEMA particles a Red blood cellsb F-DNP-HEMA particles a Red blood cells b
Peritoneal macrophages Peritoneal macrophages Splenic macrophages Splenic macrophages Peritoneal lymphocytes Peritoneal lymphocytes Splenic lymphocytes Splenic lymphocytes Peripheral lymph node lymphocytes Peripheral lymph node lymphocytes Thymocytes Thymocytes
43.6 ± 3.6 39.6 5:1.7 22. I 5:3.0 20.0 5:4.2 16.8 5:1.9 9.7 5:3.5 13.4 5:2.4 18.0 5:3.5 13.1 5:3.7 18.3 5:6.8 3.95:0.4 2.6±0.7
lgG 1 26.3 + 3.2 35.4 + 3. I 6.9 5:2.0 8.3 5:5.6 9.2 5:0.8 5.1 5:3.9 5.9 5:0.6 3.9 5:0.9 6.8 5:0.9 7.5 5:1.7 0.55:0.2 0.25:0.2
IgG2a 98.3 5 : 6 . 9 85.65:9.1 29.6 ± 1.7 40.2 5 : 4 . 6 41.2 5 : 3 . 9 30.8 5 : 5 . 1 25.6 +__ 5.2 37.0 5:11.6 16.2 5 : 1 . 8 22.8 5 : 7 . 7 6.45: 1.5 4.25:2.1
lgG2t, 6.8 5:1.2 7.5 5:1.4 5.2 5:0.9 7.3 5:1.7 13.1 5:2.1 12.8 5:5.8 2.7 5:0.9 12.4 5:1.6 14.2 5:2.3 13.9 5:2.2 3.15:0.7 2.9±0.7
IgM
Percentage of cells (mean 5: S.D.) bearing Fc-receptors
6.8 ± 1.4 7.3 5: ! .6 1.3 ± 0.2 3.2 5:2.7 8.4 ± 2.1 7.7 5:3.4 0.4 5:0.1 5. I 5:0.4 13.3 ± 0.9 17.0 ± 3.2 6.45:0.7 5.45:0.9
lgA
41.2 + 2.4 24.4 + 3.3 17.8 5:2.1 14.8 ± 5.7 4.3 ± 1.3 5.3 5:3.0 5.2 ± 0.9 10.0 5:5.3 3,3 5:0.3 2.6 ± 0.6 11.75:1.4 2.95:0.6
IgE
2.3 ± 0.8 2.9 5: 1.2 1.35:0.2 2.6 + 0.5 0.5 5:0.1 0,9 5:0.4 0.4 + 0.1 0.6 5:0.1 0.5 5:0.2 0.9 5:0.3 0.3___0.1 0.25:0.1
Control c
a F-DNP-HEMA particles were coated with maximal subagglutinating doses of respective murine monoclonal anti-TNP antibody isotypes. b Sheep red blood cells were coupled with the TNP-haptenic group and coated with maximal subagglutinating doses of respective murine anti-TNP monoclonal antibody isotypes. c Reaction with uncoated F-DNP-HEMA particles or red blood cells, respectively. No. of experiments -----3-6.
Particles used for detection
Cells
Table 1 Comparison of the use of F-DNP-HEMA particles and red blood cells for the detection of Fc-receptors on murine macrophages and lymphocytes
[10]. F - D N P - H E M A particles displaying green fluorescence were thus considered phagocytized ones, and particles with no or faint dark-red fluorescence were considered non-phagocytized ones. Because of quick penetration of the quenching agent into the cell interior, estimation had to be finished within a few minutes after addition of crystal violet or smears on microscopic slides were made immediately and dried quickly. In both types of assays control experiments with uncoated particles were carried out.
4. Results and Discussion
We tackled the development of a method for detection of Fc-receptors and for studying the phagocytosis via these structures. We intended to employ available murine monoclonal anti-TNP antibodies of different isotypes and modified H E M A copolymer particles prepared earlier in our laboratory [5,6]. Unlike many other types of polymeric particles, the non-specific adherence of H E M A particles is negligible because of their low negative charge and hydrophilic character [5]. By introducing fluorescein and the DNP-haptenic group, we obtained fluorescent particles which, when coated with antihaptenic antibody, are suitable for the detection of Fc-receptors (Fig. 1). In addition to FITC-AA, N-tetramethylrhodamine-N'-allylthiourea or N-dansylallylamine can be used. This makes possible the detection of 2
Fig. 1. A peritoneal macrophage bearing Fc-receptor for lgE as detected by F-DNP-HEMA particles coated with maximal subagglutinating dosis of murine monoclonal anti-TNP lgE. (Bar = 10 ,urn.)
32
Fig. 2. A peritoneal macrophage phagocytosing F-DNP-HEMAparticles coated with maximal subagglutinating dosis of routine monoclonal anti-TNP lgGi. Bright particles are phagocytosed and fluoresce yellow-green; ghosts are adhering (arrows) and fi-ee particles with faint dark-red fluorescence. (Bar = 10 #m.)
or 3 subtypes of Fc-receptors simultaneously (Forn~sek et al., results to be published). As can be seen from Table 1, which compares the results obtained using antibody-coated F-DNPH E M A particles with those provided by the "classical" rosette method employing antibody-coated TNP-coupled sheep red blood cells, our method gives comparable results. The advantage consists in the durability of the particles in comparison to red blood cells, in thegreater sensitivity of detection (smaller particles are less sensitive to shearing forces than larger erythrocytes) and in the larger possibility of studying the capping phenomenon. The greatest advantage of the F - D N P - H E M A particle use is the possibility to distinguish reliably phagocytosed and non-phagocytosed particles in the studies on phagocytosis (Fig. 2). The quenching effect of the crystal violet [10] shifts the fluorescence of nonphagocytosed F - D N P - H E M A particles to the dark red, while the phagocytosed particles continue to fluoresce yellow-green for a reasonable time interval (about 3-5 min). If a smear is made immediately on adding the quenching agent, the crystal violet does not succeed in penetrating to the internalized FD N P - H E M A particles. This results in the effect described above. Very recently, on completion of this manuscript, Loike and Silverstein [11] published a similar me-
thod for distinguishing between phagocytosed and non-phagocytosed particles. Unfortunately, the trypan blue used by them as the quenching agent for the fluorescence of glutaraldehyde-fixed red blood cells is unable to quench the fluorescence of F-DNPHEMA particles. Due to the fact that trypan blue is excluded from the living cells but the crystal violet is not, it is impossible to improve our method to a long-term estimation in native cell suspension. On the other hand, the possibility of obtaining durable preparations on smears as described in this paper might also be of some interest.
Acknowledgements The authors are indebted to Dr. M. Wabl (MaxPlanck-Gesellschaft, Tiibingen) for providing antiTNP hybridoma cell lines, and to Dr. M. Pegek (Institute for Research, Production and Use of Radioisotopes, Prague) for technical assistance.
References [I] Silverstein, S. C., Steinman, R. M. and Cohn, Z. A. (1977) Ann, Rev, Biochem. 46, 669-722. [2] McKeever, P. and Spicer, S. S. (1980) in: The Reticuloendothelial System l (Cart, I. and Daems, W. T. eds.) pp. 161 258, Plenum Press. [3] Unkeless, J. C., Fleit, H. and Mellman, I. S. (1981) Adv. lmmunol. 31,247 270. [4] Rembaum, A., Yen, S. P. S., Cheong, E., Wallace, S., Molday, R. S., Gordon, I. L. and Dreyer, W. J. (1976) Macromolecules 9, 328 336. [5] Forn~tsek, L., V~tvi~ka, V. and Kope~ek, J. (1981) Experientia 37,418~20. [6] V~tvi~ka, V., Forn~sek, L., Kope~ek, J., Kaminkovfi, J., Ka'spfirekl L. and Vr~,nov~,, M. (1982) Immunol. Lett. 5, 97 100. [7] Kope~ek, J., Vaclk, L. and Lira, D. (1971) J. Polymer Sci. A-l, 9, 2801-2815. [8] Rejmanovfi, P., Labsk~,, J. and Kopebek, 3. (1977) Macromolec. Chem. 178, 2159 2168. [9] Strober, W., Hague, N. E., Lure, L. G. and Henkart, P. A. (1978) J. lmmunol. 121, 2440 2445. [10] Hed, J. (1977) FEMS Lett. I, 357 361. [11] Loike, J. D. and Silverstein, S. C, (1983) J. Immunol. Meth. 57, 373 379.
33
AN ADVANTAGEOUS METHOD FOR DETECTION FOR STUDYING Fc-RECEPTOR-MEDIATED
OF Fc-RECEPTORS PHAGOCYTOSIS
AND
L. FORNUSEK*, J. KOPE~EK** and V. VETVI(~KA* Czechoslovak Academy of Sciences, *Institute of Microbiology, CS-142 20 Prague 41l and **Institute of Macrornolecular Chemistry, CS-162 06 Prague 616, Czechoslovakia (Received 24 May 1983) (Accepted 6 June 1983)
1. Summary A method employing fluorescent 2-hydroxyethyl methacrylate copolymer particles with dinitrophenyl haptenic group and anti-haptenic murine monoclonal antibodies of different isotypes is described for tracing Fc-receptors and for studies of phagocytosis via these cell surface structures. The possibility of quenching the fluorescence of non-phagocytosed particles enables us to distinguish clearly and reliably between internalized and cell surface-bound particles.
specificity, were described during the sixties and seventies [3]. In an extensive literature greatly varying data on the proportions of Fc-receptor-bearing cells in numerous cell populations have been published. Different methods used for different subtypes of Fcreceptors may be one of the most relevant causes of these discrepancies. Additionally, in studies on phagocytosis the problem of reliable distinction between internalized and cell surface-bound prey appears. The principal objective of the current paper has been to develop an additional detection technique which could be useful for Fc-receptor studies (especially for that on phagocytosis) and which would provide some advantages over methods employed so far.
2. Introduction Phagocytosis, originally a primitive feeding mechanism, developed during phyUogenesis to an important mechanism of immune defence [1]. In vertebrates specialized cell surface structures exist which render this process specific; Fc-receptors rank among these structures [2]. Many subtypes of Fc-receptors, according to their immunoglobulin isotype
Key words: Fc-receptors - fluorescence 2-hydroxyethyl methacrylate copolymers - monoclonal antibodies - phagocytosis Abbreviations: DNP, dinitrophenyl; Fc-receptor, receptor for the Fc part of an immunoglobulin; F-DNP-HEMA particles, particles based on HEMA polymer bearing fluorescent label and dinitrophenyl haptenic group; FITC-AA, N-fluorescein-N'-allylthiourea; HEMA, 2-hydroxyethyl methacrylate; MA Gly Gly-Lys-DNP, N-methacryloylglycylglycyl-N~-dinit rophenyl-Llysine; TNP, trinitrophenyl. 0165-2478 / 83 / $03.00 © Elsevier Science Publishers B.V.
3. Material and methods 3.1. Cells An arbitrary cell population where Fc-receptorbearing cells were to be detected, or in which the process of phagocytosis was to be studied was collected, washed and adjusted to 1 to 10 X 106 ml i. For the experiments documented in this paper, murine peritoneal exudate cells, spleen cells, lymph node cells and thymocytes of the 3~-month-old females of the A/J strain were used. Cell viability was checked in cell samples by the trypan blue exclusion method. 3.2. Particles Particles based on 2-hydroxyethyl methacrylate (HEMA) copolymers were prepared by gamma-ir29
radiation-activated polymerization [4-6]. Monomers: HEMA (L6~iva, Prague) and methacrylic acid (Fluka, Buchs) were purified as described earlier [7]. Methylene-bis-acrylamide (Fluka, Buchs) was recrystallized from methanol. N-methacryloylglycylglycylN~-dinitrophenyl-L-lysine (MA-Gly-Gly-Lys-DNP) was prepared by the reaction of N-methacryloylglycyl-glycine p-nitrophenyl ester [8] and N'-DNP-Llysine hydrochloride (Sigma, St. Louis) in dimethylformamide in the presence of triethylamine. N-fluorescein-N'-allylthiourea (FITC-AA) was prepared from fluorescein isothiocyanate (Isomer I; Sigma, St. Louis) and allylamine (Fluka, Buchs). Typical procedure: By a reaction of 4 mg of fluorescein isothiocyanate with an excess of allylamine in dry dioxane at 50 °C, FITC-AA was prepared. Dioxan and excess of allylamine were distilled off under vacuum. To the oil obtained 0.4 ml of mixture of monomers was added (77.9 wt. parts HEMA, 0.1 wt.p. ethylenedimethacrylate, 2.0 wt.p. methylene-bis-acrylamide, 20.0 wt.p. methacrylic acid). After a homogeneous solution was formed, 5-20 mg of crude MA-Gly-Gly-Lys-DNP was added in order to obtain fluorescent particles bearing the DNP haptenic group (F-DNP-HEMA particles). The solution obtained was diluted with 19.6 ml of distilled water, spun down (2000 g, I0 min), purged with nitrogen and irradiated (6°Co, 8 kGy, 25-30 o C; Gamma-cell, AEC, Ottawa). The F-DNP-HEMA particle suspension thus obtained was then poured into a large excess of distilled water. Occasional particle aggregates or clumps were removed by sedimentation and/or by centrifugation (300 g, 3 min). 5-10-fold thorough washing (sedimentation) followed in order to remove all detectable monomers. This procedure yielded a suspension of highly durable single particles about 2.5 #m in size. It could be stored at 4 °C for many months.
3.3. Coating of particles with monoclonal antibodies F-DNP-HEMA particles were coated (22 °C, 1 h) with maximal subagglutinating doses of murine monoclonal anti-trinitrophenyl (anti-TNP) antibodies of IgGl, IgG2a, IgG2b, IgM, IgA and IgE isotypes. All these antibodies cross-reacted strongly with the DNP haptenic group. They were products of Hy 2.15, Hy 1.2, GK 14-1, Sp 6,51-9-17, and IGELa4 hybridomas, respectively. The hybridoma 30
cell lines were kindly provided by Dr. M. Wabl (Max-Planck-Gesellschaft, Ttibingen). Particles were then washed (600 g, 10 min), resuspended, stored in refrigerator and used until deterioration due to formation of particle aggregates appeared (3-5 days at most). 3.4. Fc-receptor detection (F-DNP-HEMA assay) Cells were mixed with antibody-coated F-DNPHEMA particles (ratio l:100) and incubated under occasional gentle agitation at 22 °C for 1 h. Samples were then observed under a fluorescent microscope with epi-illumination equipment (Leitz-Orthoplan). Filters KP490 (2 pieces), KP540, K480 and K515 were used. Cells labelled with 3 and more adhering particles were considered Fc-receptor-bearing cells. 3.5. Fc-receptor detection (rosette assay) "Classical" rosette assay with antibody-coated TNP-coupled sheep red blood cells [9] was used for comparison with the technique described above. Briefly, cells were mixed (ratio 1:100) with TNPcoupled sheep red blood cells coated with maximal subagglutinating doses of murine monoclonal antiTNP antibodies of different isotypes. After a short period of centrifugation (120 g, 5 min) 1 h incubation at 22 °C followed. Cells were then gently resuspended, fixed by adding glutaraldehyde to the final concentration of 0.6%. After adding the same volume of 0.002% acridine orange (Loba Chemic, Wien) samples were estimated under fluorescent microscope. Only rosettes with a fluorescent central cell were counted; cells with 3 and more bound red blood cells were considered a rosette. In all experiments, at least 500 cells were estimated, each experiment was performed 3-6 times. 3.6. Test for Fc-receptor-mediated phagoo'tosis Cells in RPMI 1640 medium (Flow, Irvine) without serum were mixed with F-DNP-HEMA particles as decribed above and incubated for various intervals (3-12 h) under gentle agitation at 37 °C. To the withdrawn drop sample, a drop of 0.25% glutaraldehyde in phosphate-buffered saline (pH =7.4-7.6) and after a few seconds a drop of 0.1-0.05% crystal violet (Lachema, Brno) in borate buffer (pH -- 8.0-8.5) were added in order to quench the green fluorescence of non-phagocytized particles
F-DNP-HEMA particles a Red blood cellsb F-DNP-HEMA particles a Red blood cells b F-DNP-HEMA particles a Red blood cellsb F-DNP-HEMA particles a Red blood cells b F-DNP-HEMA particles a Red blood cellsb F-DNP-HEMA particles a Red blood cells b
Peritoneal macrophages Peritoneal macrophages Splenic macrophages Splenic macrophages Peritoneal lymphocytes Peritoneal lymphocytes Splenic lymphocytes Splenic lymphocytes Peripheral lymph node lymphocytes Peripheral lymph node lymphocytes Thymocytes Thymocytes
43.6 ± 3.6 39.6 5:1.7 22. I 5:3.0 20.0 5:4.2 16.8 5:1.9 9.7 5:3.5 13.4 5:2.4 18.0 5:3.5 13.1 5:3.7 18.3 5:6.8 3.95:0.4 2.6±0.7
lgG 1 26.3 + 3.2 35.4 + 3. I 6.9 5:2.0 8.3 5:5.6 9.2 5:0.8 5.1 5:3.9 5.9 5:0.6 3.9 5:0.9 6.8 5:0.9 7.5 5:1.7 0.55:0.2 0.25:0.2
IgG2a 98.3 5 : 6 . 9 85.65:9.1 29.6 ± 1.7 40.2 5 : 4 . 6 41.2 5 : 3 . 9 30.8 5 : 5 . 1 25.6 +__ 5.2 37.0 5:11.6 16.2 5 : 1 . 8 22.8 5 : 7 . 7 6.45: 1.5 4.25:2.1
lgG2t, 6.8 5:1.2 7.5 5:1.4 5.2 5:0.9 7.3 5:1.7 13.1 5:2.1 12.8 5:5.8 2.7 5:0.9 12.4 5:1.6 14.2 5:2.3 13.9 5:2.2 3.15:0.7 2.9±0.7
IgM
Percentage of cells (mean 5: S.D.) bearing Fc-receptors
6.8 ± 1.4 7.3 5: ! .6 1.3 ± 0.2 3.2 5:2.7 8.4 ± 2.1 7.7 5:3.4 0.4 5:0.1 5. I 5:0.4 13.3 ± 0.9 17.0 ± 3.2 6.45:0.7 5.45:0.9
lgA
41.2 + 2.4 24.4 + 3.3 17.8 5:2.1 14.8 ± 5.7 4.3 ± 1.3 5.3 5:3.0 5.2 ± 0.9 10.0 5:5.3 3,3 5:0.3 2.6 ± 0.6 11.75:1.4 2.95:0.6
IgE
2.3 ± 0.8 2.9 5: 1.2 1.35:0.2 2.6 + 0.5 0.5 5:0.1 0,9 5:0.4 0.4 + 0.1 0.6 5:0.1 0.5 5:0.2 0.9 5:0.3 0.3___0.1 0.25:0.1
Control c
a F-DNP-HEMA particles were coated with maximal subagglutinating doses of respective murine monoclonal anti-TNP antibody isotypes. b Sheep red blood cells were coupled with the TNP-haptenic group and coated with maximal subagglutinating doses of respective murine anti-TNP monoclonal antibody isotypes. c Reaction with uncoated F-DNP-HEMA particles or red blood cells, respectively. No. of experiments -----3-6.
Particles used for detection
Cells
Table 1 Comparison of the use of F-DNP-HEMA particles and red blood cells for the detection of Fc-receptors on murine macrophages and lymphocytes
[10]. F - D N P - H E M A particles displaying green fluorescence were thus considered phagocytized ones, and particles with no or faint dark-red fluorescence were considered non-phagocytized ones. Because of quick penetration of the quenching agent into the cell interior, estimation had to be finished within a few minutes after addition of crystal violet or smears on microscopic slides were made immediately and dried quickly. In both types of assays control experiments with uncoated particles were carried out.
4. Results and Discussion
We tackled the development of a method for detection of Fc-receptors and for studying the phagocytosis via these structures. We intended to employ available murine monoclonal anti-TNP antibodies of different isotypes and modified H E M A copolymer particles prepared earlier in our laboratory [5,6]. Unlike many other types of polymeric particles, the non-specific adherence of H E M A particles is negligible because of their low negative charge and hydrophilic character [5]. By introducing fluorescein and the DNP-haptenic group, we obtained fluorescent particles which, when coated with antihaptenic antibody, are suitable for the detection of Fc-receptors (Fig. 1). In addition to FITC-AA, N-tetramethylrhodamine-N'-allylthiourea or N-dansylallylamine can be used. This makes possible the detection of 2
Fig. 1. A peritoneal macrophage bearing Fc-receptor for lgE as detected by F-DNP-HEMA particles coated with maximal subagglutinating dosis of murine monoclonal anti-TNP lgE. (Bar = 10 ,urn.)
32
Fig. 2. A peritoneal macrophage phagocytosing F-DNP-HEMAparticles coated with maximal subagglutinating dosis of routine monoclonal anti-TNP lgGi. Bright particles are phagocytosed and fluoresce yellow-green; ghosts are adhering (arrows) and fi-ee particles with faint dark-red fluorescence. (Bar = 10 #m.)
or 3 subtypes of Fc-receptors simultaneously (Forn~sek et al., results to be published). As can be seen from Table 1, which compares the results obtained using antibody-coated F-DNPH E M A particles with those provided by the "classical" rosette method employing antibody-coated TNP-coupled sheep red blood cells, our method gives comparable results. The advantage consists in the durability of the particles in comparison to red blood cells, in thegreater sensitivity of detection (smaller particles are less sensitive to shearing forces than larger erythrocytes) and in the larger possibility of studying the capping phenomenon. The greatest advantage of the F - D N P - H E M A particle use is the possibility to distinguish reliably phagocytosed and non-phagocytosed particles in the studies on phagocytosis (Fig. 2). The quenching effect of the crystal violet [10] shifts the fluorescence of nonphagocytosed F - D N P - H E M A particles to the dark red, while the phagocytosed particles continue to fluoresce yellow-green for a reasonable time interval (about 3-5 min). If a smear is made immediately on adding the quenching agent, the crystal violet does not succeed in penetrating to the internalized FD N P - H E M A particles. This results in the effect described above. Very recently, on completion of this manuscript, Loike and Silverstein [11] published a similar me-
thod for distinguishing between phagocytosed and non-phagocytosed particles. Unfortunately, the trypan blue used by them as the quenching agent for the fluorescence of glutaraldehyde-fixed red blood cells is unable to quench the fluorescence of F-DNPHEMA particles. Due to the fact that trypan blue is excluded from the living cells but the crystal violet is not, it is impossible to improve our method to a long-term estimation in native cell suspension. On the other hand, the possibility of obtaining durable preparations on smears as described in this paper might also be of some interest.
Acknowledgements The authors are indebted to Dr. M. Wabl (MaxPlanck-Gesellschaft, Tiibingen) for providing antiTNP hybridoma cell lines, and to Dr. M. Pegek (Institute for Research, Production and Use of Radioisotopes, Prague) for technical assistance.
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