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Photometric microassay for quantitation of macrophage Fc and C3b receptor function
Journal oflmmunologicalMethods,
77 (1985) 155-163
155
Elsevier JIM03399
Photometric Microassay for Quantitation of Macrophage Fc and C3b Receptor Function John A. Rummage and Richard W. Leu The Samuel Roberts Noble Foundation, Inc., Biomedical Division, P.O. Box 2180, Ardmore, OK 73402, U.S.A.
(Received 2 October 1984, accepted 6 November 1984)
A photometric microassay has been developed to quantitate macrophage Fc and C3b receptor mediated binding and phagocytosis by measuring the absorbance of macrophage associated erythrocytes at 405 nm on an automated densitometer. The method compares favorably in sensitivity and kinetics to the 51Cr-labeled erythrocyte assay. Saturation and linear dose response kinetics were demonstrable for both total index and phagocytic index of either Fc receptor or C3b receptor. The assay allowed detection of significant differences in Fc receptor function with varying macrophage densities and between Fc receptor competent (C3HeB/FeJ) macrophages and Fc receptor deficient (C3H/HeJ) macrophages. A valid binding index was derived at 37°C by computing the difference between the total and phagocytic indices, which compared favorably with binding studies at 4°C. This new procedure provides a simple, rapid and reproducible microassay for the quantitation of Fc/C3b receptor dependent binding and phagocytosis which offers distinct advantages over the laborious rosette assay and the 51Cr-labeled erythrocyte assay. Key words: macrophage - phagocytosis - photometric quantitation - Fc receptor
Introduction A primary function of mononuclear phagocytic cells is to bind and internalize opsonized particles through Fc a n d / o r C3b receptors. Current methods for measuring macrophage Fc receptor mediated binding and phagocytosis include the standard microscopic rosette methods (Bianco et al., 1975), the 5aCr-labeled erythrocyte m e t h o d (Walker, 1977; Vogel and Rosenstreich, 1979), and radioiodinated or fluoresceinated immunoglobulin binding assays (Unkeless and Eisen, 1975; Shen et al., 1984). The 51Cr-labeled sheep erythrocyte method is widely used for quantitation of Fc and C3b mediated phagocytosis of I g G or C3b opsonized particles. A l t h o u g h this method provides a reproducible and sensitive measure of phagocytosis, certain disadvantages exist. Therefore, a simplified photometric m i c r o m e t h o d was developed in which absorbance of opsonized sheep erythrocyte associated hemoglobin was measured in microtiter plates at 405 n m by means of an automated densitometer. The novel photometric method compares favorably in sensitivity and reproducibility 0022-1759/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
156 with the 51Cr-labeling method for measuring Fc and C3b binding and phagocytosis and offers certain advantages over existing methods.
Materials and Methods
Preparation of macrophage monolayers Male C3H/HeJ and C3HeB/FeJ mice (Jackson Laboratories, Bar Harbor, ME) 8-12 weeks old were injected intraperitoneally with 2 ml of sterile paraffin oil (Fisher Scientific Co., Pittsburgh, PA). Exudate cells (PEC) were harvested 3 days after oil injection by peritoneal lavage in RPMI 1640 (Gibco, Grand Island, NY) supplemented with 200 mM L-glutamine, 25 mM Hepes, and 50/~g/ml gentamycin (Gibco). PEC were washed and counted, then resuspended at the desired concentration (1, 2, or 4 x 10 6 cells/ml) in RPMI 1640 supplemented with 10% fetal bovine serum (Hyclone Sterile Systems, Logan, UT). The PEC suspension (0.1 ml) was dispensed into individual wells of a 96-well microtiter plate (Corning Glass Works, No. 25860, Corning, NY) and incubated for 1 h at 37°C. Nonadherent cells were removed by 2 washes in 0.9% saline in which the plate was immersed vertically and moved forward and backward 3 times.
Preparation of IgG and C3b opsonized erythrocyte conjugates The methods for preparation of opsonized erythrocyte conjugates for Fc and C3b assays were described in detail previously (Bianco et al., 1975; Leu et al., 1983). Sheep erythrocytes (E) (Colorado Serum Co., Denver, CO) were washed thrice in RPMI 1640 and resuspended to a 5% (v/v) suspension. Rabbit anti-sheep erythrocyte antibody (A), IgG fraction (Cordis Laboratories, Miami, FL) was diluted 2-fold from 1 : 250 to 1 : 32,000 and incubated with an equal volume of 5% E suspension in a 37°C shaking water bath for 30 min. After incubation, the E-IgG conjugates, EA(IgG), were washed twice in cold RPMI 1640, resuspended to a 0.5% suspension and kept at 4°C until needed. For C3b receptor assays, a 5% E suspension was mixed with an equal volume of a subagglutinating dilution (approximately 1:150) of rabbit anti-E, IgM fraction (Cappel Laboratories, West Chester, PA), and incubated for 30 min in a shaking 37°C water bath. The EA(IgM) conjugates were washed twice in cold RPMI 1640 and then resuspended to a 5% suspension. A portion of the EA(IgM) conjugate was incubated with equal volumes of various dilutions of C3HeB/FeJ serum as a source of complement for 10 min in a shaking 37°C water bath. After incubation, the EA(IgM) complement (C) conjugates, EA(IgM)C, were washed twice in cold RPMI 1640 and resuspended to a 0.5% suspension and kept at 4°C until needed. E and EA(IgM) were used as controls for non-specific binding and phagocytosis by the macrophage.
Fc / C3b receptor photometric assay EA(IgG) or EA(IgM)C (0.1 ml) was added to each macrophage monolayer and incubated for 1 h at 37°C. After incubation the monolayers were washed thrice by
157 immersion in 0.9% saline as described above. The microtiter wells designated for phagocytic index determinations were individually treated with 0.2 ml of 0.09% hypotonic saline to lyse extracellular bound red cells and then washed. A 0.05 ml volume of 50% fetal bovine serum was added to each well for 2-3 min. The serum was removed by inverting the plate and the monolayers were dried at 50°C for 20 min. When dry, the monolayers were fixed with 0.1 ml absolute methanol for 2-3 min. The plate was washed in distilled H20 and dried at 50°C for 20 min. The microtiter plate was then scanned on a Dynatech MR580 microtiter plate reader at the wavelength of 405 nm. Quadruplicate samples were run for each dilution of EA(IgG) or EA(IgM)C. The mean and the standard error of the mean were calculated from the absorbance (A405nm)values. In most experiments the data were expressed as the Total Index (bound and internalized) and the Phagocytic Index (internalized) values where: 1 - (E A4os/EA(IgO) A4o5) × 100 = Index
51Cr labeling of red cells The measurement of binding and phagocytosis of 51Cr-labeled erythrocytes was performed by a modified method of Vogel and Rosenstreich (1979). A 5% suspension of washed E (109 cells/ml) in 0.9% saline was incubated with 100 /~Ci of sodium chromate (SaCr) (New England Nuclear, Boston, MA) for 1 h in a 37°C shaking water bath. After labeling, the cells were washed in 0.9% NaC1, resuspended at 5% and the EA(IgG) conjugates were prepared as described above. Adherent macrophage monolayers (2.0 × 105/well) were incubated for 1 h with 0.1 ml of the EA(IgG) dilution at 37°C in microtiter plates, washed thrice and designated wells were washed with hypotonic saline. The macrophage monolayers and associated E / E A were dissolved by addition of 0.3 ml of 0.5% SDS for 5 min. A 0.2 ml aliquot from each well was transferred to a Biovial (Beckman Instruments, Irvine, CA) and the radioactivity was counted in a gamma counter for 1 min.
Results
Comparison of the photometric and 51Cr-labelederythrocyte methods for quantitation of macrophage Fc phagocytic function The quantitation of Fc mediated phagocytic function of C3HeB/FeJ mouse peritoneal macrophages by the 51Cr-labeled erythrocyte (Fig. 1A) and the newly developed photometric (Fig. 1B) methods were compared under identical experimental conditions. Measurement of total phagocytic activity (binding and internalization) of macrophage-associated, SlCr-labeled erythrocytes or of erythrocyte hemoglobin absorbance at 405 nm yielded similar dose response kinetics of saturation and linear decrease in activity with decreasing IgG concentration. Quantitation of phagocytosis (internalized conjugates) by the 2 methods also gave comparable results. Re-expression of the A405nm data in the photometric assay (Fig. 1B) as the
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I cjG Dilution(x IC)3) Fig. 1. Comparison of (A) 51Cr-labeled erythrocyte (E) and (B) photometric assays for quantitation of Fc receptor function of C3HeB/FeJ oil elicited murine peritoneal macrophages. Adherent macrophages were incubated with EA(IgG) conjugates, prepared with increasing 2-fold dilutions of IgG (1 : 500-1 : 64,000), for 1 h at 37°C in microtiter plates. E were prelabeled with 51Cr only for the isotopic assay. Phagocytosis was quantitated either by the determination of macrophage-associated 5~C-labeled E or the photometric measurement of macrophage-associated E hemoglobin at 405 nm. See Materials and Methods for details. Each data point depicts the m e a n + SEM of Total (o o) and Phagocytic (© ©) functions of > 8 samples.
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IgG Di lution(x 163) Fig. 2. Normalization of data in the photometric assay to correct for variation in background. Re-expression of the absorbance 405 nm data (Fig. 1B) as the Total Index (O O) or Phagocytic Index
(©
O) was derived as: Index = 1 - ( E A~os/EA(IgG ) A4o5)×100.
159
Total Index or Phagocytic Index (Fig. 2) gave similar kinetics and offered the advantage of normalizing the data to correct for variation between experiments in background absorbance values.
Effects of macrophage density on photometric Fc assay The effects of macrophage density on total and phagocytic indices are shown in Fig. 3. Similar kinetics of saturation and a dose related linear decrease in total and phagocytic functions with decreasing IgG concentration were observed with 4.0 and 2.0 × l0 s cells/well, but not with 1 × l0 s cells/well. Generally the magnitude of phagocytic function was increased proportionately with increasing macrophage effector density. Effects of incubation time and temperature on Fc-mediated binding and phagocytosis The optimum time of incubation of macrophages with EA(IgG) conjugates for maximum total and phagocytic functions in the photometric assay was determined to be 60 min at 37°C. Incubation for 30, 45, or 90 min produced only minor B.
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Fig. 3. Effect of macrophage density on Fc receptor mediated phagocytic function in the photometric assay. C 3 H e B / F e J elicited peritoneal macrophages were plated at 4 × 105 (O O), 2 × 105 ( e . . . . . . e ) or 1 × 105 ( e . . . . . . e ) cells/well in microtiter plates. EA(IgG) conjugates prepared with 2-fold dilutions of IgG were incubated with the adherent macrophage monolayers for 1 h at 37°C. The Total Index (A) and the Phagocytosis Index (B) were computed from photometric A405n m values. Each point depicts the mean + SEM of > 4 experiments.
160 TABLE 1 E F F E C T OF I N C U B A T I O N T E M P E R A T U R E ON Fc D E P E N D E N T B I N D I N G A N D PHAG(3CYTOSIS BY M A C R O P H A G E S a Temperature
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1 :2
53.5 _+3.2 80.5 +0.8
2.9 _+1.2 33.4_+ 1.4
50.6 + 3.1 47.1 + 1.2
4°C 37°C
1 :4
45.3 + 3.5 70.7 + 2.8
0.7 + 0.5 26.1 + 2.2
44.6 + 3.4 44.6 + 3.4
4°C 37°C
1 :8
16.3 + 4.7 49.1 + 2.9
0.5 + 0.5 19.2 + 1.5
15.8 + 4.3 29.9 + 3.6
C 3 H e B / F e J oil elicited peritoneal macrophages (2 × 10S/well) were incubated either at 4°C or 37°C for 1 h with EA(IgG) conjugates prepared with IgG dilutions of 1:2000, 1:4000, or 1:8000 in microtiter plates using the photometric assay. See Materials and Methods for details. b Binding Index was computed as the net difference between Total Index (bound and internalized conjugates) and the Phagocytic Index (internalized conjugates). Each data point depicts the mean_+ SEM of 4 tests each in 2 separate experiments. a
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IgG Dilution (XIO3) Fig. 4. Comparison of Fc phagocytic function of Fc competent C 3 H e B / F e J (e . . . . . . e) and Fc-deficient C 3 H / H e J (e e) mouse macrophages by the photometric assay. Adherent cells (2 X 10S/well) were incubated with EA(IgG) conjugates prepared with varying dilutions of IgG for 1 h at 37°C. The Total Index (A) and the Phagocytosis Index (B) represent the mean + SEM of > 4 experiments.
161
differences in total or phagocytic indices (data not shown).To determine the effects of incubation temperature on binding and phagocytosis, macrophages were incubated for 1 h at 4°C or 37°C with EA(IgG) conjugates and the total and phagocytic indices were calculated (Table I). When incubated at 4°C the EA(IgG) conjugates were bound to Fc receptors on the macrophage, but were not internalized. Incubation at 37°C resulted in both Fc mediated binding and internalization. Comparison of these parameters indicated that the value of the Total Index at 37°C was nearly equivalent to the sum of the values for Total Index at 4°C and the Phagocytic Index at 37°C. Thus the Total Index (4°C) represents potential Fc receptor binding to the macrophage. Since this value is approximately equivalent to the net difference between the Total Index (37°C) and the Phagocytic Index (37°C) a theoretical binding index could be derived which reflects actual binding at 4°C. This relationship was shown to be valid for Total Index and phagocytic values only with higher IgG concentrations (1 : 2000 and 1 : 4000 dilutions) but not with a lower IgG concentration (1 : 8000) in which 4°C binding efficiency was markedly reduced when compared to 37°C. Binding at 37°C was approximately 60% and phagocytosis was approximately 40% of the Total Index at 37°C at IgG dilutions of 1:500 through 1 : 8000 (Fig. 2 and Table I).
Comparison of Fc competent and deficient mouse macrophage populations C 3 H / H e J mouse macrophages have reduced Fc phagocytic function as compared
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Fig. 5. Quantitation of macrophage C3b receptor dependent phagocytosis by the photometric method. EA(IgM) conjugates were coated with C3b by incubation with increasing 2-fold dilutions of homologous mouse serum (1 : 5-1 : 320 dilution). The resulting EA(IgM)C conjugates were incubated with adherent C3HeB/FeJ macrophages (2 x 105/well) for 1 h at 37°C and the Total Index (e e) and Phagocytosis (O O) quantitated by the photometric method. Each data point depicts the mean± SEM of 4 experiments. Controls which consisted of E or EA(IgM) exposure to macrophages were negligable (i.e., < 4% of Total A40~nm for peak test values).
162 to C3HeB/FeJ macrophages. This relationship was confirmed using the photometric assay (Fig. 4) in that both Total and Phagocytic functions were significantly lower with C3H/HeJ cells than with C3HeB/FeJ macrophages.
Photometric quantitation of C3b receptor dependentphagocytosis The photometric assay was applied to the measurement of macrophage C3b receptor mediated phagocytosis (Fig. 5). Conjugates prepared with EA(IgM) and various increasing dilutions of complement-containing serum yielded Total and Phagocytic Index values with linear dose response kinetics and saturation similar to that observed for Fc receptor function. Discussion
Existing quantitative methods for measuring macrophage Fc or C3b receptor dependent phagocytic functions depend largely on the use of isotopically labeled erythrocytes (Walker, 1977; Vogel and Rosenstreich, 1979) or rosette assays (Bianco et al., 1975). The 5aCr-labeled erythrocyte method offers advantages of sensitive and reproducible quantitation of phagocytic function whereas the rosette assays require laborious visual microscopic counting procedures. Disadvantages of the 51Cr-labeling method include time consuming labeling procedures, batch variation in specific activity of isotope, spontaneous shedding of label to yield variable background levels, and the problems of isotopic handling, storage, and disposal. We have developed a photometric microassay for quantitation of Fc or C3b phagocytic function which is patterned after the 51Cr_labeling method. The new method is based on measuring erythrocyte hemoglobin absorbance at 405 nm on an automated densitometer. Comparison of the 2 methods (Fig. 1) indicated that they were very similar in kinetics and sensitivity. Since the photometric method eliminates the use of isotope, it would appear to offer advantages over the isotopic labeling method. The photometric method was shown to quantitate macrophage Fc and C3b functions in a characteristic dose dependent relationship with saturation which was related to increasing IgG or C3b concentrations. To compensate for control background variation between experiments, normalization of the absorbance 405 nm data was accomplished by deriving a total and phagocytic index for expression of phagocytic activity. Increasing Fc phagocytic function with increasing macrophage effector density was demonstrated and distinct differences were detected between Fc deficient C3H/HeJ (Vogel and Rosenstreich, 1979) and Fc competent C3HeB/FeJ mouse macrophages in their Fc dependent phagocytic functions. Binding of IgG opsonized erythrocytes at 4°C, which prevents internalization of Fc receptor bound conjugates, was found to be equivalent at high IgG concentration to the net difference between total (bound and internalized complexes) and phagocytic (internalized complexes) index values derived at 37°C. A constant rate of binding and phagocytosis occurred at 37°C with higher concentrations of IgG. Thus the photometric assay allows approximation of Fc dependent binding to macrophages at 37°C without the necessity for binding assays at 4°C or the use of iodoacetic acid or other inhibitors of phagocytosis.
163
Additional advantages of the photometric assay include the ability to quantitate Fc function in fixed plates stored longer than 1 month without change in phagocytic index values as well as an accurate means of accounting for possible loss of adherent macrophages during studies requiring long-term in vitro culture.
References Bianco, C., F. Griffin and S. Silverstein, 1975, J. Exp. Med. 141, 1278. Leu, R.W., S.M. Hefley and M.J. Herriott, 1983, Cell. Immunol. 80, 31. Shen, L., P.M. Guyre and M.W. Fanger, 1984, Mol. Immunol. 21, 167. Unkeless, J.C. and H.N. Eisen, 1975, J. Exp. Med. 142, 1520. Vogel, S.N. and D.L. Rosenstreich, 1979, J. Immunol. 123, 2842. Walker, W.S., 1977, J. Immunol. 119, 367.
77 (1985) 155-163
155
Elsevier JIM03399
Photometric Microassay for Quantitation of Macrophage Fc and C3b Receptor Function John A. Rummage and Richard W. Leu The Samuel Roberts Noble Foundation, Inc., Biomedical Division, P.O. Box 2180, Ardmore, OK 73402, U.S.A.
(Received 2 October 1984, accepted 6 November 1984)
A photometric microassay has been developed to quantitate macrophage Fc and C3b receptor mediated binding and phagocytosis by measuring the absorbance of macrophage associated erythrocytes at 405 nm on an automated densitometer. The method compares favorably in sensitivity and kinetics to the 51Cr-labeled erythrocyte assay. Saturation and linear dose response kinetics were demonstrable for both total index and phagocytic index of either Fc receptor or C3b receptor. The assay allowed detection of significant differences in Fc receptor function with varying macrophage densities and between Fc receptor competent (C3HeB/FeJ) macrophages and Fc receptor deficient (C3H/HeJ) macrophages. A valid binding index was derived at 37°C by computing the difference between the total and phagocytic indices, which compared favorably with binding studies at 4°C. This new procedure provides a simple, rapid and reproducible microassay for the quantitation of Fc/C3b receptor dependent binding and phagocytosis which offers distinct advantages over the laborious rosette assay and the 51Cr-labeled erythrocyte assay. Key words: macrophage - phagocytosis - photometric quantitation - Fc receptor
Introduction A primary function of mononuclear phagocytic cells is to bind and internalize opsonized particles through Fc a n d / o r C3b receptors. Current methods for measuring macrophage Fc receptor mediated binding and phagocytosis include the standard microscopic rosette methods (Bianco et al., 1975), the 5aCr-labeled erythrocyte m e t h o d (Walker, 1977; Vogel and Rosenstreich, 1979), and radioiodinated or fluoresceinated immunoglobulin binding assays (Unkeless and Eisen, 1975; Shen et al., 1984). The 51Cr-labeled sheep erythrocyte method is widely used for quantitation of Fc and C3b mediated phagocytosis of I g G or C3b opsonized particles. A l t h o u g h this method provides a reproducible and sensitive measure of phagocytosis, certain disadvantages exist. Therefore, a simplified photometric m i c r o m e t h o d was developed in which absorbance of opsonized sheep erythrocyte associated hemoglobin was measured in microtiter plates at 405 n m by means of an automated densitometer. The novel photometric method compares favorably in sensitivity and reproducibility 0022-1759/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
156 with the 51Cr-labeling method for measuring Fc and C3b binding and phagocytosis and offers certain advantages over existing methods.
Materials and Methods
Preparation of macrophage monolayers Male C3H/HeJ and C3HeB/FeJ mice (Jackson Laboratories, Bar Harbor, ME) 8-12 weeks old were injected intraperitoneally with 2 ml of sterile paraffin oil (Fisher Scientific Co., Pittsburgh, PA). Exudate cells (PEC) were harvested 3 days after oil injection by peritoneal lavage in RPMI 1640 (Gibco, Grand Island, NY) supplemented with 200 mM L-glutamine, 25 mM Hepes, and 50/~g/ml gentamycin (Gibco). PEC were washed and counted, then resuspended at the desired concentration (1, 2, or 4 x 10 6 cells/ml) in RPMI 1640 supplemented with 10% fetal bovine serum (Hyclone Sterile Systems, Logan, UT). The PEC suspension (0.1 ml) was dispensed into individual wells of a 96-well microtiter plate (Corning Glass Works, No. 25860, Corning, NY) and incubated for 1 h at 37°C. Nonadherent cells were removed by 2 washes in 0.9% saline in which the plate was immersed vertically and moved forward and backward 3 times.
Preparation of IgG and C3b opsonized erythrocyte conjugates The methods for preparation of opsonized erythrocyte conjugates for Fc and C3b assays were described in detail previously (Bianco et al., 1975; Leu et al., 1983). Sheep erythrocytes (E) (Colorado Serum Co., Denver, CO) were washed thrice in RPMI 1640 and resuspended to a 5% (v/v) suspension. Rabbit anti-sheep erythrocyte antibody (A), IgG fraction (Cordis Laboratories, Miami, FL) was diluted 2-fold from 1 : 250 to 1 : 32,000 and incubated with an equal volume of 5% E suspension in a 37°C shaking water bath for 30 min. After incubation, the E-IgG conjugates, EA(IgG), were washed twice in cold RPMI 1640, resuspended to a 0.5% suspension and kept at 4°C until needed. For C3b receptor assays, a 5% E suspension was mixed with an equal volume of a subagglutinating dilution (approximately 1:150) of rabbit anti-E, IgM fraction (Cappel Laboratories, West Chester, PA), and incubated for 30 min in a shaking 37°C water bath. The EA(IgM) conjugates were washed twice in cold RPMI 1640 and then resuspended to a 5% suspension. A portion of the EA(IgM) conjugate was incubated with equal volumes of various dilutions of C3HeB/FeJ serum as a source of complement for 10 min in a shaking 37°C water bath. After incubation, the EA(IgM) complement (C) conjugates, EA(IgM)C, were washed twice in cold RPMI 1640 and resuspended to a 0.5% suspension and kept at 4°C until needed. E and EA(IgM) were used as controls for non-specific binding and phagocytosis by the macrophage.
Fc / C3b receptor photometric assay EA(IgG) or EA(IgM)C (0.1 ml) was added to each macrophage monolayer and incubated for 1 h at 37°C. After incubation the monolayers were washed thrice by
157 immersion in 0.9% saline as described above. The microtiter wells designated for phagocytic index determinations were individually treated with 0.2 ml of 0.09% hypotonic saline to lyse extracellular bound red cells and then washed. A 0.05 ml volume of 50% fetal bovine serum was added to each well for 2-3 min. The serum was removed by inverting the plate and the monolayers were dried at 50°C for 20 min. When dry, the monolayers were fixed with 0.1 ml absolute methanol for 2-3 min. The plate was washed in distilled H20 and dried at 50°C for 20 min. The microtiter plate was then scanned on a Dynatech MR580 microtiter plate reader at the wavelength of 405 nm. Quadruplicate samples were run for each dilution of EA(IgG) or EA(IgM)C. The mean and the standard error of the mean were calculated from the absorbance (A405nm)values. In most experiments the data were expressed as the Total Index (bound and internalized) and the Phagocytic Index (internalized) values where: 1 - (E A4os/EA(IgO) A4o5) × 100 = Index
51Cr labeling of red cells The measurement of binding and phagocytosis of 51Cr-labeled erythrocytes was performed by a modified method of Vogel and Rosenstreich (1979). A 5% suspension of washed E (109 cells/ml) in 0.9% saline was incubated with 100 /~Ci of sodium chromate (SaCr) (New England Nuclear, Boston, MA) for 1 h in a 37°C shaking water bath. After labeling, the cells were washed in 0.9% NaC1, resuspended at 5% and the EA(IgG) conjugates were prepared as described above. Adherent macrophage monolayers (2.0 × 105/well) were incubated for 1 h with 0.1 ml of the EA(IgG) dilution at 37°C in microtiter plates, washed thrice and designated wells were washed with hypotonic saline. The macrophage monolayers and associated E / E A were dissolved by addition of 0.3 ml of 0.5% SDS for 5 min. A 0.2 ml aliquot from each well was transferred to a Biovial (Beckman Instruments, Irvine, CA) and the radioactivity was counted in a gamma counter for 1 min.
Results
Comparison of the photometric and 51Cr-labelederythrocyte methods for quantitation of macrophage Fc phagocytic function The quantitation of Fc mediated phagocytic function of C3HeB/FeJ mouse peritoneal macrophages by the 51Cr-labeled erythrocyte (Fig. 1A) and the newly developed photometric (Fig. 1B) methods were compared under identical experimental conditions. Measurement of total phagocytic activity (binding and internalization) of macrophage-associated, SlCr-labeled erythrocytes or of erythrocyte hemoglobin absorbance at 405 nm yielded similar dose response kinetics of saturation and linear decrease in activity with decreasing IgG concentration. Quantitation of phagocytosis (internalized conjugates) by the 2 methods also gave comparable results. Re-expression of the A405nm data in the photometric assay (Fig. 1B) as the
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I cjG Dilution(x IC)3) Fig. 1. Comparison of (A) 51Cr-labeled erythrocyte (E) and (B) photometric assays for quantitation of Fc receptor function of C3HeB/FeJ oil elicited murine peritoneal macrophages. Adherent macrophages were incubated with EA(IgG) conjugates, prepared with increasing 2-fold dilutions of IgG (1 : 500-1 : 64,000), for 1 h at 37°C in microtiter plates. E were prelabeled with 51Cr only for the isotopic assay. Phagocytosis was quantitated either by the determination of macrophage-associated 5~C-labeled E or the photometric measurement of macrophage-associated E hemoglobin at 405 nm. See Materials and Methods for details. Each data point depicts the m e a n + SEM of Total (o o) and Phagocytic (© ©) functions of > 8 samples.
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(©
O) was derived as: Index = 1 - ( E A~os/EA(IgG ) A4o5)×100.
159
Total Index or Phagocytic Index (Fig. 2) gave similar kinetics and offered the advantage of normalizing the data to correct for variation between experiments in background absorbance values.
Effects of macrophage density on photometric Fc assay The effects of macrophage density on total and phagocytic indices are shown in Fig. 3. Similar kinetics of saturation and a dose related linear decrease in total and phagocytic functions with decreasing IgG concentration were observed with 4.0 and 2.0 × l0 s cells/well, but not with 1 × l0 s cells/well. Generally the magnitude of phagocytic function was increased proportionately with increasing macrophage effector density. Effects of incubation time and temperature on Fc-mediated binding and phagocytosis The optimum time of incubation of macrophages with EA(IgG) conjugates for maximum total and phagocytic functions in the photometric assay was determined to be 60 min at 37°C. Incubation for 30, 45, or 90 min produced only minor B.
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Fig. 3. Effect of macrophage density on Fc receptor mediated phagocytic function in the photometric assay. C 3 H e B / F e J elicited peritoneal macrophages were plated at 4 × 105 (O O), 2 × 105 ( e . . . . . . e ) or 1 × 105 ( e . . . . . . e ) cells/well in microtiter plates. EA(IgG) conjugates prepared with 2-fold dilutions of IgG were incubated with the adherent macrophage monolayers for 1 h at 37°C. The Total Index (A) and the Phagocytosis Index (B) were computed from photometric A405n m values. Each point depicts the mean + SEM of > 4 experiments.
160 TABLE 1 E F F E C T OF I N C U B A T I O N T E M P E R A T U R E ON Fc D E P E N D E N T B I N D I N G A N D PHAG(3CYTOSIS BY M A C R O P H A G E S a Temperature
IgG
Index b
dilution ( × 10 3)
Total
Phagocytic
Binding
4°C 37°C
1 :2
53.5 _+3.2 80.5 +0.8
2.9 _+1.2 33.4_+ 1.4
50.6 + 3.1 47.1 + 1.2
4°C 37°C
1 :4
45.3 + 3.5 70.7 + 2.8
0.7 + 0.5 26.1 + 2.2
44.6 + 3.4 44.6 + 3.4
4°C 37°C
1 :8
16.3 + 4.7 49.1 + 2.9
0.5 + 0.5 19.2 + 1.5
15.8 + 4.3 29.9 + 3.6
C 3 H e B / F e J oil elicited peritoneal macrophages (2 × 10S/well) were incubated either at 4°C or 37°C for 1 h with EA(IgG) conjugates prepared with IgG dilutions of 1:2000, 1:4000, or 1:8000 in microtiter plates using the photometric assay. See Materials and Methods for details. b Binding Index was computed as the net difference between Total Index (bound and internalized conjugates) and the Phagocytic Index (internalized conjugates). Each data point depicts the mean_+ SEM of 4 tests each in 2 separate experiments. a
B.
A. \
.50 o o
5o.
g. X I.O c'a 7,
\ C3 l-ieBIFed
,
t
I:0~, I:1
~i
i
~
t
i
~2
1:4 1:8 1:16 I:~
B/F'eJ I:05 I:1
1:2
1:4
1:8
1:16 I:~. 1:64
IgG Dilution (XIO3) Fig. 4. Comparison of Fc phagocytic function of Fc competent C 3 H e B / F e J (e . . . . . . e) and Fc-deficient C 3 H / H e J (e e) mouse macrophages by the photometric assay. Adherent cells (2 X 10S/well) were incubated with EA(IgG) conjugates prepared with varying dilutions of IgG for 1 h at 37°C. The Total Index (A) and the Phagocytosis Index (B) represent the mean + SEM of > 4 experiments.
161
differences in total or phagocytic indices (data not shown).To determine the effects of incubation temperature on binding and phagocytosis, macrophages were incubated for 1 h at 4°C or 37°C with EA(IgG) conjugates and the total and phagocytic indices were calculated (Table I). When incubated at 4°C the EA(IgG) conjugates were bound to Fc receptors on the macrophage, but were not internalized. Incubation at 37°C resulted in both Fc mediated binding and internalization. Comparison of these parameters indicated that the value of the Total Index at 37°C was nearly equivalent to the sum of the values for Total Index at 4°C and the Phagocytic Index at 37°C. Thus the Total Index (4°C) represents potential Fc receptor binding to the macrophage. Since this value is approximately equivalent to the net difference between the Total Index (37°C) and the Phagocytic Index (37°C) a theoretical binding index could be derived which reflects actual binding at 4°C. This relationship was shown to be valid for Total Index and phagocytic values only with higher IgG concentrations (1 : 2000 and 1 : 4000 dilutions) but not with a lower IgG concentration (1 : 8000) in which 4°C binding efficiency was markedly reduced when compared to 37°C. Binding at 37°C was approximately 60% and phagocytosis was approximately 40% of the Total Index at 37°C at IgG dilutions of 1:500 through 1 : 8000 (Fig. 2 and Table I).
Comparison of Fc competent and deficient mouse macrophage populations C 3 H / H e J mouse macrophages have reduced Fc phagocytic function as compared
x
~50 z_
1:40 1:80 1:160 1:320 S e r u m Complement Dilution
Fig. 5. Quantitation of macrophage C3b receptor dependent phagocytosis by the photometric method. EA(IgM) conjugates were coated with C3b by incubation with increasing 2-fold dilutions of homologous mouse serum (1 : 5-1 : 320 dilution). The resulting EA(IgM)C conjugates were incubated with adherent C3HeB/FeJ macrophages (2 x 105/well) for 1 h at 37°C and the Total Index (e e) and Phagocytosis (O O) quantitated by the photometric method. Each data point depicts the mean± SEM of 4 experiments. Controls which consisted of E or EA(IgM) exposure to macrophages were negligable (i.e., < 4% of Total A40~nm for peak test values).
162 to C3HeB/FeJ macrophages. This relationship was confirmed using the photometric assay (Fig. 4) in that both Total and Phagocytic functions were significantly lower with C3H/HeJ cells than with C3HeB/FeJ macrophages.
Photometric quantitation of C3b receptor dependentphagocytosis The photometric assay was applied to the measurement of macrophage C3b receptor mediated phagocytosis (Fig. 5). Conjugates prepared with EA(IgM) and various increasing dilutions of complement-containing serum yielded Total and Phagocytic Index values with linear dose response kinetics and saturation similar to that observed for Fc receptor function. Discussion
Existing quantitative methods for measuring macrophage Fc or C3b receptor dependent phagocytic functions depend largely on the use of isotopically labeled erythrocytes (Walker, 1977; Vogel and Rosenstreich, 1979) or rosette assays (Bianco et al., 1975). The 5aCr-labeled erythrocyte method offers advantages of sensitive and reproducible quantitation of phagocytic function whereas the rosette assays require laborious visual microscopic counting procedures. Disadvantages of the 51Cr-labeling method include time consuming labeling procedures, batch variation in specific activity of isotope, spontaneous shedding of label to yield variable background levels, and the problems of isotopic handling, storage, and disposal. We have developed a photometric microassay for quantitation of Fc or C3b phagocytic function which is patterned after the 51Cr_labeling method. The new method is based on measuring erythrocyte hemoglobin absorbance at 405 nm on an automated densitometer. Comparison of the 2 methods (Fig. 1) indicated that they were very similar in kinetics and sensitivity. Since the photometric method eliminates the use of isotope, it would appear to offer advantages over the isotopic labeling method. The photometric method was shown to quantitate macrophage Fc and C3b functions in a characteristic dose dependent relationship with saturation which was related to increasing IgG or C3b concentrations. To compensate for control background variation between experiments, normalization of the absorbance 405 nm data was accomplished by deriving a total and phagocytic index for expression of phagocytic activity. Increasing Fc phagocytic function with increasing macrophage effector density was demonstrated and distinct differences were detected between Fc deficient C3H/HeJ (Vogel and Rosenstreich, 1979) and Fc competent C3HeB/FeJ mouse macrophages in their Fc dependent phagocytic functions. Binding of IgG opsonized erythrocytes at 4°C, which prevents internalization of Fc receptor bound conjugates, was found to be equivalent at high IgG concentration to the net difference between total (bound and internalized complexes) and phagocytic (internalized complexes) index values derived at 37°C. A constant rate of binding and phagocytosis occurred at 37°C with higher concentrations of IgG. Thus the photometric assay allows approximation of Fc dependent binding to macrophages at 37°C without the necessity for binding assays at 4°C or the use of iodoacetic acid or other inhibitors of phagocytosis.
163
Additional advantages of the photometric assay include the ability to quantitate Fc function in fixed plates stored longer than 1 month without change in phagocytic index values as well as an accurate means of accounting for possible loss of adherent macrophages during studies requiring long-term in vitro culture.
References Bianco, C., F. Griffin and S. Silverstein, 1975, J. Exp. Med. 141, 1278. Leu, R.W., S.M. Hefley and M.J. Herriott, 1983, Cell. Immunol. 80, 31. Shen, L., P.M. Guyre and M.W. Fanger, 1984, Mol. Immunol. 21, 167. Unkeless, J.C. and H.N. Eisen, 1975, J. Exp. Med. 142, 1520. Vogel, S.N. and D.L. Rosenstreich, 1979, J. Immunol. 123, 2842. Walker, W.S., 1977, J. Immunol. 119, 367.